ALBERT

Description: Mutant mice of mi/mi genotype are osteopetrotic and deficient in tissue mast cells due to a defect in osteoclasts and mast cells. In an effort to further understand the mechanisms behind why mi/mi mouse-derived cultured mast cells (mi/mi-CMC) responded to interleukin-3 (IL-3), but not to the proliferative stimuli presented by fibroblasts, mi/mi-CMC and congenic normal (+/+) mouse-derived CMC (+/+-CMC), both of which expressed the phenotypic characteristics of immature mast cells, were cocultured with Swiss albino/3T3 fibroblasts in a medium containing IL- 3. In the in vitro CMC/fibroblast coculture, mi/mi-CMC did not acquire the phenotypes of connective tissue-type mast cells (CTMC), while +/+- CMC did. In addition, attachment of mi/mi-CMC to the fibroblasts was found to be significantly lower than that of +/+-CMC. Because the interaction of c-kit product with its ligand (stem cell factor [SCF]) is known to play an important role not only in proliferation and differentiation of mast cells but also in attachment of CMC to fibroblasts, the expression and function of c-kit were investigated in mi/mi-CMC and +/+-CMC. Recombinant rat SCF (rrSCF164) induced a dose- dependent proliferation of +/+-CMC. Also, rrSCF164 induced +/+-CMC to acquire the phenotypes of CTMC in the medium containing IL-3. By contrast, rrSCF164 did not stimulate the proliferation of mi/mi-CMC nor induce a phenotypic change of the cells from immature mast cells to mature, CTMC-like mast cells. Immunoblotting with antiphosphotyrosine antibody showed that rrSCF164 induced considerable tyrosine phosphorylation of 145- to 165-Kd protein, the product of c-kit, in +/+- CMC, whereas tyrosine phosphorylation of the protein was barely detectable in mi/mi-CMC. Northern blot and flow cytometry analyses showed that mi/mi-CMC expressed much less c-kit at both protein and message levels than +/+-CMC. Further, mi/mi-CMC were found to differ from +/+-CMC in the expression of mouse mast cell protease-6 (MMCP-6) and MMCP-2 messenger RNA transcripts. These results suggest that the gene product of the mi locus may be important in regulating the expression of gene products such as c-kit, and that mast cell deficiency of mi/mi mice appears to be due, at least in part, to impaired signaling through the c-kit receptor because of the low c-kit expression.

Description: Homozygous mutant rats at the newly found white spotting (Ws) locus were anemic and deficient in mast cells and melanocytes. Because the phenotype of Ws/Ws rats resembled the phenotype of mice possessing a double-gene dose of mutant alleles at the W locus and because the c-kit gene was mapped at the W locus of mice, we characterized the c-kit gene of Ws/Ws rats. The authentic sequence of the rat c-kit cDNA was determined by using a cDNA library prepared from the hippocampus of Sprague-Dawley rats. The c-kit cDNA of Ws/Ws and normal (+/+) control rats was obtained by reverse transcriptase modification of the polymerase chain reaction. When compared with the authentic sequence, a deletion of 12 bases was found in the c-kit cDNA of Ws/Ws rats. This change was shown to be a result of the deletion of the genomic DNA. Four amino acids encoded by the deleted 12 bases (ie, Val-Lys-Gly-Asn) were located at two amino acids downstream from the tyrosine autophosphorylation site in the c-kit kinase and were conserved not only in mouse and human c-kit kinases but also in mouse and human c-fms kinases (ie, receptors of colony-stimulating factor-1). Taken together, the Ws/Ws rat is the first characterized mutant of the c-kit gene in an animal species other than the mouse.

Description: Decreased numbers of mast cells and abnormalities in the phenotype of mast cells are observed in the skin of mi/mi mutant mice. Recently, the mi locus was identified to encode a novel member of the basic-helix- loop-helix-leucine zipper protein family of transcription factors. Since nerve growth factor (NGF) has been reported to influence the proliferation and the phenotype of cultured mast cells (CMCs), we compared the effect of NGF between mi/mi and control normal (+/+) CMCs. Addition of NGF to the suboptimal dose of recombinant murine interleukin-3 (rmIL-3) increased the plating efficiency of +/+ CMCs, but not of mi/mi CMCs. Although +/+ CMCs were berberine sulfate- negative when cultured with rmIL-3 alone, +/+ CMCs became berberine sulfate-positive when cultured in the presence of both rmIL-3 and NGF, which suggests increased heparin content. In contrast, NGF did not influence the phenotype of mi/mi CMCs. +/+ CMCs significantly bound 125I-NGF, but mi/mi CMCs did not, which suggests a defect of NGF receptors in mi/mi CMCs. Both p75 and p140 molecules are known to be involved in the formation of NGF receptors. Although the expression of p140 messenger (m)RNA was comparable between +/+ and mi/mi CMCs, the expression of p75 mRNA was significantly lower in mi/mi CMCs than in +/+ CMCs. Taken together, the poor response of mi/mi CMCs to NGF appeared to be attributable to the impaired transcription of the p75 gene.

Description: Homozygous mutant rats at the newly found white spotting (Ws) locus were anemic and deficient in mast cells and melanocytes. Because the phenotype of Ws/Ws rats resembled the phenotype of mice possessing a double-gene dose of mutant alleles at the W locus and because the c-kit gene was mapped at the W locus of mice, we characterized the c-kit gene of Ws/Ws rats. The authentic sequence of the rat c-kit cDNA was determined by using a cDNA library prepared from the hippocampus of Sprague-Dawley rats. The c-kit cDNA of Ws/Ws and normal (+/+) control rats was obtained by reverse transcriptase modification of the polymerase chain reaction. When compared with the authentic sequence, a deletion of 12 bases was found in the c-kit cDNA of Ws/Ws rats. This change was shown to be a result of the deletion of the genomic DNA. Four amino acids encoded by the deleted 12 bases (ie, Val-Lys-Gly-Asn) were located at two amino acids downstream from the tyrosine autophosphorylation site in the c-kit kinase and were conserved not only in mouse and human c-kit kinases but also in mouse and human c-fms kinases (ie, receptors of colony-stimulating factor-1). Taken together, the Ws/Ws rat is the first characterized mutant of the c-kit gene in an animal species other than the mouse.

Description: Although GATA-binding transcription factors (GATA-1 and GATA-2) are strongly expressed in cultured mast cells (CMCs), their expression in mast cells within tissues has not been reported. We examined the expression of GATA-1 and GATA-2 in skin tissues of mice using Northern blot analysis and in situ hybridization. mRNA for GATA-2 but not for GATA-1 was expressed in skin mast cells of WB-+/+ embryos between days 15 and 17 postcoitum (pc). The expression was downregulated on and after day 18 pc. Skin mast cells did not express GATA-2 after birth either. When the number of skin mast cells was compared with the number of GATA-2 mRNA-expressing cells, GATA-2 mRNA appeared to be expressed by mast cells only when the number was increasing. When the mRNA expression of high-affinity IgE receptor beta-subunit and mast cell carboxypeptidase A was used as differentiation markers, the expression of these mRNAs continued even after the downregulation of GATA-2 expression. To clarify the relationship of the proliferation and GATA-2 expression, proliferating CMCs derived from WBB6F1-+/+ mice were transplanted into the peritoneal cavity of mast cell-deficient WBB6F1- W/Wv mice. The CMCs stopped both the proliferation and GATA-2 expression after the transplantation, suggesting the association of these two parameters in mast cells within tissues of mice.

Description: The mi locus of mice encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (hereafter called MITF). Because the expression of the mouse mast cell protease 6 (MMCP-6) gene is remarkably reduced in mast cells of mi/mi mutant mice, we investigated the effect of MITF on the transcription of the MMCP-6 gene. First, we introduced the normal (+) MITF cDNA into mi/mi cultured mast cells using the retroviral vector. Overexpression of +-MITF but not mi-MITF normalized the expression of the MMCP-6 gene, indicating the involvement of +-MITF in the MMCP-6 gene transactivation. Second, we analyzed the promoter of the MMCP-6 gene by the transient cotransfection assay. The luciferase construct under the control of the MMCP-6 promoter and the cDNA encoding +-MITF or mi-MITF were cotransfected into NIH/ 3T3 fibroblasts. The coexpression of +- MITF but not mi-MITF increased the luciferase activity 10-fold. We found a CACATG and a CATCTG motif in the MMCP-6 promoter, both of which are generally recognized by bHLH-Zip-type transcription factors. We also found a GACCTG motif that was strongly bound by +-MITF. These three motifs were necessary for the 10-fold transactivation ability of the MMCP-6 promoter by +-MITF. Mutations of each motif significantly reduced the transactivation, suggesting that +-MITF directly transactivated the MMCP-6 gene through these three motifs.

Description: bcl-2 protein has been detected in surgical specimens and cultured permanent cell lines of non-Hodgkin's lymphomas and leukemias using enzyme immunohistochemistry and immunofluorescence with anti-bcl-2 monoclonal antibodies. Of 40 surgical specimens, bcl-2 protein was expressed in 50% of B-cell and 41% of T-cell lymphomas, both with and without the bcl-2 gene rearrangement. In investigations of 38 hematopoietic cell lines, bcl-2 protein was detected not only in lymphoid cell lines but also in myeloid cell lines. In situ hybridization and immunohistochemical analysis of reactive lymph nodes showed that lymphocytes in mantle zones and paracortical areas expressed bcl-2 protein consistent with the messenger RNA distribution and that germinal center cells showed abundant bcl-2 transcript, despite the absence of detectable bcl-2 protein. These results suggest that bcl-2 protein is broadly expressed in various hematopoietic neoplasms not restricted in t(14; 18) lymphomas and that germinal center cells may be involved in some arrest of bcl-2 protein expression at the posttranscriptional level.

Description: The receptor encoded by the W (c-kit) locus (W receptor) is expressed on the surface of cultured mast cells (CMC) derived from normal (+/+) mice, whereas its ligand encoded by the Sl locus (Sl ligand) is expressed on the surface of fibroblast cell lines derived from murine embryos. Involvement of W receptors and Sl ligands in attachment of CMC to fibroblasts was investigated. CMC were cocultured with fibroblasts; nonattaching CMC were removed and the remaining CMC were counted. CMC derived from mice of the W/W genotype did not express the extracellular domain of W receptors, and attachment of W/W CMC to +/+ fibroblasts was significantly impaired. Fibroblasts derived from embryos of the Sl/Sl genotype did not express Sl ligands, and the attachment of +/+ CMC to Sl/Sl fibroblasts was also impaired. The Wv and W42 alleles are point mutations at the intracellular tyrosine kinase domain. Attachment of either Wv/Wv, W/Wv, or W/W42 CMC to +/+ fibroblasts was comparable with that of +/+ CMC. Moreover, the addition of monoclonal antibody against the extracellular domain of W receptors inhibited the attachment of +/+ CMC to +/+ fibroblasts. Thus, the extracellular domain of W receptors appeared to be necessary for attachment of CMC to fibroblasts.

Description: The c-kit proto-oncogene encodes a receptor tyrosine kinase that is known to play a crucial role in hematopoiesis, especially in mast cell growth and differentiation. Although a number of dominant loss-of- function mutations of c-kit gene have been well characterized in mice, rats, and humans, little is known about the c-kit mutations contributing to ligand-independent activation of the c-kit receptor tyrosine kinase (KIT). In a murine mastocytoma cell line, P-815, KIT has been found to be constitutively phosphorylated on tyrosine and activated in a ligand-independent manner. Sequencing of the whole coding region of c-kit cDNA showed that c-kit cDNA of P-815 cells carries a point mutation in codon 814, resulting in amino acid substitution of Tyr for Asp. Murine wild-type c-kit cDNA and mutant- type c-kit cDNA encoding Tyr in codon 814 were expressed in cells of a human embryonic kidney cell line, 293T. In the transfected cells, mutant-form KITTyr814 was strikingly phosphorylated on tyrosine and activated in immune complex kinase reaction regardless of stimulation with a ligand for KIT (stem cell factor), whereas tyrosine phosphorylation and activation was barely detectable in wild-form KIT. The data presented here provide evidence for a novel activating mutation of c-kit gene that might be involved in neoplastic growth or oncogenesis of some cell types, including mast cells.

Description: To examine the regulatory mechanism of apoptosis in lymphoid cells, expression of both bcl-2 protein and Fas antigen was investigated in reactive lymph nodes, in resting lymphocytes from peripheral blood (PBLs), and in PBLs stimulated with pokeweed mitogen, interleukin-4 (IL- 4) + anti-IgM antibody, IL-2 + anti-CD3 antibody, phytohemagglutinin + phorbol myristate acetate using immunohistochemistry and flow cytometry. Germinal center cells expressed a large amount of Fas antigen, which is associated with the induction of apoptosis in lymphoid cell lines, in contrast to the lack of bcl-2 protein as an apoptosis inhibitor. On the other hand, mantle zone lymphocytes expressed a high level of bcl-2 protein and less Fas antigen. This inverse expression of bcl-2 protein and Fas antigen was also shown in activated T and B lymphocytes from peripheral blood. These lymphoblasts fell into apoptosis dose-dependently in the presence of anti-Fas monoclonal antibody, but resting lymphocytes that expressed both bcl-2 protein and Fas antigen did not undergo apoptosis. These findings suggest that bcl-2 expression prevents the apoptosis of lymphoid cells induced by the Fas antigen-dependent mechanism and that apoptosis of lymphocytes is exquisitely controlled, at least in part, by regulation of the bcl-2 and Fas genes.