Michinari Hamaguchi

Constitutive activation of mitogen-activated protein kinase pathway in acute leukemia cells

Mitogen-activated protein (MAP) kinase appears to be one of the key regulators of cell proliferation and differentiation. Very little, however, has been revealed as to how MAP kinase is involved in leukemogenesis. We have studied the activation of the MAP kinase pathway in 100 human primary leukemia cells including 73 acute myelogenous leukemias (AMLs). Forty acute leukemia samples (40% of the total), including 37 AML samples (51% of AML), showed activation of MAP kinase as revealed by the mobility shift of the phosphorylated form of the protein and by in vitro kinase assay. This activation was correlated with MAP kinase kinase activity in these cells. In contrast, none of 14 chronic myelogenous leukemia samples showed the activation of MAP kinase. These results suggest that the MAP kinase pathway is constitutively activated in a subset of primary acute leukemias, and thus indicate the possible role of the constitutively activated MAP kinase in leukemogenesis.

Tyrosine phosphorylation is crucial for growth signaling by tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2)

[3H]Thymidine (TdR) incorporation by human osteosarcoma cell line MG‐63 was significantly stimulated at as early as 3 h after the addition of either TIMP‐1 or TIMP‐2 alone. Maximum stimulation was attained at a concentration of either 20 ng/ml (0.71 nM) TIMP‐1 or 1.0 ng/ml (46 pM) TIMP‐2. Tyrosine kinase inhibitors such as genistein, erbstatin, and herbimycin A almost completely inhibited the [3H]TdR incorporation stimulated by either of the TIMPs. However, essentially no effect was observed with H‐89, H‐7, bisindolylmaleimide and K‐252a. These inhibition studies suggest a crucial role for tyrosine kinase in the signal transduction of TIMPs. Phosphotyrosine‐containing proteins were significantly elevated by the treatment with both TIMPs. We also found that either TIMP stimulated an increase in mitogen‐activated protein (MAP) kinase activity, suggesting that MAP kinase plays a role in TIMP‐dependent growth signaling.

C-RAS IS REQUIRED FOR THE ACTIVATION OF THE MATRIX METALLOPROTEINASES BY CONCANAVALIN A IN 3Y1 CELLS

Concanavalin A (Con A) is known to trigger augmented secretion and proteolytic activation of the matrix metalloproteinases (MMPs) in fibroblasts. To study the signaling pathway critical for the activation of MMPs in fibroblasts, we examined the effects of dominant negative ras (S17N ras) expression under the control of conditionally inducible promoter in Con A‐activated 3Y1 cells. We found that augmented secretion and proteolytic activation of MMP‐2 and MMP‐9 together with expression of MT1‐MMP in Con A‐activated 3Y1 were dramatically suppressed by S17N ras expression. These results strongly suggest that c‐Ras plays a critical role in the augmented expression and proteolytic activation of MMPs in fibroblasts.

Tyrosine phosphorylation of 100-130 kDa proteins in lung cancer correlates with poor prognosis.

To search for the signalling pathways in lung cancer relevant to its aggressive behaviour, we studied tyrosine phosphorylated proteins in lung cancer cell lines and surgical specimens. We found that the profiles of protein phosphorylation were closely matched among these cell lines and cancer tissues of different histological origins, and 100-130 kDa proteins were the major components of phosphorylated proteins. In surgical specimens, approximately half of the cases showed tyrosine phosphorylation of these proteins in a tumour-specific manner, and phosphorylation of these proteins showed good correlation with the survival length of patients after operation. By immunoprecipitation with specific antibodies, we found that p125FAK, p120 and beta-catenin were the major components of tyrosine-phosphorylated proteins in the surgical specimens. These results suggest that tyrosine phosphorylation of these proteins may play a role in tumour relapse and is available as a clinical marker.

Cell to substratum adhesion is involved in v-Src-induced cellular protein tyrosine phosphorylation: implication for the adhesion-regulated protein tyrosine phosphatase activity.

Protein tyrosine phosphorylation accompanies the inte-grin-mediated cell to substratum adhesion, and is essential for the progression of G1/S phase of the cell-cycle in normal fibroblasts. To examine how cellular protein tyrosine phosphatase (PTPase) activity is involved in regulating the adhesion-dependent protein tyrosine phosphorylation, we employed fibroblast cells bearing an active form of a protein tyrosine kinase (PTK), v-Src. We found that the v-Src induced tyrosine phosphorylation in certain proteins such as tensin, talin, p120, p80/85 (cortactin) and paxillin was greatly reduced when the cell to substratum adhesion was lost. Re-adhesion of the cells onto fibronectin restored these phosphorylation events, while this was inhibited by the addition of RGD peptide. The kinase activity of the v-Src was unchanged by the loss of cell to substratum adhesion. On the other hand, treatment with a protein tyrosine phosphatase inhibitor vanadate caused much the same increase in the v-Src-mediated cellular tyrosine phosphorylation between cells adhered to the culture environments and cells kept in suspension. These data suggest that PTPase(s) appears to be more critical than the v-Src PTK in determining the cell adhesion-dependent protein tyrosine phosphorylation. Moreover, most of the protein tyrosine phosphorylations that are mediated by the v-Src but still dependent on the cell adhesion were indeed greatly reduced during an anchorage-independent growth of v-Src cells. Thus our data collectively indicate that the v-Src induced high level of tyrosine phosphorylation in certain types of proteins are still under the control of the integrin(s) or the cell adhesion to culture substratum, and most of these adhesion-regulated high levels of tyrosine phosphorylations are not essential for the transformed phenotype.

Hematopoietic transcription factor GATA-2 activates transcription from HIV-1 long terminal repeat

Objectives:To study the role of the hematopoietic transcription factor GATA-2 in long terminal repeat (LTR)-directed transcriptional activation of HIV-1 in hematopoietic progenitor cells, and to investigate possible GATA-2 binding sites in HIV-1 LTR. Design and methods:Wild-type HIV-1 LTR, or mutants, ligated to a luciferase reporter gene with or without a GATA-2 expression vector, were transfected into COS cells, and standardized luciferase activity was examined. The binding activity of GATA-2 to these sites was examined by electrophoretic mobility shift assay. These wild-type or mutant reporter genes were also transfected into the murine hematopoietic progenitor cells, BAF3, in which GATA-2 was the predominantly expressed transcription factor of the GATA family, to assay LTR-directed transcription in intact hematopoietic machinery. Using a Tat expression plasmid for cotransfection, the influence of Tat protein on GATA-2-induced transactivation was determined. Results:In COS cells, LTR-dependent transactivation was highly enhanced by the coexpression of GATA-2. Experiments with mutant LTR suggested the presence of multiple GATA-2 binding sites, of which the major sites were identified. Cotransfection of Tat with GATA-2 indicated that GATA-2 and Tat synergistically enhanced the transcriptional activity. Transfection experiments in BAF3 cells showed that the disruption of these GATA sites diminished LTR-driven activity to 40% of the wild-type. Conclusions:GATA-2 may be a key host cell regulator of HIV-1 expression in hematopoietic stem cells. Manipulating this transactivation may represent a valuable approach to controlling virus production in infected hematopoietic progenitors. To elucidate the possible interaction between GATA-2 and Tat protein in vivo might give new insights to the mechanism of impaired hematopoiesis in AIDS patients.