The transcription factor nuclear factor kappa B (NFkappaB) maintains TRAIL resistance in human pancreatic cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of the TNF family of cytokines that induces apoptosis in a variety of tumor cells while sparing normal cells. However, many human cancer cell lines display resistance to TRAIL-induced apoptosis and the mechanisms contributing to resistance remain controversial. Previous studies have demonstrated that the dimeric transcription factor Nuclear Factor kappa B (NFκB) is constitutively active in a majority of human pancreatic cancer cell lines and primary tumors, and although its role in tumor progression remains unclear it has been suggested that NFκB contributes to TRAIL resistance. Based on this, I examined the effects of NFκB inhibitors on TRAIL sensitivity in a panel of nine pancreatic cancer cell lines. I show here that inhibitors of NFκB, including two inhibitors of the proteasome (bortezomib (Velcade™, PS-341) and NPI-0052), a small molecule inhibitor of IKK (PS1145), and a novel synthetic diterpene NIK inhibitor (NPI-1342) reverse TRAIL resistance in pancreatic cancer cell lines. Further analysis revealed that the expression of the anti-apoptosic proteins BclXL and XIAP was significantly decreased following exposure to these inhibitors alone and in combination with TRAIL. Additionally, treatment with NPI0052 and TRAIL significantly reduced tumor burden relative to the control tumors in an L3.6pl orthotopic pancreatic xenograft model. This was associated with a significant decrease in proliferation and an increase in caspase 3 and 8 cleavage. Combination therapy employing PS1145 or NPI-1342 in combination with TRAIL also resulted in a significant reduction in tumor burden compared to either agent alone in a Panc1 orthotopic xenograft model. My studies show that combination therapy with inhibitors of NFκB alone and TRAIL is effective in pre-clinical models of pancreatic cancer and suggests that the approach should be evaluated in patients. ^

CHARACTERIZATION OF TCL1-Tg:P53-/- MICE THAT RESEMBLE HUMAN CHRONIC LYMPHOCYTIC LEUKEMIA WITH 17P-DELETION

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United Statesand Europe. CLL patients with deletion of chromosome 17p, where the tumor suppressor p53 gene is located, often develop a more aggressive disease with poor clinical outcomes. However, the underlying mechanism remains unclear. In order to understand the underneath mechanism in vivo, I have recently generated mice with Eu-TCL1-Tg:p53-/- genotype and showed that these mice develop aggressive leukemia that resembles human CLL with 17p deletion. The Eu-TCL1-Tg:p53-/- mice developed CLL disease at 3-4 months, significantly earlier than the parental Eu-TCL1-Tg mice that developed CLL disease at 8-12 months. Flow cytometry analysis showed that the CD5+/ IgM+ cell population appeared in the peritoneal cavity, bone marrow, and the spleens of Eu-TCL1-Tg:p53-/- mice significantly earlier than that of the parental Eu-TCL1-Tg mice. Massive infiltration and accumulation of leukemia cells were found in the spleen and peritoneal cavity. In vitro study showed that the leukemia cells isolated from the Eu-TCL1-Tg:p53-/- mice were more resistant to fludarabine treatment than the leukemia cells isolated from spleens of Eu-TCL1-Tg mice. Interestingly, TUNEL assay revealed that there was higher apoptotic cell death found in the Eu-TCL1-Tg spleen tissue compared to the spleens of the Eu-TCL1-Tg:p53-/- mice, suggesting that the loss of p53 compromises the apoptotic process in vivo, and this might in part explain the drug resistant phenotype of CLL cells with 17p-deletion. In the present study, we further demonstrated that the p53 deficiency in the TCL1 transgenic mice resulted in significant down-regulation of microRNAs miR-15a and miR16-1, associated with a substantial up-regulation of Mcl-1, suggesting that the p53-miR15a/16-Mcl-1 axis may play an important role in CLL pathogenesis. Interestingly, we also found that loss of p53 resulted in a significant decrease in expression of the miR-30 family especially miR-30d in leukemia lymphocytes from the Eu-TCL1-Tg:p53-/- mice. Such down-regulation of those microRNAs and up-regulation of Mcl-1 were also found in primary leukemia cells from CLL patients with 17p deletion. To further exam the biological significance of decrease in the miR-30 family in CLL, we investigated the potential involvement of EZH2 (enhancer of zeste homolog 2), a component of the Polycomb repressive complex known to be a downstream target of miR-30d and plays a role in disease progression in several solid cancers. RT-PCR and western blot analyses showed that both EZH2 mRNA transcript and protein levels were significantly increased in the lymphocytes of Eu-TCL1-Tg:p53-/- mice relative to Eu-TCL1-Tg mice. Exposure of leukemia cells isolated from Eu-TCL1-Tg:p53-/- mice to the EZH2 inhibitor 3-deazaneplanocin (DZNep) led to induction of apoptosis, suggesting EZH2 may play a role in promoting CLL cell survival and this may contribute to the aggressive phenotype of CLL with loss of p53. Our study has created a novel CLL mouse model, and suggests that the p53/miR15a/16-Mcl-1 axis & p53/miR30d-EZH2 may contribute to the aggressive phenotype and drug resistance in CLL cells with loss of p53.

Structure-function analysis of human Integrator subunit-4

Structure-function analysis of human Integrator subunit 4 Anupama Sataluri Advisor: Eric. J. Wagner, Ph.D. Uridine-rich small nuclear RNAs (U snRNA) are RNA Polymerase-II (RNAPII) transcripts that are ubiquitously expressed and are known to be essential for gene expression. snRNAs play a key role in mRNA splicing and in histone mRNA expression. Inaccurate snRNA biosynthesis can lead to diseases related to defective splicing and histone mRNA expression. Although the 3′ end formation mechanism and processing machinery of other RNAPII transcripts such as mRNA has been well studied, the mechanism of snRNA 3′ end processing has remained a mystery until the recent discovery of the machinery that mediates this process. In 2005, a complex of 14 subunits (the Integrator complex) associated with RNA Polymerase-II was discovered. The 14subunits were annotated Integrator 1-14 based on their size. The subunits of this complex together were found to facilitate 3′ end processing of snRNA. Identification of the Integrator complex propelled research in the direction of understanding the events of snRNA 3’end processing. Recent studies from our lab confirmed that Integrator subunit (IntS) 9 and 11 together perform the endonucleolytic cleavage of the nascent snRNA 3′ end to generate mature snRNA. However, the role of other members of the Integrator complex remains elusive. Current research in our lab is focused on deciphering the role of each subunit within the Integrator complex This work specifically focuses on elucidating the role of human Integrator subunit 4 (IntS4) and understanding how it facilitates the overall function of the complex. IntS4 has structural similarity with a protein called “Symplekin”, which is part of the mRNA 3’end processing machinery. Symplekin has been thoroughly researched in recent years and structure-function correlation studies in the context of mRNA 3’end processing have reported a scaffold function for Symplekin due to the presence of HEAT repeat motifs in its N-terminus. Based upon the structural similarity between IntS4 and Symplekin, we hypothesized that Integrator subunit 4 may be behaving as a Symplekin-like scaffold molecule that facilitates the interaction between other members of the Integrator Complex. To answer this question, the two important goals of this study were to: 1) identify the region of IntS4, which is important for snRNA 3′ end processing and 2) determine binding partners of IntS4 which promote its function as a scaffold. IntS4 structurally consists of a highly conserved N-terminus with 8 HEAT repeats, followed by a nonconserved C- terminus. A series of siRNA resistant N and C-terminus deletion constructs as well as specific point mutants within its N-terminal HEAT repeats were generated for human IntS4 and, utilizing a snRNA transcriptional readthrough GFP-reporter assay, we tested their ability to rescue misprocessing. This assay revealed a possible scaffold like property of IntS4. To probe IntS4 for interaction partners, we performed co-immunoprecipitation on nuclear extracts of IntS4 expressing stable cell lines and identified IntS3 and IntS5 among other Integrator subunits to be binding partners which facilitate the scaffold like function of hIntS4. These findings have established a critical role for IntS4 in snRNA 3′ end processing, identified that both its N and C termini are essential for its function, and mapped putative interaction domains with other Integrator subunits.

Inhibitor of cytochrome c messenger RNA -protein interaction in stimulated rat skeletal muscle

The purpose of this work was to examine the possible mechanisms for the regulation of cytochrome c gene expression in response to increased contractile activity in rat skeletal muscle. The working hypothesis was that increased contractile activity enhances cytochrome c gene expression through a cis-element. A 110% increase in cytochrome c mRNA concentration was observed in tibialis anterior (TA) muscle after 9 days of chronic stimulation. Similar difference (120%) exists between soleus (SO) muscle of higher contractile activity and white vastus lateralis (WV) muscle of lower contractile activity. These results suggest that the endogenous cytochrome c gene expression is regulated by contractile activity. Cytochrome c-reporter genes were injected into skeletal muscles to identify the cis-element that is responsible for the regulation. Although the data was inconclusive, part of it suggested the importance of the 3$\sp\prime$-untranslated region (3$\sp\prime$-UTR) in mediating the response to increased contractile activity.^ RNA gel mobility shift (GMSA) and ultraviolet (UV) cross-linking assays revealed specific RNA-protein interaction in a 50-nucleotide region of the 3$\sp\prime$-UTR in unstimulated TA muscle. Computer analysis predicted a stem-loop structure of 17 nucleotides, which provides a structural basis for RNA-protein interaction. These 17 nucleotides are 100% conserved among rat, mouse and human cytochrome c genes and their 13 pseudogenes, suggesting a functional role for this region. The RNA-protein interaction was significantly less in highly active SO muscle than in inactive WV muscle and was dramatically decreased in stimulated TA muscle due to a protein inhibitor(s) associated with ribosome. It is possible that cytochrome c mRNAs undergoing translation are subject to a compartmentalized regulatory influence.^ The conclusion from these results is that increases in contractile activity induce or activate a protein inhibitor(s) associated with ribosome in rat skeletal muscle. The inhibitor decreases RNA-protein interaction in the 3$\sp\prime$-UTR of cytochrome c mRNA, which may result in increased mRNA stability and/or translation. ^

Human-robot interaction for telemanipulation in large workspaces

Independientemente de la existencia de técnicas altamente sofisticadas y capacidades de cómputo cada vez más elevadas, los problemas asociados a los robots que interactúan con entornos no estructurados siguen siendo un desafío abierto en robótica. A pesar de los grandes avances de los sistemas robóticos autónomos, hay algunas situaciones en las que una persona en el bucle sigue siendo necesaria. Ejemplos de esto son, tareas en entornos de fusión nuclear, misiones espaciales, operaciones submarinas y cirugía robótica. Esta necesidad se debe a que las tecnologías actuales no pueden realizar de forma fiable y autónoma cualquier tipo de tarea. Esta tesis presenta métodos para la teleoperación de robots abarcando distintos niveles de abstracción que van desde el control supervisado, en el que un operador da instrucciones de alto nivel en la forma de acciones, hasta el control bilateral, donde los comandos toman la forma de señales de control de bajo nivel. En primer lugar, se presenta un enfoque para llevar a cabo la teleoperación supervisada de robots humanoides. El objetivo es controlar robots terrestres capaces de ejecutar tareas complejas en entornos de búsqueda y rescate utilizando enlaces de comunicación limitados. Esta propuesta incorpora comportamientos autónomos que el operador puede utilizar para realizar tareas de navegación y manipulación mientras se permite cubrir grandes áreas de entornos remotos diseñados para el acceso de personas. Los resultados experimentales demuestran la eficacia de los métodos propuestos. En segundo lugar, se investiga el uso de dispositivos rentables para telemanipulación guiada. Se presenta una aplicación que involucra un robot humanoide bimanual y un traje de captura de movimiento basado en sensores inerciales. En esta aplicación, se estudian las capacidades de adaptación introducidas por el factor humano y cómo estas pueden compensar la falta de sistemas robóticos de alta precisión. Este trabajo es el resultado de una colaboración entre investigadores del Biorobotics Laboratory de la Universidad de Harvard y el Centro de Automática y Robótica UPM-CSIC. En tercer lugar, se presenta un nuevo controlador háptico que combina velocidad y posición. Este controlador bilateral híbrido hace frente a los problemas relacionados con la teleoperación de un robot esclavo con un gran espacio de trabajo usando un dispositivo háptico pequeño como maestro. Se pueden cubrir amplias áreas de trabajo al cambiar automáticamente entre los modos de control de velocidad y posición. Este controlador háptico es ideal para sistemas maestro-esclavo con cinemáticas diferentes, donde los comandos se transmiten en el espacio de la tarea del entorno remoto. El método es validado para realizar telemanipulación hábil de objetos con un robot industrial. Por último, se introducen dos contribuciones en el campo de la manipulación robótica. Por un lado, se presenta un nuevo algoritmo de cinemática inversa, llamado método iterativo de desacoplamiento cinemático. Este método se ha desarrollado para resolver el problema cinemático inverso de un tipo de robot de seis grados de libertad donde una solución cerrada no está disponible. La eficacia del método se compara con métodos numéricos convencionales. Además, se ha diseñado una taxonomía robusta de agarres que permite controlar diferentes manos robóticas utilizando una correspondencia, basada en gestos, entre los espacios de trabajo de la mano humana y de la mano robótica. El gesto de la mano humana se identifica mediante la lectura de los movimientos relativos del índice, el pulgar y el dedo medio del usuario durante las primeras etapas del agarre. ABSTRACT Regardless of the availability of highly sophisticated techniques and ever increasing computing capabilities, the problems associated with robots interacting with unstructured environments remains an open challenge. Despite great advances in autonomous robotics, there are some situations where a humanin- the-loop is still required, such as, nuclear, space, subsea and robotic surgery operations. This is because the current technologies cannot reliably perform all kinds of task autonomously. This thesis presents methods for robot teleoperation strategies at different levels of abstraction ranging from supervisory control, where the operator gives high-level task actions, to bilateral teleoperation, where the commands take the form of low-level control inputs. These strategies contribute to improve the current human-robot interfaces specially in the case of slave robots deployed at large workspaces. First, an approach to perform supervisory teleoperation of humanoid robots is presented. The goal is to control ground robots capable of executing complex tasks in disaster relief environments under constrained communication links. This proposal incorporates autonomous behaviors that the operator can use to perform navigation and manipulation tasks which allow covering large human engineered areas of the remote environment. The experimental results demonstrate the efficiency of the proposed methods. Second, the use of cost-effective devices for guided telemanipulation is investigated. A case study involving a bimanual humanoid robot and an Inertial Measurement Unit (IMU) Motion Capture (MoCap) suit is introduced. Herein, it is corroborated how the adaptation capabilities offered by the human-in-the-loop factor can compensate for the lack of high-precision robotic systems. This work is the result of collaboration between researchers from the Harvard Biorobotics Laboratory and the Centre for Automation and Robotics UPM-CSIC. Thirdly, a new haptic rate-position controller is presented. This hybrid bilateral controller copes with the problems related to the teleoperation of a slave robot with large workspace using a small haptic device as master. Large workspaces can be covered by automatically switching between rate and position control modes. This haptic controller is ideal to couple kinematic dissimilar master-slave systems where the commands are transmitted in the task space of the remote environment. The method is validated to perform dexterous telemanipulation of objects with a robotic manipulator. Finally, two contributions for robotic manipulation are introduced. First, a new algorithm, the Iterative Kinematic Decoupling method, is presented. It is a numeric method developed to solve the Inverse Kinematics (IK) problem of a type of six-DoF robotic arms where a close-form solution is not available. The effectiveness of this IK method is compared against conventional numerical methods. Second, a robust grasp mapping has been conceived. It allows to control a wide range of different robotic hands using a gesture based correspondence between the human hand space and the robotic hand space. The human hand gesture is identified by reading the relative movements of the index, thumb and middle fingers of the user during the early stages of grasping.

Distribution of haplotypes from a chromosome 21 region distinguishes multiple prehistoric human migrations

Despite mounting genetic evidence implicating a recent origin of modern humans, the elucidation of early migratory gene-flow episodes remains incomplete. Geographic distribution of haplotypes may show traces of ancestral migrations. However, such evolutionary signatures can be erased easily by recombination and mutational perturbations. A 565-bp chromosome 21 region near the MX1 gene, which contains nine sites frequently polymorphic in human populations, has been found. It is unaffected by recombination and recurrent mutation and thus reflects only migratory history, genetic drift, and possibly selection. Geographic distribution of contemporary haplotypes implies distinctive prehistoric human migrations: one to Oceania, one to Asia and subsequently to America, and a third one predominantly to Europe. The findings with chromosome 21 are confirmed by independent evidence from a Y chromosome phylogeny. Loci of this type will help to decipher the evolutionary history of modern humans.

Segregating the functions of human hippocampus

It is now accepted that hippocampal lesions impair episodic memory. However, the precise functional role of the hippocampus in episodic memory remains elusive. Recent functional imaging data implicate the hippocampus in processing novelty, a finding supported by human in vivo recordings and event-related potential studies. Here we measure hippocampal responses to novelty, using functional MRI (fMRI), during an item-learning paradigm generated from an artificial grammar system. During learning, two distinct types of novelty were periodically introduced: perceptual novelty, pertaining to the physical characteristics of stimuli (in this case visual characteristics), and exemplar novelty, reflecting semantic characteristics of stimuli (in this case grammatical status within a rule system). We demonstrate a left anterior hippocampal response to both types of novelty and adaptation of these responses with stimulus familiarity. By contrast to these novelty effects, we also show bilateral posterior hippocampal responses with increasing exemplar familiarity. These results suggest a functional dissociation within the hippocampus with respect to the relative familiarity of study items. Neural responses in anterior hippocampus index generic novelty, whereas posterior hippocampal responses index familiarity to stimuli that have behavioral relevance (i.e., only exemplar familiarity). These findings add to recent evidence for functional segregation within the human hippocampus during learning.

Promotion of agonist activity of antiandrogens by the androgen receptor coactivator, ARA70, in human prostate cancer DU145 cells

Although hormone therapy with antiandrogens has been widely used for the treatment of prostate cancer, some antiandrogens may act as androgen receptor (AR) agonists that may result in antiandrogen withdrawal syndrome. The molecular mechanism of this agonist response, however, remains unclear. Using mammalian two-hybrid assay, we report that antiandrogens, hydroxyflutamide, bicalutamide (casodex), cyproterone acetate, and RU58841, and other compounds such as genistein and RU486, can promote the interaction between AR and its coactivator, ARA70, in a dose-dependent manner. The chloramphenicol acetyltransferase assay further demonstrates that these antiandrogens and related compounds significantly enhance the AR transcriptional activity by cotransfection of AR and ARA70 in a 1:3 ratio into human prostate cancer DU145 cells. Our results suggest that the agonist activity of antiandrogens might occur with the proper interaction of AR and ARA70 in DU145 cells. These findings may provide a good model to develop better antiandrogens without agonist activity.

Detection of 400-year-old Yersinia pestis DNA in human dental pulp: An approach to the diagnosis of ancient septicemia

Ancient septicemic plague epidemics were reported to have killed millions of people for 2 millenniums. However, confident diagnosis of ancient septicemia solely on the basis of historical clinical observations is not possible. The lack of suitable infected material has prevented direct demonstration of ancient septicemia; thus, the history of most infections such as plague remains hypothetical. The durability of dental pulp, together with its natural sterility, makes it a suitable material on which to base such research. We hypothesized that it would be a lasting refuge for Yersinia pestis, the plague agent. DNA extracts were made from the dental pulp of 12 unerupted teeth extracted from skeletons excavated from 16th and 18th century French graves of persons thought to have died of plague (“plague teeth”) and from 7 ancient negative control teeth. PCRs incorporating ancient DNA extracts and primers specific for the human β-globin gene demonstrated the absence of inhibitors in these preparations. The incorporation of primers specific for Y. pestis rpoB (the RNA polymerase β-subunit-encoding gene) and the recognized virulence-associated pla (the plasminogen activator-encoding gene) repeatedly yielded products that had a nucleotide sequence indistinguishable from that of modern day isolates of the bacterium. The specific pla sequence was obtained from 6 of 12 plague skeleton teeth but 0 of 7 negative controls (P < 0.034, Fisher exact test). A nucleic acid-based confirmation of ancient plague was achieved for historically identified victims, and we have confirmed the presence of the disease at the end of 16th century in France. Dental pulp is an attractive target in the quest to determine the etiology of septicemic illnesses detected in ancient corpses. Molecular techniques could be applied to this material to resolve historical outbreaks.

In vitro repair of oxidative DNA damage by human nucleotide excision repair system: Possible explanation for neurodegeneration in Xeroderma pigmentosum patients

Xeroderma pigmentosum (XP) patients fail to remove pyrimidine dimers caused by sunlight and, as a consequence, develop multiple cancers in areas exposed to light. The second most common sign, present in 20–30% of XP patients, is a set of neurological abnormalities caused by neuronal death in the central and peripheral nervous systems. Neural tissue is shielded from sunlight-induced DNA damage, so the cause of neurodegeneration in XP patients remains unexplained. In this study, we show that two major oxidative DNA lesions, 8-oxoguanine and thymine glycol, are excised from DNA in vitro by the same enzyme system responsible for removing pyrimidine dimers and other bulky DNA adducts. Our results suggest that XP neurological disease may be caused by defective repair of lesions that are produced in nerve cells by reactive oxygen species generated as by-products of an active oxidative metabolism.

Paracrine stimulation of interstitial collagenase (MMP-1) in the human endometrium by interleukin 1α and its dual block by ovarian steroids

In the cycling human endometrium, the expression of interstitial collagenase (MMP-1) and of several related matrix metalloproteinases (MMPs) follows the late-secretory fall in sex steroid plasma concentrations and is thought to be a critical step leading to menstruation. The rapid and extensive lysis of interstitial matrix that precedes menstrual shedding requires a strict control of these proteinases. However, the mechanism by which ovarian steroids regulate endometrial MMPs remains unclear. We report here that, in the absence of ovarian steroids, MMP-1 expression in endometrial fibroblasts is markedly stimulated by medium conditioned by endometrial epithelial cells. This stimulation can be prevented by antibodies directed against interleukin 1α (IL-1α) but not against several other cytokines. Ovarian steroids inhibit the release of IL-1α and repress MMP-1 production by IL-1α-stimulated fibroblasts. In short-term cultures of endometrial explants obtained throughout the menstrual cycle, the release of both IL-1α and MMP-1 is essentially limited to the perimenstrual phase. We conclude that epithelium-derived IL-1α is the key paracrine inducer of MMP-1 in endometrial fibroblasts. However, MMP-1 production in the human endometrium is ultimately blocked by ovarian steroids, which act both upstream and downstream of IL-1α, thereby exerting an effective control via a “double-block” mechanism.

Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage

Stabilization of p53 in response to DNA damage is caused by its dissociation from Mdm2, a protein that targets p53 for degradation in the proteasome. Dissociation of p53 from Mdm2 could be caused by DNA damage-induced p53 posttranslational modifications. The ATM and ATR kinases, whose activation in response to ionizing radiation (IR) and UV light, respectively, is required for p53 stabilization, directly phosphorylate p53 on Ser-15. However, phosphorylation of Ser-15 is critical for the apoptotic activity of p53 and not for p53 stabilization. Thus, whether any p53 modifications, and which, underlie disruption of the p53–Mdm2 complex after DNA damage remains to be determined. We analyzed the IR- and UV light-induced stabilization of p53 proteins with substitutions of Ser known to be posttranslationally modified after DNA damage. Substitution of Ser-20 was sufficient to abrogate p53 stabilization in response to both IR and UV light. Furthermore, both IR and UV light induced phosphorylation of p53 on Ser-20, which involved the majority of nuclear p53 protein and weakened the interaction of p53 with Mdm2 in vitro. ATM and ATR cannot phosphorylate p53 on Ser-20. We therefore propose that ATM and ATR activate an, as yet unidentified, kinase that stabilizes p53 by phosphorylating it on Ser-20.

Novel Ras antagonist blocks human melanoma growth

During past decades, knowledge of melanoma biology has increased considerably. Numerous therapeutic modalities based on this knowledge are currently under investigation. Advanced melanoma, nevertheless, remains a prime example of poor treatment response that may, in part, be the consequence of activated N-Ras oncoproteins. Besides oncogenic Ras, wild-type Ras gene products also play a key role in receptor tyrosine kinase growth factor signaling, known to be of importance in oncogenesis and tumor progression of a variety of human neoplasms, including malignant melanoma; therefore, it is reasonable to speculate that a pharmacological approach that curtails Ras activity may represent a sensible approach to inhibit melanoma growth. To test this concept, the antitumor activity of S-trans, trans-farnesylthiosalicylic acid (FTS), a recently discovered Ras antagonist that dislodges Ras from its membrane-anchoring sites, was evaluated. The antitumor activity of FTS was assessed both in vitro and in vivo in two independent SCID mouse xenotransplantation models of human melanoma expressing either wild-type Ras (cell line 518A2) or activated Ras (cell line 607B). We show that FTS (5–50 μM) reduces the amounts of activated N-Ras and wild-type Ras isoforms both in human melanoma cells and Rat-1 fibroblasts, interrupts the Ras-dependent extracellular signal-regulated kinase in melanoma cells, inhibits the growth of N-Ras-transformed fibroblasts and human melanoma cells in vitro and reverses their transformed phenotype. FTS also causes a profound and statistically significant inhibition of 518A2 (82%) and 607B (90%) human melanoma growth in SCID mice without evidence of drug-related toxicity. Our findings stress the notion that FTS may qualify as a novel and rational treatment approach for human melanoma and possibly other tumors that either carry activated ras genes or rely on Ras signal transduction more heavily than nonmalignant cells.

Sustained secretion of human alpha-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors

Recombinant adeno-associated virus (AAV) vectors have been used to transduce murine skeletal muscle as a platform for secretion of therapeutic proteins. The utility of this approach for treating alpha-1-antitrypsin (AAT) deficiency was tested in murine myocytes in vitro and in vivo. AAV vectors expressing the human AAT gene from either the cytomegalovirus (CMV) promoter (AAV-C-AT) or the human elongation factor 1-α promoter (AAV-E-AT) were examined. In vitro in C2C12 murine myoblasts, the expression levels in transient transfections were similar between the two vectors. One month after transduction, however, the human elongation factor 1 promoter mediated 10-fold higher stable human AAT expression than the CMV promoter. In vivo transduction was performed by injecting doses of up to 1.4 × 1013 particles into skeletal muscles of several mouse strains (C57BL/6, BALB/c, and SCID). In vivo, the CMV vector mediated higher levels of expression, with sustained serum levels over 800 μg/ml in SCID and over 400 μg/ml in C57BL/6 mice. These serum concentrations are 100,000-fold higher than those previously observed with AAV vectors in muscle and are at levels which would be therapeutic if achieved in humans. High level expression was delayed for several weeks but was sustained for over 15 wk. Immune responses were dependent upon the mouse strain and the vector dosage. These data suggest that recombinant AAV vector transduction of skeletal muscle could provide a means for replacing AAT or other essential serum proteins but that immune responses may be elicited under certain conditions.