guanosine-triphosphate and Myeloproliferative-Disorders

The biological and clinical heterogeneity of chronic myelomonocytic leukemia features renders its classification difficult. Moreover, because of the limited knowledge of the mechanisms involved in malignant evolution, chronic myelomonocytic leukemia remains a diagnostic and therapeutic challenge and a poor prognosis disease. We aimed to verify the biological and clinical significance of the discrimination, based on the leukocyte count, between myelodysplastic chronic myelomonocytic leukemia (MD-CMML) and myeloproliferative chronic myelomonocytic leukemia (MP-CMML).. Peripheral blood samples from 22 patients classified as MD-CMML and 18 as MP-CMML were collected at different time points during disease course, and patients' clinical characteristics were examined. RAS mutational screening was done by sequencing and, for each substitution identified, a highly selective allele-specific PCR was set up to screen all specimens.. MP-CMML patients showed a significantly poorer survival (P = 0.003) and a higher frequency of RAS mutations (P = 0.033) by sequencing compared with MD-CMML. Overall, five MD-CMML patients progressed to myeloproliferative disease: in two, allele-specific PCR unveiled low levels of the RAS mutations predominating in the myeloproliferative phase at the time of myelodysplastic disease, documenting for the first time the expansion of a RAS mutated clone in concomitance with chronic myelomonocytic leukemia evolution. Moreover, one of the progressed patients harbored the FLT3-ITD and two MP-CMML patients presented with the JAK2 V617F substitution. All these lesions were mutually exclusive.. Our results strongly suggest RAS mutations to function as a secondary event that contributes to development of the chronic myelomonocytic leukemia variant with the poorer prognosis (MP-CMML) and therefore advise their detection to be implemented in chronic myelomonocytic leukemia diagnostics and monitoring.

Topics: Aged; Aged, 80 and over; Animals; Colony-Forming Units Assay; Disease Progression; Evolution, Molecular; Female; fms-Like Tyrosine Kinase 3; Genes, ras; Guanosine Triphosphate; Humans; Janus Kinase 2; Leukemia, Myelomonocytic, Chronic; Life Expectancy; Male; Mice; Middle Aged; Mutation; Myelodysplastic Syndromes; Myeloproliferative Disorders; NIH 3T3 Cells; Prognosis; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Survival Rate

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.

Topics: DNA Mutational Analysis; Guanosine Diphosphate; Guanosine Triphosphate; Hematologic Neoplasms; Humans; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Juvenile; Lymphoma; Mutation; Myeloproliferative Disorders; Oncogenes; Precursor Cell Lymphoblastic Leukemia-Lymphoma; ras Proteins; Signal Transduction; Tumor Cells, Cultured

Deoxyribonucleic acid synthesis requires adequate cellular concentrations of the four deoxyribonucleoside triphosphates. Using a sensitive enzymic assay, we have measured the concentrations (pools) of these compounds in human bone marrow cells and in lymphocytes. The mean concentrations (pmol/10(6) cells) in normal human bone marrow cells were: deoxyadenosine triphosphate (dATP) 1.5; deoxyguanosine triphosphate (dGTP) 0.4; thymidine triphosphate (dTTP) 1.4 and deoxycytidine triphosphate (dCTP) 0.6; and in normal phytohaemagglutinin (PHA)-stimulated lymphocytes (72 h cultures); dATP 3.7; dGTP 1.9; dTTP 9.4 and dCTP 2.9. The deoxyribonucleoside triphosphate concentrations were increased approximately threefold in the nucleated marrow cells from patients with leukaemia and myeloproliferative diseases. PHA-stimulation of lymphocytes caused a marked increase of the deoxyribonucleoside triphosphate concentrations, particularly of dTTP, between 24 and 48 h of culture. In PHA-stimulated lymphocytes, the antifolate drugs methotrexate, pyrimethamine and trimethoprim, all produced a fall in dTTP and a rise in dATP concentrations within 1 h. These effects could be reversed by folinic acid. 5-Fluorouracil caused a fall in dTTP and in dCTP but no consistent changes in dATP; hydroxyurea caused a fall in dATP with a rise in dTTP. BCNU caused a significant fall in dATP and dCTP. Dibutyryl cyclic 3', 5' adenosine monophosphate and theophylline had no consistent effect on the deoxyribonucleoside triphosphate concentrations. 6-Mercaptopurine caused a fall in dATP and dGTP, the fall in dATP being marked after 4 h incubation. It is concluded that measurement of the deoxyribonucleoside triphosphates in human cells provides a new method of studying DNA synthesis in human disease states and of analysing the action of antimetabolite drugs on normal and diseased cells.

Topics: Adenosine Triphosphate; Antimetabolites; Antineoplastic Agents; Bone Marrow; Bone Marrow Cells; Cells, Cultured; Cytosine Nucleotides; Deoxyribonucleotides; DNA; DNA, Neoplasm; Guanosine Triphosphate; Leukemia; Lymphocytes; Myeloproliferative Disorders; Thymidine; Thymine Nucleotides