Ras Gene Product Research Articles

A RAS renaissance: emerging targeted therapies for KRAS-mutated non-small cell lung cancer.

Of the numerous oncogenes implicated in human cancer, the most common and perhaps the most elusive to target pharmacologically is RAS. Since the discovery of RAS in the 1960s, numerous studies have elucidated the mechanism of activity, regulation, and intracellular trafficking of the RAS gene products, and of its regulatory pathways. These pathways yielded druggable targets, such as farnesyltransferase, during the 1980s to 1990s. Unfortunately, early clinical trials investigating farnesyltransferase inhibitors yielded disappointing results, and subsequent interest by pharmaceutical companies in targeting RAS waned. However, recent advances including the identification of novel regulatory enzymes (e.g., Rce1, Icmt, Pdeδ), siRNA-based synthetic lethality screens, and fragment-based small-molecule screens, have resulted in a "Ras renaissance," signified by new Ras and Ras pathway-targeted therapies that have led to new clinical trials of patients with Ras-driven cancers. This review gives an overview of KRas signaling pathways with an emphasis on novel targets and targeted therapies, using non-small cell lung cancer as a case example.

Universal intracellular transducer ras and its role in the development of drosophila

Ras genes were first identified in the 1960s as transforming oncogenes that caused tumors in rats infected with Harvey and Kirsten sarcoma viruses (Ha-ras and Ki-ras oncogenes, accordingly). Subsequently, transforming ras genes were found in human cancer cells. Further investigations of neuroblastoma cells resulted in the finding of the third ras gene in the human, which was called N-ras. Ras gene products play an important role in the processes of cellular proliferation and differentiation and are controlled by receptor tyrosine kinases. Using drosophila as a model object allowed us to perform a successful genetic analysis while studying the functions of ras genes. Three polytene chromosome bands were detected in D. melanogaster with the help of the v-Ha ras sampling. According to Bridges' map, all three bands (Dras1, Dras2, Dras3) were mapped to regions 85D, 64B, and 62B of chromosome 3. Among them, only Drasl has a common origin with ras genes of mammals. Although there are numerous investigations of the role played by ras genes in the de- velopment of insects, this problem is still not fully understood. The importance of ras gene variations in the course of the evolutionary process has been insufficiently studied as well. Currently, Ras target proteins are actively identified, their signal pathways, as well as effects of influencing these pathways in the drosophila tissues, are studied in the cells of yeast and mammals. The main functions of Ras protein is in the signaling pathways controlling mutations during drosophila's morphogenesis and the connections of ras gene with phenotypic symptoms of tumors.

Sos-mediated cross-activation of wild-type Ras by oncogenic Ras is essential for tumorigenesis

Mammalian cells contain three closely related ras genes, H-ras, K-ras and N-ras. Although, in a given tumor type, oncogenic mutations are selectively observed in only one of the ras genes, the acquisition of the transformed phenotype has been shown to require the contribution of the normal products of the other ras genes. Here we demonstrate that oncogenic K-Ras promotes the activation of wild type H- and N-Ras. This activation is mediated by oncogenic K-Ras-dependent allosteric stimulation of Sos and confers a growth advantage to oncogenic K-Ras harboring cancer cells. These findings underscore the complementary functions of oncogenic and wild type Ras in tumor cells and identify a potential new targeting strategy for Ras-driven tumors.

Functional Specificity of Ras Isoforms: So Similar but So Different

H-ras, N-ras, and K-ras are canonical ras gene family members frequently activated by point mutation in human cancers and coding for 4 different, highly related protein isoforms (H-Ras, N-Ras, K-Ras4A, and K-Ras4B). Their expression is nearly ubiquitous and broadly conserved across eukaryotic species, although there are quantitative and qualitative differences of expression depending on the tissue and/or developmental stage under consideration. Extensive functional studies have determined during the last quarter century that these Ras gene products are critical components of signaling pathways that control eukaryotic cell proliferation, survival, and differentiation. However, because of their homology and frequent coexpression in various cellular contexts, it remained unclear whether the different Ras proteins play specific or overlapping functional roles in physiological and pathological processes. Initially, their high degree of sequence homology and the observation that all Ras isoforms share common sets of downstream effectors and upstream activators suggested that they were mostly redundant functionally. In contrast, the notion of functional specificity for each of the different Ras isoforms is supported at present by an increasing body of experimental observations, including 1) the fact that different ras isoforms are preferentially mutated in specific types of tumors or developmental disorders; 2) the different transforming potential of transfected ras genes in different cell contexts; 3) the distinct sensitivities exhibited by the various Ras family members for modulation by different GAPs or GEFs; 4) the demonstration that different Ras isoforms follow distinct intracellular processing pathways and localize to different membrane microdomains or subcellular compartments; 5) the different phenotypes displayed by genetically modified animal strains for each of the 3 ras loci; and 6) the specific transcriptional networks controlled by each isoform in different cellular settings.

Marked Amplification and Diversification of Products of ras Genes from Rat Brain, Rab GTPases, in the Ciliates Tetrahymena thermophila and Paramecium tetraurelia

Small GTPase Rab (products of ras genes from rat brain) is a widely conserved molecular switch among eukaryotes and regulates membrane trafficking pathways. It is generally considered that the number of Rab encoded in the genome correlates with multicellularity; however, we found that unicellular ciliates Tetrahymena thermophila (Tt) and Paramecium tetraurelia (Pt) possess many more Rab genes in their genome than the 64 HsRab genes in the human genome. We succeeded in isolating 86 cDNA clones of 88 TtRab genes in the Tetrahymena genome. By comparing the amino acid sequence of Rab in humans and the budding yeast Saccharomyces cerevisiae, 42 TtRab belonged to subfamilies functionally characterized and designated as conventional Rab, while the remaining 44 TtRab were considered to be species-specific. To examine the diversity of Rab in ciliates, we searched for Rab genes in the genome database of P. tetraurelia. Overall, 229 PtRab genes were found and categorized as 157 conventional and 72 species-specific PtRab, respectively. Among them, nine PtRab genes showed high homology to seven TtRab, suggesting the conservation of ciliate-specific Rab. These data suggested that the range of Rab is markedly amplified and diversified in ciliates, which may support the elaborate cellular structures and vigorous phagocytosis of those organisms.

Abstract 5076: Dissection of RAS downstream pathways in melanomagenesis

Abstract Cutaneous malignant melanoma is considered one of the most deadly human cancers, based upon both its penchant for metastatic spread and its typical resistance to currently available therapy. Long known to harbor oncogenic NRAS mutations, melanomas were more recently reported to be frequent bearers of activating mutations in BRAF, one of the effectors situated downstream of wildtype NRAS. RAS gene products (HRas, KRas and NRas) are 21kd G-proteins that serve as molecular switches converting cell-surface kinase activation events to nuclear events, thus influencing cell behavior. The major downstream effectors of Ras are the Rafs (ARaf, BRaf and CRaf), phosphatidylinositol 3-kinase (PI3K) and the Ral guanine exchange factors (RalGEF). NRAS and BRAF mutations are rarely found in the same melanoma, suggesting that they may possess important overlapping oncogenic activities. Here we compare and contrast the oncogenic roles of the three major NRas downstream effectors, Raf, PI3K and RalGEF, using genetically engineered Arf-deficient immortalized mouse melanocytes as a model system. Activation of each of the three major downstream pathways of NRas has overt phenotypic effects on immortalized mouse melanocytes, many distinct. Our data indicate a prominent role for BRaf and PI3K in melanocyte senescence and invasiveness, respectively. More surprisingly, we found that constitutively activated RalGEF stimulated melanocyte invasiveness and anchorage-independent growth almost as impressively as NRasQ61K. The suppressive effects of expression of a dominant negative Ral corroborated the role of the RalGEF-Ral pathway in anchorage-independent growth. Although no single downstream pathway could recapitulate all of the consequences of oncogenic NRas expression, melanocytes expressing constitutively activated mutants of all three NRas downstream factors (RLF-CAAX plus p110α-CAAX plus BRafV600E) achieved soft agar colony forming efficiency that was fully comparable to those expressing NRasQ61K alone. In conclusion, our data suggested a distinct function for all three NRas downstream pathways - Raf, PI3K and Ral in melanocyte senescence, invasiveness and transformation, respectively. Importantly, our finding that constitutive RalGEF activation confers malignant phenotypes in melanocytes, particularly anchorage-independent growth, indicates that this often overlooked pathway should be more carefully evaluated as a possible therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5076.

Abstract 4116: Relationship between K-ras 4A and superoxide in lung adenocarcinoma cell lines

Abstract The K-ras gene is mutated in 30-50% of human lung adenocarcinomas, and even more frequently in cancers of colon and pancreas. It exists in two protein isoforms, K-ras 4A and 4B. Their amino acid sequence is very similar, except for marked differences in the hypervariable domain near the carboxyl terminus. K-ras 4B, with a polylysine tail, is unique among the ras gene products and is the dominant K-ras isoform in most tissues. Wild-type K-ras 4B has tumor suppressor activity (Zhang et al. Nature Gen. 29:25, 2001), whereas K-ras 4A was recently found to be necessary for lung tumor development in mice (To et al. Nature Gen. 40:1240, 2008). In search of an oncogenic function for K-ras 4A in lung cancer, we previously demonstrated a highly significant (P<0.01) positive correlation between K-ras 4A expression, at both mRNA and protein levels, and superoxide amounts in a panel of 11 human lung adenocarcinoma cell lines (Proc. Am. Assoc. for Cancer Research 50: (#437)). Superoxide has been widely implicated in cancer causation and progression. In the present study, we test whether there is a cause-effect relationship between expression of K-ras 4A and superoxide production. Cell line H441 expresses high levels of both superoxide and K-ras 4A. Superoxide levels were determined using a nitroblue tetrazolium (NBT) assay and K-ras 4A protein levels quantified by immunoblot with a specific antibody. Results are means ± SEMs. The effect of reduced levels of K-ras 4A on superoxide production was determined using a K-ras 4A silencing siRNA, which reduced K-ras 4A protein by 60.2 ± 3.0% after 72 hours of siRNA treatment. This reduction had no effect on superoxide (control 1.13 ± 0.08, siRNA 1.17 ± 0.08 nmol NBT reduced/ug protein). The effect of reduced levels of superoxide on K-ras 4A expression was determined using 5 mM Tiron, an electron scavenger. A 36% decrease in superoxide did not affect K-ras 4A protein levels (control 26.90 ± 0.89 vs. Tiron-treated 26.89 ± 1.91, arbitrary units). If these preliminary results are confirmed with additional cell lines and reagents, they may suggest that both expression of K-ras 4A and production of superoxide are controlled by another, pro-oncogenic factor or set of conditions. In view of the importance of both K-ras and superoxide in cancer, discovery of this putative central pro-oncogenic regulator might offer an important preventive and therapeutic target in lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4116.

Identification of ras targets using a genetic approach.

The Sevenless receptor tyrosine kinase is required for the development of the R7 photoreceptor cell in the Drosophila eye. Several components of the Sevenless signal transduction pathway have been identified in genetic screens for enhancers/suppressors of the sevenless phenotype. These studies suggest that activation of Sevenless leads to stimulation of Ras1 activity, whereas Gap1 appears to act as a negative regulator of the pathway. Inactivation of the Gap1 locus causes transformation of non-neuronal cone cells into supernumerary R7 cells. This same mutant phenotype is observed when activated Ras1 is expressed under the control of the sevenless promoter. While studies in other organisms have demonstrated a role for ras gene products in signal transduction, the effectors of Ras activity have not yet been identified. We are carrying out genetic screens for enhancers and suppressors of the Gap1 and activated Ras1 phenotypes in the hope of identifying genes encoding some of these effectors. We are conducting chemical mutagenesis screens and have also screened existing collections of P element lines. A molecular characterization of the most promising mutations is in progress.

Ras as a therapeutic target in hematologic malignancies

The RAS gene product is normally a membrane-localized G protein (N-Ras, K-Ras and H-Ras) of 21 kDa classically described as a molecular off/on switch. It is inactive when bound to guanosine diphosphate and active when bound to GTP. When mutated, the gene produces an abnormal protein resistant to GTP hydrolysis by GTPase, resulting in a constitutively active GTP-bound protein that stimulates a critical network of signal transduction pathways that lead to cellular proliferation, survival and differentiation. At least three downstream effector pathways have been described, including Raf/MEK/ERK, PI3K/AKT and RalGDS, but they are not completely understood. Ras pathways are also important downstream effectors of several receptor tyrosine kinases localized in the cell membrane, most notably the BCR-ABL fusion protein seen in patients with Philadelphia chromosome positive chronic myelogenous leukemia. An important consideration in designing strategies to block Ras stimulatory effect is that Ras proteins are synthesized in the cytosol, but require post-translational modifications and attachment to anchor proteins or membrane binding sites in the cell membrane to be biologically active. Farnesyl transferase inhibitors (FTIs) are probably the best-studied class of Ras inhibitors in hematologic malignancies. They block the enzyme farnesyl-transferase (FTase), which is essential for post-translational modification. However, it has been observed that the Ras proteins also can be geranylgeranylated in the presence of FTIs, thus allowing membrane localization and activation, which limits their effectiveness. It is now hypothesized that their mechanism of action may be through FTase inhibition involving other signal transduction pathways. S-trans, trans-farnesylthiosalicylic acid, which was first designed as a prenylated protein methyltransferase inhibitor, has shown in vitro activity against all activated Ras proteins by dislodging them from their membrane-anchoring sites. Here, Ras biology, its signaling pathways and its implications as a therapeutic target in hematologic malignancies are reviewed.

Reversible inactivation of the transcriptional function of P53 protein by farnesylation

BackgroundThe use of integrating viral vectors in Gene therapy clinical trials has pointed out the problem of the deleterous effect of the integration of the ectopic gene to the cellular genome and the safety of this strategy. We proposed here a way to induce the death of gene modified cells upon request by acting on a pro-apoptotic protein cellular localization and on the activation of its apoptotic function.ResultsWe constructed an adenoviral vector coding a chimeric p53 protein by fusing p53 sequence with the 21 COOH term amino acids sequence of H-Ras. Indeed, the translation products of Ras genes are cytosolic proteins that become secondarily associated with membranes through a series of post-translational modifications initiated by a CAAX motif present at the C terminus of Ras proteins. The chimeric p53HRCaax protein was farnesylated efficiently in transduced human osteosarcoma p53-/- cell line. The farnesylated form of p53 resided mainly in the cytosol, where it is non-functional. Farnesyl transferase inhibitors (FTIs) specifically inhibited farnesyl isoprenoid lipid modification of proteins. Following treatment of the cells with an FTI, p53HRCaax underwent translocation into the nucleus where it retained transcription factor activity. Shifting p53 into the nucleus resulted in the induction of p21waf1/CIP1 and Bax transcription, cell growth arrest, caspase activation and apoptosis.ConclusionArtificial protein farnesylation impaired the transcriptional activity of p53. This could be prevented by Farnesyl transferase inhibition. These data highlight the fact that the artificial prenylation of proteins provides a novel system for controlling the function of a transactivating factor.

SAD: A Presynaptic Kinase Associated with Synaptic Vesicles and the Active Zone Cytomatrix that Regulates Neurotransmitter Release

A serine/threonine kinase SAD-1 in C. elegans regulates synapse development. We report here the isolation and characterization of mammalian orthologs of SAD-1, named SAD-A and SAD-B, which are specifically expressed in the brain. SAD-B is associated with synaptic vesicles and, like the active zone proteins CAST and Bassoon, is tightly associated with the presynaptic cytomatrix in nerve terminals. A short conserved region (SCR) in the COOH-terminus is required for the synaptic localization of SAD-B. Overexpression of SAD-B in cultured rat hippocampal neurons significantly increases the frequency of miniature excitatory postsynaptic current but not its amplitude. Introduction of SCR into presynaptic superior cervical ganglion neurons in culture significantly inhibits evoked synaptic transmission. Moreover, SCR decreases the size of the readily releasable pool measured by applying hypertonic sucrose. Furthermore, SAD-B phosphorylates the active zone protein RIM1 but not Munc13-1. These results suggest that mammalian SAD kinase presynaptically regulates neurotransmitter release.

Implications of the network structure of cellular control circuits for the design of anti-cancer vaccine therapies

I describe the use of a statistical mechanical model of a heirarchical network of failure-prone elements as a model for the failure of a network of interconnected biological elements that are or could be involved in the causation of cancer. From its topology, such a model predicts some of the properties of observed oncogene/tumour suppressor gene networks. I show that the same model suggests that a cancer vaccine strategy that targets ‘key’ network elements, such as p53 or RAS gene products, is not the only effective strategy for a broadly effective anti-cancer therapy. A mixed antigen or whole cell strategy could be as effective as single antigen vaccines, even if the latter are targeted at the most commonly mutated gene products.

Blocking oncogenic Ras signaling for cancer therapy.

The Ras gene product is a monomeric membrane-localized G protein of 21 kd that functions as a molecular switch linking receptor and nonreceptor tyrosine kinase activation to downstream cytoplasmic or nuclear events. Each mammalian cell contains at least three distinct ras proto-oncogenes encoding closely related, but distinct proteins. Activating mutations in these Ras proteins result in constitutive signaling, thereby stimulating cell proliferation and inhibiting apoptosis. Oncogenic mutations in the ras gene are present in approximately 30% of all human cancers. K-ras mutations occur frequently in non-small-cell lung, colorectal, and pancreatic carcinomas; H-ras mutations are common in bladder, kidney, and thyroid carcinomas; N-ras mutations are found in melanoma, hepatocellular carcinoma, and hematologic malignancies. The ras-signaling pathway has attracted considerable attention as a target for anticancer therapy because of its important role in carcinogenesis. In this review, the physiologic and biochemical properties of the Ras proteins, their mechanism of cell signaling, and their relation to human cancer will be discussed. Novel cancer therapeutic approaches based on the inhibition of Ras-mediated signaling, including inhibition of Ras processing, inhibition of Ras protein synthesis, and blockage of downstream Ras effectors, will be discussed.

Overexpression of human H-ras transgene is responsible for tumors induced by chemical carcinogens in mice.

Level of human prototype H-ras transgene expression in tumors induced by chemical carcinogens (N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea) was analyzed in human H-ras transgenic mice (CB6F1-TgrasH2 Jic mice). All forestomach tumors examined revealed about 2-fold overexpression of the human H-ras transgene with or without point mutation at codon 12 or codon 61. However, endogenous mouse H- and K-ras genes exhibited neither point mutation nor overexpression. These results suggested that increased levels of ras gene products in the cell played an important role in facilitating chemical carcinogenesis in transgenic mice.

Ras Regulates NFAT3 Activity in Cardiac Myocytes

Multiple distinct signal transduction pathways have been implicated in the development of cardiac myocyte hypertrophy. These hypertrophic pathways include those regulated by the Ras superfamily of small GTPases and a separate calcineurin-regulated pathway that culminates in the activation of the transcription factor NFAT3. In this report, we demonstrate a functional interaction between Ras-regulated and calcineurin-regulated pathways. In particular, expression in neonatal myocytes of a constitutively active form of Ras (V12ras), but not activating mutants of Rac1, RhoA, or Cdc42, results in an increase in NFAT activity. Similarly, expression of an activated Ras, but not other small GTPases, results in the nuclear translocation of an NFAT3 fusion protein. Expression of a dominant negative ras gene product blocks phenylephrine-stimulated NFAT transcriptional activity and the ligand-stimulated NFAT3 nuclear localization. Ras proteins appear to function upstream of calcineurin, because cyclosporin A blocks the ability of V12ras to stimulate NFAT-dependent transcription and nuclear localization. Similarly, expression of a dominant negative ras gene inhibits phenylephrine-stimulated calcineurin activity. Pharmacological inhibition of MEK1 or expression of a dominant negative form of c-Raf or ERK2 inhibits phenylephrine-stimulated NFAT3 activation. Conversely, NFAT activity was stimulated by expression of constitutively active forms of c-Raf or MEK1. Taken together, these results imply that, in cardiac myocytes, a Ras-regulated pathway involving stimulation of mitogen-activated protein kinase regulates NFAT3 activity.

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 microM) 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.

Regulation of Endothelial Cell Adherens Junctions by a Ras-Dependent Signal Transduction Pathway

Adherens junctions, consisting of transmembrane cadherin molecules and their associated cytoplasmic α-, β-, and γ-catenin proteins, are thought to be critical for the development of stable cell adhesion and subsequent 3-dimensional tissue organization. In human endothelial cells there is a marked induction of γ-catenin levels when cells reach confluence. We demonstrate that expression of a dominant negative ras gene product (N17ras) via adenoviral mediated gene transfer inhibits the confluent-dependent rise in γ-catenin mRNA and protein levels. Consistent with its effects on overall γ-catenin levels, expression of N17ras also reduces the amount of γ-catenin associated with the adherens junction. Finally, although expression of N17ras under normal culture conditions produces no clear morphological phenotype, endothelial cells expressing a dominant negative ras gene product fail to form 3-dimensional, vascular-like structures when plated on reconstituted extracellular matrix.

Activity of SCH 66336, a tricyclic farnesyltransferase inhibitor, against human tumor colony-forming units

The ras gene product regulates transduction of growth-proliferative signals from the membrane to the nucleus. Mutationally-activated Ras is the oncogene most frequently found in human tumors. In order to perform its function in cell signaling, Ras must be farnesylated on the CAAX motif present on the carboxyl terminus of the ras protein. This reaction is catalysed by farnesyl protein transferase. In the present study, SCH 66336, an orally bioavailable nonpeptide tricyclic farnesyltransferase inhibitor, was tested against a large variety of human tumors to define its preclinical activity profile, utilizing the human tumor cloning assay. A soft agar cloning assay was used to determine the in vitro effects of SCH 66336 against primary human tumor specimens taken directly from patients. A total of 70 evaluable specimens were exposed to SCH 66336 for 14-day continuous exposure at concentrations ranging from 0.1 to 2.5 microM. In vitro responses were defined as an inhibition > or = 50% of human tumor colony forming units at a given concentration. There was a positive relationship between concentration and response to SCH 66336. With the highest concentration (2.5 microM), response was demonstrated in 50% (three of six) of breast tumors, 40% (6 of 15) of ovarian tumors, and 38% (5 of 13) of non-small-cell lung tumor colony forming units. Among the 69 specimens tested at the concentration of 2.5 microM, SCH 66336 had activity in 27% of tumor specimens that were resistant to doxorubicin, 38% of tumor specimens resistant to cisplatin, 33% of tumor specimens resistant to paclitaxel, and 27% of tumor specimens resistant to etoposide. The broad spectrum of soft agar growth inhibition by SCH 66336 in the human tumor cloning assay, and its efficacy at physiologically relevant concentrations in animal models, suggest that SCH 66336 may deserve future clinical trials in patients with ovarian, breast and non-small-cell lung cancer.

Adenovirus-mediated gene transfer of RasN17 inhibits specific CCK actions on pancreatic acinar cells.

CCK stimulates pleiotrophic responses in pancreatic acinar cells; however, the intracellular signaling pathways involved are not well understood. To evaluate the role of the ras gene product in CCK actions, a strategy involving in vitro adenoviral-mediated gene delivery of a dominant-negative mutant Ras (RasN17) was utilized. Isolated acini were infected with various titers of either a control adenovirus or an adenoviral construct expressing RasN17 for 24 h before being treated with CCK. Titer-dependent expression of RasN17 in the acini was confirmed by Western blotting. Infection with control adenovirus [10(6)-10(9) plaque-forming units/mg acinar protein (multiplicity of infection of approximately 1-1,000)] had no effect on CCK stimulation of acinar cell amylase release, extracellular-regulated kinase (ERK) or c-Jun kinase (JNK) kinases, or DNA synthesis. In contrast, infection with adenovirus bearing rasN17 increased basal amylase release, inhibited CCK-mediated JNK activation, had no effect on CCK activation of ERK, and inhibited DNA synthesis. These data demonstrate important roles for Ras in specific actions of CCK on pancreatic acinar function.

Sulindac sulfide inhibits Ras signaling.

The non-steroidal anti-inflammatory drug sulindac is used in cancer prevention and therapy, but the molecular aspects of its anti-tumor effect remain unresolved. In vivo the prodrug sulindac, is converted into the metabolite sulindac sulfide. We found that sulindac sulfide strongly inhibits Ras induced malignant transformation and Ras/Raf dependent transactivation. Sulindac sulfide decreases the Ras induced activation of its main effector, the c-Raf-1 kinase. In vitro sulindac sulfide directly binds to the Ras gene product p21ras in a non-covalent manner. Moreover, we can show that sulindac sulfide inhibits the interaction of p21ras with the p21ras binding domain of the Raf protein. In addition, sulindac sulfide can impair the nucleotide exchange on p21ras by CDC25 as well as the acceleration of the p21ras GTPase reaction by p120GAP. Due to its action at the most critical site in Ras signaling we propose sulindac sulfide as a lead compound in the search for novel anti-cancer drugs which directly inhibit Ras mediated cell proliferation and malignant transformation.