Gastrointestinal stromal tumors: past, present, and future.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. The name "GIST" was proposed in 1983, but the cell origin of GIST remained unclear until 1998, when my colleagues and I reported immunohistochemical evidence that GIST originated from interstitial cells of Cajal or their precursors. At the same time, we reported gain-of-function mutations of the Kit gene in GISTs. The Kit gene encodes KIT receptor tyrosine kinase, whose structure is similar to that of platelet-derived growth factor receptor (PDGFR). Imatinib mesylate was initially developed as an inhibitor of PDGFR. Then, it was found to be a potent inhibitor of BCR-ABL. Imatinib was successfully used for the treatment of chronic myeloid leukemia. When we reported gain-of-function mutations of the Kit gene in GISTs, the inhibitory effect of imatinib on KIT was already known. Imatinib was then successfully applied to the treatment of GISTs. The interrelationship between the type of Kit gain-of-function mutation and the therapeutic effect of imatinib has been well characterized in GISTs. Although various mutations of Kit and Pdgfr-alpha genes have been found in GISTs, most GISTs are luckily imatinibsensitive. After long-term administration of imatinib, however, new imatinib-resistant clones develop a secondary mutation of the Kit or Pdgfr-alpha gene. New drugs and adjuvant regimens against such secondary progression are now being intensively explored.

Heterophilic binding of the adhesion molecules poliovirus receptor and immunoglobulin superfamily 4A in the interaction between mouse spermatogenic and Sertoli cells.

The cell adhesion protein immunoglobulin superfamily 4A (IGSF4A) is expressed on the surfaces of spermatogenic cells in the mouse testis. During spermatogenesis, IGSF4A is considered to bind to the surface of Sertoli cells in a heterophilic manner. To identify this unknown partner of IGSF4A, we generated rat monoclonal antibodies against the membrane proteins of mouse Sertoli cells grown in primary culture. Using these monoclonal antibodies, we isolated a clone that immunostained Sertoli cells and reacted with the product of immunoprecipitation of the homogenate of mouse testis with anti-IGSF4A antibody. Subsequently, to identify the Sertoli cell membrane protein that is recognized by this monoclonal antibody, we performed expression cloning of a cDNA library from the mouse testis. As a result, we identified poliovirus receptor (PVR), which is another IGSF-type cell adhesion molecule, as the binding partner of IGSF4A. The antibodies raised against PVR and IGSF4A immunoprecipitated both antigens in the homogenate of mouse testis. Immunoreactivity for PVR was present in Sertoli cells but not in spermatogenic cells at all stages of spermatogenesis. Overexpression of PVR in TM4, a mouse Sertoli cell line, increased more than three-fold its capacity to adhere to Tera-2, which is a human cell line that expresses IGSF4A. These findings suggest that the heterophilic binding of PVR to IGSF4A is responsible, at least in part, for the interaction between Sertoli and spermatogenic cells during mouse spermatogenesis.

Development of mast cells.

Mast cells are progeny of the multipotential hematopoietic stem cell (MHSC). Mast cell-committed progenitors (MCPs) leave hematopoietic tissues, migrate in peripheral blood, invade to connective or mucosal tissue, proliferate and differentiate to morphologically identifiable mast cells. Phenotype of mast cells (connective tissue-type or mucosal type) is determined by the site of lodgment of MCPs. Most progeny of the multipotential hematopoietic stem cell lose proliferation potential after maturation, but connective tissue-type mast cells (CTMCs) possess appreciable proliferation potential after maturation. Even after functioning by degranulation, CTMCs proliferate and restore the original morphology. The most important cytokine for development and survival of mast cells is KIT ligand, and the KIT receptor tyrosine kinase is expressed through the whole developmental process of mast cells from MHSC to mature mast cells. The loss-of-function mutation of KIT gene results in depletion of mast cells, whereas its gain-of-function mutation causes mast cell tumors. Since mast cells are involved in various disease processes, intervention in development of mast cells might be beneficial to the treatment.

Molecular mechanisms of mast cell development.

Mast cells are progeny of multipotential hematopoietic stem cells (MHSCs). MHSCs commit to the mast cell lineage in the bone marrow, and the mast cell-committed progenitors leave the bone marrow, migrate in blood, invade connective or mucosal tissue, and then proliferate and differentiate to connective tissue-type or mucosal mast cell. GATA-1, GATA-2, and PU.1 transcription factors seem to be involved i the commitment to mast cells, and MITF, a basic helix-loop-helix leucine zipper-type transcription factor, seems to be involved in the migration, phenotypic expression, and survival of mast cells. KIT ligand (KITL) is the most important cytoline for development of mast cells, and KIT is the receptor of KITL. Tissues of loss-of-function mutants of KIT, KITL, or MITF are deficient in mast cells.

The spermatogenic Ig superfamily/synaptic cell adhesion molecule mast-cell adhesion molecule promotes interaction with nerves.

Nerve-mast cell interaction is involved in both homeostatic and pathologic regulations. The molecules that sustain this association have not been identified. Because synaptic cell adhesion molecule (SynCAM), alternatively named spermatogenic Ig superfamily (SgIGSF), is expressed on both nerves and mast cells and because it binds homophilically, this molecule may be a candidate. To examine this possibility, mast cells with or without SgIGSF/SynCAM were cocultured with superior cervical ganglion neurons that express SgIGSF/SynCAM, and the number of mast cells attached to neurites was counted. The attachment of mast cells with SgIGSF/SynCAM, i.e., bone marrow-derived mast cells (BMMC) from wild-type mice, was inhibited dose-dependently by blocking Ab to SgIGSF/SynCAM. Mast cells without SgIGSF/SynCAM, i.e., BMMC from microphthalmia transcription factor-deficient mice and BMMC-derived cell line IC-2 cells, were defective in attachment to neurite, and transfection with SgIGSF/SynCAM normalized this. When the nerves were specifically activated by scorpion venom, one-quarter of the attached IC-2 cells mobilized Ca(2+) after a few dozen seconds, and ectopic SgIGSF/SynCAM doubled this proportion. At points of contact between neurites and wild-type BMMC, SgIGSF/SynCAM was locally concentrated in both neurites and BMMC. SgIGSF/SynCAM on mast cells appeared to predominantly mediate attachment and promote communication with nerves.

Distinct role for c-kit receptor tyrosine kinase and SgIGSF adhesion molecule in attachment of mast cells to fibroblasts.

Binding of stem cell factor (SCF) to c-kit receptor tyrosine kinase (KIT) transduces signals essential for mast cell development via several pathways including activation of phosphatidylinositol 3-kinase (PI3-K). When cultured mast cells (CMCs) are cocultured with fibroblasts expressing membrane-bound SCF, CMCs with normal KIT adhere to fibroblasts and proliferate, whereas CMCs lacking cell surface expression of KIT do neither. Spermatogenic immunoglobulin superfamily (SgIGSF) was identified as another molecule that participates in mast cell adhesion to fibroblasts. Since the IC-2 mast cell line expressed neither KIT nor SgIGSF, the effect of ectopic expression of KIT or SgIGSF on the adhesion of IC-2 cells was examined. Three forms of KIT with the normal ectodomain were used: wild-type (KIT-WT) and two mutant types with a phenylalanine substitution at the tyrosine residue 719 (KIT-Y719F) or 821 (KIT-Y821F). KIT-Y719F does not activate PI3-K, whereas KIT-Y821F does. Firstly, KIT or SgIGSF was expressed singly in IC-2 cells. All three forms of KIT increased the adhesion level of IC-2 cells, whereas SgIGSF did not. Secondly, SgIGSF was coexpressed with one of the three forms of KIT. Coexpression of SgIGSF with KIT-WT or KIT-Y821F increased the adhesion level more markedly than was achieved by KIT-WT or KIT-Y821F alone. The effect was abolished by an antibody that blocks SCF-KIT interaction. In contrast, coexpression of SgIGSF with KIT-Y719F did not increase the adhesion level induced by KIT-Y719F alone. In adhesion of mast cells to fibroblasts, KIT appeared to behave as an adhesion molecule and as an activator of other adhesion molecules through phosphorylating PI3-K.

MITF and SgIGSF: an essential transcription factor and its target adhesion molecule for development and survival of mast cells.

MITF transcription factor is not produced in mutant mice of WBB6F1-Mitf(mi-vga9/Mitf(mi-vga9). When bone marrow cells of normal WBB6F1-+/+ micewere transplanted to the irradiated WBB6F1-KitW/KitW-v mice, which are genetically mast cell deficient, erythrocytes, neutrophils, macrophages, B cells and T cells of the donor origin developed in the recipients. On the other hand, when bone marrow cells of WBB6F1-Mitf(mi-vga9/Mitf(mi-vga9) mice were transplanted to similarly irradiated WBB6F1-KitW/KitW-v mice, erythrocytes, neutrophils, macrophages, B cells and T cells of the donor origin developed but mast cells never appeared. To identify the targets of MITF, we elaborated a subtracted cDNA library between cultured mast cells (CMCs) derived from +/+ mice and CMCs from MITF mutant mice. We obtained a clone encoding SgIGSF, which was expressed on the adhesion surface of +/+ CMCs attaching to fibroblasts. CMCs of WBB6F1-Mitf(mi-vga9/ Mitf(mi-vga9) mice did not express SgIGSF, did not attach to fibroblasts and did not survive in the peritoneal cavity of WBB6F1-KitW/KitW-v mice. When MITF or SgIGSF cDNA was transfected to WBB6F1-Mitf(mi-vga9/Mitf(mi-vga9) CMCs, they attached to fibroblasts and showed an improved survival in the peritoneal cavity of WBB6F1-KitW/KitW-v mice. MITF and SgIGSF appeared essential for the development and survival of mast cells in tissues of adult WBB6F1-KitW/KitW-v mice.

Distinct roles for the SgIGSF adhesion molecule and c-kit receptor tyrosine kinase in the interaction between mast cells and the mesentery.

Intraperitoneal injection of bone marrow-derived mast cells (BMMCs) has therapeutic efficacy against acute bacterial peritonitis. For this role, BMMCs need to settle down the mesentery from the peritoneal cavity. Interaction between BMMCs and the mesentery was examined by using mast cell deficient WBB6F1(F1)-W/Wv [c-kit receptor tyrosine kinase (KIT) mutant], F1-Sl/Sld [KIT ligand stem cell factor mutant], and F1-tg/tg [a practically microphthalmia transcription factor (MITF)-null mutant] mice. Three parameters were measured: the number of BMMCs: (1) developed in the mesentery 5 weeks after intraperitoneal injection into mast cell deficient mice, (2) adhered to mesenteric mesothelial cells, and (3) transmigrated across the mesenteric mesothelial cell monolayer when coculturing both cells for 3 and 18 h, respectively. After intraperitoneal injection, F1-wild type (+/+) BMMCs developed in the mesentery of F1-W/Wv mice but not in that of F1-Sl/Sld mice, while F1-tg/tg BMMCs did not develop, even in the mesentery of WBB6F1-W/Wv mice. In the coculture, WB-W/W BMMCs normally adhered to but poorly transmigrated across F1-+/+ mesothelial cells, and in accordance, F1-+/+ BMMCs normally adhered to but poorly transmigrated across F1-Sl/Sld mesothelial cells. F1-tg/tg BMMCs showed poor adhesion and transmigration, but both parameters were partially but significantly improved by ectopic expression of spermatogenic immunoglobulin superfamily (SgIGSF), a mast-cell adhesion molecule critically regulated by MITF. Since F1-tg/tg BMMCs expressed reduced levels of KIT, these results suggested that SgIGSF and KIT independently played a significant role in the transmigration. Among three parameters, development of mast cells in the mesentery well correlated with the transmigration. This process seemed important for mast cells to settle down from the peritoneal cavity to the mesentery.

Number of mast cells in the peritoneal cavity of mice: influence of microphthalmia transcription factor through transcription of newly found mast cell adhesion molecule, spermatogenic immunoglobulin superfamily.

The mi (microphthalmia) locus of mice encodes a transcription factor, MITF. B6-tg/tg mice that do not express any MITF have white coats and small eyes. Moreover, the number of mast cells decreased to one-third that of normal control (+/+) mice in the skin of B6-tg/tg mice. No mast cells were detectable in the stomach, mesentery, and peritoneal cavity of B6-tg/tg mice. Cultured mast cells derived from B6-tg/tg mice do not express a mast cell adhesion molecule, spermatogenic immunoglobulin superfamily (SgIGSF). To obtain in vivo evidence for the correlation of nonexpression of SgIGSF with decrease in mast cell number, we used another MITF mutant, B6-mi(vit)/mi(vit) mice that have a mild phenotype, ie, black coat with white patches and eyes of normal size. B6-mi(vit)/mi(vit) mice had a normal number of mast cells in the skin, stomach, and mesentery, but the number of peritoneal mast cells decreased to one-sixth that of +/+ mice. Cultured mast cells and peritoneal mast cells of B6-mi(vit)/mi(vit) mice showed a reduced but apparently detectable level of SgIGSF expression, demonstrating the parallelism between mast cell number and expression level of SgIGSF. The number of peritoneal mast cells appeared to be influenced by MITF through transcription of SgIGSF.

Different inhibitory effect of imatinib on phosphorylation of mitogen-activated protein kinase and Akt and on proliferation in cells expressing different types of mutant platelet-derived growth factor receptor-alpha.

Most gastrointestinal stromal tumors (GISTs) have gain-of-function mutations of the c-kit gene. Previously, we found 2 types of gain-of-function mutation of the PDGFRA gene, Val561 to Asp and Asp842 to Val, in about half of GISTs without c-kit gene mutations. Although the inhibitory effect of imatinib on various types of activating mutant KIT has been well examined, that on the activating mutant PDGFRA has not been fully investigated. In the present study, we examined the effect of imatinib on autophosphorylation of mutant PDGFRA, phosphorylation of MAPK and of Akt and in vitro cell proliferation using murine Ba/F3 cells stably transfected with one of the 2 murine-type mutated PDGFRA cDNAs. Imatinib almost completely inhibited autophosphorylation of mutant PDGFRA, phosphorylation of MAPK and Akt as well as in vitro cell proliferation at the concentration of 0.01 microM in cells expressing mutant PDGFRA with Val561 to Asp. However, in cells expressing mutant PDGFRA with Asp842 to Val, imatinib almost completely inhibited autophosphorylation of mutant PDGFRA and phosphorylation of MAPK and Akt at 1.0 microM. The concentration contributing to complete inhibition of in vitro cell proliferation was 10 microM. Ba/F3 cells expressing mutant PDGFRA are a good model to investigate the mechanism of cell proliferation or growth inhibition by imatinib in mutant PDGFRA-driven cells.

Cloning of a soluble isoform of the SgIGSF adhesion molecule that binds the extracellular domain of the membrane-bound isoform.

SgIGSF (spermatogenic immunoglobulin superfamily) is a recently identified intercellular adhesion molecule of the immunoglobulin superfamily. In a mast-cell cDNA library, we found a clone that resulted from the retention of intron 7 within the mature SgIGSF message. This clone was predicted to encode a soluble isoform of SgIGSF (sSgIGSF) with 336 amino-acid residues because its open reading frame ended just before the transmembrane domain. We constructed a plasmid expressing sSgIGSF fused to the human IgG Fc fragment at its C-terminus (sSgIGSF-Fc), and transfected it into COS-7 cells. The fusion protein was readily detectable in the culture supernatant. Solid-phase binding assay showed that sSgIGSF interacted directly the extracellular domain of membrane-bound SgIGSF (mSgIGSF). We next examined whether this interaction inhibited homophilic binding of mSgIGSF by aggregation assays using L cells that did not express mSgIGSF. A stable L-cell clone that overexpressed mSgIGSF aggregated with each other but not with mock-transfected L cells, indicating that a homophilic interaction of mSgIGSF mediated the aggregation. Addition of sSgIGSF-Fc inhibited the aggregation of L cells overexpressing mSgIGSF in a dose-dependent manner. Moreover, FACScan analyses revealed the specific binding of sSgIGSF-Fc to mSgIGSF expressed in L cells. Binding of sSgIGSF-Fc to mSgIGSF appeared to inhibit homophilic interactions of mSgIGSF.

Roles of MITF for development of mast cells in mice: effects on both precursors and tissue environments.

The mutant tg/tg mice, which do not express mi transcription factor (MITF), lack mast cells in most tissues. Since MITF is expressed in both mast cells and tissues where mast cells develop, there is a possibility that the tg/tg mice may show abnormalities in both mast cell precursors and tissue environments. We examined this possibility by bone marrow and skin transplantation. When bone marrow cells of tg/tg mice were transplanted to W/W(v) mice that possess normal tissue environment, mast cells did not develop in all tissues examined. The number of developing mast cells in the skin of W/W(v) mice was much lower when grafted to tg/tg recipients than when grafted to normal (+/+) recipients. These results indicated that mast cell precursors of tg/tg mice were defective. When bone marrow cells of +/+ mice were transplanted, the number of developing mast cells was significantly lower in examined tissues of tg/tg recipients than in those of W/W(v) recipients, suggesting that the tissue environment for mast cell development was defective in tg/tg mice. MITF appeared essential for the function of both mast cell precursors and tissue environments for their development.

A derivative of oleamide potently inhibits the spontaneous metastasis of mouse melanoma BL6 cells.

We reported previously that the abnormally augmented expression of connexin 26 (Cx26) is responsible for the enhanced spontaneous metastasis of mouse BL6 melanoma cells, and that the exogenous expression of a dominant negative form of Cx26 inhibits the spontaneous metastasis of BL6. Here we show that daily intraperitoneal (i.p.) injections of oleamide, a sleep-inducing lipid hormone, weakly inhibited the spontaneous metastasis of BL6 cells. To obtain a more effective reagent, 19 oleamide derivatives were chemically synthesized and tested for their ability to inhibit the gap junction-mediated intercellular communications (GJIC) that are formed between HeLa cells by the ectopic expression of Cx26 or Cx43. One of these, denoted metastasis inhibitor-18 (MI-18), inhibited the GJIC formed by Cx26 as well as oleamide but unlike oleamide, which is a non-selective inhibitor of connexin, it did not inhibit the GJIC formed by Cx43. Daily i.p. injections of MI-18 potently blocked the spontaneous metastasis of BL6 cells down to 15% of that in the untreated control mice. MI-18 was safe because even after >7 weeks of daily injections, the survival rate of the mice was 93%. We propose that MI-18 may serve as a novel and clinically important prototype of a potent inhibitor of spontaneous metastasis.

Contribution of the SgIGSF adhesion molecule to survival of cultured mast cells in vivo.

Spermatogenic immunoglobulin superfamily (SgIGSF) is a recently identified adhesion molecule, and the microphthalmia transcription factor (MITF) was essential for its expression in mast cells. Since the tg mutant allele is practically a null mutation of the MITF gene, cultured mast cells (CMCs) derived from (WBxC57BL/6)F(1) (F(1))-tg/tg mice did not express SgIGSF whereas CMCs from F(1)-wild-type (+/+) mice expressed it abundantly. When cocultured with NIH/3T3 fibroblasts, F(1)-tg/tg CMCs showed poor adhesion to NIH/3T3 fibroblasts. When injected intraperitoneally, F(1)-tg/tg CMCs showed poor survival in the peritoneal cavity of mast cell-deficient F(1)-W/Wv mice. SgIGSF was expressed in tg/tg CMCs ectopically through retroviral transfection and through expression of a transgene. The resulting tg/tg CMCs showed not only a better adhesion to NIH/3T3 fibroblasts but also a better survival in the peritoneal cavity than control F(1)-tg/tg CMCs. SgIGSF-mediated adhesion seemed to play a role in the survival of CMCs in the peritoneal cavity.

Identification of a cytokine-induced antiapoptotic molecule anamorsin essential for definitive hematopoiesis.

Many growth factors and cytokines prevent apoptosis. Using an expression cloning method, we identified a novel antiapoptotic molecule named Anamorsin, which does not show any homology to known apoptosis regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules. The expression of Anamorsin was completely dependent on stimulation with growth factors such as interleukin 3, stem cell factor, and thrombopoietin in factor-dependent hematopoietic cell lines, and forced expression of Anamorsin conferred resistance to apoptosis caused by growth factor deprivation in vitro. Furthermore, Anamorsin was found to act as an antiapoptotic molecule in vivo because Anamorsin-/- mice die in late gestation due to defective definitive hematopoiesis in the fetal liver (FL). Although the number of hematopoietic stem/progenitor cells in the FL did not decrease in these mice, myeloid, and particularly erythroid colony formation in response to cytokines, was severely disrupted. Also, Anamorsin-/- erythroid cells initiated apoptosis during terminal maturation. As for the mechanism of Anamorsin-mediated cell survival, a microarray analysis revealed that the expression of Bcl-xL and Jak2 was severely impaired in the FL of Anamorsin-/- mice. Thus, Anamorsin is considered to be a necessary molecule for hematopoiesis that mediates antiapoptotic effects of various cytokines.

The point mutation of tyrosine 759 of the IL-6 family cytokine receptor gp130 synergizes with HTLV-1 pX in promoting rheumatoid arthritis-like arthritis.

Rheumatoid arthritis (RA) is a polygenic autoimmune disease. The autoimmunity develops from synergistic actions of genetic and environmental factors. We generated a double-mutant mouse by crossing two murine models of RA, a gp130 mutant knock-in mouse (gp130F759/F759) and an HTLV-1 pX transgenic mouse (pX-Tg), in a C57BL/6 background, which is resistant to arthritis. The mice spontaneously developed severe arthritis with a much earlier onset than the gp130F759/F759 mice and with a much higher incidence than did the pX-Tg mice. The symptoms of gp130F759/F759 mice, including lymphoadenopathy, splenomegaly, hyper-gamma-globulinemia, autoantibody production, increases in memory/activated T cells and granulocytes in the peripheral lymphoid organs, and a decrease in the class II MHC(bright) CD11c+ population, were augmented in the double mutants. Marked reductions in incidence, severity and immunological abnormalities were seen in the triple mutant, IL-6-/-/gp130F759/F759/pX-Tg, indicating that the arthritis in the double mutant is IL-6 dependent. gp130F759/F759/pX-Tg is a unique mouse model for RA.