Decreased GTPase activity of K- ras mutants deriving from human functional adrenocortical tumours

Lin, Sheng-Fung; Hsu, Chin‐Mu; Tsai, Juei-Hsiung; Wang, J.-Y.; Hsieh, Tusty‐Jiuan; Wu, Chia-Hung

doi:10.1054/bjoc.1999.1039

Cited by 23 publications

(21 citation statements)

References 38 publications

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“…Then, a membrane array blotted with these 22 gene probes was constructed and applied to test the peripheral blood samples from patients with various cancers. The results demonstrated that the K-ras oncogene membrane array had a remarkable potential to detect activated K-ras in human cancers (14)(15)(16)(17).…”

Section: Introductionmentioning

confidence: 91%

Detection of activated K-ras in non-small cell lung cancer by membrane array: A comparison with direct sequencing

Chong

Chang²,

Sheu³

et al. 2007

Oncol Rep

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Abstract. The ability to detect K-ras oncogene may provide additional information for the management of patients with non-small cell lung cancer (NSCLC). In the present study, we detected the K-ras oncogene in 76 patients with NSCLC by two methods: direct sequencing of K-ras in tumor tissues and membrane array detection of the gene overexpression specific for activated K-ras in peripheral blood. The results showed that 28 (36.8%) of the 76 Taiwanese NSCLC patients had K-ras mutations, with a frequency of 36.4% (20/55) in adenocarcinomas and 38.1% (8/21) in squamous cell carcinomas. The K-ras mutations were more frequently found in smokers than in non-smokers (51.4 vs. 24.4%, P=0.015). The incidences of K-ras mutation in the subgroups of non-smokers and squamous cell carcinomas are relatively higher in Taiwan than in other countries. On the other hand, the membrane array method could positively detect circulating activated K-ras in all of the 27 NSCLC patients with K-ras mutations at codons 12, 13 and 61, and in 4 of the 48 patients with wild-type K-ras. Our results suggest that the K-ras oncogene membrane array serves as a sensitive and convenient tool for the detection of K-ras oncogene, and therefore, has a great potential for clinical applications.

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Section: Introductionmentioning

confidence: 91%

Detection of activated K-ras in non-small cell lung cancer by membrane array: A comparison with direct sequencing

Chong

Chang²,

Sheu³

et al. 2007

Oncol Rep

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“…The oligonucleotides used to amplify a fragment of human c-raf-1 were 5'-CACAGATGGATCCAAGACAAGCAACAC-3' and 5'-GGG-AAGAAT-TCACAGGAAATCTAC-3' (Herrmann et al, 1995) cDNA. The resulting 412-base pair fragment was cleaved with BamHI and EcoRI, purified, and cloned into the GST fusion vector pGEX-4T-2 (Amersham, Pharmacia Biotech UK, Ltd) as described previously (Lin et al, 2000). The resulting plasmid, pGEX-RBD, was transformed into E. coli strain TG1.…”

Section: Construction Of Gst-rbd Of Raf-1 Expression Plasmidmentioning

confidence: 99%

“…300 mM b-mercaptoethanol). The protein samples were separated on vertical slab gels composed of 10% polyacrylamide containing 0.1% sodium dodecyl sulphate (SDS) prepared in 3 mM Tris-HCl, pH 8.8, as described previously (Lin et al, 2000). Following electrophoresis, the protein was electroblotted to a nitrocellulose membrane (Schleicher & Schusll GmbH, Dassel, Germany).…”

Section: Precipitation and Immunoblot Of Active Form Rasgtp By Gst-rbmentioning

confidence: 99%

Mutant K-ras oncogene regulates steroidogenesis of normal human adrenocortical cells by the RAF-MEK-MAPK pathway

Chen

et al. 2002

Br J Cancer

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The result of our previous study has shown that the K-ras mutant (pK568MRSV) transfected human adrenocortical cells can significantly increase cortisol production and independently cause cell transformation. The aim of this study is to investigate the effect of the active K-ras oncogene on the cortisol production in normal human adrenocortical cells. First we used isopropyl thiogalactoside to induce the inducible mutant K-ras expression plasmid, pK568MRSV, in the stable transfected human adrenocortical cells. The result showed that the increase of RasGTP levels in transfected cells was time-dependent after isopropyl thiogalactoside induction. Additionally, results from Western blot analysis revealed significant elevation in phosphorylation of c-Raf-1 and Mitogen-activated protein kinase. We also detected the levels of mRNA encoding Cholesterol side-chain cleavage enzyme (P450 SCC ), 17a-Hydroxylase/17,20-lyase (P450 c17 ) and 3b-Hydroxysteroid dehydrogenase (3bHSD) were increased in human adrenocortical cells transfected with mutant K-ras after IPTG treatment. The increase of mRNA amount in P450 scc P450 c17 and 3bHSD and the elevation of cortisol level were inhibited with a pretreatment of PD098059, a specific extracellular signal-regulated kinase inhibitor. In our previous report, we proved that lovastatin, a pharmacological inhibitor of p21 ras function, also reversed the increase of cortisol level in mutant K-ras stably transfected human adrenocortical cells. Taken together, these findings proved that the active mutant Ras enhanced not only cell proliferation but also steroidogenesis in steroidogenic phenotype cells by activating Raf-MEK-MAPK related signal transduction pathway. Therefore, we believe that K-ras mutants influence regulation of steroidogenesis in adrenocortical cells through RAF-MEK-MAPK pathway.

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“…1,2,5,9 Additionally, mutations occur at codons 15,16,18, and 31. 24,25 These mutations result in abrogation of normal intrinsic or GAP-stimulated GTPase activity of RAS, leading to increased half-lives of mutant RAS-GTP. 5,9,26 Transformation results, at least in part, from deregulated stimulation of mitogenic signal transduction pathways.…”

Section: Introductionmentioning

confidence: 99%

Cell-cycle–dependent activation of mitogen-activated protein kinase kinase (MEK-1/2) in myeloid leukemia cell lines and induction of growth inhibition and apoptosis by inhibitors of RAS signaling

Morgan

Dolp

Reuter

2001

Blood

117

100

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IntroductionThe deregulation of RAS signal transduction has been implicated in the malignant growth of human cancer cells including myeloid leukemias. 1,2 RAS proto-oncogenes (H-RAS, N-RAS, and K-RAS) encode 21-kd G-proteins that play key roles in signal transduction, proliferation, differentiation, and malignant transformation. 3-5 RAS proteins are produced as cytoplasmatic precursors, which require several posttranslational modifications (eg, prenylation, proteolysis, carboxymethylation, and palmitoylation) for membrane binding and full biologic activity. 3-8 RAS functions as a biologic switch that relays signals from ligand-stimulated tyrosine kinase, cytokine, and heterotrimeric G-protein-coupled receptors to cytoplasmatic mitgen-activated protein kinase (MAPK) cascades. In its activated, GTP-bound state, RAS binds to and activates effector molecules such as Rafs, MEKK, PI-3K, and Ral-GEF. [3][4][5][8][9][10][11][12][13][14] Raf kinases (A-Raf, B-Raf, c-Raf-1) selectively phosphorylate and activate MAPK kinases (MAPKK) MEK-1/2 in the MAPK/ERK pathway. [13][14][15][16][17] MEK-1/2 are dual specificity kinases that activate MAPKs (ERK-1/2). 18,19 The best-characterized ERK substrates are cytoplasmic phospholipase A 2 (cPLA 2 ), ribosomal protein S6 kinases (RSKs), and transcription factors Elk-1 and CREB-1. [18][19][20] The importance of deregulation of ERK signaling in the molecular pathogenesis of myeloid leukemias is underscored by the positioning of several oncogene and tumor suppressor gene products on this pathway. 5,[21][22][23] RAS mutations are frequent genetic aberrations found in 20% to 30% of all human tumors, although the incidences in tumor type vary greatly. 1,2 The most commonly observed RAS mutations arise at sites critical for RAS regulation, namely codons 12, 13, and 61. 1,2,5,9 Additionally, mutations occur at codons 15, 16, 18, and 31. 24,25 These mutations result in abrogation of normal intrinsic or GAP-stimulated GTPase activity of RAS, leading to increased half-lives of mutant RAS-GTP. 5,9,26 Transformation results, at least in part, from deregulated stimulation of mitogenic signal transduction pathways. 1,2,5 The highest incidences of RAS mutations were detected in carcinomas of pancreas (90%), thyroid (50%), colon (50%), and lung (30%). RAS mutations are also frequently observed in myelodysplastic syndromes, acute myeloid leukemias (AML), juvenile myelomonocytic myeloid leukemia (JMML), and multiple myelomas (20%-40%). 1,2,5,[21][22][23] N-RAS is mutated in the majority of cases and presence of the mutation is not associated with any particular FAB type, cytogenetic abnormality, or clinical feature including prognosis. 22 In addition to activation by mutation, RAS is also deregulated in myeloid leukemias by constitutive activation of proto-oncogenes such as receptor or nonreceptor tyrosine kinases (RTKs and NRTKs) or inactivation of tumor suppressor genes. 5,[21][22][23] RTKs are constitutively activated by single point mutations (eg, colonystimulating factor-1 [CSF-1] receptor and c...

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Decreased GTPase activity of K- ras mutants deriving from human functional adrenocortical tumours

Cited by 23 publications

References 38 publications

Detection of activated K-ras in non-small cell lung cancer by membrane array: A comparison with direct sequencing

Detection of activated K-ras in non-small cell lung cancer by membrane array: A comparison with direct sequencing

Mutant K-ras oncogene regulates steroidogenesis of normal human adrenocortical cells by the RAF-MEK-MAPK pathway

Cell-cycle–dependent activation of mitogen-activated protein kinase kinase (MEK-1/2) in myeloid leukemia cell lines and induction of growth inhibition and apoptosis by inhibitors of RAS signaling

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