Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c-kit proto-oncogene

The human mast cell line (HMC)-1 cell line is growth-factor independent because of a constitutive activity of the receptor tyrosine kinase Kit. Such deregulated Kit activity has also been suggested causative in gastrointestinal stromal tumours (GISTs) and mastocytosis. HMC-1 is the only established continuously growing human mast cell line and has therefore been widely employed for in vitro studies of human mast cell biology. In this paper we describe two sublines of HMC-1, named HMC-1(560 ) and HMC-1(560,816 ), with different phenotypes and designated by the locations of specific mutations in the c-kit proto-oncogene. Activating mutations in the Kit receptor were characterized using the pyrosequencing trade mark method. Both sublines have a heterozygous T to G mutation at codon 560 in the juxtamembrane region of the c-kit gene causing an amino acid substitution of Gly-560 for Val. In contrast, only HMC-1(560,816) cells have the c-kitV816 mutation found in mast cell neoplasms causing an Asp-->Val substitution in the intracellular kinase domain. Kit was constitutively phosphorylated on tyrosine residues and associated with phosphatidylinositol 3'-kinase (PI 3-kinase) in both variants of HMC-1, but this did not lead to a constitutive phosphorylation of Akt or extracellular regulated protein kinase (ERK), which are signalling molecules normally activated by the interaction of stem cell factor (SCF) with Kit. The documentation and characterization of two sublines of HMC-1 cells provides both information on the biological consequences of mutations in Kit and recognition of the availability of what in reality are two distinct cultured human mast cell lines.     

The stem cell factor-c-kit system and mast cells in human pancreatic cancer

Stem cell factor (SCF) and its receptor c-kit take part in the regulation of developmental processes of mast cells, hematopoietic stem cells, and melanocytes, as well as in the growth control of human malignancies. To explore the possible role of the SCF-c-kit system and of mast cells in pancreatic cancer, the concomitant expression and distribution of the two molecules were examined in 17 normal and 26 cancerous human pancreatic tissues and in 6 cultured pancreatic cancer cell lines. Mast cell distribution was also evaluated in the same tissue samples. In addition, the effects of SCF and of the c-kit tyrosine-kinase inhibitor STI571 on the growth of the cancer cell lines and of the normal pancreatic ductal cell line TAKA-1 were assessed. SCF immunoreactivity was absent in acinar, ductal, and islet cells of the normal pancreas and faint in pancreatic cancer tissues and cell lines. In contrast, c-kit was clearly present in some normal and hyperplastic ducts of the normal pancreas, in the cancer cells of 73% of the tumor samples, and in all the cell lines tested. Mast cells, identified by tryptase and chymase immunostaining on consecutive tissue sections, showed immunoreactivity for SCF and c-kit in both normal and cancerous specimens and their number was significantly increased (p = 0.03) in pancreatic cancer compared with the normal pancreas. SCF showed a dose-dependent growth inhibitory effect on TAKA-1 cells (p < 0.001), whereas pancreatic cancer cells were resistant to the SCF-induced growth inhibition. Nonetheless, the growth of TAKA-1 cells and pancreatic cancer cells was inhibited by the c-kit tyrosine kinase inhibitor STI571. In conclusion, the SCF-c-kit system, possibly with the contribution of mast cells, may have a growth-regulating role in the normal pancreas, which is altered during malignant transformation.     

STI571 inhibits growth and adhesion of human mast cells in culture

Stem cell factor (SCF)/c-kit system is critical for human mast cell development. We thus examined the effects of STI571, an inhibitor of the c-kit tyrosine kinase receptor, on the proliferation and function of human mast cells. STI571 at concentrations of 10(-6) M or higher almost completely abolished the SCF-dependent progeny generation from cord blood-derived cultured mast cells through an inhibition of the tyrosine phosphorylation of c-kit. The compound also suppressed the early phase of mast cell development. The extinction of mast cell growth induced by STI571 may be due largely to apoptosis according to the flow cytometric analysis and gel electrophoresis. Two-hour exposure to STI571 that failed to influence the total viable cell number suppressed adhesion of the cells to fibronectin in the presence of SCF without altering the expressions of integrin molecules. Our results may provide a fundamental insight for the clinical application of STI571 in allergic disorders.     

Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice--evidence for an impaired c-kit kinase in mutant mice

The proto-oncogene c-kit, a transmembrane tyrosine protein kinase receptor for an unknown ligand, was shown recently to map to the dominant white spotting locus (W) of the mouse. Mutations at the W locus affect various aspects of hematopoiesis, as well as the proliferation and/or migration of primordial germ cells and melanoblasts during development. Here, we show that c-kit is expressed in tissues known to be affected by W mutations in fetal and adult erythropoietic tissues, mast cells, and neural-crest-derived melanocytes. We demonstrate that the c-kit associated tyrosine-specific protein kinase is functionally impaired in W/WV mast cells, thus providing a molecular basis for understanding the developmental defects that result from these mutations.     

KIT expression in angiosarcomas and fetal endothelial cells: lack of mutations of exon 11 and exon 17 of C-kit.

C-kit proto-oncogene product (KIT, CD117) is a tyrosine kinase growth factor receptor for stem cell factor. This receptor is important for the development and maintenance of hematopoietic stem cells, mast cells, germ cells, melanocytes, and interstitial cells of Cajal and is constitutively expressed in them. Among mesenchymal tumors, KIT seems to be specific for the gastrointestinal stromal tumors, which consistently express this protein. Activating mutations in the tyrosine kinase or juxtamembrane domains of c-kit gene have been found in mastocytoma, seminoma, and gastrointestinal stromal tumors. Following up our initial observation of KIT expression in one angiosarcoma, we examined 50 angiosarcomas, 13 Kaposi sarcomas, 10 epithelioid hemangioendotheliomas, and 31 hemangiomas of different types for KIT expression using a polyclonal antiserum specific to KIT. Adult and fetal tissues and neovascular endothelia in 20 carcinomas were studied for comparison. More than half (56%) of the angiosarcomas representing different clinicopathologic and histologic subtypes and 2 of 13 Kaposi sarcoma were KIT positive. All epithelioid hemangioendotheliomas and hemangiomas were negative, with the exception of two infantile hemangiomas that showed KIT reactivity. The fetal capillary endothelia of lungs, placenta, and soft tissues were also KIT positive, although in soft tissues and placenta, KIT positivity was more prominent in the first trimester. However, endothelia of adult vessels and neovascular capillaries of carcinomas were negative. None of the four KIT-positive angiosarcomas and one KIT-positive Kaposi sarcomas that were studied showed mutations in the juxtamembrane or tyrosine kinase domains of the c-kit gene. These results indicate that KIT expression occurs in a subset of angiosarcomas, and the expression probably represents oncofetal expression (i.e., reversion of the tumor cell phenotype to that of fetal endothelial cells that may show KIT expression).     

Juxtamembrane-type c-kit gene mutation found in aggressive systemic mastocytosis induces imatinib-resistant constitutive KIT activation

Aggressive systemic mastocytosis (ASM) is a very rare form of mast cell neoplasm that does not benefit from conventional chemotherapy. The majority of adult mast cell neoplasms and gastrointestinal stromal tumors (GISTs) have mutations in the proto-oncogene c-kit, which encodes the KIT receptor tyrosine kinase. The c-kit gene mutations are generally confined to the tyrosine kinase II domain in mast cell neoplasms, but are often observed at the juxtamembrane domain in GISTs. We found a case of ASM with a juxtamembrane-type mutation, Val559Ile, and in this report the mutation was characterized through transfection of the mutated c-kit cDNA into human embryonic kidney cells. Phosphorylation of KIT and its possible downstream signaling molecules were examined in the presence or absence of imatinib, a selective tyrosine kinase inhibitor. Ligand-independent autophosphorylation was observed in the mutant KIT with Val559Ile as well as that with Val559Asp, as found in GISTs. Imatinib, at a concentration of 10 microM, inhibited autophosphorylation of the mutant KIT with Val559Asp, but not that with the Val559Ile. Phosphorylation of MAPK and STAT5 was also inhibited by imatinib at the same concentration, in cells expressing Val559Asp but not in those expressing Val559Ile. These results suggest that different mutations, even at the same codon, in juxtamembrane domain of the c-kit gene show different inhibitory effects of imatinib, and that patients with GISTs or mast cell neoplasms possessing this Val559Ile mutation are resistant to imatinib therapy.     

C-kit protein expression correlated with activating mutations in KIT gene in oral mucosal melanoma

C-kit is a trans-membrane receptor tyrosine kinase (RTK) encoded by the proto-oncogene KIT located at 4q11-12. Gain-of-function mutations arising to c-kit activation independent of its ligand were observed in various tumors related to germ cells, mast cells, and interstitial cells of Cajal. C-kit also participates in melanocyte development; hence, its involvement in oral mucosal melanoma (OMM) tumorigenesis was investigated. Immunohistochemistry and mutation analysis were performed using 18 cases of human primary OMM. Results revealed 16 cases positive to c-kit protein. Atypical melanocytes expressed c-kit. All in situ components expressed c-kit, but only four cases exhibited intense expression in the invasive component. Missense mutations were observed in four cases, and two of those correlated with increased protein expression. C-kit expression in atypical melanocytes suggests the role of c-kit in the early stage of OMM tumorigenesis. C-kit protein expression correlated with activating mutations indicating the pertinent role of the proto-oncogene KIT in the tumorigenesis of OMM.     

The c-kit ligand, stem cell factor, promotes mast cell survival by suppressing apoptosis.

Stem cell factor (SCF) and its receptor (SCFR), a member of the receptor tyrosine kinase III family that is encoded by the c-kit gene, critically regulate several complex biological programs including hematopoiesis, mast cell development, cutaneous pigmentation, and gametogenesis. We show herein that mouse mast cells die rapidly after the withdrawal of SCF in vivo or in vitro, and provide morphological evidence that such mast cells undergo programmed cell death or apoptosis. We also show that when in vitro-derived mouse mast cells maintained in SCF are removed from SCF-containing medium for only 5 or 6 hours, the cells' genomic DNA exhibits the ladder-like pattern of oligonucleosome-sized fragments typical of apoptosis. These findings demonstrate that SCF can regulate the survival of a cellular lineage which expresses the SCFR by suppressing apoptosis. They also identify a mechanism that can result in striking and rapid reductions in the size of tissue mast cell populations without histological evidence of the concomitant induction of a significant inflammatory response.     

Lack of c-kit (CD117) expression in CD30+ lymphomas and lymphomatoid papulosis.

c-Kit receptor (CD117) is expressed by erythroid, megakaryocytic, and myeloid precursors and mature mast cells and has been reported to be expressed in CD30+ lymphomas such as Hodgkin's disease and anaplastic large-cell lymphoma. Imatinib mesylate, a well-established inhibitor of bcr-abl tyrosine kinase, and currently used for the treatment of patients with chronic myeloid leukemia, also inhibits c-kit receptor kinase activity. In view of the possible use of imatinib as experimental therapy for patients with c-kit-positive tumors, we assessed c-kit expression in CD30+ cell lines and lymphomas. The cell lines were assessed using multiple methods (RT-PCR, flow cytometry, and Western blot). c-Kit expression was also immunohistochemically assessed in 168 CD30+ lymphomas including 87 classical Hodgkin's disease, 63 anaplastic large-cell lymphoma, and 15 cutaneous anaplastic large-cell lymphoma. We also studied 18 cases of lymphomatoid papulosis, a CD30+ lesion closely related to cutaneous anaplastic large-cell lymphoma. Neither c-kit mRNA nor protein was detected in any of the cell lines assessed. Furthermore, treatment with imatinib did not inhibit proliferation of cell lines in vitro. Using immunohistochemistry, only one of 183 (0.5%) lesions was positive for c-kit, the positive case being an ALK-negative anaplastic large-cell lymphoma. Our data demonstrate that expression of c-kit receptor is exceedingly rare among CD30+ lymphomas and lymphomatoid papulosis, suggesting that c-kit receptor is unlikely to be an appropriate target for therapeutic options such as imatinib in patients with these tumors.     

The c-kit receptor, stem cell factor, and mast cells. What each is teaching us about the others.

Many years ago, alert observers noticed among thousands of laboratory mice a few individuals that, unlike their littermates, exhibited areas of white spotting on their fur. No one could have predicted then that an effort to understand the basis for these abnormalities would ultimately contribute to the characterization of a receptor (c-kit) and a corresponding ligand (stem cell factor, SCF) that are critical not only to the migration and development of melanocytes, but also to hematopoiesis, gametogenesis, mast cell development, and, perhaps, development of the central nervous system. Nor could anyone have foretold then that this receptor and ligand would be shown to regulate the development of multiple distinct cellular lineages not only in mice, but also in humans and other primates, or that c-kit and its ligand would be found to influence the secretory function of cells bearing this receptor, as well as their development. Investigation of the effects of SCF on a single cell type, the mast cell, has produced the most complete picture of the spectrum of biological processes that can be regulated by interactions between c-kit and its ligand. This work shows that SCF critically regulates the migration and survival of mast cell precursors, promotes the proliferation of both immature and mature mast cells, enhances mast cell maturation, directly induces secretion of mast cell mediators, and can regulate the extent of mediator release in mast cells activated by IgE-dependent mechanisms. Indeed, SCF may well prove to be one of the most important of the factors influencing mast cell numbers, phenotype, and function in both health and disease. It now seems virtually certain that further studies of c-kit and SCF will produce important new insights into problems as diverse as the regulation of lineage commitment during normal hematopoiesis or the development and function of the central nervous system. And even though an effect on mast cell development was one of the last phenotypic abnormalities to be recognized in mice with mutations affecting the genes encoding c-kit or SCF, mast cells will continue to represent an important model system for analyzing the biology of c-kit and its ligand.     

Pathology of gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors in the gastrointestinal tract. It was found that most GIST expressed KIT, a receptor tyrosine kinase encoded by protooncogene c-kit. In normal gastrointestinal wall, KIT is expressed by interstitial cells of Cajal (ICC), which are a pacemaker for autonomous gastrointestinal movement. Because both GIST and ICC are double-positive for KIT and CD34, and because familial and multiple GIST appear to develop from diffuse hyperplasia of ICC, GIST are considered to originate from ICC or their precursor cells. It was also found that approximately 90% of the sporadic GIST have somatic gain-of-function mutations of the c-kit gene, and that the patients with familial and multiple GIST have germline gain-of-function mutations of the c-kit gene. These facts strongly suggest that the c-kit gene mutations are a cause of GIST. Approximately half of the sporadic GIST without c-kit gene mutations were demonstrated to have gain-of-function mutations in platelet-derived growth factor receptor-alpha (PDGFRA) gene that encodes another receptor tyrosine kinase. Because KIT is immunohistochemically negative in a minority of GIST, especially in PDGFRA gene mutation-harboring GIST, mutational analyses of c-kit and PDGFRA genes may be required to diagnose such GIST definitely. Imatinib mesylate was developed as a selective tyrosine kinase inhibitor. It inhibits constitutive activation of mutated KIT and PDGFRA, and is now being used for KIT-positive metastatic or unresectable GIST as a molecular target drug. Confirmation of KIT expression by immunohistochemistry is necessary for application of the drug. The effect of imatinib mesylate is different in various types of c-kit and PDGFRA gene mutations, and the secondary resistance against imatinib mesylate is often acquired by the second mutation of the identical genes. Mutational analyses of c-kit and PDGFRA genes are also significant for prediction of effectiveness of drugs including newly developed agents.     

Growth suppression of human mast cells expressing constitutively active c-kit receptors by JNK inhibitor SP600125

Activation of c-jun N-terminal kinase (JNK) through c-kit-mediated phosphatidylinositol 3 (PI3) and Src kinase pathways plays an important role in cell proliferation and survival in mast cells. Gain-of-function mutations in c-kit are found in several human neoplasms. Constitutive activation of c-kit has been observed in human mastocytosis and gastrointestinal stromal tumor. In the present study, we demonstrate that an anthrapyrazole SP600125, a reversible ATP-competitive inhibitor of JNK inhibits proliferation of human HMC-1 showed constitutive activation of JNK/c-Jun, and the inhibitory effect of SP600125 on cell proliferation was associated with cell cycle arrest at the G1 phase and apoptosis accompanied by the cleavage of caspase-3 and PARP. Caspase-3 inhibitor Z-DEVD-FMK almost completely inhibited SP600125-induced apoptosis of HMC-1 cells. In contrast, caspase-9 inhibitor Z-LEHD-FMK failed to block SP600125-induced apoptosis. Following Sp600125 treatment, down-regulation of cyclin D3 protein expression, but not p53 was also observed. Thus, JNK/c-Jun is essential for proliferation and survival of HMC-1 cells. The results obtained from the present study suggest the possibility that JNK/c-Jun may be a therapeutic target in diseases associated with mutations in the catalytic domain of c-kit.     

Effects of interleukin-3 with or without the c-kit ligand, stem cell factor, on the survival and cytoplasmic granule formation of mouse basophils and mast cells in vitro.

We assessed the ultrastructure and the cell-surface expression of receptors for immunoglobulin E (Fc epsilon R), and c-kit, the receptor for stem cell factor (SCF), in mouse basophils and mast cells present in short-term cultures of mouse bone marrow cells in interleukin-3 (IL-3) with or without SCF. Basophils did not develop increased numbers of cytoplasmic granules and underwent apoptosis in cultures containing IL-3 and SCF, whereas mast cells thrived and developed increased numbers of granules. Basophils were nearly all Fc epsilon R+ c-kit- when sorted after culture in IL-3 and SCF; most mast cells were Fc epsilon R+ c-kit+. However, a second population of Fc epsilon R+ c-kit- mast cells was present after culture in IL-3 and SCF. These c-kit receptor-negative mast cells were less mature than c-kit+ mast cells and contained significantly fewer cytoplasmic granules than the c-kit+ mast cells present in the same cultures (P < 0.001). Thus, mouse basophils express little or no c-kit receptor on their surface, nor can they survive for long periods in SCF-supplemented cultures. By contrast, mouse mast cells seem to express the Fc epsilon R early in their development, even before they express detectable c-kit receptors on their surface. IL-3 promotes cytoplasmic granule formation in immature mast cells, but even more granules are formed when c-kit receptor-positive immature mast cells are cultured in both SCF and IL-3.     

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.