tretinoin and 3-3--4-5--tetrahydroxystilbene

Vav1 plays an important role in the all-trans retinoic acid (ATRA)-induced completion of the differentiation program of acute promyelocytic leukemia (APL)-derived cells, in which it strengthens the drug effects and is involved in the regulation of maturation-related proteins, such as the CD11b surface antigen. In both myeloid and lymphoid cells, accumulating data attribute to the multidomain protein Vav1 a functional relevance in the control of gene expression, by direct interaction with chromatin remodeling and/or transcriptional proteins. The present study provides evidence that, in the APL-derived NB4 cell line, Vav1 and the transcription factor PU.1 cooperate in regulating the ATRA-induced CD11b expression. Both chromatin immunoprecipitation (ChIP) experiments and electrophoretic mobility shift assays (EMSA) indicate that Vav1 and PU.1 are recruited to CD11b promoter. Even if the two proteins may participate in diverse protein/DNA complexes, the amounts of complexes including PU.1 seem to be dependent on the interaction of this transcription factor with tyrosine-phosphorylated Vav1. The reported data suggest that the ATRA-induced increase of Vav1 expression and tyrosine phosphorylation may be involved in recruiting PU.1 to its consensus sequence on the CD11b promoter and, ultimately, in regulating CD11b expression during the late stages of neutrophil differentiation of APL-derived promyelocytes.

Topics: CD11b Antigen; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Chromatin Immunoprecipitation; DNA; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Neoplastic; Granulocyte Precursor Cells; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Promyelocytic, Acute; Neutrophils; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-vav; RNA, Small Interfering; Stilbenes; Syk Kinase; Trans-Activators; Tretinoin

Vav is one of the genetic markers that correlate with the differentiation of hematopoietic cells. In T and B cells, it appears crucial for both development and functions, while, in non-lymphoid hematopoietic cells, Vav seems not involved in cell maturation, but rather in the response of mature cells to agonist-dependent proliferation and phagocytosis. We have previously demonstrated that the amount and the tyrosine phosphorylation of Vav are up-regulated in both whole cells and nuclei of tumoral promyelocytes induced to granulocytic maturation by ATRA and that tyrosine-phosphorylated Vav does not display any ATRA-induced GEF activity but contributes to the regulation of PI 3-K activity. In this study, we report that Vav accumulates in nuclei of ATRA-treated APL-derived cells and that the down-modulation of Vav prevents differentiation of tumoral promyelocytes, indicating that it is a key molecule in ATRA-dependent myeloid maturation. On the other hand, the overexpression of Vav induces an increased expression of surface markers of granulocytic differentiation without affecting the maturation-related changes of the nuclear morphology. Consistent with an effect of Vav on the transcriptional machinery, array profiling shows that the inhibition of the Syk-dependent tyrosine phosphorylation of Vav reduces the number of ATRA-induced genes. Our data support the unprecedented notion that Vav plays crucial functions in the maturation process of myeloid cells, and suggest that Vav can be regarded as a potential target for the therapeutic treatment of myeloproliferative disorders.

Topics: Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Enzyme Inhibitors; Gene Expression; Gene Expression Regulation, Leukemic; Granulocytes; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Myeloid Progenitor Cells; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-vav; RNA, Small Interfering; Stilbenes; Transfection; Tretinoin; Tumor Cells, Cultured

Our previous data demonstrated that cellular and nuclear tyrosine-phosphorylated Vav associate with phosphoinositide 3-kinase during all-trans-retinoic acid-dependent granulocytic differentiation of HL-60 cells. In this study, aimed to analyze the mechanism by which Vav is recruited and activated, we report that the Src homology 2 domain of Vav interacts with tyrosine-phosphorylated proteins in a differentiation-dependent manner. Two adaptor proteins, Cbl and SLP-76, were identified, showing a discrete distribution inside the cells, with Cbl absent from the nuclei and SLP-76 particularly abundant in the nuclear compartment. Of note, Vav interacts with the tyrosine kinase Syk, which is also present in the nuclear compartment and may phosphorylate Vav in vitro when cells differentiate. Inhibition of Syk activity by piceatannol prevents both in vitro and in vivo Vav tyrosine phosphorylation, its association with the regulatory subunit of phosphoinositide 3-kinase, and the nuclear modifications typically observed during granulocytic differentiation of this cell line. These findings suggest that tyrosine-phosphorylated Vav and its association with phosphoinositide 3-kinase play a crucial role in all-trans-retinoic acid-induced reorganization of the nucleoskeleton, which is responsible for the changes in nuclear morphology observed during granulocytic differentiation of HL-60 cells.

Topics: Adaptor Proteins, Signal Transducing; Cell Compartmentation; Cell Cycle Proteins; Cell Differentiation; Cell Nucleus; Enzyme Inhibitors; Enzyme Precursors; Granulocytes; Hematopoietic Stem Cells; Humans; Intracellular Signaling Peptides and Proteins; Oncogene Protein v-cbl; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-vav; Retroviridae Proteins, Oncogenic; Stilbenes; Syk Kinase; Tretinoin; Tumor Cells, Cultured; Tyrosine