OBJECTIVETo investigate the clinicopathological and prognostic significance of the c-kit protein in gastrointestinal stromal tumor (GIST).

METHODSParaffin embedded materials from 53 benign GISTs, 13 potentially malignant and 55 malignant cases were analysed for c-kit expression by immunohistochemical method, while using leiomyomas and schwannomas as controls. Positive signals were shown in cytoplasma and cell membrane.

RESULTSOf 122 GISTs, 118 (97%) were positive for c-kit. Localization of positive signals was accurate. The rate of c-kit protein in benign, potentially malignant and malignant cases was 98% (53/54), 93% (12/13), 96% (53/55) respectively. Compared with benign GIST, the positivity of c-kit in metastasis or recurrent cases decreased, but c-kit protein expression rate was not significantly different between the three patterns of GIST (chi(2) = 1.167, P > 0.05). Leiomyomas and schwannomas were typically c-kit negative.

CONCLUSIONAs a sensitive and specific marker of GIST, c-kit seems to be a useful antibody in the diagnosis and differential diagnosis of GIST, but it may not be used as a prognostic index.

Biomarkers, Tumor ; biosynthesis ; genetics ; Diagnosis, Differential ; Gastrointestinal Neoplasms ; diagnosis ; metabolism ; Humans ; Immunohistochemistry ; Mutation ; Prognosis ; Proto-Oncogene Proteins c-kit ; biosynthesis ; genetics

OBJECTIVETo express the first three immunoglobulin-like domains of human stem cell factor receptor (c-Kit/Ig1-3) in E. coli and HEK293 ET cells and study their binding activity for stem cell factor (SCF).

METHODSIn prokaryotic expression system, a double mutant form of c-Kit /Ig1-3 (c-Kit /Ig1-3(DM) was produced by overlap PCR and cloned into pET16b. The recombinant protein was expressed in E. coli BL21 (DE3) and refolded by dilution. In eukaryotic expression system, the gene of c-Kit/Igl13 with eight histidine segments was cloned into pEAK12 and the recombinant plasmid was transfected into HEK293 ET cells. The fusion protein was harvested from the growth medium and purified on Ni-NTA agarose column. The recombinant protein was tested for the receptor binding activity with his-tag pull-down and enzyme-linked immunosorbent binding assay.

RESULTSIn E. coli c-Kit /Ig1-3(DM) as produced as an inclusion body and showed low binding activity for SCF after refolding. Two HEK293 ET cell clones that express high levels of c-Kit/Ig1-3 were produced and each clone secreted 2p micro/ml of recombinant protein, whose relative molecular mass was about 58,000. Eukaryotically expressed c-Kit/Ig1-3 had specific binding activity for SCF, and the dissociation constant (Kd) was 9.39 nmol/L.

CONCLUSIONc-Kit/Ig1-3 with high receptor binding activity is successfully produced in HEK293 ET cells.

Cells, Cultured ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulins ; biosynthesis ; genetics ; isolation & purification ; Ligands ; Plasmids ; Proto-Oncogene Proteins c-kit ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Transfection

This study sought to isolate and purify C-kit+ cells from rat 2-AAF/PH model and to investigate the proliferation and differentiation of C-kit+ cells in vitro. C-kit positive oval cells were enriched by using magnetic activated cell sorting (MACS). The sorted oval cells were cultured in a low density, and then colony formation was observed. The capacity of proliferation and differentiation of C-kit positive cells were examined in vitro by immunocytochemistry and RT-PCR. By using C-kit antibody in conjunction with MACS, we developed a rapid oval cell isolation protocol. The sorted cells formed colony when cultured in vitro. Cells in the colony expressed albumin or cytokeratin 19 (CK19) or coexpressed both, and BrdU incorporation test was positive. RT-PCR on colony showed expression of albumin and CK19 gene. The results demonstrate that by means of MACS we have established a method to isolate oval cells. The sorted hepatic oval cells can form colony in vitro which expresses different combinations of phenotypic markers and genes from both hepatocytes and cholangiocyte lineage.
2-Acetylaminofluorene ; pharmacology ; Animals ; Animals, Newborn ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Hepatocytes ; cytology ; metabolism ; Male ; Proto-Oncogene Proteins c-kit ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; metabolism

The aim of study is to investigate the expression of hematopoietic cell phosphatase (SHP-1) gene and c-kit pro-oncogene in acute leukemia (AL) and its impact on prognosis in AL. Semi-quantity reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of SHP-1 mRNA and c-kit mRNA in 60 AL patients and 33 normal controls (NC). The results showed that the positive rates of SHP-1 expression from high to low level were found orderly in complete remission group, newly diagnosed group and relapsed group, there was significance difference between each group and NC group (P < 0.05). The positive rates of c-kit expression were opposite order in each groups as compared with SHP-1. there was also significance difference between each group and NC group (P < 0.05). The positive rate of SHP-1 and c-kit expressions in AML was higher than that in ALL (P < 0.05), there was negative correlation between expressions of SHP-1 and c-kit (r = -0.502, P < 0.05); The difference between the complete remission rate in SHP-1 positive and in SHP-1 negative patients from 30 newly diagnosed AML patients was significant (P < 0.05), the same result was found between c-kit(+) complete remission and c-kit(-) complete remission. It is concluded that SHP-1 gene is a potentially anti-oncogene and inhibits the growing of tumor by negatively modulating c-kit gene. Simultaneous detection of SHP-1 and c-kit gene may act as a factor for predicting prognosis in AL.
Adolescent ; Adult ; Aged ; Female ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; Middle Aged ; Polymerase Chain Reaction ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; metabolism ; Prognosis ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; biosynthesis ; genetics ; metabolism ; Proto-Oncogene Proteins c-kit ; biosynthesis ; genetics

Coexpression of Kit ligand and c-kit has been reported in some gynecologic tumors. To determine whether imatinib mesylate is useful in ovarian epithelial tumors, we performed immunohistochemical and mutational analysis. The cases consisted of 33 cases, which included 13 serous cystadenocarcinomas, 1 borderline serous tumor, 8 mucinous cystadenocarcinomas, 6 borderline mucinous tumors and 5 clear cell carcinomas. Five cases of serous cystadenoma and 5 cases of mucinous cystadenoma were also included. In the immunohistochemical study, 3 cases (3/6, 50%) of borderline mucinous cystic tumor and two cases (2/8, 25%) of mucinous cystadenocarcinoma show positive staining for KIT protein. Only one case (1/13, 7.7%) of serous cystadenocarcinoma had positive staining. On mutational analysis, no mutation was identified at exon 11. However, two cases of borderline mucinous tumors and one case of mucinous cystadenocarcinoma had mutations at exon 17. In these cases, the immunohistochemistry also shows focal positive staining at epithelial component. Although, KIT protein expression showed higher incidence in mucinous tumors than serous tumors, they lack KIT-activating mutations in exon 11. Thus, ovarian surface epithelial tumors are unlikely to respond to imatinib mesylate.
Adult ; Aged ; Cystadenocarcinoma, Mucinous/genetics/metabolism/pathology ; Cystadenoma, Mucinous/genetics/metabolism/pathology ; Cystadenoma, Serous/genetics/metabolism/pathology ; DNA Mutational Analysis ; DNA, Neoplasm/chemistry/genetics ; Epithelial Cells/chemistry/metabolism/pathology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Middle Aged ; Mutation ; Ovarian Neoplasms/genetics/metabolism/*pathology ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Proto-Oncogene Proteins c-kit/biosynthesis/*genetics