Therapeutic potential of p53 restoration in spontaneous tumors

Over 80% of p53 mutations found in human cancers are p53 missense mutations. Recent studies have shown that p53 restoration leads to tumor regression in mice with p53 deletions, but the therapeutic efficacy of p53 restoration in tumors containing p53 missense mutations has not been evaluated. Since p53 mutant such as p53R172H has gain-of-function activities and dominant-negative effect that repress wild type p53, the activity of restored wild-type p53 might be compromised by the mutant p53 in tumors. We hypothesized that p53 restoration in tumors with the p53R172H mutation may be less therapeutically effective as p53 restoration in tumors null for p53. I tested this hypothesis by comparison of the therapeutic outcomes of p53 restoration in mice with spontaneous tumors that either lacked p53 or contained the p53R172H mutation. While p53 restoration causes tumor regression in mice lacking p53, the same p53 restoration halts tumor progression in mice with the p53R172H mutation. This phenotypic difference suggests a dominant-negative activity of the mutant p53. Moreover, I showed that the mutant p53 only inhibits part of the activity of the restored wild-type p53 and that the remaining wild-type activity still causes a delay in tumor progression. We conclude that p53 restoration has therapeutic potential in p53R172H tumors via suppression of tumor progression. This knowledge is of critical importance for p53 targeted cancer therapy because many patients with cancers harbor p53 missense mutations rather p53-null mutations. Since p53R172H mutation represents one of the most frequent and potent p53 missense mutations observed in human cancers, the current findings implicates that p53 restoration may be therapeutically important not only in human cancers characterized by loss of p53 alleles but also in those in which p53 missense mutations play an important pathogenetic role. ^

Therapeutic Targeting of ATP7B in Ovarian Carcinoma.

PURPOSE: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. EXPERIMENTAL DESIGN: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). RESULTS: ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC(50) levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH(2)-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. CONCLUSION: These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance.

Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression.

BACKGROUND: TRAIL plays an important role in host immunosurveillance against tumor progression, as it induces apoptosis of tumor cells but not normal cells, and thus has great therapeutic potential for cancer treatment. TRAIL binds to two cell-death-inducing (DR4 and DR5) and two decoy (DcR1, and DcR2) receptors. Here, we compare the expression levels of TRAIL and its receptors in normal oral mucosa (NOM), oral premalignancies (OPM), and primary and metastatic oral squamous cell carcinomas (OSCC) in order to characterize the changes in their expression patterns during OSCC initiation and progression. METHODS: DNA microarray, immunoblotting and immunohistochemical analyses were used to examine the expression levels of TRAIL and its receptors in oral epithelial cell lines and in archival tissues of NOM, OPM, primary and metastatic OSCC. Apoptotic rates of tumor cells and tumor-infiltrating lymphocytes (TIL) in OSCC specimens were determined by cleaved caspase 3 immunohistochemistry. RESULTS: Normal oral epithelia constitutively expressed TRAIL, but expression was progressively lost in OPM and OSCC. Reduction in DcR2 expression levels was noted frequently in OPM and OSCC compared to respective patient-matched uninvolved oral mucosa. OSCC frequently expressed DR4, DR5 and DcR1 but less frequently DcR2. Expression levels of DR4, DR5 and DcR1 receptors were not significantly altered in OPM, primary OSCC and metastatic OSCC compared to patient-matched normal oral mucosa. Expression of proapoptotic TRAIL-receptors DR4 and DR5 in OSCC seemed to depend, at least in part, on whether or not these receptors were expressed in their parental oral epithelia. High DR5 expression in primary OSCC correlated significantly with larger tumor size. There was no significant association between TRAIL-R expression and OSSC histology grade, nodal status or apoptosis rates of tumor cells and TIL. CONCLUSION: Loss of TRAIL expression is an early event during oral carcinogenesis and may be involved in dysregulation of apoptosis and contribute to the molecular carcinogenesis of OSCC. Differential expressions of TRAIL receptors in OSCC do not appear to play a crucial role in their apoptotic rate or metastatic progression.

CHARACTERIZING AND TREATING THE NEUROPATHOLOGY OF TUBEROUS SCLEROSIS COMPLEX IN THE MOUSE

Tuberous sclerosis complex (TSC) is a multisystem, autosomal dominant disorder affecting approximately 1 in 6000 births. Developmental brain abnormalities cause substantial morbidity and mortality and often lead to neurological disease including epilepsy, cognitive disabilities, and autism. TSC is caused by inactivating mutations in either TSC1 or TSC2, whose protein products are known inhibitors of mTORC1, an important kinase regulating translation and cell growth. Nonetheless, neither the pathophysiology of the neurological manifestations of TSC nor the extent of mTORC1 involvement in the development of these lesions is known. Murine models would greatly advance the study of this debilitating disorder. This thesis will describe the generation and characterization of a novel brain-specific mouse model of TSC, Tsc2flox/ko;hGFAP-Cre. In this model, the Tsc2 gene has been removed from most neurons and glia of the cortex and hippocampus by targeted Cre-mediated deletion in radial glial neuroprogenitor cells. The Tsc2flox/ko;hGFAP-Cre mice fail to thrive beginning postnatal day 8 and die from seizures around 23 days. Further characterization of these mice demonstrated megalencephaly, enlarged neurons, abnormal neuronal migration, altered progenitor pools, hypomyelination, and an astrogliosis. The similarity of these defects to those of TSC patients establishes this mouse as an excellent model for the study of the neuropathology of TSC and testing novel therapies. We further describe the use of this mouse model to assess the therapeutic potential of the macrolide rapamycin, an inhibitor of mTORC1. We demonstrate that rapamycin administered from postnatal day 10 can extend the life of the mutant animals 5 fold. Since TSC is a neurodevelopmental disorder, we also assessed in utero and/or immediate postnatal treatment of the animals with rapamycin. Amazingly, combined in utero and postnatal rapamycin effected a histologic rescue that was almost indistinguishable from control animals, indicating that dysregulation of mTORC1 plays a large role in TSC neuropathology. In spite of the almost complete histologic rescue, behavioral studies demonstrated that combined treatment resulted in poorer learning and memory than postnatal treatment alone. Postnatally-treated animals behaved similarly to treated controls, suggesting that immediate human treatment in the newborn period might provide the most opportune developmental timepoint for rapamycin administration.

Transplantation of human embryonic stem cell-derived alveolar epithelial type II cells abrogates acute lung injury in mice.

Respiratory diseases are a major cause of mortality and morbidity worldwide. Current treatments offer no prospect of cure or disease reversal. Transplantation of pulmonary progenitor cells derived from human embryonic stem cells (hESCs) may provide a novel approach to regenerate endogenous lung cells destroyed by injury and disease. Here, we examine the therapeutic potential of alveolar type II epithelial cells derived from hESCs (hES-ATIICs) in a mouse model of acute lung injury. When transplanted into lungs of mice subjected to bleomycin (BLM)-induced acute lung injury, hES-ATIICs behaved as normal primary ATIICs, differentiating into cells expressing phenotypic markers of alveolar type I epithelial cells. Without experiencing tumorigenic side effects, lung injury was abrogated in mice transplanted with hES-ATIICs, demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Therefore, transplantation of hES-ATIICs shows promise as an effective therapeutic to treat acute lung injury.

THE HISTONE DEACETYLASE INHIBITOR, MS-275, SENSITIZES METASTATIC OSTEOSARCOMA TO FASL-INDUCED CELL DEATH: A ROLE FOR C-FLIP

The purpose of this study was to determine the effects of the histone deacetylase inhibitor, MS-275, on the Fas signaling pathway and susceptibility of osteosarcoma (OS) to Fas ligand (FasL)-induced cell death. OS metastasizes almost exclusively to the lungs. We have shown that Fas expression in OS cells is inversely correlated with their metastatic potential. Fas+ cells are rapidly eliminated when they enter the lungs via interaction with FasL, which is constitutively expressed in the lungs. Fas- OS cells escape this FasL-induced apoptosis and survive in the lung microenvironment. Moreover, upregulation of Fas in established OS lung metastases results in tumor regression. Therefore, agents that upregulate Fas expression or activate the Fas signaling pathway may have therapeutic potential. Treatment of Fas- metastatic OS cell lines with 2 μM MS-275 sensitized cells to FasL-induced cell death in vitro. We found that MS-275 did not alter the expression of Fas on the cell surface; rather it resulted in increased levels of Fas within the membrane lipid rafts, as demonstrated by an increase in Fas expression in detergent insoluble lipid raft fractions. We further demonstrated that following MS-275 treatment, Fas colocalized with GM1+ lipid rafts and that there was a decrease in c-FLIP (cellular FLICE-inhibitory protein) mRNA and protein. Downregulation of c-FLIP correlated with caspase activation and apoptosis induction. Transfection of cells with shRNA to c-FLIP also resulted in the localization of Fas to lipid rafts. These studies indicate that MS-275 sensitizes OS cells to FasL by upregulating the expression of Fas in membrane lipid rafts, which correlated with the downregulation of c-FLIP. Treatment of nu/nu-mice with established OS lung metastases with oral MS-275 resulted in increased apoptosis, a significant inhibition of c-FLIP expression in tumors and tumor regression. Histopathological examination of mice showed no significant organ toxicity. Overall, these results suggest that the mechanism by which MS-275 sensitizes OS cells and lung metastases to FasL-induced cell death may be by a reduction in the expression of c-FLIP.

THE EFFECT OF GABAMIMETICS ON NEUROTRANSMITTER RECEPTOR BINDING AND FUNCTION IN RAT BRAIN

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and alterations in central GABAergic transmission may contribute to the symptoms of a number of neurological and psychiatric disorders. Because of this relationship, numerous laboratories are attempting to develop agents which will selectively enhance GABA neurotransmission in brain. Due to these efforts, several promising compounds have recently been discovered. Should these drugs prove to be clinically effective, they will be used to treat chronic neuropsychiatric disabilities and, therefore, will be administered for long periods of time. Accordingly, the present investigation was undertaken to determine the neurochemical consequences of chronic activation of brain GABA systems in order to better define the therapeutic potential and possible side-effect liability of GABAmimetic compounds.^ Chronic (15 day) administration to rats of low doses of amino-oxyacetic acid (AOAA, 10 mg/kg, once daily), isonicotinic acid hydrazide (20 mg/kg, b.i.d.), two non-specific inhibitors of GABA-T, the enzyme which catabolizes GABA in brain, or (gamma)-acetylenic GABA (10 mg/kg, b.i.d.) a catalytic inhibitor of this enzyme, resulted in a significant elevation of brain and CSF GABA content throughout the course of treatment. In addition, chronic administration of these drugs, as well as the direct acting GABA receptor agonists THIP (8 mg/kg, b.i.d.) or kojic amine (18 mg/kg, b.i.d.) resulted in a significant increase in dopamine receptor number and a significant decrease in GABA receptor number in the corpus striatum of treated animals as determined by standard in vitro receptor binding techniques. Changes in the GABA receptor were limited to the corpus striatum and occurred more rapidly than did alterations in the dopamine receptor. The finding that dopamine-mediated stereotypic behavior was enhanced in animals treated chronically with AOAA suggested that the receptor binding changes noted in vitro have some functional consequence in vitro.^ Coadministration of atropine (a muscarinic cholinergic receptor antagonist) blocked the GABA-T inhibitor-induced increase in striatal dopamine receptors but was without effect on receptor alterations seen following chronic administration of direct acting GABA receptor agonists. Atropine administration failed to influence the drug-induced decreases in striatal GABA receptors.^ Other findings included the discovery that synaptosomal high affinity ('3)H-choline uptake, an index of cholinergic neuronal activity, was significantly increased in the corpus striatum of animals treated acutely, but not chronically, with GABAmimetics.^ It is suggested that the dopamine receptor supersensitivity observed in the corpus striatum of animals following long-term treatment with GABAmimetics is a result of the chronic inhibition of the nigrostriatal dopamine system by these drugs. Changes in the GABA receptor, on the other hand, are more likely due to a homospecific regulation of these receptors. An hypothesis based on the different sites of action of GABA-T inhibitors vis-a-vis the direct acting GABA receptor agonists is proposed to account for the differential effect of atropine on the response to these drugs.^ The results of this investigation provide new insights into the functional interrelationships that exist in the basal ganglia and suggest that chronic treatment with GABAmimetics may produce extrapyramidal side-effects in man. In addition, the constellation of neurochemical changes observed following administration of these drugs may be a useful guide for determining the GABAmimetic properties of neuropharmacological agents. ^

The mechanism of the pharmacological actions of dexrazoxane on different tissue types

Approximately 6,600 people die from acute myelogenous leukemia (AML) on an annual basis. During the past 10 to 15 years, there has been gradual overall improvements in the therapy of this disease, yet the majority of patients with AML succumb to this disease. In an attempt to improve current therapeutic strategies for AML, we became interested in a commercially available drug, dexrazoxane, which protects against anthracycline-induced cardiotoxicity. We have investigated dexrazoxane's (DEX) effects on different tissue types in an effort to determine its unique mechanism of action. Colony forming assays were used to evaluate stem-cell renewal of myeloid cells in vitro and median effect analysis was used to evaluate antagonism, synergism, or additivity. The anthracyclines, doxorubicin, daunorubicin, and idarubicin were individually combined with DEX in leukemic myeloid models to determine if the combination of the two drugs resulted in a synergistic, additive or antagonistic effect. Etoposide and cytosine arabinoside were also evaluated in combination with DEX using the same in vitro model and evaluated similarly. ^ Dexrazoxane in combination with any of the anthracyclines was schedule dependent. The combination of DEX and anthracycline resulted in a greater antitumor effect than anthracycline alone except for DEX administered 24 hours before doxorubicin or daunorubicin. These data were corroborated through median effect analysis. Etoposide in combination with dexrazoxane was synergistic for all combinations, and the combination of cytosine arabinoside and DEX was schedule dependent. In contrast, using an in vivo gastrointestinal model, DEX in combination with doxorubicin was antagonistic for almost all of the ratios used, except for the highest. A Withers' assay was used to evaluate toxicity on jejunal crypt cells. No effect was apparent for the combination of idarubicin and DEX, however, as seen with RZ, DEX in addition to radiation greatly potentiated the cytotoxic effects of radiation on crypts. These paradoxical effects of dexrazoxane were initially enigmatic, but after additional investigation, we propose a model that explains our findings. We conclude that DEX in combination with anthracyclines produces an additive to synergistic antileukemic response and may have therapeutic potential clinically. Additionally, DEX protects the gastrointestinal tract from doxorubicin toxicity, which could have clinical implications for the administration of greater doses of doxorubicin. ^

The characterization of PKC-isozyme specific EGFR-dependent Erk1/2 activation in androgen-independent prostate cancer cell lines

One growth factor receptor commonly altered during prostate tumor progression is the epidermal growth factor receptor (EGFR). EGFR signaling regulates Erk1/2 phosphorylation through multiple mechanisms. We hypothesized that PKC isozymes play a role in EGFR-dependent signaling, and that through PKC isozyme selective inhibition, EGFR-dependent Erk1/2 activation can be attenuated in AICaP cells. ^ To test the hypothesis, PKC activation was induced by 12-O-tetradecanoyi-phorbol-13-acetate (TPA) in PC-3 cells. As a result, Erk1/2 was activated similarly to what was observed upon EGF stimulation. EGF-induced Erk1/2 activation in PC-3 cells was PKC-dependent, as demonstrated through use of a selective PKC inhibitor, GF109203X. This provides evidence for PKC regulatory control over Erk1/2 signaling downstream of EGFR. Next, we demonstrated that when PKC was inhibited by GF109203X, EGF-stimulated Erk1/2 activation was inhibited in PC-3, but not DU145 cells. TPA-stimulated Erk1/2 activation was EGFR-dependent in both DU145 and PC-3 cells, demonstrated through abrogation of Erk1/2 activation by a selective EGFR inhibitor AG1478. These data support PKC control at or upstream of EGFR in AICaP cells. We observed that interfering with ligand/EGFR binding abrogated Erk1/2 signaling in TPA-stimulated cells, revealing a role for PKC upstream of EGFR. ^ Next, we determined which PKC isozymes might be responsible for Erk1/2 regulation. We first determined that human AICaP cell lines express the same PKC isozymes as those observed in clinical prostate cancer specimens (α, ϵ, &zgr;, ι and PKD). Isozyme-selective methods were employed to characterize discrete PKC isozyme function in EGFR-dependent Erk1/2 activation. Pharmacologic inhibitors implicated PKCα in TPA-induced EGFR-dependent Erk1/2 activation in both PC-3 and DU145 cells. Further, the cPKC-specific inhibitor, Gö6976 decreased viablilty of DU145 cells, providing evidence that PKCα is necessary for growth and survival. Finally, resveratrol, a phytochemical with strong cancer therapeutic potential inhibited Erk1/2 activation, and this correlated with selective inhibition of PKCα. These results demonstrate that PKC regulates pathways critical to progression of CaP cells, including those mediated by EGFR. Thus, PKC isozyme-selective targeting is an attractive therapeutic strategy, and understanding the role of specific PKC isozymes in CaP cell growth and survival may aid in development of effective, non-toxic PKC-targeted therapies. ^

Bortezomib modulates TRAIL sensitivity in human bladder and prostate cancer

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a member of the TNF superfamily of cytokines that can induce cell death through engagement of cognate death receptors. Unlike other death receptor ligands, it selectively kills tumor cells while sparing normal cells. Preclinical studies in non-human primates have generated much enthusiasm regarding its therapeutic potential. However, many human cancer cell lines exhibit significant resistance to TRAIL-induced apoptosis, and the molecular mechanisms underling this are controversial. Possible explanations are typically cell-type dependent, but include alterations of receptor expression, enhancement of pro-apoptotic intracellular signaling molecules, and reductions in anti-apoptotic proteins. We show here that the proteasome inhibitor bortezomib (Velcade, PS-341) produces synergistic apoptosis in both bladder and prostate cancer cell lines within 4-6 hours when co-treated with recombinant human TRAIL which is associated with accumulation of p21 and cdk1/2 inhibition. Our data suggest that bortezomib's mechanism of action involves a p21-dependent enhancement of caspase maturation. Furthermore, we found enhanced tumor cell death in in vivo models using athymic nude mice. This is associated with increases in caspase-8 and caspase-3 cleavage as well as significant reductions in microvessel density (MVD) and proliferation. Although TRAIL alone had less of an effect, its biological significance as a single agent requires further investigations. Toxicity studies reveal that the combination of bortezomib and rhTRAIL has fatal consequences that can be circumvented by altering treatment schedules. Based on our findings, we conclude that this strategy has significant therapeutic potential as an anti-cancer agent. ^

Identification and functional characterization of novel phosphotyrosine and phosphoserine residues in the interleukin-2 receptor complex

Interleukin-2 (IL-2) is a major T cell growth factor and plays an essential role in the development of normal immune responses. The Janus kinases (Jaks) and Signal transducers and activators of transcription (Stats) are critical for transducing signals from the IL-2 receptors (IL2Rs) to the nucleus to control cell growth and differentiation. In recent years there has been increasing evidence to indicate that the IL-2 activated Jak3/Stat5 pathway provides a new molecular target for immune suppression. Thus, understanding the regulation of this effector cascade has important therapeutic potential.^ One objective of this work was to identify and define the role and molecular mechanism of novel phosphorylation sites in Jak3. Using functional proteomics, three novel Jak3 phosphorylation sites, Y904, Y939 and S574 were identified. Phosphospecific antibodies confirmed that phosphorylation of Y904 and Y939 were mediated by IL-2 and other IL-2 family cytokines in distinct cell types. Biochemical analysis demonstrated that phosphorylation of both Y904 and Y939 positively regulated Jak3 enzymatic activity, while phosphorylation of S574 did not affect Jak3 in vitro kinase activity. However, a gain-of-function mutation of S574 in Jak3 abrogated IL-2 mediated Stat5 activation, suggesting that phosphorylation of this residue might serve a negative role to attenuate IL-2 signaling. Furthermore, mechanistic analysis suggested that phosphorylation of Y904 in Jak3 affects the KmATP of Jak3, while phosphorylation of Y939 in Jak3 was required to bind one of its substrates, Stat5.^ The second objective was to determine the role of serine/threonine phosphatases in the regulation of the IL2R complex. Activation of Jak3 and Stat5 by IL-2 is a transient event mediated by phosphorylation. Using a specific PP1/PP2A inhibitor, we observed that inhibition of PP1/PP2A negatively regulated the IL-2 activated Jak3/Stat5 signaling pathway in a human NK cell line (YT) and primary human T cells. More importantly, coimmunoprecipitation assays indicated that inhibition of PP1/PP2A blocked the formation of an active IL2R complex. Pretreatment of cells with the inhibitor also reduced the electrophoretic mobility of the IL2Rβ and IL2Rγ subunits in YT cells, suggesting that inhibition of PP1/PP2A directly or indirectly regulates undefined serine/threonine kinases which phosphorylate these proteins. Based on these observations, a model has emerged that serine/threonine phosphorylation of the IL2Rβ and IL2Rγ subunits causes a conformational change of these proteins, which disrupts IL2R dimerization and association of Jak3 and Stat5 to these receptors.^

INDUCED-PLURIPOTENT STEM-DERIVED NEURONAL PROGENITOR CELLS AS A NOVEL TREATMENT FOR NEURODEGENERATIVE DISEASES

Cellular therapies, as neuronal progenitor (NP) cells grafting, are promising therapies for patients affected with neurodegenerative diseases like Creutzfeldt-Jakob Disease (CJD). At this time there is no effective treatment or cure for CJD. The disease is inevitably fatal and affected people usually die within months of the appearance of the first clinical symptoms. Compelling evidence indicate that the hallmark event in the disease is the conversion of the normal prion protein (termed PrPC) into the disease-associated, misfolded form (called PrPSc). Thus, a reasonable therapeutic target would be to prevent PrP misfolding and prion replication. This strategy has been applied with poor results since at the time of clinical intervention substantial brain damage has been done. It seems that a more effective treatment aimed at patients with established symptoms of CJD would need to stop further brain degeneration or even recover some of the previously lost brain tissue. The most promising possibility to recover brain tissue is the use of NPs that have the potential to replenish the nerve cells lost during the early stages of the disease. Advanced cellular therapies, beside their potential for cell replacement, might be used as biomaterials for drug delivery in order to stimulate cell survival or the resolution the disease. Also, implanted cells can be genetically manipulated to correct abnormalities causing disease or to make them more resistant to the toxic microenvironments present in damaged tissue. In recent years cell engineering has been within the scope of the scientific and general community after the development of technologies able to “de-differentiate” somatic cells into induced-pluripotent stem (IPS) cells. This new tool permits the use of easy-to-reach cells like skin or blood cells as a primary material to obtain embryonic stem-like cells for cellular therapies, evading all ethical issues regarding the use of human embryos as a source of embryonic stem cells. The complete work proposes to implant IPS-derived NP cells into the brain of prion-infected animals to evaluate their therapeutic potential. Since it is well known that the expression of prion protein in the cell membrane is necessary for PrPSc mediated toxicity, we also want to determine if NPs lacking the prion protein have better survival rates once implanted into sick animals. The main objective of this work is to develop implantable neural precursor from IPS coming from animals lacking the prion protein. Specific aim 1: To develop and characterize cellular cultures of IPS cells from prp-/- mice. Fibroblasts from prp-/- animals will be reprogrammed using the four Yamanaka factors. IPS colonies will be selected and characterized by immunohistochemistry for markers of pluripotency. Their developmental capabilities will be evaluated by teratoma and embryoid body formation assays. Specific aim 2: To differentiate IPS cells to a neuronal lineage. IPS cells will be differentiated to a NP stage by the use of defined media culture conditions. NP cells will be characterized by their immunohistochemical profile as well as by their ability to differentiate into neuronal cells. Specific aim 3: Cellular labeling of neuronal progenitors cells for in vitro traceability. In order to track the cells once implanted in the host brain, they will be tagged with different methods such as lipophilic fluorescent tracers and transduction with GFP protein expression.

Examination of the engraftment of exogenous mesenchymal stem cells in ovarian tumors and their potential use as delivery vehicles for therapeutic genes

Interactions between neoplastic cells and the host stroma play a role in both tumor cell migration and proliferation. Stromal cells provide structural support for malignant cells, modulate the tumor microenvironment, and influence phenotypic behavior as well as the aggressiveness of the malignancy. In response, the tumor provides growth factors, cytokines, and cellular signals that continually initiate new stromal reactions and recruit new cells into the microenvironment to further support tumor growth. Since growing tumors recruit local cells, as well as supplemental cells from the circulation, such as fibroblasts and endothelial precursors, the question arises if it would be possible to access circulating stromal cells to modify the tumor microenvironment for therapeutic benefits. One such cell type, mesenchymal stem cells (MSC), could theoretically be engrafted into stroma. MSC are pluripotent cells that have been shown to form stromal elements such as myofibroblasts, perivascular tissues and connective tissues. Several reports have demonstrated that MSC can incorporate into sites of wound healing and tissue repair, due to active tissue remodeling and local paracrine factors, and given the similarity between wound healing and the carcinoma induced stromal response one can hypothesize that MSC have the potential to be recruited to sites of tumor development. In addition, gene-modified MSC could be used as cellular vehicles to deliver gene products into tumors. My results indicate that MSC home to and participate in tumor stroma formation in ovarian tumor xenografts in mice. Additionally, once homed to tumor beds, MSC proliferate rapidly and integrate. My studies aim at understanding the fate of MSC in the tumor microenvironment, as well as utilizing them for cellular delivery of therapeutic genes into the stroma of ovarian carcinomas. ^

The survival role and the potential therapeutic targeting of MET in multiple myeloma

Multiple myeloma (MM) is a debilitating and incurable B-cell malignancy. Previous studies have documented that the hepatocyte growth factor (HGF) plays a role in the pathobiology of MM. The receptor tyrosine kinase MET induced signaling initiates when its ligand HGF binds to the MET receptor. However, the direct importance of MET in MM has not been elucidated. The present work used three different but complementary approaches to reduce MET protein levels or its activity to demonstrate the importance of MET in MM. ^ In the first approach, MET transcript and protein levels were reduced by directly targeting the cellular MET transcripts using shRNA retroviral infection techniques. This direct reduction of MET mRNA leads to a reduction of MET protein levels, which caused an inhibition of growth and induction of cell death. ^ In the second approach, a global transcription inhibitor flavopiridol was used as a potential pharmacological tool to reduce MET levels. MET has a short half-life of 30 min for mRNA and 4 hours for protein; therefore using a RNA pol II inhibitor such as flavopiridol would be a viable option to reduce MET levels. When using flavopiridol in MM cell lines, there was a reduction of MET transcript and protein levels, which was associated with the induction of cell death. ^ Finally in the last strategy, MET kinase activity was suppressed by MP470, a small molecule inhibitor that binds to the ATP binding pocket in the kinase domain. At concentrations where phosphorylation of MET was inhibited there was induction of cell death in MM cell lines and primary cells from patients. In addition, in MM cell lines there was a decrease in phosphorylation of AKT (ser473) and caspase-9 (ser196); downstream of MET, suggesting that the mechanism of action for survival may be through these cascade of events. ^ Overall, this study provides a proof-of-principle that MET is important for the survival of MM cell lines as well as primary plasma cells obtained from patients. Therefore, targeting MET therapeutically may be a possible strategy to treat patients with this debilitating disease of MM. ^

Thoracic aortic disease in tuberous sclerosis complex: molecular pathogenesis and potential therapies in Tsc2+/- mice.

Tuberous sclerosis complex (TSC) is a genetic disorder with pleiotropic manifestations caused by heterozygous mutations in either TSC1 or TSC2. One of the less investigated complications of TSC is the formation of aneurysms of the descending aorta, which are characterized on pathologic examination by smooth muscle cell (SMC) proliferation in the aortic media. SMCs were explanted from Tsc2(+/-) mice to investigate the pathogenesis of aortic aneurysms caused by TSC2 mutations. Tsc2(+/-) SMCs demonstrated increased phosphorylation of mammalian target of rapamycin (mTOR), S6 and p70S6K and increased proliferation rates compared with wild-type (WT) SMCs. Tsc2(+/-) SMCs also had reduced expression of SMC contractile proteins compared with WT SMCs. An inhibitor of mTOR signaling, rapamycin, decreased SMC proliferation and increased contractile protein expression in the Tsc2(+/-) SMCs to levels similar to WT SMCs. Exposure to alpha-elastin fragments also decreased proliferation of Tsc2(+/-) SMCs and increased levels of p27(kip1), but failed to increase expression of contractile proteins. In response to artery injury using a carotid artery ligation model, Tsc2(+/-) mice significantly increased neointima formation compared with the control mice, and the neointima formation was inhibited by treatment with rapamycin. These results demonstrate that Tsc2 haploinsufficiency in SMCs increases proliferation and decreases contractile protein expression and suggest that the increased proliferative potential of the mutant cells may be suppressed in vivo by interaction with elastin. These findings provide insights into the molecular pathogenesis of aortic disease in TSC patients and identify a potential therapeutic target for treatment of this complication of the disease.