The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells was not detectable by means of PCR after reverse transcription (RT-PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells.

/pdf/identification-of-mutations-in-the-coding-sequence-of-the-3qbmxnf2de.pdf

Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.

Publisher Summary This chapter focuses on the importance of stem cell factor (SCF) and the stem cell factor receptor (SCFR) in hematopoiesis and other complex developmental programs affecting melanocytes, germ cells, and mast cells that are predicted based on an analysis of the phenotype of mice with mutations affecting SCF or its receptor. 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 the SCFR and SCF. This work shows that SCF critically regulates the migration and survival of mast cell precursors and mast cell adherence, promotes the proliferation of both immature and mature mast cells, enhances mast cell maturation, directly induces secretion of mast cell mediators, and regulates the extent of mediator release in mast cells activated by immunoglobulin (IgE)-dependent mechanisms. SCF also has a major role in lymphohematopoiesis. Notably, many of the actions of SCF in hematopoiesis and in lymphocyte development, including effects on the survival and proliferation of early hematopoietic cells, as well as actions that augment the maturation/differentiation of diverse lineages of hematopoietic or lymphoid cells, are expressed in large part through synergistic interactions with other growth factors.

The kit ligand, stem cell factor

Mice carrying certain mutations in the white spotting (W) locus (ie, c-kit) exhibit reduced c-kit tyrosine kinase-dependent signaling that results in mast cell deficiency and other phenotypic abnormalities. The c-kit mutations in KitW/W-v mice impair melanogenesis and result in anemia, sterility, and markedly reduced levels of tissue mast cells. In contrast, KitW-sh/W-sh mice, bearing the W-sash (Wsh) inversion mutation, have mast cell deficiency but lack anemia and sterility. We report that adult KitW-sh/W-sh mice had a profound deficiency in mast cells in all tissues examined but normal levels of major classes of other differentiated hematopoietic and lymphoid cells. Unlike KitW/W-v mice, KitW-sh/W-sh mice had normal numbers of TCRγδ intraepithelial lymphocytes in the intestines and did not exhibit a high incidence of idiopathic dermatitis, ulcers, or squamous papillomas of the stomach, but like KitW/W-v mice, they lacked interstitial cells of Cajal in the gut and exhibited bile reflux into the stomach. Systemic or local reconstitution of mast cell populations was achieved in nonirradiated adult KitW-sh/W-sh mice by intravenous, intraperitoneal, or intradermal injection of wild-type bone marrow-derived cultured mast cells but not by transplantation of wild-type bone marrow cells. Thus, KitW-sh/W-sh mice represent a useful model for mast cell research, especially for analyzing mast cell function in vivo.

Mast Cell-Deficient W-sash c-kit Mutant KitW-sh/W-sh Mice as a Model for Investigating Mast Cell Biology in Vivo

:CD117 (KIT) is a type III receptor tyrosine kinase operating in cell signal transduction in several cell types. Normally KIT is activated (phosphorylated) by binding of its ligand, the stem cell factor. This leads to a phosphorylation cascade ultimately activating various transcription fact

KIT (CD117): a review on expression in normal and neoplastic tissues, and mutations and their clinicopathologic correlation.

Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by recombinant human stem cell factor/kit-ligand in long-term culture.

Characterization of Ws mutant allele of rats: a 12-base deletion in tyrosine kinase domain of c-kit gene.

Anemia and mast cell depletion in mutant rats that are homozygous at "white spotting (Ws)" locus.

Inbreeding of Japanese wild rats (Rattus norvegicus) resulted in five new strains. The conception rates of wild rat strains were lower than those of inbred laboratory strains, and maternal aggressiveness was observed in some wild strains. The body size of domesticated animals was larger than that of captured ancestors. Two strains have been established as inbred strains; the MITA strain has been tamed to the level of laboratory rats, while the nature of the MITD strain is wild. The MITB strain was derived from the MITA strain as a coat color variant. The MITC and MITE strains have been bred to the F15 and F11 generations, respectively. The new strains can provide new genetic variations against a wild type background and might be useful for physiological and psychological experiments.

Establishment of new inbred strains derived from Japanese wild rats (Rattus norvegicus).

The dsinezumi shrew (Crocidura dsinezumi), a small insectivore, has been bred for the first time as a laboratory animal. The original animals were captured using Sherman's live traps and transferred into wooden cages. After several generations they were housed in plastic cages. Their diet consisted of trout pellets, cat food, and water provided ad libitum. Monogamous pairs were housed together for 2-3 weeks for mating, and the male was separated from the female during delivery and nursing. In captivity, the reproductive activity was observed throughout the year and the gestation period was estimated at 28-30 days with a litter size of between 1 and 4 pups. Pups grew very rapidly, and reached adult body size (mean: male, 9.7 g; female, 8.3 g) and sexual maturation at 6-8 weeks of age. The reproductive life was estimated at one and a half years, while the longevity was approximately 2 years.

/pdf/the-domestication-of-crocidura-dsinezumi-as-a-new-laboratory-3fkn4agfkq.pdf

Yoshiki Niwa

Papers

Characterization of Ws mutant allele of rats: a 12-base deletion in tyrosine kinase domain of c-kit gene.

Anemia and mast cell depletion in mutant rats that are homozygous at "white spotting (Ws)" locus.

Establishment of new inbred strains derived from Japanese wild rats (Rattus norvegicus).

The domestication of Crocidura dsinezumi as a new laboratory animal.