guanosine-diphosphate and Leukemia-Lymphoma--Adult-T-Cell

Adult T-cell leukemia/lymphoma (ATLL) is a distinct form of peripheral T-cell lymphoma with poor prognosis, which is caused by the human T-lymphotropic virus type 1 (HTLV-1). In contrast to the unequivocal importance of HTLV-1 infection in the pathogenesis of ATLL, the role of acquired mutations in HTLV-1 infected T cells has not been fully elucidated, with a handful of genes known to be recurrently mutated. In this study, we identified unique RHOA mutations in ATLL through whole genome sequencing of an index case, followed by deep sequencing of 203 ATLL samples. RHOA mutations showed distinct distribution and function from those found in other cancers. Involving 15% (30/203) of ATLL cases, RHOA mutations were widely distributed across the entire coding sequence but almost invariably located at the guanosine triphosphate (GTP)-binding pocket, with Cys16Arg being most frequently observed. Unexpectedly, depending on mutation types and positions, these RHOA mutants showed different or even opposite functional consequences in terms of GTP/guanosine diphosphate (GDP)-binding kinetics, regulation of actin fibers, and transcriptional activation. The Gly17Val mutant did not bind GTP/GDP and act as a dominant negative molecule, whereas other mutants (Cys16Arg and Ala161Pro) showed fast GTP/GDP cycling with enhanced transcriptional activation. These findings suggest that both loss- and gain-of-RHOA functions could be involved in ATLL leukemogenesis. In summary, our study not only provides a novel insight into the molecular pathogenesis of ATLL but also highlights a unique role of variegation of heterologous RHOA mutations in human cancers.

Topics: Adult; Amino Acid Sequence; Binding Sites; DNA Mutational Analysis; Guanosine Diphosphate; Guanosine Triphosphate; High-Throughput Nucleotide Sequencing; Humans; Leukemia-Lymphoma, Adult T-Cell; Models, Molecular; Molecular Sequence Data; Mutation; Protein Structure, Tertiary; rhoA GTP-Binding Protein

The possible involvement of G-proteins in T cell antigen-receptor complex (TCR)-mediated inositol phosphate production was investigated in HPB-ALL T-cells, which were found to express the phospholipase C gamma 1 and beta 3 isoforms. Cross-linking the CD3 antigen on streptolysin-O-permeabilized cells stimulated a dose-dependent increase in inositol phosphate production, as did addition of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or vanadate, a phosphotyrosine phosphatase inhibitor. It was possible, therefore, that the CD3-antigen-mediated production of inositol phosphates was either via a G-protein-dependent mechanism or by stimulation of protein tyrosine phosphorylation. The CD3-induced inositol phosphate production was potentiated by addition of vanadate, but not by addition of GTP[S]. Guanosine 5'-[beta-thio]diphosphate (GDP[S]) inhibited the rise in inositol phosphates induced by GTP[S], vanadate or cross-linking the CD3 antigen. The increase in protein tyrosine phosphorylation stimulated by vanadate or the OKT3 monoclonal antibody was not observed in the presence of GDP[S], showing that in permeabilized HPB-ALL cells, GDP[S] inhibits the actions of tyrosine kinases as well as G-protein function. Addition of either ADP[S] or phenylarsine oxide inhibited CD3- and vanadate-mediated increases in both tyrosine phosphorylation and inositol phosphate production, but did not inhibit GTP[S]-stimulated inositol phosphate production. On the other hand, pretreatment of cells with phorbol 12,13-dibutyrate inhibited subsequent GTP[S]-stimulated inositol phosphate production but did not inhibit significantly inositol phosphate production stimulated by either OKT3 F(ab')2 fragments or vanadate. Our results are consistent with the CD3 antigen stimulating inositol phosphate production by increasing the level of protein tyrosine phosphorylation, but not by activating a G-protein.

Topics: CD3 Complex; Cell Membrane Permeability; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Isoenzymes; Kinetics; Leukemia-Lymphoma, Adult T-Cell; Muromonab-CD3; Phospholipases; T-Lymphocytes; Thionucleotides; Tumor Cells, Cultured; Vanadates