Raf1 Is a Validated Target in Cancer Therapy

Raf-1 (c-Raf), B-Raf, and A-Raf are members of a family of serine-threonine kinases known to regulate extracellular signal-regulated kinase -mediated mito-genic signal transduction pathways (11). Numerous reports from our laboratory and others have shown that Raf-1 plays a key role in cell proliferation, survival, In Vivo Delivery Systems of Antisense Oligonucleotides3 Molecular Formulation I.D. and target ASO parameters in vivo6 Cationic liposomes (first generation) Cationic liposomes (second...

Gti2040

3PS, phosphorothioate MBO, mixed-backbone minimally modified, one base at 5 - and 3 -ends has PS-linkage MM, multiple myeloma AML, acute myeloid leukemia HRPC, hormone-refractory prostate cancer. 3PS, phosphorothioate MBO, mixed-backbone minimally modified, one base at 5 - and 3 -ends has PS-linkage MM, multiple myeloma AML, acute myeloid leukemia HRPC, hormone-refractory prostate cancer. Fig. 10. Plasma pharmacokinetics of liposomal nanoparticle containing minimally modified raf antisense...

Vegf

Cdk inhibitors (R-roscovitine, rapamycin) Dominant-negative In vitro, xenograft mouse model In vitro In vitro In vitro, xenograft mouse model In vitro, xenograft mouse model In vitro In vitro, xenograft mouse model In vitro In vitro, xenopus model, xenograft mouse model In vitro, xenograft mouse model In vitro, xenograft mouse model In vivo In vitro, phase I clinical study In vitro, xenograft mouse model In vitro

Ribozymes Targeting Survivin

We first demonstrated the possibility to efficiently inhibit survivin expression through the use of ribozymes. Specifically, we designed two hammerhead ribozymes targeting the 3'-end of the CUA110 (RZ7) and the GUC294 (RZ1) triplets in the survivin mRNA and transfected them into the JR8 human melanoma cell line over expressing survivin. Stably transfected clones proven to endogenously express the active ribozyme RZ1 or RZ7 were characterized by a markedly lower survivin protein level than JR8...

And Short Hairpin RNA Sequences for Mammalian RNA Interference

Design for Small-Interfering RNA Highly functional class Ia siRNAs with maximal target-specificity for mammalian RNAi can be designed by using the web-based online software system, siDirect (http design.RNAi.jp ) (33), which is driven by algorithm I (23) and an accelerated off-target search algorithm developed by Yamada and Morishita (35). In siDirect, functional siRNAs for human and mouse RNAi (see Note 3) are easily designed under default conditions. 1. First, enter the accession...

Targeting Downstream Effectors of PCatenin

P-catenin Tcf regulate the expression of multiple genes some of which are known to promote tumor growth, including c-Myc, cyclin D1, c-Jun, VEGF, and COX-2. Therefore, targeting these genes in cancers with deregulated Wnt signaling might be a promising alternative to the previously mentioned concepts. The oncogene c-Myc is overexpressed in a variety of human cancers including leukemia, lymphoma, melanoma, breast cancer, and colorec-tal cancer (189). Being a direct target gene of P-catenin,...

Ribozymes Targeting Telomerase

Telomerase is an exploitable target for strategies based on the use of antisense oligonucleotides. In fact, the template region of hTR, which naturally binds to the 3' single-strand overhang of the telomere end to add new telomeric repeats, is inherently accessible to incoming nucleic acids and represents a suitable target site for these approaches. A number of studies on experimental human tumor models have shown the possibility to obtain efficient inhibition of telomerase through the use of...

References

C., Jr., Bock, P., et al. (1995) CSF-1 treatment promotes angiogenesis in the metaphysis of osteopetrotic (toothless, tl) rats. Bone 16, 315-324. 2. Aharinejad, S., Abraham, D., Paulus, P., et al. (2002) Colony-stimulating factor-1 antisense treatment suppresses growth of human tumor xenografts in mice. Cancer Res. 62, 5317-5324. 3. Aharinejad, S., Paulus, P., Sioud, M., et al. (2004) Colony-stimulating factor-1 blockade by antisense oligonucleotides and small...

Introduction

Although several factors may influence individual drug responses, including age, gender, disease state, and oigan function, there is increasing evidence that a large portion of variability in drug response is genetically determined (1,2). In addition to DNA polymorphisms that represent common variation in a DNA sequence leading to either reduced or increased activities of the encoded protein (3), gene expression profiling has enhanced our understanding of disease mechanism, classification of...

Info

Target for pharmacological intervention in cancer. MEK is a crucial intracellular kinase for many mitogene-signaling pathways activated by oncogenes (46). Although MEK is not identified as an oncogene, its constitutive activation can result in cellular transformation (46). In this respect, small molecular compounds that inhibit the activity of MEK have been developed (46). PD09059 is a synthetic inhibitor, which inhibits the activation of MEK1 and in lesser extent MEK2 (47). PD184352 is a...

Analysis of Cell Responses

Proliferative responses may be measured by incorporation of 3H thymidine in response to stimulation of lymph node cells with type II collagen. Alternatively, cytokines in culture supernatants can be measured by ELISA. 2. Make up stock solution of type II collagen in 0.05 M Tris-HCl, 0.2 M NaCl, pH 7.4, at 1 mg mL. Collagen in solution can be kept at 4 C for up to 3 mo (for stimulation of T cells only see Note 2). 3. Remove draining (inguinal) lymph nodes from collagen-immunized mice. 4. Push...

Isis2503

Inhibiting Ha-Ras protein expression In addition to quinazoline compounds, monoclonal antibodies (mAb), which block receptor activation, were developed. These antibodies bind to the receptors with affinity comparable to the natural ligands, compete with ligand binding, and thereby block the tyrosine kinase-receptor activity (5). Cetuximab (IMC-225) and Erbitux are two anti-EGFR (ErbB1) mAb, which are in phase II III studies. mAb 225 induces antibody-mediated receptor dimerization (without...

Methyl Cytosine

Epigenetics refers to alterations in gene expression that occur without a change in DNA sequence. The chemistry behind one of these events is simple. It involves the covalent addition of a methyl group to cytosine. Of the four bases that make up DNA adenine, thymine, cytosine, and guanine only cytosine has the potential to be methylated in humans and most mammals. The methylation reaction involves flipping the target cytosine out of the intact double helix, so that the transfer of the...

The Influence of Genetic Variations on Drug Effects in Other Diseases

The increasing focus on the role of genetic variation in drug targets and their downstream signaling pathways will extend the pharmacogenetic knowledge platform from the field of drug metabolizing enzymes into the clinically important target area. Thus, a number of genetic polymorphisms have been detected in drug targets and signaling elements, some of which have attained interest with regard to pharmacological treatment responses. The group of G protein-coupled receptors (GPCR), of which there...

Neurological Diseases

Certain data suggest that G protein pathways may play a role in neurological diseases including epilepsy and Alzheimer's disease. Various experimental models used in studies of epilepsy shed light on mechanisms involving GPCRs and their heterotrimeric G proteins. In mice it was found that the neuropeptide Y receptor, Y5R, mediated antiepileptic actions of neuropeptide Y regulating limbic seizures (104). Limbic status epilepticus in a rat model was also recently reported to be counteracted by...

In Vivo Applications of the Aptamers

To be of practical use, in vivo aptamers must possess defined molecular properties, for instance, adequate stability in the biological situation in which it will be employed, or sufficient systemic clearance in the case of aptamers used as imaging reagents. One of the major limitations of the use of aptamers, especially RNA-based aptamers, in cell culture and animal models, is their rapid degradation by nucle-ases. To date, several chemical modifications have been employed to overcome this...

Chuanbo Zhang Jin Pei Deepak Kumar Isamu Sakabe Howard E Boudreau Prafulla C Gokhale and Usha N Kasid

Antisense oligonucleotides (ASO) against specific molecular targets (e.g., Bcl-2 and Raf-1) are important reagents in cancer biology and therapy. Phosphorothioate modification of the ASO backbone has resulted in an increased stability of ASO in vivo without compromising, in general, their target selectivity. Although the power of antisense technology remains unsurpassed, dose-limiting side effects of modified ASO and inadequate penetration into the tumor tissue have necessitated further...

Personalized Medicine Based on Monitoring miRNA Expression

Several studies have shown that some diseases, such as cancer, are associated with a change in miRNA expression levels (63,70). But whether these changes are the cause or the effect of cancer remains to be determined. As for viral infections, miRNAs expressed from viruses like Epstein-Barr virus (80) and Kaposi's sarcoma-associated herpesvirus (81) may influence viral and host gene expression. These observations suggest that monitoring of miRNA expression levels in an affected organ or tissue...

Other Promising Potential Therapeutic Targets

Several inhibitory mechanisms can act at various levels of signal transduc-tion pathways. At least three additional main strategies hold potential for inhibiting signal transduction pathways. One strategy consists in inhibiting an enzyme that activates a signal transduction pathway by administering a pharmacological inhibitor. First, Imatinib Mesylate (Gleevec ) is a tyrosine kinase inhibitor that is highly effective in several haematological malignancies. Second, the role of p38...

Cellular Responses to Extracellular HMGB1

The cellular responses to extracellular HMGB1 vary considerably depending upon the cell type. As such, cell-type specific responses to HMGB1 stimulation, and their relative contributions to inflammatory responses are addressed specifically (Fig. 3). Immune cells HMGB1 is a proinflammatory cytokine that acts as a very potent activator of macrophages and monocytes. HMGB1 stimulates the migration and phagocytosis of these cells (24,25), and activates the production and secretion of a battery of...

Marjorie P Perron Vincent Boissonneault Lise Andre Gobeil Dominique L Ouellet and Patrick Provost

With potentially up to 1000 microRNAs (miRNAs) present in the human genome, altogether regulating the expression of thousands of genes, one can anticipate that miRNAs will play a significant role in health and disease. Deregulated protein expression induced by a dysfunctional miRNA-based regulatory system is thus expected to lead to the development of serious, if not lethal, genetic diseases. A relationship among miRNAs, Dicer, and cancer has recently been suggested. Further investigations will...

Intraportal Delivery of siRNA in Mice

The hydrodynamic tail-vein injection technique leads to high and reproducible siRNA uptake into the liver. It offers the possibility to investigate the effects of siRNA-mediated gene knockdown in the target organ liver in different physiological and pathophysiological settings (see Note 3). However, a criticism against hydrodynamic tail-vein injection is that it is an experimental procedure that is not feasible in the clinical situation in humans. Therefore, it is desirable to have an...

Laura Cerchia and Vittorio de Franciscis Summary

Isolated through combinatorial libraries by an iterative in vitro selection process, small single-stranded nucleic acid compounds, named aptamers, have been developed as high-affinity ligands for a variety of targets, ranging from small chemical compounds to large proteins. In the last years, an increasing number of aptamers has been generated that represent potential antagonists of the disease-associated target proteins. These molecules have been shown to discriminate between even closely...

A

Acute promyelocyte leukemia (APL), 43 Adenomatous polyposis coli (APC), 67 Albright's hereditary osteodystrophy, 131 Allegro high throughput screening system, 301 Angiotensin-converting enzyme (ACE), 332 Anti-CD40 ligand therapy, 291 Anti-CD154 antibody therapy, 292 Anti-TNFa antibodies, 272 Anticancer drugs, 6, 9, 12, 13, 14, 15, 18 Anticollagen IgG, 277 Antitumor-necrosis-a therapies in RA, 290 Antisense, 72 treatment, 231 Antisense oligonucleotides (ASO), 163 cancer biology and therapy, 165...

Contents of Volume

Emerging Strategies for Targets and Biomarker Discovery 1 Main Approaches to Target Discovery and Validation Mouldy Sioud 2 Bioinformatics Approaches to Cancer Gene Discovery Ramaswamy Narayanan 3 Analysis of Gene Networks for Drug Target Discovery and Validation Seiya Imoto, Yoshinori Tamada, Christopher J. Savoie, and Satoru Miyano 4 Target Discovery and Validation in Pancreatic Cancer Robert M. Beaty, Mads Gronborg, Jonathan R. Pollack, 5 Molecular Classification of Breast Tumors Toward...

CSF1 and Macrophages

The production of macrophages is regulated by colony-stimulating factor (CSF)-1 also called macrophage-CSF (M-CSF) (19). CSF-1 is produced by a variety of cell types such as fibroblasts or macrophages and prevents the death of monocytes and promotes their differentiation into macrophages (20,21). CSF-1 also induces or augments the production of a variety of cytokines by macrophages such as tumor necrosis factor-a (22). Macrophages most likely enhance tumor progression through paracrine circuits...

Receptors and Cytokine Activity of HMGB1

HMGB1 is considered a cytokine because it is secreted by activated immune cells, transduces signals through cell surface receptors, and induces conventional inflammatory responses in immune and endothelial cells (1,2). Extracellular HMGB1 binds to the cellular receptor for advanced glycation end-products (RAGE) in a concentration-dependent manner (36). RAGE is a transmembrane protein that belongs to the immunoglobulin super-family, and acts as a receptor for diverse ligands including RAGE,...

Concluding Remarks and Potentials for Therapeutic Options

Heterotrimeric G proteins transduce signals in response to a number of hormones and neurotransmitters. Furthermore, about 50 of all drugs used in therapy act through GPCRs, thus emphasizing the importance of GPCR G protein signaling pathways in medical treatment. In addition to the role of G proteins in signaling mechanisms, the identification of their localization also intracellu-larly as well as the discovery of novel receptor-independent mechanisms for their activation, suggest that G...

Normalizing the Stroma Through Targeting NG2HMP 31 mAbDirected Therapy

Antibody-based immunotherapy has been greatly investigated as a therapeutic strategy against NG2 HMP-expressing melanomas. This method utilizes a molecular vehicle, mAb, to selectively deliver radionuclides or toxins to tumor cells (100). Several studies have used various mAbs that recognize the NG2 HMP because the majority of melanomas extensively express it. There is limited intra- and intertumor heterogeneity, where it has been detected on > 90 of melanoma cells (49,101,102). Furthermore,...

Preclinical Development and Validation of Liposome Entrapped raf Antisense Oligonucleotide

A schematic diagram of LErafAON formulation is shown in Fig. 4A. The rafAON is a 15-mer ASO with phosphorothioate linkage limited to one base at 5'- and 3'-ends (30). The rafAON sequence is targeted against the translation initiation region of c-raf-1 mRNA. The liposome formulation consists of a mixture of a cationic lipid (dimethyldioctadecyl ammonium bromide), egg phos-phatidylchloline, and cholesterol in a molar ratio of 1 3.2 1.6. The rafAON to lipid ratio is 1 15 (w w). The particle size...

Andreas Herbst and Frank Thomas Kolligs

The Wnt p-catenin signaling pathway is tightly regulated and has important functions in development, tissue homeostasis, and regeneration. Deregulation of Wnt p-catenin signaling is frequently found in various human cancers. Eighty percent of colorectal cancers alone reveal activation of this pathway by either inactivation of the tumor-suppressor gene adenomatous polyposis coli or mutation of the proto-oncogene P-catenin. Activation of Wnt p-catenin signaling has been found to be important for...

Hydrodynamic Intravascular Injection

The large volume hydrodynamic delivery technique was a major advance in the in vivo delivery of nucleic acids into mice and rats. In principle, it means the injection of a high volume into the vascular system within a short period of time. The first report that this delivery method can be even more effective than direct injection into the target organ is from Budker et al. (13), who injected high volumes of either lacZ or luciferase reporter plasmids into the portal vein of mice. Using this...

CSF1 Antisense Treatment Suppresses Growth of Human Embryonic and Colon Carcinoma Xenografts in Mice

Human Embryonic Cancer Cells Upregulate Host CSF-1 Production Human embryonic cancer cells (CRL-2073) show no detectable mRNA or protein for human CSF-1 or CSF-1R in vitro. When these cells are xenografted into the testis of SCID mice, however, mouse tissue CSF-1 gene and protein expression increases significantly compared to untreated mice. Associated with increasing CSF-1 tissue expression is an enhanced infiltration of macrophages within and surrounding the tumor. These findings...

The Brain Stroma as the Glioma Backdrop

The human brain is made up of numerous cell types that interact physically through cell-cell, and cell-extracellular matrix (ECM) contacts, and biochemically via soluble and insoluble signaling molecules. Neuroepithelial cells of the central nervous system (CNS) constitute neuroglia (astrocytes, oligodendro-cytes, and microglia) and neurons, which are derived from the neuroectoderm. These cells produce the ECM, an important feature of the normal stroma, which provides structural scaffolding as...

Influence of Genetic and Other Factors on Drug Effects in Cancers

Cancer progression is characterized by the accumulation of multiple genetic mutations, chromosomal instabilities, and or epigenetic changes that cooperate to drive malignancy (7). Also, changes in gene expression are further affected by the microenvironment. Indeed, it is now well established that the microenvironment of the tumor-host interface plays a proactive role during malignant disease progression, including the transition from carcinoma in situ to invasive cancer, tumor cell...

Heterotrimeric G Proteins and Disease 0yvind Melien

Heterotrimeric G proteins attached to the cell membrane convey signals from G proteincoupled receptors in response to stimulation by a number of hormones, neurotransmitters, chemokines, and pharmacological agents to intracellular signaling cascades. The heterotrimeric G proteins are also located in the cell interior, and receptor-independent mechanisms may elicit their activation. Thus, G proteins may possibly exert cellular functions other than acting as signaling transducers. There is also...

NG2 at the Tumor Stroma Interface

Glial Cell Differentiation

NG2 is a 300-kDa membrane spanning CSPG that was originally identified as a surface epitope on a subset of tumor cell lines derived from rat embryos after ethylnitrosourea administration (6). The cell lines were characterized by the presence of Na+ and K+ ion channels, but an inability to generate full-fledged action potentials, features characteristic of neural precursor cells. NG2 positive neural precursors can differentiate into either neuroglia or neurons, hence the designation nerve glial...

Expression wtLfLHttf

Anomalies) is transmitted as an autosomal recessive trait. It is characterized by immune deficiency in association with unstable paracentromeric heterochromatin instability (extensively related with hypomethylated genomic regions) and facial dysmorphism. Patients are affected by recurrent respiratory infections beginning in childhood. The syndrome directly results from mutations in the gene encoding for DNA-methyltransferase 3B. This may explain the hypomethylation in the pericentromeric...

Mouldy Sioud and 0yvind Melien

Although several drug targets are identified, current strategies in therapy do not take into account that patients vary in their response to drugs, both with respect to efficacy and toxic side effects. Whereas both clinical and histopathologic predictors of prognosis are established in some diseases, a better understanding of the molecular mechanisms that determine treatment response should play an important role in the development of individualized medicine. Treatment optimization will rely on...

Therapeutical Use of siRNA to Prevent and Treat Acute Liver Failure in Mice

As the first demonstrations of a therapeutic application of siRNA in vivo were done in mouse models of acute liver failure (ALF), the next paragraphs will give a brief introduction into this disease, followed by a discussion of the therapeutic use of siRNA to suppress apoptosis in this model. ALF is defined as a dramatic clinical syndrome in which a previously normal liver fails within days or weeks. Three subgroups of ALF can be distinguished, hyperacute, acute, and subacute liver failure....

DNA Methylation and Histone Modifications in Patients With Cancer

Acetlation Histone

Potential Prognostic and Therapeutic Targets Michel Herranz and Manel Esteller Epigenetics, a combination of DNA modifications, chromatin organization, and variations in its associated proteins, configure a new entity that regulates gene expression throughout methylation, acetylation, and chromatin remodeling. In addition to silencing as a result of mutations, loss of heterozygosity, or classical genetic events epigenetic modification symbolizes essential early events during carcinogenesis and...

CSF1 in Breast Cancer

The mechanism by which mammary epithelial cells undergo genetic changes that result in acquisition of the ability to invade and colonize distant sites is complex (2,41,42). Normal and malignant mammary epithelium and the surrounding stromal cells produce and respond to various growth factors. Among the stromal cells, macrophages play a unique role because they are recruited into mammary gland carcinomas (43,44). The fact that in the absence of such tumor-associated macrophages, metastatic...

Hydrodynamic High Volume Tail Vein Injection in Mice see Note

In principle, any mouse strain can be used for this technique. BALB c or other strains with white fur are preferred as visualization and puncture of the tail vein, especially with bigger needles, is more difficult in black mice. We prefer to perform standard hydrodynamic tail-vein injection without anesthesia, as the combination of the high-volume injection together with anesthesia can lead to complications in some mice. If anesthesia is used, a gentle gas-anesthesia (e.g., isoflurane) should...

Lars Zender and Stefan Kubicka

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by double-stranded RNA. RNAi was shown to allow transient or stable knockdown of gene expression in a broad range of species and has been used successfully for functional genomic screens in mammalian cells and Caenorhabditis elegans. Standard therapeutic use of RNAi in clinical settings in humans has been hampered by the lack of effective methods to deliver the small-interfering RNAs (siRNAs) or short-hairpin RNA...

In Vivo Delivery of Antisense Oligonucleotides

The criteria of a clinically and commercially compatible ASO delivery system include nanoparticle size (< 1 in diameter), biodegradability, high encapsulation entrapment efficiency, ASO stability and favorable pharmaco-kinetics, tumor-specific drug delivery, easy formulation, scalability, and safety profiles in animals and humans. Such an optimal in vivo delivery system offers merits that may complement not only the chemically modified ASO but also natural ASO. First, a suitable vehicle may...

Druggable Signaling Proteins

In normal cells, signaling pathways are tightly regulated. However, when they are aberrantly activated, certain pathways are capable of causing diseases. In many tumors, the aberrantly activated signaling proteins include members of the epidermal growth factor receptor family, the Ras proteins, protein kinase C isoenzymes, BCR-ABL fusion protein as well as transcription factors such as signal transducers and activators of transcriptions and Myc. Accordingly, deregulation of these signaling...

AntiCD40 Ligand Therapy

Production of pathogenic antibodies in SLE requires T-cell help along with ligation of the B-cell surface immunoglobulin receptor by antigen. It is likely that macrophages, dendritic cells, and endothelial cells are also activated by interaction with T-cells and contribute to lupus pathology. CD40 ligand (CD40L, CD154), a member of TNF family of cell surface molecules, mediates these contact-dependent signals delivered by CD4+ T helper cells to CD40+ target cells (46). Several tissue injuries...

Efficient Delivery the Holy Grail of Gene Therapeutic Approaches

The holy grail of all in vivo gene therapeutic approaches is to deliver effective amounts of the therapeutic gene into the desired target cells. Currently, viral vectors are most widely used for delivery of therapeutic genes in vivo. However, vector-associated side effects limit their value as a tool for clinical use. Systemic application of plasmid DNA or antisense oligonucleotides ASO , however, leads to very low uptake in most tissues. In part, this is owing to the fact that nucleic acids...

Martha Chekenya and Heike Immervoll

Neuroepithelial cells of the central nervous system constitute neuroglia astrocytes, oligodendrocytes, and microglia , ependyma, and neurons, which make up the stromal cells of the brain. The stromal tissue organization of the brain is tightly regulated, but occasionally the signals that define the normal contexts become disrupted and result in cancer. Malignant progression is then maintained by cross-talks between the tumor and its stroma, where the activated stroma nurtures the proliferative...