201

Molecular Control Of Articular Cartilage Degeneration By Transforming Growth Factor Alpha

Tom Appleton, PhD, CIHR Group in Skeletal Development & Remodeling, London, ON, Canada; Shirine Usmani, HBSc, CIHR Group in Skeletal Development & Remodeling, London, ON, Canada; Frank Beier, PhD, CIHR Group in Skeletal Development & Remodeling, London, ON, Canada

Articular cartilage degeneration is a hallmark of osteoarthritis (OA). We identified increased expression of transforming growth factor alpha (TGF α) in degenerating OA knee cartilage (1,2). We determined that increased TGFα signaling modified chondrocyte cytoskeletal organization, increased catabolic and decreased anabolic gene expression and suppressed Sox9. We hypothesized that these effects are mediated by Rho/ROCK and MEK/ERK signaling pathways. Primary cultures of chondrocytes and articular ostechondral explants were treated with pharmacological inhibitors of MEK1/2 (U0126), ROCK (Y27632), Rho (C3), p38 MAPK (SB202190) and PI3K (LY294002) to elucidate pathway involvment. Using ELISA we determined that stimulation of primary chondrocytes with TGFα activates RhoA. Reciprocally, inhibition of RhoA/ROCK but not other signaling pathways prevents modification of the actin cytoskeleton in response to TGFα. Inhibition of MEK/ERK signaling rescued the TGFα-induced abrogation of anabolic gene expression including SOX9 mRNA and protein levels. Inhibition of MEK/ERK, Rho/ROCK, p38 MAPK and PI3K signaling pathways differentially controlled the induction of MMP13 and TNFα gene expression. TGFa also induced the expression of CCL2 specifically through MEK/ERK activation. In turn, CCL2 treatment induced the expression of MMP3 and TNFα. In conclusion, Rho/ROCK signaling mediates TGFα-induced actin cytoskeleton reorganization, while MEK/ERK signaling mediates signals resulting in the suppression of Sox9, anabolic genes activated by Sox9 and CCL2. CCL2, in turn, induces the expression of MMP3 and TNFα, two potent catabolic factors known to be involved in OA. These pathways may represent strategic targets for interventional approaches to treating cartilage degeneration in osteoarthritis.

REFERENCES: 1C.T.G. Appleton et al. (2007). Arthritis Rheum 56(6): 1854-1868. 2C.T.G. Appleton et al. (2007), Arthritis Rheum 56(11):3693-705.

202

Hyperglycemia Induced Oxidative Stress and MnSOD Deficiency of Endothelial Progenitor Cells Contribute to Impaired Angiogenesis and Wound Healing in Type 2 Diabetes

Eric Marrotte, MS, Michigan State University, East Lansing, MI, USA; Jeffrey Hakim, BS, Michigan State University, East Lansing, MI, USA; Dandan Chen, MD, Michigan State University, East Lansing, MI, USA; Alex Chen, MD, PhD, Michigan State University, East Lansing, MI, USA

Introduction: Circulating endothelial progenitor cells (EPCs) are both reduced and dysfunctional in diabetes, but the mechanisms are incompletely understood. Normal EPCs express intrinsically high MnSOD levels and are thus resistant to oxidative stress. We hypothesized that oxidative stress reduces EPCs and their angiogenesis function, resulting in refractory wound healing in type 2 diabetic mice. Methods and Results: Adult male (C57BLKS/J, 10–14 weeks) type 2 diabetic db/db and their normal littermates db/+ mice (glucose 270.4±38.3 vs. 153.6±4.8 mg/dL, n=5, p<0.01) were used. Circulating EPC number measured by flow cytometry was significantly lower in db/db vs. db/+ mice (1.45±0.5 vs. 3.6±0.4% sca-1/flk-1 double-positive cells, n=4–5, p<0.05). Mitochondrial oxidative stress was increased in both db/+ and db/db mice EPCs incubated in high glucose EGM-2 (MitoSox, P<0.05). The levels of superoxide anion (O2-) were markedly elevated in circulating db/db EPCs vs. db/+ EPCs (Flk-1/DHE-positive cells n=4, p<0.05). MnSOD mRNA and protein expressions in EPCs were decreased by >50% (real-time RT-PCR, p<0.05) and >60% (Western Blot, p<0.01) in db/db vs. db/+ mice, respectively. The angiogenesis capacity of EPCs from db/db type 2 diabetic mice was significantly decreased (Matrigel tube formation assay, tubes/high powered field, n=4, p<0.05 vs. db/+). The impaired angiogenesis in diabetic EPCs was significantly improved following adenoviral vector-mediated MnSOD gene transfer (transduced db/db EPCs vs. non-transduced db/db EPCs, n=4, p<0.05). In db/db mice, the course of excisional wound closure (6-mm punch biopsy, n=10, p<0.01 vs. db/+) was markedly delayed, paralleled with impaired wound angiogenesis (CD31+ capillary number, n=4, p<0.05 vs. db/+). Cell therapy of db/+ EPCs (106 cells) onto the wounds of db/db diabetic mice induced a marked acceleration of wound closure rate vs. db/db EPC (106 cells) wound therapy (n=10, p<0.05). Conclusion: MnSOD deficiency in EPCs and hyperglycemia induced oxidative stress contribute to impaired angiogenesis and wound healing in type 2 diabetic mice. Gene transfer of MnSOD augments diabetic EPC angiogenesis function, and cell therapy of db/+ EPCs accelerates excisional wound healing more rapidly than db/db EPC therapy in type 2 diabetic mice.

203

Median Eminence Compression: A Model for the Evaluation of Neuroprotective Molecules and its Application for Assessment of the Neuroprotective Efficacy of Ciliary Neurotrophic Factor

Nathan Herman, B.S., University of North Dakota, Grand Forks, ND, USA; John Watt, PhD, University of North Dakota, Grand Forks, ND, USA

Poor neuron survival and functional recovery following injury is the underlying basis of the devastating effects of traumatic brain injury. Common in the industrialized world, traumatic brain injury is largely a condition of young adults, with smaller peaks in young children and the elderly. The high incidence of long-term disability following brain injury is associated with exorbitant economic and human costs. A multitude of experimental treatment strategies have arisen to prevent neuron loss, enhance post-injury recovery, and minimize the occurrence of long-term complications. OBJECTIVE: The purpose of this work was to establish a simple and reliable in vivo model to assess the effects of experimental therapies on survival and function of injured neurons. The median eminence compression model was applied to evaluate the ability of the neurotrophic cytokine CNTF to improve neuron survival following injury. METHODS: Adult male Sprague-Dawley rats underwent surgical compression of the median eminence, sham lesion, compression with continuous intracerebral recombinant CNTF infusion (0.05, 0.5, 5 ng/hr) or compression with infusion of artificial cerebrospinal fluid vehicle. To characterize the effect of lesion on magnocellular neuron function, water intake, urine output and urine osmolality were measured and recorded daily. Neuron survival was assessed by cell counts performed on brain sections collected from animals sacrificed 7, 14, and 28 days following surgery. RESULTS: Median eminence compression resulted in a syndrome of polydipsia with copius production of dilute urine, mimicking human post-traumatic diabetes insipidus. When compared to sham lesioned rats, median eminence compression was found to result in a 40-50% reduction in magnocellular neurons. At the doses administered, continuous intracerebral infusion of recombinant rat CNTF did not improve survival of magnocellular neurons following median eminence compression. CONCLUSION: Median eminence compression presents a convenient model to assess therapeutic potential of neuron sparing drugs to enhance neuron survival following axon injury.

204

Non-Invasive Evaluation of Breast Cancer Response to Neoadjuvant Chemotherapy using Diffuse Optical Spectroscopic Imaging (DOSI)

Shwayta Kukreti, PhD, University of Illinois-Urbana,Champaign/University of California-Irvine, Urbana, IL, USA; Vaya Tanamai, BS, University of California-Irvine, Irvine, CA, USA; Saurabh Kukreti, HS, University of Illinois, Urbana, IL, USA; Albert Cerussi, PhD, University of California, Irvine, CA, USA; David Hsiang, MD, University of California, Irvine, CA, USA; John Butler, MD, University of California, Irvine, CA, USA; Rita Mehta, MD, University of California, Irvine, CA, USA; Bruce Tromberg, PhD, University of California, Irvine, CA, USA; Enrico Gratton, PhD, University of California, Irvine, CA, USA

Near Infrared (NIR) optical methods provide contrast based on quantitative functional changes in tissue.  NIR techniques have shown to be sensitive to changes in breast physiology from disease by quantifying oxyhemoglobin, deoxyhemoglobin, oxygen saturation, fat and water content. We have already shown that tumors display changes in these parameters; however, it is debatable whether they are specific signatures of cancer. Recently it was shown that double-differential analysis of breast tissue absorption spectra from cancer-containing tissue reveals specific tumor components (STC). The STC absorption signature is an intrinsic spectroscopic biomarker characterized by specific NIR absorption bands. Here we apply the double-differential analysis to spectra from tissue of patients on neaoadjuvant chemotherapy. We ask the following question: Can residual disease after neoadjuvant chemotherapy be detected using Near Infrared optical biomarkers?

            A Diffuse Optical Spectroscopic Imaging (DOSI) instrument was used to non-invasively obtain the absorption and scattering spectra from 650-1000 nm from the breast tissues of patients pre and post-neoadjuvant chemotherapy. 6 patients (5 complete and 1 partial pathological responses), ages 32-53 years, with stage 2 or 3 cancers obtained the following neoadjuvant chemotherapy regimen: 2-4 cycles of doxorubicin-cyclophosphamide (AC), followed by 2-4 cycles of carboplatin-taxol/abraxane-avastin (nabTC). We used the double-differential method to analyze the NIR absorption spectra. Briefly, this is a self-referencing method to reveal only the spectral differences between normal and tumor tissue.

Results show characteristic STC spectral signatures were present in all tumor-containing regions in the pre-chemotherapy measurements. For the patients classified as exhibiting complete pathological response, the final STC spectrum was dissimilar from the pre-treatment spectrum. Upon further analysis, we found that this spectrum is more similar to the normal tissue than the partial. For the patient classified as exhibiting partial response, we observe that the final STC spectrum shared some features with the pre-treatment STC spectrum. Here we present pilot data suggesting NIR optical methods can potentially be used to non-invasively evaluate tumor specific changes metabolic states to neoadjuvant chemotherapy. Future work will determine the prognostic utility of the STC biomarker as well as investigate other therapy regimens.

205

Wnt/B-catenin Signaling In Diabetic Retinopathy

Michael Grassi, MD, University of Chicago, Chicago, IL, USA; Xiangqian Song, PhD, University of Chicago, Chicago, IL, USA; Soo-yuong Park, PhD, University of Chicago, Chicago, IL, USA; Qingbei Zhang, Phd, University of Chicago, Chicago, IL, USA; Yves Lussier, PhD, University of Chicago, Chicago, IL, USA; Rosie Xing, PhD, University of Chicago, Chicago, IL, USA; Rosie Xing, PhD, University of Chicago, Chicago, IL, USA

 Purpose: Wnt/β-catenin signaling has been found to regulate angiogenesis an important sight threatening complication of diabetic retionpathy. In this study, we examine the effects of high glucose on Wnt/β-catenin signaling in a conditionally immortalized rat retinal endothelial cell line (TRiBRB).
Methods: TRiBRB cells were cultured in 5mM glucose/DMEM/10%FBS medium at 33 degrees. Cells were treated with 15mM glucose/DMEM and 0.5% human albumin for 30 minutes to 48 hours. Western blots were performed and membranes were blocked with anti-β-catenin, anti-GSK3β, anti-phosphorylated GSK3β and anti-active β-catenin antibodies. Total RNA was isolated from cells with a Qiagen RNeasy Mini Kit. For real-time PCR, 100 ng of reverse transcribed total RNA was used in a 25 μl volume. The cells were cultured in Matrigel under 5mM or 15mM glucose/DMEM/0.5% human albumin for two weeks.
Results: Stimulation of TRiBRB cells with 15 mM glucose produced initial GSK3 inhibition followed by activation and a corresponding biphasic response in β-catenin protein levels. Western blot revealed that at 4 hours post high glucose exposure, total β-catenin protein levels were increased then reduced below control levels from 18 hours to 48 hours. Corresponding with the changes in -catenin, western blot analysis of active GSK3β (de-phosphorylated form) demonstrated that there was a reduction in the active form of GSK3β at 4 hours, which then increased by 18 hours. To examine the transcriptional activity associated with -catenin activation, real-time PCR demonstrated that mRNA expression of cyclin D1 and VEGF, two transcriptional targets of Wnt/β-catenin signaling, increased initially following high glucose exposure. In 3-D culture, cells grown in 15 mM glucose demonstrated decreased sprouting and impaired tube formation by seven days of culture.
Conclusions: High glucose exposure results in a biphasic response in both β-catenin and GSK3β signaling in TRiBRB cells. Further, high glucose treatment inhibited TRiBRB cell sprouting and formation of tube-like structures in Matrigel 3-D culture. These findings suggest that Wnt/β-catenin signaling may be an important player in regulating retinal angiogenesis in response to hyperglycemia seen in diabetic patients.

206

Cognitive Impact Of Interictal Spikes In A Modified Model Of Temporal Lobe Epilepsy

Jonathan Kleen, BS, Dartmouth Medical School, Hanover, NH, USA; Pierre-Pascal Lenck-Santini, PhD, Dartmouth Medical School, Lebanon, NH, USA; Gregory Holmes, MD, Dartmouth Medical School, Lebanon, NH, USA

EEGs recordings of epileptic patients during periods between seizures (interictal periods) often show bursts of high-amplitude spikes that stem from groups of aberrant neurons discharging synchronously. These spikes are exclusively present in epileptic syndromes, and thus are clinically useful in that they can assist in diagnosing many of these conditions, including temporal lobe epilepsy (TLE). However, depending on the brain region where they originate, interictal spikes may briefly disrupt neural processes occurring in that particular region, and are thus hypothesized to contribute to cognitive slowing over time. We are investigating interictal spikes with a modified rat model of temporal lobe epilepsy. Pilocarpine infusion into the ventral CA1 region of the hippocampus, a temporal lobe structure, produces well-delineated interictal spikes. With depth electrodes in both hippocampi as well as the ipsilateral right prefrontal cortex, we observed the time course of the development of spike-generating foci at the infusion site (n=5), with spikes emergent at 11-14 days post-infusion. All rats developed interictal spikes, though two rats required a second infusion. Spontaneous seizures are generated at the infusion site, but have been somewhat infrequent in this model. We have also observed the establishment of independent spike-generating mirror foci in the contralateral hippocampus and similar spike foci in the prefrontal cortex. The autonomous spike foci may have synergistic impact on working memory processes concerting between these regions. To examine our hypothesis of functional neural circuitry disruption by spikes, we are utilizing an operant behavior task called delayed-match-to-sample (DMTS), which employs hippocampal-dependent short-term memory. We are assessing whether animals make more errors on trials with spikes than trials without, which avoids the confounding variables of other behavioral paradigms, such as structural damage and long-term changes in cognitive function. We are also analyzing the consequences of spikes in specific epochs of the task, to isolate memory encoding vs. recall, etc. Additionally, we are weighing the influence of the autonomous spikes engendered in the each hippocampi versus the ipsilateral PFC. Preliminary data indicates that spikes briefly obliterate transient memory storage in the hippocampus, and that certain subtypes of spikes have differential impacts, possibly implicating multiple aberrant network reorganizations in this TLE model.

207

Targeted Regulation of Specific Hematopoietic Stem Cell Subsets by Prostaglandin E2

Rebecca Porter, B.S., University of Rochester, Rochester, NY, USA; Rebecca Porter, BS, University of Rochester, Rochester, NY, USA; Benjamin Frisch, BS, University of Rochester, Rochester, NY, USA; Laura Calvi, MD, University of Rochester, Rochester, NY, USA

Hematopoietic stem cells (HSC) are pluripotent cells responsible for the establishment and renewal of the entire hematopoietic system. HSCs can be subdivided into long-term (LT-HSC), short-term (ST-HSC) and multipotent progenitors (MPP) based on quiescence and repopulating capability. Specific regulation of these subsets is poorly defined and a better understanding would be useful in clinical situations necessitating bone marrow reconstitution. Parathyroid hormone (PTH) expands all three subpopulations of HSC by activating osteoblasts in the HSC niche. Since PTH increases local production of prostaglandin E2 (PGE2) by osteoblasts, we investigated the effect of PGE2 on HSC. PGE2 treatment demonstrated a time and dose dependent 2.75-fold increase in the HSC-enriched lineage- Sca-1+ c-kit+ (LSK) population of bone marrow cells compared with vehicle treated mice. PGE2 dependent HSC increase was confirmed by competitive repopulation analysis. Surprisingly, superior repopulation was only found in the myeloid lineage for 6 weeks and in the lymphoid lineage for 16 weeks after transplantation, suggesting that PGE2 specifically expands the less quiescent but limited self-renewing ST-HSC and MPP. Further, flow cytometric analysis of subpopulations showed significant increases in Flt3+ ST-HSC (0.0273 vs. 0.0140%, p=0.0307, n=4 per group) and MPP (0.0305 vs. 0.0195%, p=0.0070) cells from PGE2 treated compared to vehicle treated mice. LT-HSC remained unchanged, (0.0123 vs. 0.0078%, p=0.1069) consistent with the competitive repopulation data. Colony Forming Unit-Spleen (CFU-S) assays demonstrated a significant increase in CFU-S at day 8 (corresponding to ST-HSC) with BMMC from PGE2 treated vs. vehicle treated mice. There was no difference in CFU-S at day 10-12 (LT-HSC), further confirming the selective ST-HSC expansion. To investigate the mechanism of PGE2 mediated expansion of ST-HSC, we assayed the proliferation of each subset in vitro by BrdU labeling and DAPI analysis of DNA content. Whole bone marrow was treated with 10-6 M PGE2 or vehicle in the presence of BrdU for 90 minutes. The percentage of cells that underwent S phase was higher in the PGE2 treated compared to the vehicle treated wells in the ST-HSC subpopulation (1.48 fold) and the MPP subpopulation (1.27 fold). Like the in vivo results, the LT-HSC proliferation was not increased. Next, expression of the four PGE2 receptors, EP1-EP4, was analyzed by quantitative RT-PCR. While expression of EP2 and EP4 was barely detectable in Flt3- LT-HSC but was high in Flt3+ ST-HSC and MPP, EP1 and EP3 showed the opposite effect. Taken together, these results suggest that PGE2 causes an increase in proliferation of ST-HSC and MPP, which may be explained in part by the differential expression of the EP receptors across subpopulations. These findings demonstrate a novel regulatory mechanism of specific subsets of HSCs and could be exploited in clinical situations requiring rapid bone marrow reconstitution.

208

Activated MSC: A New Method for Suppression of Alloreactive T cells

GW Douglas, BS, University of Illinois at Chicago, Chicago, IL, USA; David Polchert, BS, University of Illinois at Chicago, Chicago, IL, USA; Amelia Bartholomew, MD, University of Illinois at Chicago, Chicago, IL, USA

MSC reliably suppress large scale T cell proliferation in response to polyclonal stimulation in vitro. In contrast, with allogeneic mixed lymphocyte cultures of variable stimulation, MSC suppression is also variable; MSC do not completely abrogate lymphocyte proliferative responses between all donor and recipient pairs. We hypothesized that the magnitude of MSC-induced T cell suppression may be dependent on the degree of MHC matching between MSC and antigen presenting cell (APC). To test this hypothesis, MSC of different MHC were tested for their ability to suppress anti-host T cell activity in a lethal graft versus host disease model. B6 (H2b) recipients underwent lethal irradiation and reconstitution with Balb (H2d) bone marrow and Balb splenocytes. MSC of Balb, B6, C3H (H2k), or CAF1/J (F1 hybrid H2d/a) were administered 2 days after bone marrow transplant and tested for their ability to prevent GVHD mortality. Balb and B6 MSC provided the best suppression of T cell activity with 70% 40 day-survival, a statistically significant increase from control animals that did not receive MSC, (30%, p

209

Sensitive Detection of Drug-Resistant BCR-ABL Mutations Using Picoliter Pyrosequencing

Ralph DeBiasi, B.A., Memorial Sloan Kettering Cancer Center, New York, NY, USA; Neil Shah, MD, Univerisity of California San Francisco, San Francisco, CA, USA; Agnes Viale, PhD, The Sloan Kettering Institute, New York, NY, USA; Ronald Paquette, MD, Univerisity of California Los Angeles, David Geffen School of Medicine, Los Angeles, CA, USA; Charles Sawyers, MD, Memorial Sloan Kettering Cancer Center, New York, NY, USA

Chronic myeloid leukemia (CML) is caused by a chromosomal translocation that results in the BCR-ABL oncogene.  The tyrosine kinase inhibitor imatinib is effective as a first-line therapy for CML, as it blocks the activity of the oncogenic BCR-ABL fusion protein.   Unfortunately many patients develop resistance, whereupon treatment with dasatinib, a “second generation” kinase inhibitor, effectively blocks most mutant forms of BCR-ABL.  Resistance to dasatinib may also develop due to the evolution of new and compound mutations, but it is not known when these mutations begin to emerge or how diverse these mutations are. 

Determining the molecular mechanisms of mutation evolution may help to elucidate whether combination therapy would be more effective than current sequential treatment plans.

Our goals were to determine if picoliter pyrosequencing is effective in detecting low-frequency mutations in BCR-ABL in patients receiving targeted cancer therapy, to determine if compound resistance mutations are present before relapse, and to determine if the patients with compound mutations are different from those without compound mutations.  

Picoliter pyrosequencing was performed at ~12,000x coverage to detect evolving and compound mutations in BCR-ABL in patients receiving dasatinib treatment.  This technique allowed us to detect allele frequencies down to 0.5%, a clear advantage over traditional "Sanger" sequencing's lower limit of ~ 25%.

We detected low-frequency dasatinib-resistant mutations in patient samples before clinical presentation of secondary relapse.  This indicates that targeted cancer therapies may be more effective when administered in combination rather than sequentially so as to prevent the outgrowth of mutant drug-resistant clones.

210

Nanoscale Sensors for Multi-Modal Detection of Genotoxic Agents

Daniel Heller, BA, Massachusetts Institute of Technology, Cambridge, MA, USA

Reactive oxygen species and active alkylating chemotherapeutics are important bioanalytes but difficult to measure selectively, in vivo, or in real time.  A new nanoscale optical sensor platform under development can now detect as well as identify these analytes in situ. 

Carbon nanotube-DNA complexes, formed by individually encapsulating nanotubes with oligonucleotides, respond in real time to these agents via multiple optical modes, giving each analyte a distinct optical signature.  Single-walled carbon nanotubes are environmentally sensitive and emit tissue-transparent near-infrared fluorescence.  By their encapsulation in short strands of synthetic DNA, we introduce a selective handle for changes in their emission, which responds to the polarity of the solvent medium.  The complexes consequently respond to DNA-damaging oxygen species or alkylating agents, by red-shifts in emission energy as well as optical quenching, giving multiple detection modes which result in analyte specificity.  

The nanotube-DNA complexes exhibit uptake into mammalian cells via endocytosis without cytotoxic effects and emit from within live tissues. Upon introduction of alkylating agents and oxidative species, the complexes transduce analyte activity information live and in situ.  This platform is being developed as a research and diagnostic tool for free radical biology, drug discovery, and pharmacodynamics.

211

Development Of A Specific Assay For And Characterization Of The Microtubule-Inhibitor Activated Bcl-Xl Kinase, A Key Determinant Of Anti-Mitotic Drug Sensitivity

David Terrano, Bachelor of Arts, University of Arkansas for Medical Sciences, Little Rock, AR, USA; Timothy Chambers, PhD, University of Arkansas for Medical Sciences, Little Rock, AR, USA

Microtubule inhibitors (MTIs) are among the most actively used cancer chemotherapeutic agents. Resistance is a major hurdle to MTI efficacy. The anti-apoptotic protein Bcl-xL is a key determinant of chemotherapeutic resistance in multiple tumor cells. Treatment with MTIs induces Bcl-xL phosphorylation during mitotic arrest, which is followed by dephosphorylation and subsequent apoptosis. Current evidence suggests that phosphorylation disables Bcl-xL’s anti-apoptotic activity, which may represent a previously unappreciated determinant of drug resistance and/or sensitivity. The kinase responsible is unknown. Our goal was to better characterize this key signal transduction pathway. We developed a highly specific in vitro Bcl-xL kinase assay using a novel peptide substrate, FL62, harboring the vinblastine-induced phosphorylation site Ser62. Significant in vitro phosphorylation of FL62 was observed only in extracts and size separated protein from KB-3 tumor cells treated with MTIs. Following vinblastine-treatment for 48 h, the point at which cellular Bcl-xL is dephosphorylated and apoptosis occurs, or doxorubicin, a DNA-damaging agent, kinase activity was at the level of controls, further validating the assay. The same size separation fractions with FL62 kinase activity also phosphorylated full length Bcl-2, which supports previous data that the same kinase phosphorylates Bcl-xL and Bcl-2 following MTI-treatment. As predicted, FL62 phosphorylation also correlated with the dose dependent increase in G2/M arrest using the dose range of 0-300 nM vinblastine. Thus, the FL62 kinase assay detects cellular Bcl-xL kinase active only during MTI-induced mitotic arrest. To start purification with a less complex mixture, we determined the subcellular location of kinase activity. FL62 phosphorylation was maximal in the mitochondrial fraction, which is expected as Bcl-xL is predominantly mitochondrial. Following salt extraction of mitochondria, robust FL62 phosphorylation was maintained in the soluble fraction. Thus, purification can be initiated with the less complex soluble salt extracted mitochondrial protein. Basic characterization studies indicated that the Bcl-xL kinase has a native molecular weight in the range 150-190 kDa and it does not co-elute with the c-Jun NH2-terminal kinase (JNK) isozymes, which were the primary candidates. Furthermore, recombinant, active JNK1 did not phosphorylate FL62, further ruling out JNK as the Bcl-xL kinase. In summary, we have a robust in vitro Bcl-xL kinase assay that detects the cellular Bcl-xL kinase activity only from G2/M arrested cells.  The kinase also phosphorylates Bcl-2 and that is not JNK. Furthermore, the Bcl-xL kinase is in the mitochondrial fraction, which will provide a less complex mixture from which to begin purification and identify this key enzyme linking MTI-induced G2/M arrest and apoptosis.

212

Oncolytic Potential of a Replication-Competent rVSV In a Rat Model of Intracranial Glioma

Erika Dillard, BS, University of Tennessee Health Science Center Dept of Molecular Science; Neurosu, Memphis, TN, USA; Michael Whitt, PhD, University of Tennessee Health Science Center Department of Molecular Science, Memphis, TN, USA; Himangi Jayakar, PhD, University of Tennessee Health Science Center Department of Molecular Science, Memphis, TN, USA; Christopher Duntsch, MD, PhD, University of Tennessee Health Science Center Department of Neurosurgery, Memphis, TN, USA; Qihong Zhou, MD, PhD, University of Tennessee Health Science Center Department of Neurosurgery, Memphis, TN, USA

Vesicular stomatitis virus (VSV) is a neurotropic, enveloped, negative-strand RNA virus currently being evaluated for its use as an oncolytic agent in treating a variety of tumors. VSV infection is largely obstructed by the antiviral actions of the interferon (IFN) pathway, a pathway known to be defective in many cancers as this provides a survival advantage for tumor cells while rendering them defenseless against many viral infections. While VSV has shown good safety and efficacy in a number of different animal model systems, it has not been sufficiently evaluated as a potential therapeutic for brain tumors in immunocompetent animals primarily due to viral-induced toxicity permitted by the unique immune environment of the CNS. One approach to circumvent this toxicity is to enhance vector safety by introducing mutations in the matrix (M) protein of VSV, a viral protein responsible for VSV's cytopathic effects including inhibition of host cell gene expression and of nucleocytoplasmic transport. Such mutations diminish the ability of M to block gene expression, allowing genes important in the IFN antiviral pathway to be expressed hence protecting cells with functioning IFN responses. We are investigating if rVSV vectors possessing specific mutations in the M protein at sites shown to be involved in cytopathogenesis could be used to treat high-grade astrocytic tumors. We examined a replication-competent M mutant vector, NCP12.1, in an intracranial rat glioma model using immunocompetent rats. Tumor size and survival were determined and compared to untreated tumor-bearing animals.Our results show that in animals sacrificed at 15 days post-infection, one direct intracranial injection of 109 pfu of NCP12.1 significantly reduced tumor load by 66% in rats with established F98 intracranial gliomas. Survival of NCP 12.1 treated animals was increased by 9 days compared to untreated animals, however, tumor size of these animals was not significantly different demonstrating that effects on tumor load are not permanent. These results indicate that M mutant rVSV's such as NCP 12.1 are relatively safe and may provide an alternative for the treatment of intracranial glioma. However, in comparing our results to studies using immunocompromised animals, it is likely that the immune system creates an obstacle in reaching the optimal efficacy of VSV in the treatment of glioma. Further studies need to be done to fully address the role of the immune system in rVSV therapy in order to achieve full oncolytic potential.

213

Where The HEK Does Piggybac Insert Itself In The Human Genome?

Tara Albano, BA - Biochemistry, Mount Sinai School of Medicine, New York, NY, USA; Peter Ward, PhD, Mount Sinai School of Medicine, New York, NY, USA; Christopher Walsh, MD/PhD, Mount Sinai School of Medicine, New York, NY, USA

Background: Vectors capable of efficient, site-specific integration are needed for human gene transfer. Transposons are mobile genetic elements that integrate transgenes into host cell genomes. The piggybac transposon system shows a higher integration activity than other transposases when tested in human cell lines. Objective: To determine the integration sites of piggybac-mediated transposition in the human-derived cell line, HEK293. Methods: Target cells were transduced with a plasmid containing the piggybac transposase and carrying green fluorescent protein (GFP) cDNA. Integration analysis was performed using Ligation-Mediated polymerase chain reaction (LM-PCR). Briefly, this protocol comprises: 1) a restriction digest of genomic DNA from transduced cells; 2) ligation of a known adapter sequence onto the digested DNA; 3) a nested PCR with primers designed to anneal to the adapter and a known sequence of integrated DNA. Amplification of DNA between these two sites should yield a sequence with a junction between plasmid and genomic DNA, thus indicating where the transposase inserted the transgene in the genome. Results: A junction was formed between the 3 end of the inserted plasmid DNA and genomic DNA. The 25-base genomic sequence was analyzed using NCBIs Basic Local Alignment Search Tool (BLAST), and matched up with a sequence on chromosome two. This locus of insertion is in a non-coding region of the chromosome, over one million bases away from any known gene in either direction. This suggests that, in the HEK293 cell line, the piggybac transposon system acts in a region that lacks functional genes. Conclusions: This work will provide the foundation for further work on the use of the piggybac system for the delivery of an assortment of cDNAs in an integrated and site-specific fashion. Future studies should seek to determine piggybacs integration pattern in different cell types.

214

Igm Against Cryptococcus Neoformans Glucosylceramide Is Found In Sera Prior To The Dissemination Of The Fungus To The Central Nervous System

Ryan Rhome, MD, PhD Student, Medical University of South Carolina, Charleston , SC, USA; Maurizio Del Poeta, MD, Medical University of South Carolina, Charleston , SC, USA; Maurizio Del Poeta, MD, Medical University of South Carolina, Charleston , SC, USA

Cryptococcus neoformans (Cn) is an environmental fungal pathogen that, once inhaled, can disseminate to the central nervous system where it causes life-threatening meningo-encephalitis. Current methods for the diagnosis of infection are unable to identify patients at risk of developing a disseminated disease. In previous studies we showed that a mutant strain of Cn that can not produce glucosylceramide (Cn delta gcs1) does not cause meningo-encephalitis in mice because it does not grow in the lung. Thus, by favoring fungal growth in the lung, glucosylceramide (GlcCer) is required for Cn to cause a disseminated disease. Since this fungal glycolipid is immunogenic, we reasoned that it would stimulate an antibody response. Thus, we monitored the IgM and IgG levels against Cn GlcCer in sera of infected mice. The results show that in contrast to IgG, IgM anti-GlcCer is found in sera of infected mice when Cn cells are exclusively localized in the lung and not in other organs. We produced IgM monoclonal antibody against Cn GlcCer and showed that it has high specificity against fungal but not mammalian GlcCer. This study suggests that the presence of IgM against Cn GlcCer in sera can predict the dissemination of the fungus from the lung to the brain, promoting this test as an early diagnostic method for fungal infection in patients at risk of developing a disseminated disease.

PRE Median*

POST Median*

p-value

Actual codes attended

3-5 (1,9)

3-5 (3,9)

0.061

Actual codes with an active role

1-2 (0,5)

1-2 (1,5)

0.093

Mock codes observed

1-2 (1,2)

3-5 (1,5)

<0.001

Mock codes participated

1-2 (0,2)

3-5 (1,5)

<0.001

Kristen J. Nadeau, MD, University of Colorado Health Sciences/The Children’s Hospital (Kristen.nadeau@uchsc.edu)