RESUMO
Prognostic gene expression signatures have been proposed as clinical tools to clarify therapeutic options in acute myeloid leukemia (AML). However, these signatures rely on measuring large numbers of genes and often perform poorly when applied to independent cohorts or those with older patients. Long intergenic non-coding RNAs (lincRNAs) are emerging as important regulators of cell identity and oncogenesis, but knowledge of their utility as prognostic markers in AML is limited. Here we analyze transcriptomic data from multiple cohorts of clinically annotated AML patients and report that (i) microarrays designed for coding gene expression can be repurposed to yield robust lincRNA expression data, (ii) some lincRNA genes are located in close proximity to hematopoietic coding genes and show strong expression correlations in AML, (iii) lincRNA gene expression patterns distinguish cytogenetic and molecular subtypes of AML, (iv) lincRNA signatures composed of three or four genes are independent predictors of clinical outcome and further dichotomize survival in European Leukemia Net (ELN) risk groups and (v) an analytical tool based on logistic regression analysis of quantitative PCR measurement of four lincRNA genes (LINC4) can be used to determine risk in AML.
Assuntos
Leucemia Mieloide Aguda/genética , RNA Longo não Codificante/genética , Transcriptoma/genética , Adolescente , Adulto , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Medição de Risco , Fatores de Risco , Adulto JovemRESUMO
Heterozygous GATA2 mutations underlie an array of complex hematopoietic and lymphatic diseases. Analysis of the literature reporting three recurrent GATA2 germline (g) mutations (gT354M, gR396Q and gR398W) revealed different phenotype tendencies. Although all three mutants differentially predispose to myeloid malignancies, there was no difference in leukemia-free survival for GATA2 patients. Despite intense interest, the molecular pathogenesis of GATA2 mutation is poorly understood. We functionally characterized a GATA2 mutant allelic series representing major disease phenotypes caused by germline and somatic (s) mutations in zinc finger 2 (ZF2). All GATA2 mutants, except for sL359V, displayed reduced DNA-binding affinity and transactivation compared with wild type (WT), which could be attributed to mutations of arginines critical for DNA binding or amino acids required for ZF2 domain structural integrity. Two GATA2 mutants (gT354M and gC373R) bound the key hematopoietic differentiation factor PU.1 more strongly than WT potentially perturbing differentiation via sequestration of PU.1. Unlike WT, all mutants failed to suppress colony formation and some mutants skewed cell fate to granulocytes, consistent with the monocytopenia phenotype seen in GATA2-related immunodeficiency disorders. These findings implicate perturbations of GATA2 function shaping the course of development of myeloid malignancy subtypes and strengthen complete or nearly complete haploinsufficiency for predisposition to lymphedema.
Assuntos
Diferenciação Celular/genética , Fator de Transcrição GATA2/genética , Sistema Hematopoético/patologia , Mutação/genética , Transcrição Gênica/genética , Animais , Células COS , Chlorocebus aethiops , Feminino , Predisposição Genética para Doença/genética , Genótipo , Células HEK293 , Haploinsuficiência/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , FenótipoRESUMO
We describe a novel ERBB1/EGFR somatic mutation (p. C329R; c.985 T > C) identified in a patient with JAK2V617F Polycythaemia Vera (PV). This substitution affects a conserved cysteine residue in EGFR domain 2 and leads to the formation of a ligand-independent covalent receptor dimer, associated with increased transforming potential. Aberrant signalling from the EGFRC329R receptor is cell type-dependent and in the TF1.8 erythroid cell line expression of this mutant suppresses EPO-induced differentiation. Clonal analysis shows that the dominant JAK2V617F-positive clone in this PV patient harbors EGFRC329R, thus this mutation may contribute to clonal expansion. Somatic mutations affecting other ERBB and related receptor tyrosine kinases are observed in myeloproliferative neoplasms (MPN), and we show elevated EGFR levels in MPN samples, consistent with previous reports. Thus activation of this group of receptors, via multiple mechanisms, may contribute to clonal growth and survival of the JAK2V617F disease clone in MPN.
Assuntos
Janus Quinase 2/genética , Leucemia Eritroblástica Aguda/genética , Mutação , Policitemia Vera/genética , Mielofibrose Primária/genética , Sequência de Aminoácidos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Clonais , Receptores ErbB/genética , Receptores ErbB/metabolismo , Eritroblastos/efeitos dos fármacos , Eritroblastos/metabolismo , Eritroblastos/patologia , Eritropoetina/farmacologia , Expressão Gênica , Humanos , Janus Quinase 2/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patologia , Policitemia Vera/metabolismo , Policitemia Vera/patologia , Mielofibrose Primária/metabolismo , Mielofibrose Primária/patologia , Multimerização Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de SinaisAssuntos
Janus Quinase 2/genética , Policitemia Vera/genética , Policitemia Vera/patologia , Trombocitemia Essencial/genética , Trombocitemia Essencial/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Doenças Assintomáticas , Estudos de Coortes , Progressão da Doença , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Fenilalanina/genética , Polimorfismo de Nucleotídeo Único , Sintomas Prodrômicos , Valina/genéticaAssuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Animais , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Transdução de Sinais/fisiologia , Células Tumorais CultivadasRESUMO
Hypoxia-inducible factor (HIF)-1α accumulation promotes hematopoietic stem cells' quiescence and is necessary to maintain their self-renewal. However, the role of HIF-2α in hematopoietic cells is less clear. We investigated the role of HIF-2α in leukemia and lymphoma cells. HIF-2α expression was high in subsets of human and mouse leukemia and lymphoma cells, whereas it was low in normal bone marrow leukocytes. To investigate the role of HIF-2α, we transduced human HIF-2α cDNA in mouse syngeneic models of myeloid preleukemia and a transgenic model of B lymphoma. Ectopic expression of HIF-2α accelerated leukemia cell proliferation in vitro. Mice transplanted with cells transduced with HIF-2α died significantly faster of leukemia or B lymphoma than control mice transplanted with empty vector-transduced cells. Conversely, HIF-2α knockdown in human myeloid leukemia HL60 cells decreased proliferation in vitro and significantly prolonged animal survival following transplantation. In human acute myeloid leukemia (AML), HIF-2α mRNA was significantly elevated in several subsets such as the t(15;17), inv(16), complex karyotype and favorable cytogenetic groups. However, patients with high HIF-2α expression had a trend to higher disease-free survival in univariate analysis. The different effects of HIF-2α overexpression in mouse models of leukemia and human AML illustrates the complexity of this mutliclonal disease.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/patologia , Linfoma/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Western Blotting , Hipóxia Celular , Células Cultivadas , Estudos de Coortes , Progressão da Doença , Feminino , Seguimentos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Técnicas Imunoenzimáticas , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Linfoma/genética , Linfoma/mortalidade , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Adulto JovemAssuntos
Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fator de Transcrição STAT5/genética , Fatores de Transcrição/metabolismo , HumanosAssuntos
Crise Blástica/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação/genética , Proteínas Nucleares/genética , Estudos de Coortes , DNA de Neoplasias/genética , Progressão da Doença , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Nucleofosmina , Transportador 1 de Cátions Orgânicos/genética , Reação em Cadeia da Polimerase , Prognóstico , Taxa de SobrevidaAssuntos
Proteínas de Ciclo Celular/genética , DNA (Citosina-5-)-Metiltransferases/genética , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Proteínas Nucleares/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Metilação de DNA/fisiologia , DNA Metiltransferase 3A , Feminino , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Adulto JovemRESUMO
BACKGROUND: The human organic cation transporter-1 (OCT-1) is the primary active protein for imatinib uptake into target BCR-ABL-positive cells. Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used by chronic myeloid leukaemia (CML) patients on imatinib to manage musculoskeletal complaints. METHODS: Here we investigated the impact of NSAIDs on functional activity of the OCT-1 (OCT-1 activity; OA) in CML cells. RESULTS: Although ten of twelve NSAIDs tested had no significant impact on OA (P>0.05), we observed increased OA (27% increase in K562; 22% increase in KU812 cells, P<0.05) and reduced IC50(imatinib) when treated with diclofenac. Co-incubation with imatinib and diclofenac resulted in a significantly lower viable cell number compared with imatinib alone. In contrast, ibuprofen led to a significant decrease in OA, an increase in IC50(imatinib) and thus reduced the cytotoxicity of imatinib. In primary CML samples, diclofenac significantly increased OA, particularly in patients with low OA (<4 ng per 200 000 cells), and significantly decreased IC50(imatinib). Ibuprofen induced significant decreases in OA in CML samples and healthy donors. CONCLUSION: On the basis of the expected impact of these two drugs on OA, ibuprofen should be avoided in combination with imatinib. Further studies are warranted regarding the potential benefit of diclofenac to improve OA in a clinical setting.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/metabolismo , Diclofenaco/farmacologia , Ibuprofeno/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Transportador 1 de Cátions Orgânicos/efeitos dos fármacos , Piperazinas/metabolismo , Pirimidinas/metabolismo , Benzamidas , Linhagem Celular Tumoral , Interações Medicamentosas/fisiologia , Humanos , Mesilato de Imatinib , Concentração Inibidora 50Assuntos
Biomarcadores Tumorais/genética , Cromossomos Humanos Par 8/genética , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Leucemia Mieloide Aguda/genética , Trissomia , Biomarcadores Tumorais/metabolismo , Medula Óssea/metabolismo , Medula Óssea/patologia , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Regulação para CimaRESUMO
In chronic myeloid leukemia (CML) cell lines, brief exposure to pharmacologically relevant dasatinib concentrations results in apoptosis. In this study, we assess the impact of intensity and duration of Bcr-Abl kinase inhibition on primary CD34(+) progenitors of chronic phase CML patients. As CML cells exposed to dasatinib in vivo are in a cytokine-rich environment, we also assessed the effect of cytokines (six growth factors cocktail or granulocyte-macrophage colony-stimulating factor (CSF) or granulocyte-CSF) in combination with dasatinib. In the presence of cytokines, short-term intense Bcr-Abl kinase inhibition (>or=90% p-Crkl inhibition) with 100 nM dasatinib did not reduce CD34(+) colony-forming cells (CFCs). In contrast, without cytokines, short-term exposure to dasatinib reduced CML-CD34(+) CFCs by 70-80%. When cytokines were added immediately after short-term exposure to dasatinib, CML-CD34(+) cells remained viable, suggesting that oncogene dependence of these cells can be overcome by concomitant or subsequent exposure to cytokines. Additional inhibition of Janus tyrosine kinase (Jak) activity re-established the sensitivity of CML progenitors to intense Bcr-Abl kinase inhibition despite the presence of cytokines. These findings support the contention that therapeutic strategies combining intense Bcr-Abl kinase inhibition and blockade of cytokine signaling pathways can be effective for eradication of CML progenitors.
Assuntos
Apoptose/efeitos dos fármacos , Citocinas/antagonistas & inibidores , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Células-Tronco Hematopoéticas/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Antígenos CD34/metabolismo , Western Blotting , Citocinas/metabolismo , Dasatinibe , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Janus Quinases/antagonistas & inibidores , Janus Quinases/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/farmacologia , Tiazóis/farmacologia , Células Tumorais CultivadasRESUMO
The tumor suppressor Gadd45alpha was earlier shown to be a repressed target of sustained receptor-mediated ERK1/2 signaling. We have identified Gadd45alpha as a downregulated gene in response to constitutive signaling from two FLT3 mutants (FLT3-ITD and FLT3-TKD) commonly found in AML, and a leukemogenic GM-CSF receptor trans-membrane mutant (GMR-V449E). GADD45A mRNA downregulation is also associated with FLT3-ITD(+) AML. Sustained ERK1/2 signaling contributes significantly to receptor-mediated downregulation of Gadd45alpha mRNA in FDB1 cells expressing activated receptor mutants, and in the FLT3-ITD(+) cell line MV4;11. Knockdown of Gadd45alpha with shRNA led to increased growth and survival of FDB1 cells and enforced expression of Gadd45alpha in FDB1 cells expressing FLT3-ITD or GMR-V449E resulted in reduced growth and viability. Gadd45alpha overexpression in FLT3-ITD(+) AML cell lines also resulted in reduced growth associated with increased apoptosis and G(1)/S cell cycle arrest. Overexpression of Gadd45alpha in FDB1 cells expressing GMR-V449E was sufficient to induce changes associated with myeloid differentiation suggesting Gadd45alpha downregulation contributes to the maintenance of receptor-induced myeloid differentiation block. Thus, we show that ERK1/2-mediated downregulation of Gadd45alpha by sustained receptor signaling contributes to growth, survival and arrested differentiation in AML.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Leucemia Mieloide Aguda/patologia , Mutação/fisiologia , Proteínas Nucleares/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/fisiologia , Tirosina Quinase 3 Semelhante a fms/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo/genética , Leucemia Mieloide Aguda/etiologia , Camundongos , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Nucleares/genética , RNA Mensageiro/análise , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Sphingosine kinase (SK) catalyses the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in mediating such fundamental biological processes as cell growth and survival. Very little is currently known regarding the structure or mechanisms of catalysis and activation of SK. Here we have tested the functional importance of Gly(113), a highly conserved residue of human sphingosine kinase 1 (hSK), by site-directed mutagenesis. Surprisingly, a Gly(113)-->Ala substitution generated a mutant that had 1.7-fold greater catalytic activity than wild-type hSK (hSK(WT)). Our data suggests that the Gly(113)-->Ala mutation increases catalytic efficiency of hSK, probably by inducing a conformational change that increases the efficiency of phosphoryl transfer. Interestingly, hSK(G113A) activity could be stimulated in HEK293T cells by cell agonists to a comparable extent to hSK(WT).
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Alanina/genética , Sequência de Aminoácidos , Ácido Aspártico/genética , Sequência Conservada , Ativação Enzimática , Estabilidade Enzimática , Glicina/genética , Humanos , Mutagênese Sítio-Dirigida , Mutação Puntual , Dobramento de Proteína , Esfingosina/metabolismoRESUMO
We have isolated a previously unknown human homeobox-containing cDNA, VENT-like homeobox-2 (VENTX2), using PCR with a bone marrow cDNA library and primers designed from the VENTX1 (alias HPX42) homeobox sequence. Here we describe the molecular cloning, chromosomal localization to 10q26.3, and functional analysis of this gene. The 2.4-kb human VENTX2 cDNA encoded a protein with a predicted molecular weight of 28 kDa containing a homeodomain with 65% identity to the Xenopus laevis ventralizing gene Xvent2B. VENTX2 antisera detected a 28-kDa protein in cells transfected with a VENTX2 expression construct, in a human erythroleukemic cell line and in bone marrow samples obtained from patients in recovery phase after chemotherapy. The similarity of the homeodomains from VENTX2 and the X. laevis Vent gene family places them in the same homeodomain class. Consistent with this structural classification, overexpression of VENTX2 in zebrafish embryos led to anterior truncations and failure to form a notochord, which are characteristics of ventralization.
Assuntos
Antígenos de Neoplasias , Clonagem Molecular/métodos , Genes Homeobox/genética , Proteínas de Homeodomínio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 10/genética , Humanos , Dados de Sequência Molecular , Proteínas Repressoras/genética , Alinhamento de Sequência , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
A mutant form of the common beta-subunit of the GM-CSF, interleukin-3 (IL3) and IL5 receptors is activated by a 37 residue duplicated segment which includes the WSXWS motif and an adjacent, highly conserved, aliphatic/basic element. Haemopoietic expression of this mutant, hbeta(c)FIDelta, in mice leads to myeloproliferative disease. To examine the mechanism of activation of this mutant we targetted the two conserved motifs in each repeat for mutagenesis. Here we show that this mutant exhibits constitutive activity in BaF-B03 cells in the presence of mouse or human GM-CSF receptor alpha-subunit (GMRalpha) and this activity is disrupted by mutations of the conserved motifs in the first repeat. In the presence of these mutations the receptor reverts to an alternative conformation which retains responsiveness to human IL3 in a CTLL cell line co-expressing the human IL3 receptor alpha-subunit (hIL3Ralpha). Remarkably, the activated conformation is maintained in the presence of substitutions, deletions or replacement of the second repeat. This suggests that activation occurs due to insertion of extra sequence after the WSXWS motif and is not dependent on the length or specific sequence of the insertion. Thus hbeta(c) displays an ability to fold into functional receptor conformations given insertion of up to 37 residues in the membrane-proximal region. Constitutive activation most likely results from a specific conformational change which alters a dormant, inactive receptor complex, permitting functional association with GMRalpha and ligand-independent mitogenic signalling.
Assuntos
Ligantes , Peptídeos/química , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/química , Receptores de Interleucina-3/química , Receptores de Interleucina/química , Sequência de Aminoácidos , Animais , Divisão Celular , Linhagem Celular , Sequência Conservada , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Células-Tronco Hematopoéticas/metabolismo , Humanos , Interleucina-3/farmacologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Interleucina-5 , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
The stoichiometry of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor complex is still unresolved. We have utilised a sensitive, functional assay for receptor homodimerisation to show that GM-CSF induces dimerisation of the common signalling subunit, hbeta(c). We generated a chimeric cytokine receptor in which the extracellular and transmembrane domains of hbeta(c)are fused to the cytoplasmic domain of erythropoietin receptor (EPO-R). Given that to induce EPO-R activation and mitogenic signalling there is a requirement for formation of a specific homodimeric complex, we reasoned that the cytoplasmic domain of EPO-R could be utilised as a highly sensitive reporter for functional homodimer formation. We show that, in the presence of a cytoplasmically truncated GM-CSF alpha-subunit, the hbetac-EPO receptor chimera transduces a mitogenic signal in BaF-B03 in response to GM-CSF. This is consistent with formation of a hbeta(c)homodimer following GM-CSF binding and implies that ligand stimulation induces formation of a higher order complex that contains the hbeta(c)homodimer.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Animais , Linhagem Celular , Subunidade beta Comum dos Receptores de Citocinas , Dimerização , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Humanos , Camundongos , Ligação Proteica/fisiologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Sphingosine kinase (SphK) is a highly conserved lipid kinase that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). S1P/SphK has been implicated as a signalling pathway to regulate diverse cellular functions [1-3], including cell growth, proliferation and survival [4-8]. We report that cells overexpressing SphK have increased enzymatic activity and acquire the transformed phenotype, as determined by focus formation, colony growth in soft agar and the ability to form tumours in NOD/SCID mice. This is the first demonstration that a wild-type lipid kinase gene acts as an oncogene. Using a chemical inhibitor of SphK, or an SphK mutant that inhibits enzyme activation, we found that SphK activity is involved in oncogenic H-Ras-mediated transformation, suggesting a novel signalling pathway for Ras activation. The findings not only point to a new signalling pathway in transformation but also to the potential of SphK inhibitors in cancer therapy.