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

摘要

The 21-kD protein Ras of the low-molecular-weight 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 N- and 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.