Expression Levels Of C-kit Research Articles

Expression of c-Kit and PDGFRα in epithelial ovarian tumors and tumor stroma

The purpose of this study was to investigate the expression of c-Kit and platelet-derived growth factor receptor α (PDGFRα) in epithelial ovarian tumor cells and tumor stroma. The expression of c-Kit and PDGFRα in 71 malignant or benign epithelial ovarian tumor tissues and 20 normal ovarian tissues was evaluated by immunohistochemical staining. The expression of c-Kit and PDGFRα in 71 malignant epithelial ovarian tumors and tumor stroma tissue samples was analyzed. A significant increase (P<0.01) of c-Kit expression was observed in malignant ovarian tumors (50.7%) when compared to normal ovarian tissues (10.0%) or benign ovarian tumors (20.0%). The PDGFRα expression rate in malignant ovarian tumors (63.4%) was also significantly higher (P<0.01) than that in normal ovarian tissues (15.0%) or benign ovarian tumors (25.0%). c-Kit was expressed in only 4.2% of the tumor stroma samples, which was significantly lower than the expression of malignant ovarian tumors (P<0.01), whereas the PDGFRα expression in tumor stroma (87.3%) was significantly higher than that of the malignant ovarian tumors (P<0.01). The expression levels of c-Kit and PDGFRα are higher in the malignant ovarian tumors than in the benign ovarian tumors or normal tissues. In the malignant ovarian tumor stroma, c-Kit expression is low and PDGFRα expression is high, and the differential changes of c-kit and PDGFRα suggest distinct roles in ovarian cancer.

Targeted Therapy in Lung Cancer; Lessons Learned from Past Experiences

Targeted Therapy in Lung Cancer; Lessons Learned from Past Experiences

Abnormal development of intrinsic innervation in murine embryos with anorectal malformations

Constipation, soiling, and incontinence are common problems after definitive repair of anorectal malformations (ARMs) in children. We studied the expression of substance P (SP), vasoactive intestinal peptide (VIP), and c-kit in the rectum of murine embryos with or without ARMs at later developmental stages. On the 9th embryonic day (E9), pregnant Institute of Cancer Research mice were fed etretinate, a synthetic vitamin A analogue (60 mg/kg), whereas controls were fed only with sesame oil. Embryos were excised between E14 and E18, and prepared for histological examination. The SP, VIP, and c-kit expressions were examined by immunohistochemical staining for the SP, VIP, and c-kit antigens, respectively. On E14 and E15, the expression levels of the anti-SP and anti-VIP antibodies in the rectum did not differ between the control and etretinate-treated group. However, as compared to the controls, a decreased SP and VIP immunoreactivity was observed in the circular muscle layer of the rectum between E16 and E18. On the other hand, on E14 and E15, the expression of anti-c-kit antibody in the rectum did not differ between the etretinate-treated and control group. However, c-kit immunoreactivity was slightly higher in the circular muscle layer of the rectum in the controls on E16 and E17, and considerably higher on E18 than that of the muscle layer in the etretinate-treated group. At later developmental stages, the expression levels of SP, VIP, and c-kit reduced in the circular muscle layer of the rectum in mice with etretinate-induced ARMs. This result indicates that reduced SP, VIP, and c-kit expression levels in the circular muscle layer may cause severe constipation in children who develop severe ARMs after definitive surgery.

The expression levels of stem cell markers importin13, c-kit, CD146, and telomerase are decreased in endometrial polyps

SummaryBackgroundTo investigate the expression levels of importin13 (IPO13), c-kit, CD146, telomerase, caspase-3, bcl-2 and bax in endometrial polyps (EPs).Material/MethodsWe detected the mRNA expression levels of IPO13, c-kit, bcl-2 and bax in endometrial polyps (EPs) using real-time PCR. We detected the protein expression levels of IPO13, telomerase, CD146, caspase-3, bcl-2 and bax in EPs using S-P (Streptavidin-Peroxidase) immunohistochemistry. Western blotting was performed to determine the levels of importin13 and bcl-2 proteins in EPs.ResultsThe expression levels of IPO13, c-kit, telomerase, caspase3, and bax were lower in the EP tissue compared to normal endometrial tissue during the proliferation and secretion phases of the menstrual cycle (p<0.05). The expression of CD146 was decreased in the EP tissue compared to the normal endometrial tissue during the proliferation phase of the menstrual cycle (p<0.05). The expression of bcl-2 was increased in the EP tissue compared to the normal endometrial tissue during the proliferation and secretion phases of the menstrual cycle (p<0.05).ConclusionsThe expression levels of IPO13, c-kit, telomerase, caspase3, and bax were decreased; however, the expression of bcl-2 was increased in the EP tissue compared to the normal endometrial tissue. These findings suggest that the development of EPs is associated with the deregulated activities of the endometrial stem/progenitor cells and the decreased apoptosis of endometrial cells, with the latter being the major factor involved in the development of EPs.

Decreased expression of c-kit and telomerase in a rat model of chronic endometrial ischemia

SummaryBackgroundIt was unclear whether chronic endometrial ischemia contributed to the pathogenesis of thin endometrium and was associated with decreased endometrial stem/progenitor cell. Thus, we explored the role of chronic endometrial ischemia in the pathogenesis of thin endometrium and its effect on endometrial stem/progenitor cells apoptosis.Material/MethodsIn vitro, endometrial side population (ESP) cell apoptosis models were built, and apoptosis was quantified by fluorescence-activated cell sorter (FACS) analysis, pou5f1, and c-kit mRNA was detected by qPCR. In vivo, a rat model of chronic endometrial ischemia was induced by performing bilateral uterine artery ligation. TERT and caspase3 were detected by immunohistochemistry. Pou5f1 and c-kit mRNA was examined by qPCR. C-kit, caspase3 and telomerase were detected by Western blot.ResultsIn the in vitro endometrial SP (ESP) cells apoptosis model, we found that the apoptotic rate was gradually increased with time, prolonging the expression of TERT, and c-kit mRNA was gradually decreased. In the in vivo endometrial SP (ESP) cells apoptosis model, we found that endometrial thickness, luminal epithelium thickness, gland epithelium thickness and the number of glands in the experiment group were significantly decreased compared with those in the control group (P<0.05). The expression levels of c-kit, pou5f1 and telomerase was significantly lower in the experimental group than those in the control group (P<0.05). The expression level of caspase3 was significantly higher in the experimental group compared with that in the control group (P<0.05).ConclusionsThe present work shows that chronic ischemia and chronic endometrial ischemia-associated stem/progenitor cells apoptosis may be responsible for the pathogenesis of thin endometrium.

Stem cell-related proteins C-KIT, C-MYC and BMI-1 in juvenile nasopharyngeal angiofibroma—do they have a role?

Juvenile nasopharyngeal angiofibroma (JNA) is a highly vascular tumour, occurring almost exclusively in adolescent males. Histogenesis of JNA remains unclear, two optional theories proposing either fibrous or vascular tissue as the tissue of origin. Stem cell-related proteins have been discussed to possibly participate in the growth of these tumours. In our study, we reviewed retrospective clinicopathological data of 26 JNA patients. By immunohistochemistry, we investigated the cellular distribution and expression levels of stem cell-related proteins C-KIT, C-MYC and BMI-1 and their correlation with cell and vessel density of the tumour. Contrary to earlier reports, we detected C-KIT expression in addition to stromal cells also in endothelial cells. The C-KIT expression was more dominant in slit vessels than large vessels. A significant correlation was found between endothelial immunoexpression of C-KIT and cellular density of the tumour. C-MYC and BMI-1 expression was detected in stromal cells only. Due to our finding of C-KIT expression in both stromal and endothelial cells and the strong correlation between the endothelial C-KIT expression and cellular density, we suggest that, besides the stromal tissue, the vascular component might take part in the neoplastic growth of JNA.

Candidemia-Induced Emergency Granulopoiesis Consists of Successive Dual Waves Triggered by the Shift From C/EBPalpha to C/EBPbeta Dependency.

AbstractAbstract 3778 Introduction:Granulocyte is a major cellular component in the front line of host defense. The number of granulocytes must be tightly tuned to meet the demand, because both the shortage and the excess of granulocytes can be harmful to the host. During emergency situations such as infections, granulocytes are replenished from peripheral pools and bone marrow production. As the half-life of granulocytes is quite short, granulopoiesis, de novo production of granulocytes in bone marrow, plays an important role during emergency. We have previously shown that granulopoiesis at steady state is largely dependent on a transcription factor, C/EBPalpha, whereas emergency granulopoiesis is dependent on C/EBPbeta (Hirai H, et al. Nature Immunol., 2006). However, the precise developmental stage where the shift from C/EBPalpha dependency to C/EBPbeta dependency takes place is almost unknown. The aim of this study is to dissect the process of granulopoiesis by a novel flow cytometric method and to elucidate the molecular mechanisms involved in the regulation of emergency granulopoiesis. Methods:4 ▪ ≂ 106 cfu Candida albicans were intravenously injected to induce emergency granulopoiesis. Mouse bone marrow cells were harvested and stained with a combination of fluorescent-conjugated antibodies including anti-c-kit, anti-CD34, anti-Ly6G antibodies and markers for other lineages. Then the stained cells were analyzed or sorted by flow cytometry. After eliminating the cells which lost potential to give rise to granulocytes, the remaining cells were dissected into five subpopulations (#1≂ #5) according to the expression levels of c-kit and Ly6G. #1 is c-kithigh Ly6Glow cells, ▪@ #2: c-kitint Ly6Glow, #5: c-kitlow Ly6Ghigh, and the cells residing between #2 and #5 are divided into #3 and #4. Cell number, gene expressions and cell cycle status of each population were analyzed before and after inducing emergency granulopoiesis. ▪@ Results and Discussions:Wright-Giemsa staining and qRT-PCR for granule proteins (cathepsin G, myeloperoxidase, neutrophil elastase2, lactoferrin and MMP9) in each population indicated that lower c-kit expression and higher Ly6G expression correlated well with granulocytic differentiation and that the granulopoiesis progresses from # 1 to #5 in this order both at steady state and during emergencies (Figure 1). Then we applied this method to candidemia-induced emergency granulopoiesis. In vivo BrdU incorporation analysis showed immediate acceleration of the cell cycle in the most immature population (#1) and in one of the intermediate populations (#2). Chronological monitoring of each population after inducing candidemia revealed that rapid increase in mature granulocytes (#5) preceded the replenishment from the most immature population (#1). These results suggested that there are two distinct ▪gwaves▪h in granulopoiesis at the early phase of infection, a rapid supply (first ▪gwave▪h) of granulocytes from relatively mature population (#2≂ #4), and a further and sustained supply (second ▪gwave▪h) originated from more immature populations (#1) including hematopoietic stem/progenitor cells (Figure 1). Transcripts of C/EBPalpha were significantly downregulated in #1≂ #4 at the early phase of infection, while those of C/EBPbeta were maintained in all the subpopulation (Figure 2), suggesting that shift from C/EBPalpha dependency to C/EBPbeta dependency took place at multiple developmental steps in granulopoiesis. C/EBPbeta has less inhibitory effects on cell cycle than C/EBPalpha while their abilities to induce granulocytic differentiation are similar (Hirai H, et al. Nature Immunol., 2006). The shift toward C/EBPbeta dependency may trigger the dual waves in emergency granulopoiesis, which demands both differentiation and proliferation of granulocytic precursors. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

KGF Promotes Paracrine Activation of the SCF/c-KIT Axis from Human Keratinocytes to Melanoma Cells

The paracrine networks of the human melanoma microenvironment are able to influence tumor growth and progression. Among the paracrine growth factors involved in skin homeostasis, the KGF/FGF7 secreted by dermal fibroblasts promotes the epidermal proliferation and differentiation as well as the release from keratinocytes of other paracrine mediators. To evaluate the possible role played by KGF in affecting the behavior of different subtypes of melanoma carrying activating mutations or overexpression of the SCF receptor c-KIT, we used human melanoma cell lines, characterized by different expression levels of c-KIT and opposing responsivity to SCF, and HaCaT keratinocytes. Quantitative real-time reverse transcription-polymerase chain reaction assay and ELISA test on KGF-treated keratinocytes showed enhanced expression and secretion of SCF in response to KGF and dependent on functional KGF receptor. Immunofluorescence microscopy and biochemical analysis showed, in one of the selected melanoma cell models, SCF-dependent c-KIT activation induced by stimulation with the culture supernatants collected from KGF-treated keratinocytes. In keratinocyte-melanoma cocultures stained for the Ki67 proliferation marker, incubation with KGF induced enhanced growth not only of the keratinocytes but also of the melanoma cells, which could be blocked by the c-KIT inhibitor imatinib, demonstrating the establishment of a KGF-induced paracrine signaling network owing to the coexpression of biologically active SCF released from keratinocytes and functional c-KIT on melanoma cells.

The Epithelioid Variant of Angiomyolipoma in Kidney Expresses a Higher Level of Epidermal Growth Factor Receptor Than Its Conventional Type

The epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase that plays an important role in tumor growth, invasion and metastasis. EGFR Positive immunoreactivity ranged from focal to diffuse in overexpression has been shown in many different tumors and multiple EGFR-targeted therapies are currently being investigated. Renal epithelioid angiomyolipoma (epiAML) is an aggressive and potentially malignant variant of conventional angiomyolipoma (cAML). Currently, the status of EGFR expression in epiAML is unknown. We compared EGFR protein expression, as well as expression levels of several other tumor regulatory proteins including c-Kit, p53, alpha-catenin, beta-catenin and osteopontin in 6 cases of cAML and 10 cases of epiAML by semiquantitative immunohistochemistry (IHC). EGFR gene amplification was analyzed by fluorescent in situ hybridization (FISH). Within epiAML, EGFR immunostaining was significantly higher when compared to cAML (90% vs. 16.7%, p<0.01). epiAML, whereas no diffuse reactivity was found in cAML. EGFR gene amplification was not identified in either cAML or epiAML as illustrated by FISH. Expression levels of c-Kit, osteopontin, p53, alpha-catenin

Radiosensitization Effect of STI-571 on Pancreatic Cancer Cells In Vitro

To examine STI-571-induced radiosensitivity in human pancreatic cancer cells in vitro. Three human pancreatic cancer cell lines (Bxpc-3, Capan-1, and MiaPaCa-2) exhibiting different expression levels of c-Kit and platelet-derived growth factor receptor beta (PDGFRbeta) and showing different K-ras mutation types were used. For evaluation of the antitumor activity of STI-571 in combination with radiation, clonogenic survival assays, Western blot analysis, and the annexin V/propidium iodide assay with microscopic evaluation by 4',6-diamidino-2-phenylindole were conducted. Dramatic phosphorylated (p)-c-Kit and p-PDGFRbeta attenuation, a modest dose- and time-dependent growth inhibition, and significant radiosensitization were observed after STI-571 treatment in view of apoptosis, although the levels of growth inhibition and increased radiosensitization were different according to cell lines. The grades of radiosensitivity corresponded to the attenuation levels of p-c-Kit and p-PDGFRbeta by STI-571, particularly to those of p-c-Kit, and the radiosensitivity was partially affected by K-ras mutation in pancreatic cancer cells. Among downstream pathways associated with c-Kit or PDGFRbeta, p-PLCgamma was more closely related to radiosensitivity compared with p-Akt1 or p-extracellular signal-regulated kinase 1. STI-571 enhances radiation response in pancreatic cancer cells. This effect is affected by the attenuation levels of p-c-Kit or p-PDGFRbeta, and K-ras mutation status. Among them, p-c-Kit plays more important roles in the radiosensitivity in pancreatic cancer compared with p-PDGFRbeta or K-ras mutation status.

MRNA Expression of Platelet-Derived Growth Factor Receptor-β and C-KIT: Correlation With Pathologic Response to Cetuximab-Based Chemoradiotherapy in Patients With Rectal Cancer

Deviant expression of platelet-derived growth factor receptor-beta (PDGFRbeta) and c-kit was shown in patients with colorectal cancer. In the present study, mRNA expression of PDGFRbeta and c-kit in 33 patients with locally advanced rectal cancer undergoing preoperative chemoradiotherapy with cetuximab/capecitabine/irinotecan in correlation with the tumor regression rate was investigated. Pretherapeutic biopsy cores and tumor material from the resected specimens were collected in parallel with normal rectal mucosa. The expression levels of PDGFRbeta and c-kit were measured by quantitative polymerase chain reaction. Tumors were classified as good responders (tumor regression grade [TRG], 2-3) or poor responders (TRG, 0-1). The TRG evaluation of the resected specimen was TRG 0-1 in 11 and TRG 2-3 in 22. The median normalized ratios in the pretreatment mucosa vs. tumor biopsy cores was as follows: PDGFRbeta ratio of 15.2 vs. 49.5 (p <0.0001) and c-kit ratio of 0.94 vs. 0.67 (p = 0.014). The same tendency was observed for the median PDGFRbeta ratios after chemoradiotherapy completion: 34.2 vs. 170.0 (p <0.0001). The PDGFRbeta and c-kit mRNA expression values in the pretreatment tumor biopsy cores were lower than the values in the resected specimens: PDGFRbeta ratio 49.5 vs. 170.0 (p = 0.0002) and c-kit ratio 0.67 vs. 1.1 (p = 0.0003). Nevertheless, no correlation was seen between the pretherapeutic PDGFRbeta and c-kit mRNA expression and the pathologic regression rate. Cetuximab-based chemoradiotherapy increased PDGFRbeta levels even further compared with the pretreatment samples and deserves further investigation.

Change and significance of the expression of c-kit and SCF following recovery from unilateral testicular torsion in rats

To investigate the change in expression levels of c-kit and SCF, and the protective effects of FSH on ischemia-reperfusion injury due to testicular torsion-detorsion. 24 adult male SD rats were divided into three groups of 8: control group, testicular torsion group and FSH-treated group. The control group was treated with sham-operation. Animals in the testicular torsion and FSH-treated groups were subjected to unilateral 720 degrees counterclockwise testicular torsion for 2 hours and then reperfusion was allowed after detorsion. The FSH-treated group received intraperitoneal injection of FSH 15min before detorsion. Then, the rats were sacrificed and the testes were harvested. Histopathological changes were observed by light microscope, and the expression levels of c-kit, SCF in testicular tissue in the different groups were detected by Immunohistochemical assay and Quantitative Real-time RT-PCR analysis. Finally, the relative proportions of germ cells were measured by FCM. c-kit and SCF were positive expressed in 52.58% and 61.16% of testicular cells of control tissues, respectively. Decreases of c-kit and SCF positive cells (15.01% and 9.18%) were found in the testicular torsion group. After being treated by FSH, the number of positive cells increased (31.25% and 20.01%). Moreover, the c-kit and SCF mRNA expression was increased dramatically (P < 0.01) in response to FSH stimulation. Furthermore, the number of haploid, diploid and tetraploid cells has also increased significantly in drug-treated testes (P < 0.01). The mechanism of tissue damage in the testicular torsion model, includes changes in the expression of c-kit and SCF following torsion. Also, FSH has a protective effect on germ cells after unilateral testicular torsion, which was reflected by increased c-kit and SCF levels.

Baseline and stimulated turnover of cell surface c‐Kit expression in different types of human mast cells

The receptor tyrosine kinase c-Kit is fundamental to mast cell (MC) development and maintenance. Its regulation can occur at various levels, but nothing is known about how this is accomplished in normal human tissue MC. Likewise, the baseline turnover of c-Kit has not been addressed yet. We used mature MC from human skin, along with the MC lines LAD-2 and HMC-1 and treated them with stem cell factor (SCF), cycloheximide, actinomycin D (AD) and combinations thereof, and determined expression levels of c-Kit and other surface receptors by flow cytometry. Ligand-induced internalization of c-Kit was found to be a universal mechanism and detectable in all MC subtypes. By Western blot analysis of LAD-2 cells, c-Kit was found to nearly disappear 3 h after the addition of SCF to slowly recover thereafter. Investigations into the baseline turnover of c-Kit expression revealed that c-Kit is strongly affected by the inhibition of de novo translation in all MC subsets, while a suppression of transcription had a weaker effect and displayed greater cell-to-cell variation. Only a minor impact on other cell surface receptors (CD29, CD50 and CD54) was noted. On combined treatment, cycloheximide, AD and SCF displayed additive effects, resulting in a complete disappearance of c-Kit from the cell surface. In conclusion, c-Kit represents a rapidly cycling cell surface receptor. It is not only immediately internalized upon binding of its ligand, but it is also heavily affected by the inhibition of translation or transcription when viewed against an average background. Interestingly, c-Kit regulation seems largely independent of the MC subtype.

Fetal Hematopoietic Development in the Mouse Aorta-Gonad-Mesonephros Region Is Inhibited by Homeodomain-Interacting Protein Kinase 2.

Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of mouse embryo. Our previous studies showed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine 727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in the hematopoietic development. HIPK2 transcripts were found in the fetal hematopoietic tissues such as the AGM region at embryonic day (E) 10.5 and E11.5 and the E14.5 fetal liver as detected by RT-PCR and whole mount in situ hybridization. The functional analysis of HIPK2 was done by introducing wild-type and its mutant constructs into the primary culture of the mouse AGM region. Retrovirus mediated forced expression of HIPK2 with green fluorescent protein (GFP) in the AGM culture remarkably inhibited the emergence of GFP+ nonadherent cells. In coculture of virus-infected GFP+ nonadherent cells with OP9 stromal cells, no cobblestone-like area-forming colonies were detected in the HIPK2-infected cells, while control GFP virus had no effect on the colony formation. Similarly, when GFP+ nonadherent cells were sorted and cultured in semisolid medium, colonies were not formed from the HIPK2-GFP virus-infected AGM culture. These data suggest that HIPK2 suppresses differentiation of adherent endothelial-like cells to colony-forming hematopoietic cells or, alternatively HIPK2 inhibits proliferation of newly developed hematopoietic progenitors. Development of cells positive for CD45 (an immature hematopoietic cell maker) cells was suppressed by forced expression of HIPK2 in the AGM culture. To determine the domain important for the inhibitory activity of HIPK2, we constructed a series of point and deletion mutants. Overexpression of HIPK2 mutants in the AGM culture showed that a certain structure of the kinase domain as well as its C-terminally located structure but not the kinase activity is essential for the HIPK2-mediated inhibition of hematopoiesis. When nonadherent cells in the AGM culture were fractionated for their expression levels of CD45 and c-Kit (a receptor for stem cell factor), CD45lowc-Kit+ cells have a potential to form hematopoietic cell colonies. In the HIPK2-overexpressed AGM culture, emergence of CD45lowc-Kit+ cells was significantly inhibited. These data indicated that HIPK2 plays a negative regulatory role in the development of AGM hematopoiesis in mouse embryo.

Aberrant Overexpression of the Wilms Tumor Gene (WT1) in Human Leukemia

AbstractTo clarify whether the expression of the WT1 gene in leukemic cells is aberrant or merely reflects that in normal counterparts, the expression levels of the WT1 gene were quantitated for normal hematopoietic progenitor cells. Bone marrow (BM) and umbilical cord blood (CB) cells were fluorescence-activated cell sorting (FACS)-sorted into CD34+ and CD34− cell populations, and the CD34+ cells into nine subsets (CD34+CD33−, CD34+CD33+, CD34+CD38−, CD34+CD38+, CD34+HLA-DR−, CD34+HLA-DR+, CD34+c-kithigh, CD34+c-kitlow, and CD34+c-kit−) according to the expression levels of CD34, CD33, CD38, HLA-DR, and c-kit. Moreover, acute myeloid leukemic cells were also FACS-sorted into four populations (CD34+CD33−, CD34+CD33+, CD34− CD33+, and CD34− CD33−). FACS-sorted normal hematopoietic progenitor and leukemic cells and FACS-unsorted leukemic cells were examined for the WT1 expression by quantitative reverse transcriptase-polymerase chain reaction. The WT1 expression in the CD34+ and CD34− cell populations and in the nine CD34+ subsets of BM and CB was at either very low (1.0 to 2.4 × 10−2) or undetectable (<10−2) levels (the WT1 expression level of K562 cells was defined as 1.0), whereas the average levels of WT1 expression in FACS-sorted and -unsorted leukemic cells were 2.4 to 9.3 × 10−1. Thus, the WT1 expression levels in normal hematopoietic progenitor cells were at least 10 times less than those in leukemic cells. Therefore, we could not find any normal counterparts of BM or CB that expressed the WT1 at levels comparable with those in leukemic cells. These results indicate an aberrant overexpression of the WT1 gene in leukemic cells and imply the involvement of this gene in human leukemogenesis.

Separation of Myeloid and Erythroid Progenitors Based on Expression of CD34 and c-kit

In this report, a novel approach is described to physically separate erythroid progenitors from monocyte and granulocyte progenitors, based on the expression of CD34 and Kit. Using biotin-labeled human Kit ligand (KL) and flow cytometry, Kit was detectable on 2% to 3% of the nucleated cells in rhesus monkey bone marrow. Combination of biotin-KL with CD34 monoclonal antibodies (MoAb) showed that Kit was expressed on subsets of CD34low and CD34pos cells. Our data clearly demonstrate that CD34pos cells are more heterogeneous with respect to Kit expression than observed in studies using Kit MoAb. A small cluster, approximately 7% of the CD34pos cells, expressed CD34 at submaximal levels and stained brightly with biotinylated KL. This CD34pos/kithi fraction contained predominantly erythroid progenitors (burst-forming units-erythroid; BFU-E). The majority of the granulocytic and monocytic progenitors (colony-forming units-granulocyte/macrophage; CFU-GM) were CD34pos/kitmed. Some BFU-E were also detected in the CD34pos/kitmed and CD34low/kitpos fractions at low frequency. In the latter subset, most erythroid colony-forming units (CFU-E) were recovered. Using three-color flow cytometry, we analyzed expression of Kit in relation to that of CD34 and the class II major histocompatibility antigen, RhLA-DR. The most immature bone marrow cells that can be identified in vitro, ie, CD34pos/RhLA-DRlow cells, were kitmed. The CD34pos/kithi and CD34pos/kitneg subsets predominantly contained the more mature RhLA-DRbright cells. Our results demonstrate that erythroid precursors express c-kit at much higher levels than monomyeloid precursors and pluripotent progenitors. The difference in expression levels of CD34 and c-kit can be exploited to isolate BFU-E populations that are virtually devoid of nonerythroid cells.