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Background: Esophageal cancer is a lethal malignancy with a poor prognosis. The incidence of esophageal adenocarcinoma, which develops from Barrett's esophagus (BE), has recently been increasing. In a previous study, we found that PDZK1 expression is higher in long segment BE compared to that in short-segment BE. However, the function of PDZK1 in the mucosa of BE is unclear., Aims: Clarify the role of PDZK1 in BE mucosa using PDZK1 overexpressed cells., Methods: Human adenocarcinoma-derived OE33 cells were used as a parental cell line and transfected to generate PDZK1 overexpressed OE33 cells (PC cells) or transfected with empty vector as control cells (NC cells). Cell growth of NC and PC cells in 10% fetal bovine serum was evaluated by cell counting. The effect of PDZK1 on proteasome inhibitor (PSI)-induced apoptosis was qualified by fluorescence microscopy and quantified by flow cytometry. Expression of apoptosis-related proteins was evaluated by western blotting., Results: There were no significant differences in cell growth between NC and PC cells. PSI significantly increased apoptosis in NC cells, but not in PC cells. In response to PSI, increased levels of cleaved-caspase3 and decreased pro-caspase3 levels were found in NC cells, but not in PC cells. In NC cells, PSI significantly decreased Bcl-2 expression without affecting Bax levels. In contrast, high expression of both Bcl-2 and Bax was observed in PC cells., Conclusion: Overexpression of PDZK1 protein induces an apoptosis-resistant phenotype in BE cells, which may be a potential therapeutic target.

Background/aim: Sorafenib is a multikinase inhibitor approved as a first-line therapy for hepatocellular carcinoma. This study examined the sorafenib resistance mechanism., Materials and Methods: Hepatoma HepG2 cells were exposed to sorafenib, and the biological activity of the conditioned media was analyzed using cell proliferation/apoptosis assays, multiplex immunoassays, ELISA, and western blot analyses. The effect of urokinase-type plasminogen activator (uPA) inhibitors or siRNA-mediated gene silencing was examined in culture experiments and a mouse xenograft tumor model., Results: Sorafenib increased uPA secretion, which was abrogated by an Akt inhibitor. The growth-inhibitory effect of sorafenib was significantly enhanced by the uPA inhibitors UK122 and amiloride. Sorafenib-induced apoptosis was increased 2.4-fold in uPA siRNA-transduced cells (p<0.05). Combined therapy with sorafenib and amiloride significantly decreased tumor volumes [mean volume: 759 mm 3 (sorafenib) vs. 283 mm 3 (sorafenib plus amiloride), p<0.05]., Conclusion: uPA may play a critical role in sorafenib resistance., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The structures of many membrane proteins have been analyzed in detergent micelles. However, the environment of detergent micelles differs somewhat from that of the lipid bilayer, where membrane proteins exhibit physiological functions. Therefore, a more membrane-like environment has been awaited for structural analysis of membrane proteins. Nanodiscs are "hockey-puck"-shaped lipid bilayer particles that distribute in a monodispersed manner in aqueous solution. We review how nanodiscs or protein-reconstituted nanodiscs are prepared and how they are utilized to analyze protein structure, dynamics, and interactions with lipid molecules using solution NMR and cryo-electron microscopy.

Fukuyama congenital muscular dystrophy (FCMD), which is caused by mutations in the fukutin gene, is the second most common form of childhood muscular dystrophy in Japan. The founder haplotype is the most prevalent in the chromosomes of Japanese FCMD patients, and corresponds to an SVA retrotransposal insertion in the 3'-untranslated region of fukutin. Although other mutations have been reported, the mutation corresponding to the second most prevalent haplotype in Japanese FCMD patients remained unknown. Recently a deep-intronic point mutation c.647+2084G>T was identified in Korean patients with congenital muscular dystrophy. Here, we performed mutational analysis of 10 patients with the second most prevalent haplotype and found that all of them were compound-heterozygous for the SVA insertion and this c.647+2084G>T mutation. The fukutin mRNA of these patients contained a pseudoexon between exon 5 and exon 6, which was consistent with the previous Korean study. As expected, the mutated fukutin protein was smaller than the normal protein, reflecting the truncation of fukutin due to a premature stop codon. Immunostaining analysis showed a decrease in the signal for the glycosylated form of α-dystroglycan. These findings indicated that this mutation is the second most prevalent loss-of-function mutation in Japanese FCMD patients.

Chemical exchange processes of proteins on the order of microseconds (μs) to milliseconds (ms) play critical roles in biological functions. Developments in methyl-transverse relaxation optimized spectroscopy (methyl-TROSY), which observes the slowly relaxing multiple quantum (MQ) coherences, have enabled the studies of biologically important large proteins. However, the analyses of μs to ms chemical exchange processes based on the methyl-TROSY principle are still challenging, because the interpretation of the chemical exchange contributions to the MQ relaxation profiles is complicated, as significant chemical shift differences occur in both (1)H and (13)C nuclei. Here, we report a new methyl-based NMR method for characterizing chemical exchanges, utilizing differential MQ relaxation rates and a heteronuclear double resonance pulse technique. The method enables quantitative evaluations of the chemical exchange processes, in which significant chemical shift differences exist in both the (1)H and (13)C nuclei. The versatility of the method is demonstrated with the application to KirBac1.1, with an apparent molecular mass of 200 kDa.

The calcium (Ca(2+)) ion is a universal signalling messenger which plays vital physiological roles in all eukaryotes. To decode highly regulated intracellular Ca(2+) signals, cells have evolved a number of sensor proteins that are ideally adapted to respond to a specific range of Ca(2+) levels. Among many such proteins, calmodulin (CaM) is a multi-functional cytoplasmic Ca(2+) sensor with a remarkable ability to interact with and regulate a plethora of structurally diverse target proteins. CaM achieves this 'multi-talented' functionality through two EF-hand domains, each with an independent capacity to bind targets, and an adaptable flexible linker. By contrast, stromal interaction molecule-1 and -2 (STIMs) have evolved for a specific role in endoplasmic reticulum (ER) Ca(2+) sensing using EF-hand machinery analogous to CaM; however, whereas CaM structurally adjusts to dissimilar binding partners, STIMs use the EF-hand machinery to self-regulate the stability of the Ca(2+) sensing domain. The molecular mechanisms underlying the Ca(2+)-dependent signal transduction by CaM and STIMs have revealed a remarkable repertoire of actions and underscore the flexibility of nature in molecular evolution and adaption to discrete Ca(2+) levels. Recent genomic sequencing efforts have uncovered a number of disease-associated mutations in both CaM and STIM1. This article aims to highlight the most recent key structural and functional findings in the CaM and STIM fields, and discusses how these two Ca(2+) sensor proteins execute their biological functions., (Copyright © 2015. Published by Elsevier Inc.)

Transcriptome analysis of the epidermis of Hes1(-/-) mouse revealed the direct relationship between Hes1 (hairy and enhancer of split-1) and BNIP3 (BCL2 and adenovirus E1B 19-kDa-interacting protein 3), a potent inducer of autophagy. Keratinocyte differentiation is going along with activation of lysosomal enzymes and organelle clearance, expecting the contribution of autophagy in this process. We found that BNIP3 was expressed in the suprabasal layer of the epidermis, where autophagosome formation is normally observed. Forced expression of BNIP3 in human primary epidermal keratinocytes (HPEKs) resulted in autophagy induction and keratinocyte differentiation, whereas knockdown of BNIP3 had the opposite effect. Intriguingly, addition of an autophagy inhibitor significantly suppressed the BNIP3-stimulated differentiation of keratinocytes, suggesting that BNIP3 plays a crucial role in keratinocyte differentiation by inducing autophagy. Furthermore, the number of dead cells increased in the human epidermal equivalent of BNIP3 knockdown keratinocytes, which suggests that BNIP3 is important for maintenance of skin epidermis. Interestingly, although UVB irradiation stimulated BNIP3 expression and cleavage of caspase3, suppression of UVB-induced BNIP3 expression led to further increase in cleaved caspase3 levels. This suggests that BNIP3 has a protective effect against UVB-induced apoptosis in keratinocytes. Overall, our data provide valuable insights into the role of BNIP3 in the differentiation and maintenance of epidermal keratinocytes.

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Fukutin is the gene responsible for Fukuyama-type congenital muscular dystrophy (FCMD), an autosomal recessive disease associated with central nervous system (CNS) and eye anomalies. Fukutin is involved in basement membrane formation via the glycosylation of α-dystroglycan (α-DG), and hypoglycosylation of α-DG provokes the muscular, CNS and eye lesions of FCMD. Astrocytes play an important role in the pathogenesis of the CNS lesions, but the post-transcriptional regulation of fukutin mRNA has not been elucidated. In this study, we investigated the characteristics of fukutin mRNA using an astrocytoma cell line that expresses fukutin and glycosylated α-DG. The glycosylation of α-DG was considered to be increased by over-expression of fukutin and decreased by knockdown of fukutin. Knockdown of Musashi-1, one of the RNA-binding proteins involved in the regulation of neuronal differentiation, induced a decrease in fukutin mRNA. Immunoprecipitation and ELISA-based RNA-binding assay demonstrated possible binding between fukutin mRNA and Musashi-1 protein. A relationship between fukutin mRNA and vimentin protein was also proposed. In situ hybridization for fukutin mRNA showed a positive cytoplasmic reaction including cytoplasmic processes. From these results, fukutin mRNA is suggested to be a localized mRNA up-regulated by Musashi-1 and to be a component of a mRNA-protein complex which includes Musashi-1 and (presumably) vimentin proteins., (© 2012 The Authors. International Journal of Experimental Pathology © 2012 International Journal of Experimental Pathology.)

Fukuyama type congenital muscular dystrophy (FCMD) is an autosomal recessive disease, exhibiting muscular dystrophy, and central nervous system (CNS) and ocular malformations. It is included in alpha-dystroglycanopathy, a group of muscular dystrophy showing reduced glycosylation of alpha-dystroglycan. alpha-Dystroglycan is one of the components of dystrophin-glycoprotein complex linking extracellular and intracellular proteins. The sugar chains of alpha-dystroglycan are receptors for extracellular matrix proteins such as laminin. Fukutin, a gene responsible for FCMD, is presumably related to the glycosylation of alpha-dystroglycan like other causative genes of alpha-dystroglycanopathy. The CNS lesion of FCMD is characterized by cobblestone lissencephaly, associated with decreased glycosylation of alpha-dystroglycan in the glia limitans where the basement membrane is formed. Astrocytes whose endfeet form the glia limitans seem to be greatly involved in the genesis of the CNS lesion. Fukutin is probably necessary for astrocytic function. Other components of the CNS may also need fukutin, such as migration and synaptic function in neurons. However, roles of fukutin in oligodendroglia, microglia, leptomeninges and capillaries are unknown at present. Fukutin is expressed in various somatic organs as well, and appears to work differently between epithelial cells and astrocytes. In the molecular level, since the dystrophin-glycoprotein complex is linked to cell signaling pathways involving c-src and c-jun, fukutin may be able to affect cell proliferation/survival. Fukutin was localized in the nucleus on cancer cell lines. With the consideration that mutations of fukutin give rise to wide spectrum of the clinical phenotype, more unknown functions of fukutin besides the glycosylation of alpha-dystroglycan can be suggested. Trials for novel treatments including gene therapy are in progress in muscular dystrophies. Toward effective therapies with minimal side effects, precise evaluation of the pathomechanism of FCMD and the function of fukutin would be required.

We have developed a system for producing tubular multilamellar liposomes that incorporate the protein FtsZ on the inside. We start with a mixture of spherical multilamellar liposomes with FtsZ initially on the outside. Shearing forces generated by applying a coverslip most likely distort some of the spherical liposomes into a tubular shape, and causes some to leak and incorporate FtsZ inside. We describe protocols for liposome preparation, and for preparing membrane-targeted FtsZ that can assemble contractile Z rings inside the tubular liposomes. We also describe the characterization of the multilamellar liposomes in terms of the permeability or leakiness for a small fluorescent dye and larger protein molecules. These liposomes may be useful for reconstitution of other biological systems.

Fukutin, a gene responsible for Fukuyama type congenital muscular dystrophy (FCMD), is presumably related to the glycosylation of alpha-dystroglycan (alpha-DG), involved in basement membrane formation. Hypoglycosylation of alpha-DG plays a key role for the pathogenesis of FCMD. On the other hand, fukutin and alpha-DG are also expressed in various non-neuromuscular tissues. Recently, a role of alpha-DG as a cancer suppressor has been proposed, because of a decrease of glycosylated alpha-DG in cancers. In this study, function of fukutin was investigated in two cancer cell lines, focusing on whether fukutin is involved in the glycosylation of alpha-DG in cancer cells and has any possible roles related to a cancer suppressor. Localization of fukutin and a result of laminin-binding assay after RNA interference suggest that fukutin may be involved in the glycosylation of alpha-DG in a small portion in these cancer cell lines. In Western blotting and immuno-electron microscopy, localization of fukutin in the nucleus was suggested in addition to the Golgi apparatus and/or endoplasmic reticulum. Immunohistochemically, there were more Ki-67-positive cells and more nuclear staining of phosphorylated c-jun after knockdown of fukutin in two cell lines. Fukutin appears to suppress cell proliferation through a system involving c-jun, although it is unclear this process is related to alpha-DG or not at present. The result may propose a possibility of another function of fukutin in addition to the glycosylation of alpha-DG in cancer cells.

FtsZ is a tubulin homolog and the major cytoskeletal protein in bacterial cell division. It assembles into the Z ring, which contains FtsZ and a dozen other division proteins, and constricts to divide the cell. We have constructed a membrane-targeted FtsZ (FtsZ-mts) by splicing an amphipathic helix to its C terminus. When mixed with lipid vesicles, FtsZ-mts was incorporated into the interior of some tubular vesicles. There it formed multiple Z rings that could move laterally in both directions along the length of the liposome and coalesce into brighter Z rings. Brighter Z rings produced visible constrictions in the liposome, suggesting that FtsZ itself can assemble the Z ring and generate a force. No other proteins were needed for assembly and force generation.

Objective: The fukutin gene (FKTN) is the causative gene for Fukuyama-type congenital muscular dystrophy, characterized by rather homogeneous clinical features of severe muscle wasting and hypotonia from early infancy with mental retardation. In contrast with the severe dystrophic involvement of skeletal muscle, cardiac insufficiency is quite rare. Fukuyama-type congenital muscular dystrophy is one of the disorders associated with glycosylation defects of alpha-dystroglycan, an indispensable molecule for intra-extra cell membrane linkage., Methods: Protein and functional analyses of alpha-dystroglycan and mutation screening of FKTN and other associated genes were performed., Results: Surprisingly, we identified six patients in four families showing dilated cardiomyopathy with no or minimal limb girdle muscle involvement and normal intelligence, associated with a compound heterozygous FKTN mutation. One patient died by rapid progressive dilated cardiomyopathy at 12 years old, and the other patient received cardiac implantation at 18 years old. Skeletal muscles from the patients showed minimal dystrophic features but have altered glycosylation of alpha-dystroglycan and reduced laminin binding ability. One cardiac muscle that underwent biopsy showed altered glycosylation of alpha-dystroglycan similar to that observed in a Fukuyama-type congenital muscular dystrophy patient., Interpretation: FKTN mutations could cause much wider spectrum of clinical features than previously perceived, including familial dilated cardiomyopathy and mildest limb girdle muscular dystrophy.

Aggrus (T1alpha/podoplanin, known as a specific marker for type I alveolar cells or lymphatic endothelial cells) is a transmembrane sialoglycoprotein that aggregates platelets. Previously, we showed that upregulated expression of Aggrus occurs in colorectal tumors or testicular tumors and could be associated with platelet-aggregating activity and metastatic ability. In testicular tumors, Aggrus is specifically expressed in seminoma. The present study investigates Aggrus expression in human primary lung cancer tissues of different types. Microarray analysis demonstrated that aggrus was significantly expressed in squamous cell carcinoma (10/15; 66.7%). Immunohistochemical analysis also showed that the incidence of positive staining in sections of squamous cell carcinoma (7/8; 87.5%) was higher than that in adenocarcinoma (2/13; 15.4%). Furthermore, Aggrus expression was detected in a squamous cell carcinoma cell line, NCI-H226, by real-time PCR. These findings indicated that overexpression of Aggrus occurred in squamous cell carcinoma of the lung. Therefore, Aggrus could be a useful diagnostic marker for squamous cell carcinoma of the lung., (Copyright (c) 2005 S. Karger AG, Basel.)

Aggrus (also known as T1alpha/podoplanin) is a membrane sialoglycoprotein whose function in tumors is unknown. We recently determined that Aggrus possessed the ability of inducing platelet aggregation and that its expression was frequently upregulated in colorectal tumors. Thus, Aggrus expression might be associated with tumor-induced platelet aggregation and tumor metastasis. Here we show, by means of cancer profiling array and real-time PCR, that aggrus mRNA expression is frequently upregulated in testicular germ cell tumors when compared with the surrounding normal tissue. Immunohistochemical staining revealed that Aggrus protein expression was detected in 10 of 11 seminomas (90.9%), but its expression was not observed in embryonal carcinomas (0/4; 0%). Specific markers for seminomas have not been reported, and Aggrus is a potential diagnostic marker for seminomas and may be associated with malignancies of the testis.

Aggrus, also called T1alpha and podoplanin, is a novel platelet aggregation-inducing factor that is expressed in various carcinoma cells. Aggrus/T1alpha/podoplanin is known to be expressed in lung type I alveolar cells or lymphatic endothelial cells. However, its physiological role has not been clarified. To assess the attribution of glycosylation to Aggrus platelet aggregation activity, recombinant molecules were stably expressed in a series of Chinese hamster ovary (CHO) cell mutants, N-glycan-deficient Lec1, CMP-sialic acid transporter-deficient Lec2, and UDP-galactose transporter-deficient Lec8. A new anti-human Aggrus monoclonal antibody, YM-1, was established to detect the expression of human Aggrus on these CHO cell mutants. Aggrus on Lec1 cells induced platelet aggregation, but those on Lec2 and Lec8 cells did not. Further, the glycans on Aggrus were analyzed by lectin blotting. Aggrus expressed in CHO and Lec1 cells showed Wheat-germ agglutinin, Jacalin, and Vicia villosa lectin bindings. Lectin blotting results indicated that sialylated core 1 structures, sialic acid plus Galbeta1,3GalNAc-Ser/Thr, were critical for the platelet aggregation activity. This oligosaccharide structure is known as tumor-associated antigen, which is potentially related to the metastasis process of cancer cells.

Platelets play an important role in hemostasis, thrombosis, and antimicrobial host defense and are also involved in the induction of inflammation, tissue repair, and tumor metastasis. We have previously characterized the platelet aggregation-inducing sialoglycoprotein (Aggrus/gp44) overexpressed on the surface of tumor cells. Because a platelet aggregation-neutralizing 8F11 monoclonal antibody that could specifically recognize Aggrus suppressed tumor-induced platelet aggregation, we have previously purified Aggrus by 8F11-affinity chromatography and found that purified Aggrus possessed the ability to induce aggregation of platelets. Here we show that Aggrus is identical to the T1alpha/gp38P/OTS-8 antigen, the function of which in tumors is unknown. Expression of mouse Aggrus and its human homologue (also known as T1alpha-2/gp36) induced platelet aggregation without requiring plasma components. Using the 8F11 antibody, we identified the highly conserved platelet aggregation-stimulating domain with putative O-glycosylated threonine residues as the critical determinant for exhibiting platelet aggregation-inducing capabilities. We compared the expression level of human aggrus mRNA using an array containing 160 cDNA pair samples derived from multiple human tumorigenic and corresponding normal tissues from individual patients. We found that expression level of aggrus was enhanced in most colorectal tumor patients. To confirm the protein expression, we generated anti-human Aggrus polyclonal antibodies. Immunohistochemical analysis revealed that Aggrus expression was frequently up-regulated in colorectal tumors. These results suggest that Aggrus/T1alpha is a newly identified, platelet aggregation-inducing factor expressed in colorectal tumors.

To facilitate searching for genes encoding cell membrane proteins, we developed a method for isolating cDNAs that contain sequences for hydrophobic transmembrane runs. This cloning strategy, termed the "transmembrane (TM) trap method," utilizes a vector that directs the cell surface expression of mouse CD4 fusion protein when an insert encoding hydrophobic transmembrane sequences is cloned in-frame with correct orientation. We applied this novel method to isolation of cytokine receptor cDNAs. Our strategy enabled efficient isolation of relatively rare species encoding receptors such as IL-2Rgamma, IL-3Rbeta, IL-4Ralpha, IL-5Ralpha, and IL-6Ralpha. This method also could be used to isolate cDNAs for intracellular molecules with a transmembrane region, e.g., bcl-2. These results indicate that the TM trap method provides an efficient cloning strategy for identification of various families of genes encoding proteins with one or more transmembrane regions.

Dendritic cells (DCs) are the most potent antigen-presenting cells and play an essential role for triggering T-cell-mediated immune responses. In search for novel cell surface molecules expressed on DCs involved in T cell priming by representational differential analysis, we identified a mouse homologue of Tspan-3 (mTspan-3), a novel member of the tetraspanin superfamily. The mTspan-3 consists of four hydrophobic, putative transmembrane regions, forming a small and a large extracellular loop, with short intracellular amino and carboxil tails. Although the mTspan-3 is expressed on a variety of immune cell types including resting DCs, its expression on DCs is downregulated during activation induced by cross-linking CD40 with anti-CD40 monoclonal antibody. These results suggest that mTspan-3 may be involved in the function of DCs in association with T cell stimulation., (Copyright 2001 Academic Press.)

In an attempt to identify novel transmembrane molecules expressed on hematopoietic cells, we identified a novel transmembrane protein gene, M83. Cloning of the full-length cDNAs of human and mouse M83 revealed that M83 encodes a type I transmembrane protein with a region containing five hydrophobic segments within the C-terminal part of the protein, suggesting that M83 is a five-span transmembrane molecule. The M83 protein was expressed on the cell surface as a glycosylated protein with a molecular mass of 84 kDa. The M83 gene was localized to human chromosome 16p13.3, mouse chromosome 17B1, and rat chromosome 10q12.3 distal. In human, M83 mRNA was highly expressed in placenta, pancreas, and lymphohematopoietic tissues including peripheral blood, spleen, and bone marrow. Among hematopoietic cells, it was highly expressed in resting T lymphocytes and was downregulated by cell activation, suggestive of its biological role related to the T cell resting status., (Copyright 2000 Academic Press.)

T lymphocyte development requires a series of interactions between developing thymocytes and thymic epithelial (TE) cells. In this paper we show that TE cells in the developing thymus express Pref-1, a Delta-like cell-surface molecule. In fetal thymus organ cultures (FTOC), thymocyte cellularity was increased by the exogenous dimeric Pref-1 fusion protein, but was reduced by the soluble Pref-1 monomer or anti-Pref-1 Ab. Dimeric Pref-1 in FTOC also increased thymocyte expression of the HES-1 transcription factor. Thymocyte cellularity was increased in FTOC repopulated with immature thymocytes overexpressing HES-1, whereas FTOC from HES-1-deficient mice were hypocellular and unresponsive to the Pref-1 dimer. We detected no effects of either Pref-1 or HES-1 on developmental choice among thymocyte lineages. These results indicate that Pref-1 expressed by TE cells and HES-1 expressed by thymocytes are critically involved in supporting thymocyte cellularity.

1q21 is frequently involved in different types of translocation in many types of cancers. Jumping translocation (JT) is an unbalanced translocation that comprises amplified chromosomal segments jumping to various telomeres. In this study, we identified a novel gene human JTB (Jumping Translocation Breakpoint) at 1q21, which fused with the telomeric repeats of acceptor telomeres in a case of JT. hJTB (human JTB) encodes a trans-membrane protein that is highly conserved among divergent eukaryotic species. JT results in a hJTB truncation, which potentially produces an hJTB product devoid of the trans-membrane domain. hJTB is located in a gene-rich region at 1q21, called EDC (Epidermal Differentiation Complex). This is the first report identifying the gene involved in unbalanced translocations at 1q21.

The long term marrow-repopulating ability of mouse bone marrow c-Kit+ Sca-1+ Lin(low/-) cells was studied. Injection of as few as 100 c-Kit+ Sca-1+ Lin(low/-) cells could rescue a lethally irradiated recipient mouse and reconstitute both myeloid and lymphoid cells in their peripheral blood for at least 10 mo. When limiting dilution analysis was performed in the presence of host-derived rescue cells, as low as five c-Kit+ Sca-1+ Lin(low/-) cells were shown to be capable of repopulating lymphohemopoietic cells. Subsequently, we examined whether c-Kit+ Sca-1+ Lin(low/-) cells had a capacity for self-renewal in vivo. After transplantation of 500 c-Kit+ Sca-1+ Lin(low/-) Ly-5.1+ cells into lethally irradiated Ly-5 congenic mice, the expansion of cells with the same phenotypes as the injected cells was monitored. By day 28 post-transplantation, more than 50,000 donor type c-Kit+ Sca-1+ Lin(low/-) Ly-5.1+ cells were found in the spleen and over 18,000 cells were found in bone marrow. The expansion in spleen, however, was transient in that by day 60 cells of the donor phenotype were found only in bone marrow. The c-Kit+ Sca-1+ Lin(low/-) LY-5.1+ cells expanded in spleen or bone marrow contained as many high proliferative potential colony-forming cells as the originally injected cells. They also contained cells with LTRA, but the frequency appeared to be less compared with naive c-Kit+ Sca-1+ Lin(low/-) cells. These data provide evidence that c-Kit+ Sca-1+ Lin(low/-) cells in bone marrow are capable of multilineage differentiation as well as self-renewal and that spleen is a primary site of stem cell expansion after transplantation.