Ras-mediated Signal Transduction Research Articles

Identification of long non-coding RNAs in two anthozoan species and their possible implications for coral bleaching

Long non-coding RNAs (lncRNAs) have been shown to play regulatory roles in a diverse range of biological processes and are associated with the outcomes of various diseases. The majority of studies about lncRNAs focus on model organisms, with lessened investigation in non-model organisms to date. Herein, we have undertaken an investigation on lncRNA in two zoanthids (cnidarian): Protolpalythoa varibilis and Palythoa caribaeorum. A total of 11,206 and 13,240 lncRNAs were detected in P. variabilis and P. caribaeorum transcriptome, respectively. Comparison using NONCODE database indicated that the majority of these lncRNAs is taxonomically species-restricted with no identifiable orthologs. Even so, we found cases in which short regions of P. caribaeorum’s lncRNAs were similar to vertebrate species’ lncRNAs, and could be associated with lncRNA conserved regulatory functions. Consequently, some high-confidence lncRNA-mRNA interactions were predicted based on such conserved regions, therefore revealing possible involvement of lncRNAs in posttranscriptional processing and regulation in anthozoans. Moreover, investigation of differentially expressed lncRNAs, in healthy colonies and colonial individuals undergoing natural bleaching, indicated that some up-regulated lncRNAs in P. caribaeorum could posttranscriptionally regulate the mRNAs encoding proteins of Ras-mediated signal transduction pathway and components of innate immune-system, which could contribute to the molecular response of coral bleaching.

Abstract PR09: A systems biology approach to elucidate novel drug targets in KRAS mutant tumors

Abstract Mutations of RAS GTPase family oncogenes affect more than 20% of human cancers. Of the three major RAS isoforms (HRAS, KRAS, and NRAS), KRAS is the most frequently mutated, affecting 57% of pancreatic adenocarcinomas, 33% of colorectal cancers, 17% of lung adenocarcinomas, and many other tumor subtypes. Most RAS mutations abrogate its GTPase activity, thus activating aberrant downstream signals associated with worst prognosis and tumor resistance to both chemotoxic and targeted therapy. Therapies against RAS have failed, in part, because of our limited understanding of RAS-mediated signal transduction pathways. Therefore, we hypothesize that the systematic and complete dissection of RAS relevant regulatory interactions can help elucidate druggable mechanisms to abrogate its aberrant activity or elicit synthetic lethality in RAS mutated tumors. Here, we have employed computational systems biology approaches to begin to dissect KRAS regulatory pathways. Specifically, we reverse-engineered a cell context specific KRAS regulatory network from TCGA transcriptome-wide expression data. The KRAS regulatory network was used to predict KRAS activation levels across all colon and lung adenocarcinoma samples available in TCGA in order to generate a patient-by-patient estimate of KRAS activity. Genome-wide somatic mutations and copy-number variations were then tested for significant association with the predicted KRAS activity. Consistent with known KRAS biology, samples harboring KRAS or BRAF activating mutations were significantly associated with increased KRAS activity. However, we also observed that mutations in the DEAD box helicase DDX6 and the Glycolipid Transfer-domain protein GLTPD2 to be significantly associated, suggesting that these proteins may be involved in KRAS pathways. We dissected the effects of KRAS mutations on the activity of all transcription factors, identifying those more closely activated or repressed in a KRAS-specific context. KRAS is activated by its own mutations, along with known KRAS interactors, such as SOS1 and RAPGEF6. Interestingly, we found that these activating KRAS mutations significantly correlate with increased activities of the Rho-associated kinases ROCK1 and ROCK2. Moreover, the protein-protein interaction database PrePPI predicts KRAS to homodimerize, and surprisingly, to interact with ROCK1/2 through a new PPI interface. We are currently confirming these results through biochemical and biophysical analyses. This abstract is also presented as Poster B34. Citation Format: David A. Wah, Federico M. Giorgi, Donald Petrey, Mariano J. Alvarez, Tonya Silkov, Josh Broyde, Gabrielle E. Rieckhof, Barry Honig, Andrea Califano. A systems biology approach to elucidate novel drug targets in KRAS mutant tumors. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr PR09. doi: 10.1158/1557-3125.RASONC14-PR09

In vitro Scleroderma laeve and Eucalyptus grandis mycorrhization and analysis of atp6, 17S rDNA, and ras gene expression during ectomycorrhizal formation.

The interaction between fungi and plants that form ectomycorrhizae (ECM) promotes alterations in the gene expression profiles of both organisms. Fungal genes expression related to metabolism were evaluated at the pre-symbiotic stage and during the ECM development between Scleroderma laeve and Eucalyptus grandis. Partial sequences of ATP synthase (atp6), translation elongation factor (ef1α), the RAS protein (ras), and the 17S rDNA genes were isolated. The expression of the atp6 and 17S rDNA genes during the pre-symbiotic stage showed an approximately threefold increase compared to the control. During ECM development, the expression of the 17S rDNA gene showed a 4.4-fold increase after 3 days of contact, while the expression of the atp6 gene increased 7.23-fold by the 15th day, suggesting that protein synthesis and respiratory chain activities are increased during the formation of the mantle and the Hartig net. The ras gene transcripts were only detected by RT-PCR 30 days after fungus-plant contact, suggesting that RAS-mediated signal transduction pathways are functional during the establishment of symbiosis. The present study demonstrates that alterations in gene expression occur in response to stimuli released by the plant during ECM association and increases the understanding of the association between S. laeve and E. grandis.

Network quantification of EGFR signaling unveils potential for targeted combination therapy

The epidermal growth factor receptor (EGFR) signaling network is activated in most solid tumors, and small-molecule drugs targeting this network are increasingly available. However, often only specific combinations of inhibitors are effective. Therefore, the prediction of potent combinatorial treatments is a major challenge in targeted cancer therapy. In this study, we demonstrate how a model-based evaluation of signaling data can assist in finding the most suitable treatment combination. We generated a perturbation data set by monitoring the response of RAS/PI3K signaling to combined stimulations and inhibitions in a panel of colorectal cancer cell lines, which we analyzed using mathematical models. We detected that a negative feedback involving EGFR mediates strong cross talk from ERK to AKT. Consequently, when inhibiting MAPK, AKT activity is increased in an EGFR-dependent manner. Using the model, we predict that in contrast to single inhibition, combined inactivation of MEK and EGFR could inactivate both endpoints of RAS, ERK and AKT. We further could demonstrate that this combination blocked cell growth in BRAF- as well as KRAS-mutated tumor cells, which we confirmed using a xenograft model.

Titrated extract of Centella asiatica provides a UVB protective effect by altering microRNA expression profiles in human dermal fibroblasts

The titrated extract of Centella asiatica (TECA) is a reconstituted mixture comprising of asiatic acid, madecassic acid, asiaticoside and madecassoside, and is used as a therapeutic agent in wound healing and also as an anti-microbial, anticancer and anti-aging agent. Although these properties and the associated cell signaling pathways have been elucidated, the cellular mechanism of anti-photoaging upon ultraviolet (UV) exposure in normal human dermal fibroblasts (NHDFs) remains unknown. In this study, we investigated the photoprotective role of TECA via microRNA (miRNA) expression profiling analysis. Low dose of TECA did not exhibit toxicity and showed a protective effect against UVB irradiation in NDHFs. miRNA microarray experiments revealed that specific miRNAs were altered by TECA stimulation in UVB-irradiated NHDFs. Functional bioinformatic analysis showed that the putative target genes of the altered miRNAs were associated with the positive regulation of cell proliferation, anti-apoptosis, small GTPase- and Ras-mediated signal transduction and activation of MAPKK. Therefore, these results suggest that TECA may serve as a potential natural chemoprotective agent against UVB-mediated damage in NHDFs through changes in the expression of specific miRNAs.

Photochemical modulation of Ras-mediated signal transduction using caged farnesyltransferase inhibitors: activation by one- and two-photon excitation.

The creation of caged molecules involves the attachment of protecting groups to biologically active compounds such as ligands, substrates and drugs that can be removed under specific conditions. Photoremovable caging groups are the most common due to their ability to be removed with high spatial and temporal resolution. Here, the synthesis and photochemistry of a caged inhibitor of protein farnesyltransferase is described. The inhibitor, FTI, was caged by alkylation of a critical thiol group with a bromohydroxycoumarin (Bhc) moiety. While Bhc is well established as a protecting group for carboxylates and phosphates, it has not been extensively used to cage sulfhydryl groups. The resulting caged molecule, Bhc-FTI, can be photolyzed with UV light to release the inhibitor that prevents Ras farnesylation, Ras membrane localization and downstream signaling. Finally, it is shown that Bhc-FTI can be uncaged by two-photon excitation to produce FTI at levels sufficient to inhibit Ras localization and alter cell morphology. Given the widespread involvement of Ras proteins in signal transduction pathways, this caged inhibitor should be useful in a plethora of studies.

Ras-Mediated Signal Transduction and Virulence in Human Pathogenic Fungi.

Signal transduction pathways regulating growth and stress responses are areas of significant study in the effort to delineate pathogenic mechanisms of fungi. In-depth knowledge of signal transduction events deepens our understanding of how a fungal pathogen is able to sense changes in the environment and respond accordingly by modulation of gene expression and re-organization of cellular activities to optimize fitness. Members of the Ras protein family are important regulators of growth and differentiation in eukaryotic organisms, and have been the focus of numerous studies exploring fungal pathogenesis. Here, the current data regarding Ras signal transduction are reviewed for three major pathogenic fungi: Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus. Particular emphasis is placed on Ras-protein interactions during control of morphogenesis, stress response and virulence.

Receptor for activated C-kinase (RACK1) plays a central role in Ras-mediated signal transduction

Presented at the 16th International Charles Heidelberger Symposium on Cancer Research, Coimbra, Portugal. 26–28 September 2010

Phase II Study of the Oral MEK Inhibitor AZD6244 in Advanced Acute Myeloid Leukemia (AML).

Abstract 2081Poster Board II-58AZD6244 is an orally bioavailable small molecule inhibitor of the MEK kinase. MEK is downstream of the RAS/RAF pathway, which is activated by mutations occurring in RAS as well as mutations and/or overexpression of upstream receptor tyrosine kinases such as FLT3 and c-KIT in AML. In addition, elevated levels of phosphorylated-ERK (p-ERK), the only known substrate of MEK, have been demonstrated in >75% of patients(pts) with AML, and MEK inhibition of primary AML cells in vitro results in growth arrest. AZD6244 was well tolerated in phase I trials in advanced solid tumors and had a favorable pharmacokinetic profile; the recommended phase II dose was 100mg twice daily. We hypothesized that in AML, a MEK inhibitor would lead to inhibition of RAS-mediated signal transduction, with subsequent antiproliferative effects and inhibition of the leukemia clone. We report our experience with the first clinical trial utilizing a MEK inhibitor in advanced AML. Methods:47 pts were enrolled on a Phase II multicenter study of AZD6244 in relapsed/refractory AML. Median age was 69 years (range, 26-83 yrs) with 57% males. ECOG performance status at baseline was 0, 1 and 2, respectively, in 12, 27 and 8 pts. 14 pts (30%) were previously untreated for AML and >60 yrs; 11 of these pts had received prior therapy for an antecedent hematologic disorder (AHD); 1 had therapy-related AML (t-AML) and only 2 pts (4%) had previously untreated de novo AML. 6 pts (13%) had AML in first relapse, 14 (30%) had AML beyond first relapse, and 13 (27%) had primary refractory disease. 4% had good risk cytogenetics, 28% intermediate risk, 49% poor risk, and 19% had other or unknown cytogenetics. Overall, 53% had AML that had evolved from an AHD and/or t-AML. Ten pts had a FLT3 ITD or TKD, 36 had no FLT3 mutation detected, and FLT3 mutational status was unknown in 1. Median number of prior therapies for AML and/or MDS was 2 (range, 0-6).AZD6244 was given at 100mg twice daily without interruption; cycles were repeated every 28 days. Dose modifications and/or delays occurred for grade 3&4 non-hematologic toxicities, or prolonged grade1&2 toxicities. Peripheral blood and/or marrow samples were obtained at baseline for mutational analysis (RAS/c-KIT/FLT3), and at serial time points to measure p-ERK. Results: Daily AZD6244 was tolerable. 42 pts are evaluable for efficacy and safety. Median number of cycles administered was 1 (range, 1-9). 19 pts (40%) received ≥2 cycles. 4 pts required dose reduction. The most common drug-related toxicities were grade 1&2 diarrhea, nausea, fatigue and vomiting, occurring in 43%, 36%, 31% and 24%, respectively. Grade 3&4 adverse events possibly related to AZD6244 included fatigue, nausea and dehydration, occurring in 7%, 5% and 5%, respectively. 4 pts had a minor response (defined as >50% decline in peripheral blood and/or marrow blasts lasting 4 weeks). 2 additional patients also had >50% decline in marrow blasts but did not have a follow up confirmatory biopsy. In 1 of these pts, the decline in blasts was associated with sustained improvement in platelets (>100K/uL) lasting 4 months. 6 additional pts had evidence of disease stabilization, lasting a median of 34 days (range, 21-222+ days). Analysis of p-ERK by flow cytometry has just been intiated, and in the first 3 pts analyzed, baseline p-ERK levels were low, and none of these pts responded. In contrast, p-mTOR levels (downstream of the PI3 Kinase pathway) were significantly elevated in these same pts. Conclusions: Administration of the oral MEK inhibitor AZD6244 is feasible in AML. Modest evidence of antileukemic activity has been observed, consistent with the predicted cytostatic activity of this class of drugs. Analysis of the effect of AZD6244 on p-ERK and signaling intermediates of the PI3 Kinase pathway such as p-mTOR is ongoing. Given its modest toxicity profile, AZD6244 should be investigated further in combination with drugs that target other critical signaling pathways and/or dysregulated transcriptional pathways in AML. Sponsored by NCI grant NO1-CM-62201 Disclosures:No relevant conflicts of interest to declare.

Effect of antisense oligomer in controlling c-raf.1 overexpression during diethylnitrosamine-induced hepatocarcinogenesis in rat

In ras-mediated signal transduction pathway, c-raf.1 is believed to have predominant oncogenic potential and has been found to be highly expressed in certain human and animal malignancies including hepatocellular carcinoma. In the present study, anticancer efficacy of antisense c-raf.1 oligomer on the inhibition of c-raf.1 mRNA overexpression during hepatocarcinogenesis was determined. Initially antiproliferating effect of the antisense oligomers was studied in vitro by measuring the rate of tritiated thymidine incorporation into DNA in rat hepatocellular carcinoma cells in culture medium. Based on the findings, the antisense treatment was carried out in rat hepatocarcinogenesis model-initiated with diethylnitrosamine and promoted using 2-acetylaminoflourene. Different drug-metabolizing enzymes, lipid peroxidation, liver morphology and histopathological studies along with c-raf.1 gene expression by in situ hybridization were performed. c-raf.1 antisense oligomers exhibited an inhibitory effect (approximately 68%) on cancer cell proliferation in vitro. Gross and microscopic examination of liver showed fewer (29%) and smaller hyperplastic nodules and preneoplastic lesions (30%) in carcinogen and antisense oligomer-treated group as compared with carcinogen control group. Treatment of antisense c-raf.1 oligomers enhanced cytochrome P-450 content (81%) and reduced glutathione S-transferase activity (33%), UDP glucuronosyltransferase activity (74%) and MDA concentration (30%) in carcinogen and antisense oligomer-treated group as compared with carcinogen control animals. The oligomer treatment also resulted in less expression in terms of c-raf.1 expressed lesion count as compared to carcinogen control group. The study demonstrates that the antisense oligomer targeted against c-raf.1 mRNA inhibits the overexpression of c-raf.1 gene during hepatocellular carcinoma in rats.

A phase I trial of romidepsin in combination with gemcitabine in patients with pancreatic and other advanced solid tumors

2567 Background: Romidepsin is a histone deacetylase (HDAC) inhibitor that inhibits ras-mediated and MAP kinase signal transduction causing cell cycle arrest. Preclinical studies of romidepsin and gemcitabine in xenografts of ras transformed pancreatic cancer demonstrated significant antitumor activity for the combination compared to gemcitabine alone. Long-term effects were observed on tumor cell growth and cell cycle regulators following treatment cessation, suggesting down regulation of HDAC activity. This trial evaluated the safety and tolerability of romidepsin in combination with gemcitabine. Methods: Premedications consisted of a serotonin antagonist plus dexamethasone. Romidepsin was administered as a 4 hour infusion followed by gemcitabine over 30 minutes on days 1, 8, and 15 of a 28 day cycle. Results: Thirty-three patients (9 pancreatic, 8 breast, 7 NSCLC, 3 ovarian, 6 other; 23 F/10M) have received 104+ cycles (median 2, range 1 - 8). The initial romidepsin/gemcitabine dose level was 10/800 mg/m2, but 4/7 patients required dose omissions on days 8 or 15 due to thrombocytopenia (<75,000) or neutropenia (≤ 1,000) and were considered to have dose-limiting toxicity (DLT). Subsequently, the romidepsin was decreased to 7 mg/m2. At this dose level, 6/7 patients missed doses due to DLTs of thrombocytopenia and/or neutropenia. As a result, the day 8 dose was eliminated. Three dose levels have been explored on the every other week schedule. Ten patients were enrolled at 10/800 mg/m2 and 2/10 experienced a DLT (grade 4 thrombocytopenia, ↑LFTs). Six patients were enrolled at 10/1,000 mg/m2 with 2/6 experiencing dose-limiting grade 4 thrombocytopenia. Concurrently, the romidepsin was increased to 12 mg/m2 in combination with gemcitabine 800 mg/m2 and no DLTs were reported in 3 patients. Nonhematologic toxicities have been mild to moderate and consist primarily of nausea, vomiting, and fatigue. One patient with ovarian cancer experienced a minor response (29% ↓) and 12 pts experienced stable disease for ≥ 4 cycles (5 pancreatic, 4 breast, 1 NHL, 1 ovarian, 1 ampullary cancer). Conclusions: The recommended phase II dose (romidepsin 12 mg/m2 and gemcitabine 800 mg/m2 every other week) is being expanded to further assess the safety and activity of the regimen. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Gloucester Pharmaceuticals Gloucester Pharmaceuticals

Solution structure and backbone dynamics of the AF‐6 PDZ domain/Bcr peptide complex

The human AF-6, a scaffold protein between cell membrane-associated proteins and the actin cytoskeleton, plays an important role in special cell-cell junctions and signal transduction. It can be phosphorylated by the protein kinase Bcr, which allows efficient binding of the C terminus of Bcr to the PDZ domain of AF-6 and consequently enhances the binding affinity of AF-6 to Ras. Formation of the AF-6, Bcr, and Ras ternary complex results in down-regulation of the Ras-mediated signal transduction pathway. To better understand the molecular basis for the recognition of the AF-6 PDZ domain and Bcr, we solve the solution structure of the AF-6 PDZ domain complexed with the C-terminal peptide of Bcr and explore the interactions between them in detail. Compared with previously reported structures, the complex exhibits a noncanonical binding mode of PDZ/peptide. Owing to the distinct residues involved in the AF-6 PDZ domain and Bcr peptide interaction, the interaction mode does not adapt to the existing classification rules that have been put forward, based on the ligand or the PDZ domain specificity. Furthermore, the PDZ domain of AF-6 can bind to the C terminus of Bcr efficiently after phosphorylation of AF-6 by the Bcr kinase. The phosphorylation may induce a conformational change of AF-6, which makes the binding surface on the PDZ domain accessible to Bcr for efficient binding. This study not only characterizes the structural details of the AF-6 PDZ/Bcr peptide complex, but also provides a potential target for future drug design and disease therapy.

Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by heterotrimeric G-protein alpha subunits and thus inhibit signaling by many G protein-coupled receptors. Several RGS proteins have a multidomain architecture that adds further complexity to their roles in cell signaling in addition to their GTPase-accelerating activity. RGS12 contains a tandem repeat of Ras-binding domains but, to date, the role of this protein in Ras-mediated signal transduction has not been reported. Here, we show that RGS12 associates with the nerve growth factor (NGF) receptor tyrosine kinase TrkA, activated H-Ras, B-Raf, and MEK2 and facilitates their coordinated signaling to prolonged ERK activation. RGS12 is required for NGF-mediated neurite outgrowth of PC12 cells, but not outgrowth stimulated by basic fibroblast growth factor. siRNA-mediated knockdown of RGS12 expression also inhibits NGF-induced axonal growth in dissociated cultures of primary dorsal root ganglia neurons. These data suggest that RGS12 may play a critical, and receptor-selective, role in coordinating Ras-dependent signals that are required for promoting and/or maintaining neuronal differentiation.

Pharmacological effects of novel agents on Ras mediated signal transduction

Pharmacological effects of novel agents on Ras mediated signal transduction

Bimolecular Fluorescence Complementation (BiFC) in Interactions Analysis in Ras-mediated signal transduction

Bimolecular Fluorescence Complementation (BiFC) in Interactions Analysis in Ras-mediated signal transduction

Neurofibromatosis-Noonan syndrome: Molecular evidence of the concurrence of both disorders in a patient

Noonan syndrome (NS) is an autosomal dominant disorder characterized by short stature, facial anomalies, webbed neck, sternal deformity, heart defects, and, in males, cryptorchidism. PTPN11 encodes SHP2, an important component of several signal transduction pathways that acts as a positive regulator of RAS-mitogen activated protein kinase signaling. Neurofibromatosis type 1 (NF1) is another autosomal dominant disorder characterized by hamartomas in multiple organs. The NF1 gene encodes a GAP-related protein, which acts as a negative regulator of the Ras-mediated signal transduction pathway. Clinical overlap between both syndromes, neurofibromatosis-Noonan syndrome (NFNS) is well known. We studied a female patient with typical findings of NFNS and found two mutations: a novel PTPN11 transversion, 1909A --> G, resulting in Gln510Arg, and an NF1 transversion, 2531A --> G, resulting in Leu844Arg. She inherited the PTPN11 mutation from her father and had a de novo NF1 mutation. This is the first report of molecular concurrence of both disorders in the same patient.

Effects of an inhibitor of Acyl Protein Thioesterase 1 on Ras-mediated signal transduction

Effects of an inhibitor of Acyl Protein Thioesterase 1 on Ras-mediated signal transduction

Omega-3 fatty acids in colorectal cancer prevention.

Omega-3 fatty acids in colorectal cancer prevention.

Role of reactive oxygen species and Cr(VI) in Ras-mediated signal transduction.

Previous studies have shown that a constitutively active isoform of Ras is able to produce superoxide radical (O2(-)). The present study investigate the mechanisms by which O2(-) radical mediates signals from Ras protein to the nucleus, leading to cellular responses such as apoptosis in Cr(VI)-stimulated cells. Two human prostate tumor cell lines, Ras(+), which overexpresses Ras, and Ras(-), which has a normal Ras level, were utilized. Compared to Ras(-) cells, Ras(+) cells exhibited higher susceptibility to apoptosis induced by Cr(VI). Catalase, sodium formate, and deferoxamine inhibited Cr(VI)-induced apoptosis. Similar differences were observed in both cellular DNA damage and the activation of p53 protein. The differences in Cr(VI)-induced cell responses in Ras(+) and Ras(-) cells were due to differences in the generation of free radicals between these two cells. ESR spin trapping measurements showed that Ras(+) cells generated more hydroxyl radical ((.)OH), O2(-) radical, and Cr(V) than Ras(-) cells following Cr(VI) stimulation. The generation of the reactive oxygen species (ROS) can be abolished by the addition of superoxide dismutase (SOD) or if the experiment were carried out in an argon atmosphere. Catalase inhibited spin adduct signals but was much less potent than SOD. The mechanism of ROS generation in Cr(VI)-stimulated Ras(+) cells involves the reduction of molecular oxygen to O2(-) radical by a flavoenzyme-containing NADPH oxidase complex as shown by oxygen consumption and diphenylene iodonium (DPI) inhibition. Results shown above support the following conclusions: (a) Ras protein mediates O2(-) radical generation through reduction of molecular oxygen by NADPH oxidase in Cr(VI)-stimulated cells. (b) The O2(-) radical and Cr(VI) produce other reactive species, including H2O2, OH radical, and Cr(V) through O2(-) dismutation and Haber-Weiss type of reactions. (c) Among these reactive species, (.)OH radical is responsible for the further transduction of signals from Ras to the nucleus, leading to various cell responses.

Molecular regulation of cholesterol biosynthesis: implications in carcinogenesis.

Cholesterol synthesis was demonstrated to be mandatory for cellular growth and serves to supply one of the necessary building blocks for new membranes demanded by dividing cells during growth. The mevalonate pathway, which is regulated through a finely tuned mechanism, is responsible mainly for cholesterol enrichment to cells. Among the various steps, the production of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is the most critically regulated step catalyzed by HMG-CoA reductase. The ability of sterols to regulate both the transcriptional rates of the reductase gene and the degradative machinery for the reductase protein provides a multilevel system for controlling the expression of this enzyme. Much convincing evidence indicates that cells manifest a higher flux through the mevalonate pathway when proliferating than when they are in the cell cycle arrest condition; furthermore, tumors undergo deregulated cholesterogenesis mainly at the critical rate-controlling juncture (i.e., the reaction catalyzed by HMG-CoA reductase). The mevalonate component of the cholesterol biosynthesis plays a key role in controlling cell proliferation by generating prenyl intermediates, particularly farnesyl and geranyl-geranyl moieties. These isoprenoids covalently modify and thus modulate the biological activity of signal transducing proteins, such as that of the Ras superfamily. The prenylated Ras-mediated signal transduction pathway provides much of the molecular information needed to trigger cell proliferation. Therefore, depletion of mevalonate can block both the processing and the transforming activities of Ras, indicating that drugs such as lovastatin and compactin, which had previously been exploited for lowering cholesterol levels, may be useful chemotherapeutic agents for treating tumors harboring oncogenic Ras mutation. In addition, Ras prenylation, which provides much of the molecular information needed to trigger cell proliferation, represents an inviting target for the design of chemotherapeutic drugs that would interrupt such signaling events and arrest tumor cell proliferation.