Determination of Ras-GTP and Ras-GDP in patients with acute myelogenous leukemia (AML), myeloproliferative syndrome (MPS), juvenile myelomonocytic leukemia (JMML), acute lymphocytic leukemia (ALL), and malignant lymphoma: assessment of mutational and indirect activation

Abstract

The 21-kD protein Ras of the low-molecularweight GTP-binding (LMWG) family plays an important role in transduction of extracellular signals. Ras functions as a 'molecular switch' in transduction of signals from the membrane receptors of many growth factors, cytokines, and other second messengers to the cell nucleus. Numerous studies have shown that in multiple malignant tumors and hematopoietic malignancies, faulty signal transduction via the Ras pathway plays a key role in tumorigenesis. In this work, a non-radioactive assay was used to quantify Ras activity in hematologic malignancies. Ras activation was measured in six different cell lines and 24 patient samples, and sequence analysis of Nand K-ras was performed. The 24 patient samples comprised of seven acute myelogenous leukemia (AML) samples, five acute lymphocytic leukemia (ALL) samples, four myeloproliferative disease (MPD) samples, four lymphoma samples, four juvenile myelomonocytic leukemia (JMML) samples, and WBC from a healthy donor. The purpose of this study was to compare Ras activity determined by percentage of Ras-GTP with the mutational status of the Ras gene in the hematopoietic cells of the patients. Mutation analysis revealed ras mutations in two of the seven AML samples, one in codon 12 and one in codon 61; ras mutations were also found in two of the four JMML samples, and in one of the four lymphoma samples (codon 12). We found a mean Ras activation of 23.1% in cell lines with known constitutively activating ras mutations, which was significantly different from cell lines with ras wildtype sequence (Ras activation of 4.8%). Two of the five activating ras mutations in the patient samples correlated with increased Ras activation. In the other three samples, Ras was probably activated through "upstream" or "downstream" mechanisms.

Tumor-selective effects of active RAS inhibition in pancreatic ductal adenocarcinoma

“…This approach showed that inhibition of physiological MYC activity reduced proliferation in most epithelial tissues, but that key epithelial functions were broadly maintained for extended periods of time. Systemic RAS inhibition may prove to be similar in nature, with tolerability enabled by the relatively low levels of active RAS-GTP (the target for RMC-7977) in normal tissues 35 and the somewhat reduced affinity of RMC-7977 for wild type RAS as compared to mutant variants 14 . This is consistent with prior work showing that normal cells can rapidly restore homeostasis following RAS pathway inhibition in contrast to RAS-addicted tumor cells 36 and that normal RAS WT skin cells behave differently following wounding stimuli as compared to RAS MUT skin cells 37 .…”

Diabetic Retinopathy, Superoxide Damage and Antioxidants

“…H-Ras serves as a “molecular switch” converting signals from cell membrane to nucleus and serves as a key regulator of the signaling cascade triggered by oxidative stress [98]. H-Ras exists as either inactive GDP-bound form or an active GTP-bound form [99], and guanine nucleotide exchange factor in ERK and JNK pathway activates Ras by converting Ras-GDP to Ras-GTP [100]. This activated Ras stimulates the production of super-oxide radicals by NADPH oxidase-like activity [101], and also increase in intracellular ROS triggers H-Ras activation, which further stimulates ROS level by activating NADPH oxidase [98, 101].…”

Enablingin vivoAnalysis Via Nanoparticle-mediated Intracellular Assay Probe Delivery: Using RAS as the Prototype

“…Many GTP analogs are suitable to act as as probes for analyses of RAS and other G-proteins, but their cell impermeability make it impossible to do the analysis in vivo in live cells (16)(17)(18)(19)(20)(21). Currently, RAS activity measurement methods are mostly in vitro (22). One popular approach is the GST-RBD pull-down assay -quantification of the amount of the RAS protein in the cell lysate that binds to a recombinant Glutathione S-transferases (GST) tagged RAS-binding domain (RBD) of a RAS effector protein, usually the RAFs.…”