Interobserver agreement in endoscopic evaluation of reflux esophagitis using a modified Los Angeles classification incorporating grades N and M: a validation study in a cohort of Japanese endoscopists.

The Los Angeles classification system is the most widely employed criteria associated with the greatest interobserver agreement among endoscopists. In Japan, the Los Angeles classification system has been modified (modified LA system) to include minimal changes as a distinct grade of reflux esophagitis, rather than as auxiliary findings. This adds a further grading M defined as minimal changes to the mucosa, such as erythema and/or whitish turbidity. The modified LA system has come to be used widely in Japan. However, there have been few reports to date that have evaluated the interobserver agreement in diagnosis when using the modified LA classification system incorporating these minimal changes as an additional grade. A total of 100 endoscopists from university hospitals and community hospitals, as well as private practices in the Osaka-Kobe area participated in the study. A total of 30 video clips of 30-40 seconds duration, mostly showing the esophagocardiac junction, were created and shown to 100 endoscopists using a video projector. The participating endoscopists completed a questionnaire regarding their clinical experience and rated the reflux esophagitis as shown in the video clips using the modified LA classification system. Agreement was assessed employing kappa (kappa) statistics for multiple raters. The kappa-value for all 91 endoscopists was 0.094, with a standard error of 0.002, indicating poor interobserver agreement. The endoscopists showed the best agreement on diagnosing grade A esophagitis (0.167), and the poorest agreement when diagnosing grade M esophagitis (0.033). The kappa-values for the diagnoses of grades N, M, and A esophagitis on identical video pairs were 0.275-0.315, with a standard error of 0.083-0.091, indicating fair intraobserver reproducibility among the endoscopists. The study results consistently indicate poor agreement regarding diagnoses as well as fair reproducibility of these diagnoses by endoscopists using the modified LA classification system, regardless of age, type of practice, past endoscopic experience, or current workload. However, grade M reflux esophagitis may not necessarily be irrelevant, as it may suggest an early form of reflux disease or an entirely new form of reflux esophagitis. Further research is required to elucidate the pathophysiological basis of minimal change esophagitis.

A mouse model of a human multiple GIST family with KIT-Asp820Tyr mutation generated by a knock-in strategy.

Several families exhibiting multiple gastrointestinal stromal tumours (GISTs) and germline c-kit gene mutations at exons 8, 11, 13, or 17 have been reported. These patients also exhibit diffuse hyperplasia of the interstitial cells of Cajal (ICCs) as a pre-existing lesion of multiple GISTs. We generated a mouse model of a family with germline c-kit gene mutation at exon 17, and compared the phenotypes between the mice and humans. The mouse counterpart (KIT-Asp818Tyr) of the human KIT-Asp820Tyr mutation was transmitted into germline by a knock-in strategy. Mating of male and female heterozygotes (KIT-Asp818Tyr/+) resulted in the generation of homozygotes (KIT-Asp818Tyr/KIT-Asp818Tyr). Histological examination revealed that all heterozygotes had both a small KIT-positive mesenchymal tumour at the caecum, consistent with GIST, and KIT-positive diffuse spindle-shaped cell proliferation in the distal oesophagus, stomach, proximal duodenum, and colon consistent with ICC hyperplasia. All homozygotes exhibited a larger caecal tumour and more prominent spindle-shaped cell proliferation compared with the heterozygous mice, and they usually died within 10 weeks after birth, likely due to ileus. The small intestine of both genotypes showed no apparent morphological abnormality, and autonomous contraction of the ileal segments appeared normal. Western blotting demonstrated that the caecal tumours expressed phosphorylated KIT, MAPK, Stat1, and Stat5. These mutant mice are considered to be useful for further investigation of the mechanism of GIST development as a result of ICC hyperplasia and for assessment of the in vivo effects of drugs against molecular targets.

Differences in signaling pathways and expression level of the phosphoinositide phosphatase SHIP1 between two oncogenic mutants of the receptor tyrosine kinase KIT.

Oncogenic mutations of the receptor tyrosine kinase KIT are encountered in myeloid leukemia and various solid tumors, including gastrointestinal stromal tumors. We previously identified the human oncogenic germ line mutant KIT(K642E), a substitution in the tyrosine kinase 1 domain (TK1D) in a familial form of gastrointestinal stromal tumors. The effects of oncogenic KIT mutants on cell signaling and regulation are complex. Cellular models are valuable basic tools to tailor novel strategies on specific cellular and molecular bases for tumors expressing KIT oncogenic mutants. Murine KIT(WT) and the murine homologues of human KIT oncogenic mutants, further referred to as KIT(K641E) and KIT(del559), a point deletion in the juxtamembrane domain (JMD), were stably expressed in IL-3-dependent Ba/F3 cells. Major differences in the constitutively activation of Akt/PKB, MAP kinases and STATs pathways were observed between KIT(K641E) and KIT(del559), whereas KIT ligand elicited responses in both mutants. Noteworthy, the protein level of the phosphoinositide phosphatase SHIP1, but not SHIP2 and PTEN, was reduced in KIT(K641E) only while inhibition of KIT phosphorylation reversibly raised SHIP1 level in both JMD and TK1D oncogenic mutants, unraveling the control of SHIP protein level by KIT phosphorylation.

Gain-of-Function Mutations of Receptor Tyrosine Kinases in Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in human gastrointestinal tract. We first found that most GISTs expressed KIT, a receptor tyrosine kinase encoded by protooncogene c-kit and that approximately 90% of the sporadic GISTs had somatic gain-of-function mutations of the c-kit gene. Since both GISTs and interstitial cells of Cajal (ICCs) were double-positive for KIT and CD34, GISTs were considered to originate from ICCs or their precursor cells. We also found that germline gain-of-function mutations of the c-kit gene resulted in familial and multiple GISTs with diffuse hyperplasia of ICCs as the preexisting lesion. Moreover, we found that about half of the sporadic GISTs without c-kit gene mutations had gain-of-function mutations of platelet-derived growth factor receptor alpha (PDGFRA) gene that encodes another receptor tyrosine kinase. Imatinib which is known to inhibit constitutively activated BCR-ABL tyrosine kinase in chronic myelogenous leukemia also inhibits constitutive activation of mutated KIT and PDGFRA, and is now being used for metastatic or unresectable GISTs as a molecular target drug. Mutational analyses of c-kit and PDGFRA genes are considered to be significant for prediction of effectiveness of imatinib and newly developed/developing other agents on GISTs. Some mouse models of familial and multiple GISTs have been genetically created, and may be useful for further investigation of GIST biology.

Polyclonal nature of diffuse proliferation of interstitial cells of Cajal in patients with familial and multiple gastrointestinal stromal tumours.

BACKGROUND: Diffuse proliferation of interstitial cells of Cajal (ICCs) in the myenteric plexus layer of the intestine has been described in patients with familial and multiple gastrointestinal stromal tumours (GISTs). However, it is not fully understood whether proliferation is polyclonal or monoclonal. AIMS: To evaluate the clonal nature of diffuse ICC proliferation in familial and multiple GIST cases, we carried out clonal analysis using inactivation at the human androgen receptor (HUMARA) locus. MATERIALS AND METHODS: Diffuse ICC proliferation tissues from three female patients were microdissected using a laser capture microdissection (LCM) system. Normal intestinal mucosal tissues were also microdissected for polyclonal controls and GIST tissues for monoclonal controls from the same patients, and genomic DNA was extracted. After digestion by restriction enzyme HhaI, the HUMARA locus was amplified by a fluorescent polymerase chain reaction (PCR) procedure and the PCR products were analysed. RESULTS: One case was uninformative because it was homozygous at the HUMARA locus. In the two other cases, PCR products from the diffuse ICC proliferation showed two alleles as well as those from normal intestinal mucosal tissues, indicating that ICC proliferation was polyclonal. In contrast, PCR products from associated GIST tissues showed only one allele, indicating that GISTs were monoclonal. CONCLUSION: The results suggested that diffuse ICC proliferation in familial and multiple GIST cases was non-neoplastic hyperplasia.

Gain-of-function mutation at the extracellular domain of KIT in gastrointestinal stromal tumours.

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the human gastrointestinal tract. Previous studies of GISTs found gain-of-function mutations of the c-kit gene, which encodes a receptor tyrosine kinase (KIT). All the mutations were confined to exon 11, which encodes the juxtamembrane domain. By further examination of the whole coding region of c-kit complementary DNA in 35 GISTs, two were found to show the identical mutation at exon 9, which encodes the extracellular domain. The aims of the present study were to examine the frequency of the extracellular domain mutation and to determine whether the mutation is a gain-of-function type or not. Genomic DNA was extracted from paraffin-embedded tissues of 133 GISTs and exon 9 of the c-kit gene was amplified by polymerase chain reaction. Screening of the mutation was carried out by single-strand conformation polymorphism analysis and direct sequencing was done. Mutant c-kit cDNA was transfected into 293T human embryonic kidney cells and the magnitude of autophosphorylation of the mutant KIT was examined with or without the ligand of KIT, stem cell factor (SCF). In total, seven GIST cases (approximately 5%) were found with the identical mutation at exon 9. The mutant KIT exhibited constitutive autophosphorylation without SCF stimulation. The prognosis of the patients with the extracellular domain mutation was comparable to that of the patients with the juxtamembrane domain mutation.

Germline-activating mutation in the kinase domain of KIT gene in familial gastrointestinal stromal tumors.

The proto-oncogene KIT encodes the receptor tyrosine kinase KIT. Gain-of-function mutations in the juxtamembrane domain of KIT have been reported in human gastrointestinal stromal tumors. In a family with multiple gastrointestinal stromal tumors and diffuse hyperplasia of myenteric plexus layer, we have identified another mutation of KIT, a single base mutation, resulting in the substitution of Glu for Lys(642) in the kinase I domain, and studied its biological effect in a cellular system. The mouse homologue of the human KIT mutant was generated by site-directed mutagenesis and stably transfected into the interleukin-3-dependent Ba/F3 murine cell line. The oncogenic potential of the mutated KIT was assessed in vitro by a proliferation assay and in vivo by transplantation into nude mice. Transfected Ba/F3 cells grew autonomously in absence of growth factors and formed tumors in nude mice. Substitution of Glu for Lys(642) is an oncogenic mutation in the tyrosine kinase domain of KIT. As germline heterozygous mutation, it causes a diffuse hyperplasia of myenteric interstitial cells of Cajal during embryonic development and occurrence of multiple gastrointestinal stromal tumors at adulthood.

Expression of hepatocyte growth factor and c-met in ulcerative colitis.

OBJECTIVE AND DESIGN: This study was designed to determine if the hepatocyte growth factor (HGF)-Met system is involved in the repair process of inflamed mucosa of ulcerative colitis (UC) and in the development of UC-associated colorectal cancer. MATERIALS AND METHODS: HGF and c-met gene expressions were quantified in colonic mucosal specimens from healthy control subjects, patients with UC and patients with UC-associated colorectal cancer, using the competitive reverse transcription-polymerase chain reaction. Expression of HGF protein was determined by immunoblot analysis. Expression of c-Met protein was analyzed immunohistochemically. RESULTS: HGF and c-met gene expressions were increased in inflamed mucosa of UC, compared with control subjects. Gene expression of HGF was also increased in the surrounding inflamed mucosa of UC-associated cancers. In cases in which the HGF gene expression was increased, an apparent increase in protein levels of HGF in inflamed mucosa of UC were observed by immunoblot analysis. The c-met gene was overexpressed in UC-associated cancers and a high level of immunoreactivity of the c-Met protein was immunohistochemically detected within the cancer cells. CONCLUSION: We showed that HGF and c-met expression is increased in the inflamed mucosa of UC and that c-met is overexpressed in UC-associated colorectal cancers. These observations suggest HGF-Met system is involved in the repair process of the inflamed mucosa of UC and provide further support for the view that the inappropriate expressions of both HGF and c-met genes predispose to the development of colorectal cancer in patients with UC.

Effects of loss-of-function and gain-of-function mutations of c-kit on the gastrointestinal tract.

Protooncogene c-kit encodes a receptor tyrosine kinase, KIT. Interstitial cells of Cajal (ICCs) that are important for the autonomous movement of the gastrointestinal tract essentially require the normal function of the KIT for their development. Therefore, germline loss-of-function mutations of the c-kit gene cause deficiency of ICCs that results in disturbed gastrointestinal movement. On the other hand, somatic gain-of-function mutations of the c-kit gene induce gastrointestinal stromal tumors (GIST) that are considered to originate from ICCs. Moreover, germline gain-of-function mutations of the c-kit gene are a cause of familial development of multiple GISTs.

Effect of c-kit mutation on prognosis of gastrointestinal stromal tumors.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. Gain-of-function mutations in the juxtamembrane domain of the c-kit gene have been found in several GISTs. In this study, we examined the correlation between the presence of c-kit mutation and prognosis in 124 cases of GIST. DNA samples were extracted from paraffin sections. Exon 11 of the c-kit gene encoding the juxtamembrane domain and exon 17 encoding the kinase domain were amplified by PCR and sequenced. Most GISTs (89%) express the KIT protein, and missense mutations of exon 11 were found in 71 of 124 GISTs (57%). No mutations were detectable in exon 17. These 71 mutation-positive GISTs were larger in size and had more frequently invaded adjacent tissues than did the 53 mutation-negative GISTs. Histologically, the mutation-positive GISTs showed higher mitotic figures and more necrosis and hemorrhage. The patients with mutation-positive GISTs showed more frequent recurrences (P = 0.0005) and higher mortality (P = 0.0001) than did those with mutation-negative GISTs. The c-kit mutation was an independent prognostic factor for overall and cause-specific survival of the patients with GISTs. These results suggest that GISTs may be divided into mutation-positive and -negative subtypes. The prognosis was worse in patients with mutation-positive GISTs than in those with mutation-negative GISTs. Thus, mutation of the c-kit gene may be a good prognostic marker of GISTs.

A novel gain-of-function mutation of c-kit gene in gastrointestinal stromal tumors.

BACKGROUND & AIMS: The c-kit gene encodes a receptor tyrosine kinase (KIT). Recently, we found gain-of-function mutations of the c-kit gene in gastrointestinal stromal tumors (GISTs). All mutations were confined within the 11 amino acids (Lys-550 to Val-560) in the juxtamembrane domain, but one GIST showed a novel deletion-type mutation at codon 579 (Asp) in the juxtamembrane domain. The aim of this study was to clarify whether the mutation is activating. METHODS: Mutant c-kit cDNA was transfected into an interleukin 3 (IL-3)-dependent Ba/F3 murine lymphoid cell line, and the magnitude of autophosphorylation of the mutant KIT was examined with or without stem cell factor (SCF), a ligand of KIT. An in vitro kinase assay was also performed. The biological behavior of the transfectant was estimated by both an in vitro proliferation assay and in vivo transplantation to nude mice. RESULTS: The mutant KIT exhibited constitutive phosphorylation and strong kinase activity without SCF. The transfectant grew autonomously without IL-3 and SCF, and it formed tumors in nude mice. CONCLUSIONS: Deletion at codon 579 (Asp) in the juxtamembrane domain of the c-kit gene is a novel gain-of-function mutation other than the region between Lys-550 and Val-560.

High Fas ligand expression on lymphocytes in lesions of ulcerative colitis.

BACKGROUND: The pathogenesis of ulcerative colitis is unclear, but cytotoxic T lymphocytes infiltrating the mucosa have been implicated in mucosal damage. The Fas ligand (FasL), expressed on cytotoxic T lymphocytes, induces apoptosis in cells expressing Fas. AIM: To analyse FasL expression in affected colonic mucosa to ascertain Fas-FasL interaction in ulcerative colitis. METHODS: FasL mRNA was quantified in colonic mucosal specimens from healthy subjects and patients with ulcerative colitis or Crohn's disease, using the competitive reverse transcription polymerase chain reaction. FasL mRNA localisation was determined by in situ hybridisation. Expression of Fas in colonic mucosa was analysed immunohistochemically. Phenotypes of lamina propria lymphocytes that expressed FasL were analysed by flow cytometry. RESULTS: FasL mRNA was strongly expressed in active ulcerative colitis lesions, but not in those associated with active Crohn's disease or active proctitis-type ulcerative colitis. In situ hybridisation showed that FasL mRNA expression occurred in mononuclear cells infiltrating lesions. Fas was expressed in epithelial cells in ulcerative colitis and Crohn's disease, and in normal subjects. Cytometry showed that FasL was expressed in CD3 lymphocytes infiltrating the lamina propria in active lesions. CONCLUSIONS: FasL is expressed in CD3 lymphocytes infiltrating into ulcerative colitis but not Crohn's disease lesions, suggesting that Fas-FasL induced apoptosis participates in the mucosal damage of ulcerative colitis.

Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the human digestive tract, but their molecular etiology and cellular origin are unknown. Sequencing of c-kit complementary DNA, which encodes a proto-oncogenic receptor tyrosine kinase (KIT), from five GISTs revealed mutations in the region between the transmembrane and tyrosine kinase domains. All of the corresponding mutant KIT proteins were constitutively activated without the KIT ligand, stem cell factor (SCF). Stable transfection of the mutant c-kit complementary DNAs induced malignant transformation of Ba/F3 murine lymphoid cells, suggesting that the mutations contribute to tumor development. GISTs may originate from the interstitial cells of Cajal (ICCs) because the development of ICCs is dependent on the SCF-KIT interaction and because, like GISTs, these cells express both KIT and CD34.

Deficiency of c-kit+ cells in patients with a myopathic form of chronic idiopathic intestinal pseudo-obstruction.

OBJECTIVES: Chronic idiopathic intestinal pseudo-obstruction (CIIP) is a syndrome characterized by a failure of intestinal movement, but the cause of dysmotility remains unknown. Because interstitial cells of Cajal (ICCs) are believed to initiate basic contractile activity of the gastrointestinal tract, there is a possibility that changes in ICCs are involved in the development of CIIP. ICCs express c-kit in mice, and it has been reported that the c-kit+ cells, the location and shape of which resemble those in mice, are detected in the human gastrointestinal muscular layer using immunohistochemistry. In the present study, we counted the number of c-kit+ cells in the affected intestine of two patients with myopathic form of CIIP and compared this number with the number of c-kit+ cells in the normal intestine. METHODS: The c-kit+ cells in the external muscle layer were detected by immunohistochemistry, and the number of them was counted under the microscope. Mast cells, which are known to express c-kit, were detected by staining with Alcian blue, and the number of them was also counted. RESULTS: Immunohistochemistry revealed that the distribution pattern of c-kit+ cells resembles that of ICCs in the external muscle layer of normal control subjects. The numbers of c-kit+ cells apart from mast cells in two patients with myopathic form of CIIP decreased to about 3% of those in normal subjects. CONCLUSIONS: The failure of intestinal movement in patients with CIIP, at least in a subpopulation, might be related to a deficiency of c-kit+ cells, probably ICCs.

Expression of heparin-binding epidermal growth factor-like growth factor in neointimal cells induced by balloon injury in rat carotid arteries.

Balloon catheter injury of rat carotid arteries induces migration and proliferation of smooth muscle cells (SMCs), with subsequent neointimal formation. Several growth factors, such as platelet-derived growth factor and basic fibroblast growth factor, have been shown to be involved in this process, but the mechanisms that modulate the growth and/or migratory properties of SMCs remain unclear. In this study, we investigated whether heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is known to be a potent SMC stimulator from in vitro study, is associated with the proliferative response of SMCs to arterial injury. Northern blot analysis showed that the transcript levels of HB-EGF increased rapidly approximately 12-fold within 2 hours after injury and declined by 2 days but remained 3-fold at 14 days. In situ hybridization analysis demonstrated that the transcript of HB-EGF remained strongly expressed in the neointima, especially near the luminal surface, at 14 days after injury. Immunohistochemical staining showed that HB-EGF protein was positive in the endothelium and only faintly visible in medial SMCs in uninjured vessels. In contrast, 2 days after injury, positive HB-EGF immunostaining was detected in the medial SMCs along the luminal surface. At 7 days, the neointimal SMCs exhibited strong immunostaining for HB-EGF, and at 14 days, they exhibited a gradient of HB-EGF expression with strong immunoreactivity in the most luminal cells. SMCs labeled with 5-bromo-2'-deoxyuridine in their nuclei showed strong immunostaining for HB-EGF protein. Furthermore, the epidermal growth factor receptor to which HB-EGF can bind was also immunostained positively in neointimal SMCs. These data suggest that HB-EGF may play an important role of the proliferation and migration of SMCs in the process of neointimal accumulation induced by arterial injury, probably in an autocrine, paracrine, and/or juxtacrine manner.

Over-expression of bcl-xL gene in human gastric adenomas and carcinomas.

The present study was designed to clarify whether bcl-xL is involved in the development of carcinoma in the stomach. Levels of bcl-xL and bcl-2 mRNA were determined by a reverse-transcription/polymerase-chain reaction in endoscopic gastric biopsy specimens from 10 control subjects, 11 patients with adenomas and 14 patients with carcinomas. In 6 of 11 adenomas, 5 of 8 early carcinomas and 3 of 6 advanced carcinomas, the bcl-xL gene was over-expressed. In carcinomas, over-expression of the bcl-xL gene was observed in 6 of 9 intestinal-type carcinomas and 2 of 5 diffuse-type carcinomas. No correlation was observed between bcl-xL and bcl-2 gene expression. In cases in which the bcl-xL gene was over-expressed, an apparent increase in the protein level of Bcl-xL was observed by immunoblot analysis and intense Bcl-x immunoreactivity was detected immunohistochemically within the tumor cells. In conclusion, we showed that bcl-xL is over-expressed in gastric carcinomas at both the RNA and protein levels, suggesting that over-expression of bcl-xL may play a role in gastric carcinogenesis.

Localization of heparin-binding epidermal growth factor-like growth factor in human gastric mucosa.

BACKGROUND & AIMS: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been recently identified as a member of the EGF family. EGF receptors to which HB-EGF can bind have been detected in some types of gastric epithelial cells. The aim of this study was to investigate whether HB-EGF is produced in gastric epithelial cells to maintain normal gastric mucosa. METHODS: Gene expression and production of HB-EGF protein were investigated using Northern hybridization and immunohistochemistry, and the types of cells producing this protein were determined in human gastric mucosa. RESULTS: HB-EGF messenger RNA was detected in the body and antrum. Immunohistochemical staining showed that HB-EGF was localized mainly in parietal cells of fundic glands and in gastrin cells of pyloric glands. Also, the immunoreactivity of EGF receptors was observed in parietal cells and gastrin cells and faintly in surface epithelial cells and mucous neck cells of the proliferative zone. CONCLUSIONS: The results suggest that HB-EGF is synthesized mainly in parietal cells and gastrin cells and may act in an autocrine and/or paracrine manner in the regulation of proliferation and differentiation of the gastric mucosal cells through their surface EGF receptors.

Disturbed intestinal movement, bile reflux to the stomach, and deficiency of c-kit-expressing cells in Ws/Ws mutant rats.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICCs) are believed to initiate the basic contractile activity of the gastrointestinal tract. Because ICCs in the intestine of mice express c-kit receptor tyrosine kinase and because rats are more commonly used than mice for pathophysiological investigations of the gastrointestinal tract, the number of the c-kit messenger RNA-expressing cells was compared with gastrointestinal movement in rats. METHODS: The c-kit messenger RNA-expressing cells were detected by in situ hybridization. The autonomous contraction of excised segments of the ileum was recorded. The function of the pyloric sphincter was evaluated by measuring the content of bile acids in the stomach. RESULTS: The c-kit messenger RNA-expressing cells were not detectable in the stomach of Ws/Ws mutant rats with a small deletion at the tyrosine kinase domain of c-kit, and the number of c-kit messenger RNA-expressing cells decreased to 7% that of normal control rats in the ileum of Ws/Ws rats. The contractile activity of the ileum was apparently impaired, and the content of bile acids in the stomach was significantly increased in Ws/Ws rats. CONCLUSIONS: The abnormalities in the ileal movement and pyloric sphincter function in Ws/Ws rats were attributable to the deficiency of c-kit messenger RNA-expressing cells.

Inhibition of attachment between cultured mast cells and fibroblasts by phorbol 12-myristate 13-acetate and stem cell factor.

Cultured mast cells (CMC) derived from the bone marrow of mice express the receptor encoded by the W (c-kit) locus (W receptor), and the WCB6F1(+/+)-3T3 fibroblasts express the ligand encoded by the Sl locus (stem cell factor [SCF]). CMC attach to the fibroblasts through the W receptors and cell-bound SCF. We investigated the effect of phorbol 12-myristate 13-acetate (PMA) and recombinant murine SCF (rmSCF) on the attachment. PMA induced both the internalization and shedding of W receptors, whereas rmSCF induced only the internalization. Moreover, both PMA and rmSCF reduced the expression of c-kit mRNA levels in CMC. Addition of either PMA or rmSCF to the coculture of CMC and fibroblasts resulted in the inhibition of attachment. Since the magnitude of the attachment between CMC and fibroblasts may be manipulated by changing the doses of either PMA or rmSCF, the present experimental system may be useful as a model for the attachment between blood cells and stromal cells.