Stem cells which are able to selfrenew and give rise to differentiated progeny are required in all the tissues where there exists a recurring need to replace differentiated cells that can not themselves proliferate. Recent studies have indicated that c-kit receptor and its ligand Steel Factor (SLF) is an essential signalling system for the activation of a number of stem cell systems. By injecting an anti-c-kit monoclonal antibody (mAb), ACK2 which is able to block c-kit function in vivo, we have found three distinct modes of stem cell activation in these c-kit/SLF-dependent systems. The first is the system where stem cells are constitutively activated, thus ACK2-injection results in depletion of the stem cells and their progenies. This group includes hematopoietic tissue and testis. The second is the system where stem cells are activated cyclically. This group includes melanoblasts in the hair follicles of adult mice and oocytes in the ovary. In such systems, ACK2-injection is effective only on stem cells that are being activated at the time of the injection. The third is some processes induced by artificial stimulations. This includes re-activa-tion of splenic hematopoietis by ionizing radiation and melanocyte activation in the hair follicles following shaving.
We want to understand the molecular mechanism determining the difference in the mode of stem cell activation in c-kit/SLF systems. Since all the processes listed above are dependent on c-kit/SLF irrespective of the mode of activation, differential regulation of c-kit or SLF in each tissue may account for a part of this difference.
To test this possibility, we have taken following three approaches. First, both anti-c-kit and anti-SLF mAbs are established to specify the localization and timing of SLF- and c-kit-ex-pression. We will show a highly contiguous pattern of c-kit- and SLF-expression in these stem cell systems. Second, genomic organization of c-kit and SLF genes were investigated, and some regulatory regions required for tissue specific expression of each genes were studied by CAT-assay. Although some more work is necessary for identifying the regulatory region of c-kit expression, we have identified a region of SLF gene which renders it expressed in fibroblast and stromal cell lines but does not so in hematopoietic cell lines. This region was ligated to bacterial β-gal gene and introduced to fertilized eggs, which eventually gave rise to a numbers of mouse strains. Actually, some strains express the β-gal gene in a tissue specific manner. These strains will provide powerful tools to understand the molecular basis of stem cell vs microenvironment interaction in c-kit/SLF-dependent system. Finally, some W or Sl alleles which may bear mutations in these regulatory regions were selected from many W and Sl alleles and analyzed at both molecular and phenotype levels. In light of our findings, we will discuss on how stem cell activation is regulated constitu-tively on one hand and inducibly on the other hand in c-kit/SLF system.

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Despite the importance of germ cells to the survival of species, little is known about their embryological origin, proliferation, migration and entry into mitotic arrest or meiosis. We have studied the effect of Steel factor (c-kit ligand), LIF (leukemia inhibitory factor) and bFGF on cultured murine primordial germ cells (PGCs) . We have found that Steel factor and LIF synergistically promote the proliferation of PGCs. However, under these conditions, PGCs have a finite proliferative capacity that correlates with their cessation of division in vivo. In the presence of bFGF, LIF and membrane associated Steel factor but not soluble Steel factor, PGCs continue to grow over the finite proliferative capacity and give rise to colonies of cells resembling undiffer-entiated embryonic stem (ES) cells which can be subcultured. The PGC derived cell lines are indeed pluripotential and can give rise to chimeras when they are introduced into blastocysts. The long term culture of PGCs and the derivation of ES cells from them have implications for germ cell biology, the induction of teratocarcinoma and transgenic technology.

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In order to define the role of steel factor encoded by the mouse steel (Sl) gene in spermatogenesis, we have examined its production in Sertoli cells. The ability to support proliferation and maintenance of mast cells in co-culture with primary mouse Sertoli cells were used for steel factor bioactivity. Sertoli cells derived from +/+ and W^v/W^v but not Si^d/Si^d mutant mice produce biologically active steel factor as a membrane-bound form.
Testicular cells prepared from neonatal (2 days and 5 days after birth) mice by enzymatic digestion were cultured in serum-supplemented medium to investigate c-kit mediated germ cell proliferation. The addition of antibody to the c-kit receptor inhibited the proliferation of cultured spermatogonia from 5-day-old mice but not from 2-day-old mice in a dose dependent manner indicating that the differentiated but not undifferentiated germ cell growth is c-kit mediated. Furthermore, the proliferation of undifferentiated primitive type A spermatogonia in 2 day testis may depend on some other supporting cell-derived factor (s) .

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Human papillomavirus type 16 (HPV16) is frequently found in carcinoma and precancerous lesions of the uterine cervix and is thought to be closely associated with carcinogenesis in these regions. However, the transforming activity of the E6 and E7 genes in vivo has not been characterised. To investigate this function, we produced transgenic mice carrying HPV16 E6 and E7 open reading frames. We obtained five transgenic founders and established three transgenic lineages. We observed a characteristic testicular tumor in mice of all three lineages. In one lineage, all of the male mice developed this tumor. This tumor appeared to be a type of germ cell tumor (seminoma) on histopathological analysis and performed a plant rectin PNA binding and reaction to some kinds of anti-germ cell antibodies. We also observed significant expression of some enzymes for androgene metagolism at the same time. To know the mechanism of tumori-genesis, we searched for some host genetical alterations cooperating with HPV transgene and finally found marked expression of Steel/kit.

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Production of mast cells is regulated by fibroblast-dependent and T-cell-dependent mechanisms. The fibroblast-dependent mechanism is mediated by the c-kit receptor tyrosine kinase encoded by the mouse W locus and its ligand encoded by the mouse Sl locus. Mice possessing a double gene dose of mutant alleles at either W (c-kit) or Sl locus are deficient in mast cells. We recently found that homozygous mutant rats at the Ws locus are also deficient in mast cells. Because the phenotype of Ws/Ws rats resembled the phenotype of the W (c-kit) mutant mice, we characterized the c-kit gene of Ws/Ws rats. When compared with the c-kit cDNA of control +/+ rats, a deletion of 12 bases was found in the c-kit cDNA of Ws/Ws rats.
Control +/+ and Ws/Ws rats were infected with Nippostrongylus brasil iensis (NB) . A 60-fold increase of basophils and a 10-fold increase of mucosal-type mast cells (MMC) occurred in the +/+ rats. A 60-fold increase of basophils was observed in the Ws/Ws rats as well. Although no MMC were detectable in the small intestine of non-infected Ws/Ws rats, MMC did develop after the infection of NB. However, the peak value of MMC in the small intestine of infected Ws/ Ws rats was about 10 % that of the infected +/+ rats. This suggests that only T cell-dependent mechanism is enough for production of basophils and that T cell-dependent and fibroblast-dependent mechanisms are necessary for production of MMC. In contrast with the marked increase of basophils and MMC, connective tissue-type mast cells (CTMC) in the skin and bone marrow did not increase in the + / + rats and did not develop in the Ws/ Ws rats. This suggests that fibroblast-dependent mechanism is indispensable for development of CTMC. Taken together, the Ws/Ws rats are useful model for discrimination of differentiation mechanisms between basophils, MMC, and CTMC.

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To clarify the phenotypes of various classes of human hematopoietic progenitor cells, we used a multicolor staining protocol in conjunction with CD34 and a newly developed mouse antihuman c-kit proto-oncogene product (KIT) monoclonal antibody (MoAb) . We characterized three cell fractions in CD34⁺ cells which express KIT^low and KJT^high cells in addition to KIT^-cells.
A clonogenic assay demonstrated that most granulocyte-macrophage colony-forming cells (GM-CFC) were present in CD34⁺KIT^high populations, while erythroid burst forming cells (BFU-e) were detected mainly in CD34⁺KIT^low population. CD34⁺KIT^-fraction contained a small number of BFU-e. Morphological analysis showed that blast-like cells were more enriched in the CD34⁺KIT^low fraction, KIT^low cells contained CD34⁺CD38^-cells which were considered to be very primitive progenitor cells, determined by a replating assay.
In order to clarify the biological differences between both fractions, we examined the more primitive progenitor cell functions by assessing long-term culture-initiating cells (LTC-IC) on the stromal cells. At week 2, more colony forming cells (CFC) recovered from the culture in the fraction initiated with a CD34⁺KIT^high population. However, more LTC-IC during weeks 5 to 9 were present in CD34⁺KIT^low population. These results indicate that primitive progenitors are more enriched in KIT^low population and that the KIT^high population contains many GM-com-mitted progenitor cells. We also demonstrated that anti-KIT MoAb inhibited the ability of CD34⁺ cells to generate CFC on the stromal layer in the LTC system. This suppressive effect was more evident in the generation of BFU-e by CD34⁺ KIT^low cells. Moreover, we confirmed that some CD34⁺KIT^low cells differentiate into CD34⁺KIT^high cells during coculture with allogeneic stromal cells. These results emphasize the pivotal role of the KIT and SCF interaction in hematopoiesis and indicate that KIT^low cells are more primitive than KIT^high cells.

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The c-kit proto-oncogene encodes a receptor tyrosine kinase that is considered to play imp portant roles in hematopoiesis. The proto-oncogene c-kit product was expressed on various types of human cell lines derived from leukemic cells of erythroid, megakaryocytic and mast cell lineages. Also, the c-kit product was detectable in blast cells in most cases of acute myeloblastic leukemia (AML) . Tyrosine phosphorylation and activation of the c-kit product was induced by treatment with a ligand for c-kit, stem cell factor (SCF), resulting in proliferation of some human leukemia cell lines, such as M07E, and blast cells in most cases of AML. In addition, SCF appeared to have an activity in inducing differentiation of human leukemia cells. In some cases, further, the c-kit product was activated even before the stimulation with SCF, and the c-kit activation was found to be due to point mutations of c-kit gene in a human leukemia cell line. These results suggest that c-kit proto-oncogene may be involved in excessive proliferation and aberrant differentiation of human leukemia cells, and possibly in leukemogenesis.

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