Stem Cell Factor Receptor Research Articles

Expression of stem cell factor receptor c-kit in human nontumoral and tumoral hepatic cells

The aim of this study was to examine and compare the stem cell factor receptor c-kit expression in hepatocellular carcinoma (HCC) with the corresponding peritumoral tissue. To confirm the immunohistochemical results in the investigated HCC tissues, HCC cell lines were analyzed for c-kit expression. Expression of c-kit (SCF receptor) has been evaluated in 72 HCC and in the corresponding surrounding nontumorous tissue. Additionally, immunohistochemical analysis reverse transcription-polymerase chain reaction was also used to examine the mRNA expression of c-kit protooncogene in tumor homogenates. Furthermore, three HCC cell lines (HUH-7, HepG2, and SK-Hep1) were used for gene-expression analysis of c-kit mRNA. C-kit expression was detected immunohistochemically in 70% of HCC with different degrees of intensity. Moreover, c-kit expression could also be found in about 90% of the corresponding peritumoral noncirrhotic as well as in cirrhotic liver tissues. C-kit mRNA was detectable in 83% of HCC and in 75% of the corresponding peritumoral noncirrhotic as well as in 100% of corresponding peritumoral cirrhotic samples. In addition, two of the three HCC cell lines (HUH-7 and SK-Hep1) showed a well detectable PCR-product for c-kit. Hepatocytes express the c-kit receptor at different grade of intensity under normal and altered pathological conditions. The presence of c-kit on HCC cell lines supports the assumption that SCF might play a role in the regulation of proliferative activity of tumorous and nontumorous hepatic cells.

Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT

BackgroundThe stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT.Methodology/Principal Findings In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC50) of 200±40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC50 of 150±80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant.ConclusionsMasitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity.

MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

Early events in kidney organogenesis involve reciprocal interactions between the ureteric bud and the metanephric mesenchyme, which lead to remodeling of the extracellular matrix. This remodeling involves matrix metalloproteases (MMPs), but the specific roles of individual MMPs in kidney development are not completely understood. Here, we analyzed MMP9-deficient mice at the first step of kidney development and found that MMP9 deficiency delayed embryonic kidney maturation and increased apoptosis ex vivo by 2.5-fold. These early defects resulted in a 30% decrease in nephron number, a 20% decrease in adult kidney weight, and altered kidney function and morphology at 12 mo. The membrane form of stem cell factor (SCF) increased, whereas the activated form of the SCF receptor, c-kit, decreased in MMP9-deficient embryonic kidneys. In organotypic culture, MMP9-deficient kidneys failed to secrete SCF, and addition of recombinant SCF partially rescued both apoptosis and the branching defect. In conclusion, these data show that MMP9 protects mesenchymal cells from apoptosis during kidney development and stimulates ureteric bud branching morphogenesis, most likely by releasing the soluble form of SCF, suggesting that normal renal development requires MMP9.

Phase II study of motesanib in Japanese patients with advanced gastrointestinal stromal tumors with prior exposure to imatinib mesylate

PurposeMotesanib (AMG 706) is a multitargeted anticancer agent with an inhibitory action on the human vascular endothelial growth factor receptor, the platelet-derived growth factor receptor, and the cellular stem-cell factor receptor (KIT). The aim of this single-arm phase II clinical study was to assess the efficacy and safety of single-agent motesanib in Japanese patients with advanced gastrointestinal stromal tumors with prior exposure to imatinib mesylate.MethodsAll patients had experienced progression or relapse while undergoing with imatinib as 400 mg/day or higher. The patients were administered 125 mg of motesanib once daily. The primary endpoint was overall response. Efficacy was evaluated according to the Response Evaluation Criteria in Solid Tumor, and safety was assessed according to the Common Terminology Criteria for Adverse Events (version 3).ResultsOf 35 enrolled and treated patients, no patient showed a complete response, and one patient showed a partial response (PR). Seven had stable disease (SD) for at least 24 months, two of whom continued to have SD for more than 2 years. The median progression-free survival time was 16.1 weeks. Motesanib was well tolerated; commonly reported treatment-related adverse events were hypertension, diarrhea, and fatigue. Anemia was the only hematological toxicity that was reported.ConclusionsOne patient showed PR, and seven patients showed SD more than 24 weeks. Motesanib was found to be safe and well tolerated. The observed toxicities were consistent with Phase I study findings.

Reply to Chimenti: c-kit cardiovascular progenitors and post-infarct myogenesis

The letter from Chimenti et al. (1) makes 2 points regarding our manuscript (2). First, they point out that their previous in vitro study of cardiospheres (3) suggested the importance of c-kit as a predictor of cardiovascular precursor status. We acknowledge this contribution, which was not referenced because of space constraints. Second, the letter references the carefully controlled conditional recombination study of Hsieh et al. (4), which provided evidence of cardiac myogenesis after heart injury or stress but not during normal aging. With respect to the findings reported in that manuscript (4), we would point out that our study (2) does not exclude post-infarct myogenesis. Rather, the data reported in our study (2) indicate that any new heart cell formation is unlikely to involve the expression of c-kit and that c-kit expression itself does not reflect myogenesis in this context, because the stem cell factor receptor is expressed within vascular cells and in committed cardiac myocytes. This finding is likely to explain the result reported by Hsieh et al. (4) that c-kit mRNA increases >5-fold within the infarct zone, which might otherwise be interpreted as providing a link between c-kit-expressing cardiovascular precursor cells and post-infarct myogenesis. In their manuscript, Hsieh et al. (4) point out that their data do not directly implicate precursor cells in cardiac repair. Our study (2) narrows the molecular characteristics of the cell pool responsible for this putative post-infarct myogenesis and more specifically clarifies the role of c-kit-expressing cardiovascular precursors in that process.

Masitinib, a c‐kit/PDGF receptor tyrosine kinase inhibitor, improves disease control in severe corticosteroid‐dependent asthmatics

Masitinib is a tyrosine kinase inhibitor targeting stem cell factor receptor (c-kit) and platelet-derived growth factor (PDGF) receptor, which are expressed on several cell types including mast cells and bronchial structural cells, respectively. We hypothesized that c-kit and PDGF receptor inhibition may decrease bronchial inflammation and interfere with airway remodeling, which are crucial features of severe asthma. The primary endpoint was the percent change from baseline in oral corticosteroids after 16 weeks of treatment. Change in asthma control (asthma control questionnaire), exacerbation rate, pulmonary function tests, rescue medication requirement and safety were secondary endpoints. A 16-week randomized, dose-ranging (3, 4.5, and 6 mg/kg/day), placebo-controlled study was undertaken in 44 patients with severe corticosteroid-dependent asthma who remained poorly controlled despite optimal asthma management. At 16 weeks of treatment, a comparable reduction in oral corticosteroids was achieved with masitinib and placebo (median reduction of -78% and -57% in the masitinib and placebo arms, respectively). Despite this similar reduction, the Asthma Control Questionnaire score was significantly better in the masitinib arm as compared to placebo with a reduction by 0.99 unit at week 16 (P < 0.001) vs 0.43 unit in the placebo arm. Masitinib therapy was associated with more transient skin rash and edema. Masitinib, a c-kit and PDGF-receptor tyrosine kinase inhibitor, may represent an innovative avenue of treatment in corticosteroid-dependent asthma. These preliminary results warrant further long-term clinical studies in severe asthma

Screening of Kit inhibitors: suppression of Kit signaling and melanogenesis by emodin

In search of novel antipigmentation agents, a set of 3,840 compounds with natural-like synthetic or natural origin were screened against Kit (stem cell factor receptor). Emodin from the seed of Cassia tora and baicalin from Scutellariae radix showed potent inhibitory effects (IC(50) = 4.9 and 9.0 microM, respectively) on the phosphorylation of Kit. Emodin also blocked other receptor tyrosine kinase activities, such as epithelial growth factor receptor (EGFR), vascular endothelial growth factor receptor 2 (VEGFR-2), fibroblast growth factor receptor 1 (FGFR-1), platelet-derived growth factor receptor b (PDGFR-b). In contrast to emodin, aloe-emodin did not inhibit Kit activity at all. Emodin also blocked the cellular kinase activities of Kit and its down-stream p44/42 mitogen activated protein kinase (MAPK) in MO7e cells and human primary melanocytes. Emodin strongly suppressed the melanin synthesis triggered by stem cell factor (SCF) treatment. Also, emodin showed almost no toxicity up to 10 microM on cultured melanocytes as reported previously by other researchers. The results indicate that emodin is a good candidate for the development of antipigmentation agents since it can radically block the differentiation and proliferation of pigment cells by reducing Kit signaling.

Erythropoietin Down-Regulates Stem Cell Factor Receptor (Kit) Expression in the Leukemic Proerythroblast: Role of Lyn Kinase

Overexpression of the transcription factor Spi-1/PU.1 by transgenesis in mice induces a maturation arrest at the proerythroblastic stage of differentiation. We have previously isolated a panel of spi-1 transgenic erythroleukemic cell lines that proliferated in the presence of either erythropoietin (Epo) or stem cell factor (SCF). Using these cell lines, we observed that EpoR stimulation by Epo down-regulated expression of the SCF receptor Kit and induced expression of the Src kinase Lyn. Furthermore, enforced expression of Lyn in the cell lines increased cell proliferation in response to Epo, but reduced cell growth in response to SCF in accordance with Lyn ability to down-regulate Kit expression. Together, the data suggest that Epo-R/Lyn signaling pathway is essential for extinction of SCF signaling leading the proerythroblast to strict Epo dependency. These results highlight a new role for Lyn as an effector of EpoR in controlling Kit expression. They suggest that Lyn may play a central role in during erythroid differentiation at the switch between proliferation and maturation.

Kit Inhibitor APcK110 Induces Apoptosis and Inhibits Proliferation of Acute Myeloid Leukemia Cells

Kit is a membrane-bound tyrosine kinase and receptor for stem cell factor (SCF) with a crucial role in hematopoiesis. Mutations of KIT occur in almost half of patients with core-binding factor leukemias, in which they have been associated with worse outcome. Development of new compounds targeting Kit may therefore hold promise for therapy. We investigated the activity and mechanism of action of APcK110, a novel Kit inhibitor, in the mastocytosis cell line HMC1.2 (KITV560G and KITD816V), acute myeloid leukemia (AML) lines OCIM2 and OCI/AML3 (both wild-type), and primary samples from patients with AML. We show that (a) APcK110 inhibits proliferation of the mastocytosis cell line HMC1.2 and the SCF-responsive cell line OCI/AML3 in a dose-dependent manner; (b) APcK110 is a more potent inhibitor of OCI/AML3 proliferation than the clinically used Kit inhibitors imatinib and dasatinib and at least as potent as cytarabine; (c) APcK110 inhibits the phosphorylation of Kit, Stat3, Stat5, and Akt in a dose-dependent fashion, showing activity of APcK110 on Kit and its downstream signaling pathways; (d) APcK110 induces apoptosis by cleavage of caspase-3 and poly(ADP-ribose) polymerase; and (e) APcK110 inhibits proliferation of primary AML blasts in a clonogenic assay but does not affect proliferation of normal colony-forming cells. Although APcK110 activity may partly depend on cytokine responsiveness (e.g., SCF) and not exclusively KIT mutation status, it remains a potent inhibitor of AML and mastocytosis cell lines and primary AML samples. APcK110 and similar compounds should be evaluated in clinical trials of patients with AML.

In Vitro Metabolism of the Novel, Highly Selective Oral Angiogenesis Inhibitor Motesanib Diphosphate in Preclinical Species and in Humans

Motesanib diphosphate is a novel, investigational, highly selective oral inhibitor of the receptor tyrosine kinases vascular endothelial growth factor receptors 1, 2, and 3, the platelet-derived growth factor receptor, and the stem cell factor receptor (Kit). The in vitro metabolic profiles of [(14)C]motesanib were examined by using microsomes and hepatocytes from preclinical species and humans. Several oxidative metabolites were observed and characterized by tandem mass spectrometry, nuclear magnetic resonance spectroscopy, and coinjection with authentic standards. Cytochrome P450 (P450) 3A4 is the major isozyme involved in the oxidative biotransformation of motesanib, but the CYP2D6 and CYP1A isozymes also make minor contributions. In hepatocyte incubations, oxidative and conjugative pathways were observed for all species examined, and indoline N-glucuronidation was the dominant pathway. Three less common and novel phase II conjugates of the indoline nitrogen were detected in hepatocytes and in microsomes supplemented with specific cofactors, including N-carbamoyl glucuronide, N-glucose, and N-linked beta-N-acetylglucosamine. An N-glucuronide metabolite was the most frequently observed phase II conjugate in liver microsomes of all species, whereas the N-acetylglucosamine conjugate was observed only in monkey liver microsomes. Incubations with recombinant human UDP-glucuronosyltransferases (UGTs) and inhibition by the UGT1A4 and UGT1A1 substrates/inhibitors imipramine and bilirubin suggested that UGT1A4 is the major UGT isozyme catalyzing the N-glucuronidation of motesanib, with a minor contribution from UGT1A1. The in vitro metabolic profiles were similar between the human and preclinical species examined. All metabolites found in humans were also detected in other species.

SCF and IL-25 induced Th2 associated pathophysiology in chronic cockroach antigen induced asthma (79.19)

Abstract Asthma is a chronic inflammatory airway disease with a nationwide prevalence of ~6%. It is associated with goblet cell metaplasia and airway hyperreactivity (AHR). Models of allergen-induced asthma exhibit increased levels of stem cell factor (SCF) and the accumulation of myeloid derived SCF receptor (c-kit)+ cells in the airway. Anti-SCF antibody treatments into the airway attenuate Th2 cytokine responses and reduce IL-25 responsiveness in CRA challenged mice in a T cell independent manner. Additional data have identified that an IL-25R+ myeloid cell accumulates in the lung during chronic allergen responses and contributes to Th2 cytokine production. When W/Wv mice were sensitized and challenged with allergen, AHR and mucous hypersecretion responses were significantly attenuated along with overall inflammatory responses. A significant reduction in IL-25R+ myeloid cells was a prominent feature that correlated with reduced Th2 cytokine responses. Subsequent experiments will further define the responses using adoptive transfer of wildtype myeloid progenitor cells to define whether the IL-25R+ myeloid cell population is reconstituted along with Th2 cytokine responses and pathophysiology. This work was supported by NIH grant HL59178

Effects of imatinib mesylate in osteoblastogenesis

Imatinib mesylate (IM), a tyrosine kinase inhibitor currently used in chronic myeloid leukemia (CML), may also affect the growth of other cellular systems besides CML cells. Because it has been reported that IM may affect bone tissue remodeling, we evaluated the effects of IM on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). After 21 days of culture, hBM-MSCs treated with IM (1 microM) alone or osteogenic medium (OM) + IM showed changes in morphology with evidence of extracellular mineralization and increased mRNA expression of osteogenic markers, such as RUNX2, osteocalcin (OCN), and bone morphogenetic protein (BMP-2). We also observed that levels of OCN and the osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL) ratio (OPG/RANKL ratio) were increased in the surnatant of the 21-day culture with IM or OM + IM compared to controls (p<0.005). In addition, we found that in 46 serum samples collected from CML patients treated with IM for 3 to 24 months, the OPG/RANKL ratio increased after 3 and 6 months (p<0.004) returning back to the basal level after 24 months of IM treatment. In these patients, OCN levels were low at diagnosis but they increased throughout the IM treatment, approaching normal levels at 24 months of IM therapy. In summary, our data show that IM increases mRNA expression of osteogenic markers in hBM-MSCs and increases the OPG/RANKL ratio and the OCN levels both in surnatant of hBM-MSCs cultured with IM and in serum of patients treated with IM, thus indicating that IM potentially favors osteoblastogenesis.

D011 Mesenchymal stem cells engineered to overexpress stem cell factor improve cardiac function but have malignant potential

Myocardial infarction (MI) in mice with mutations in the stem cell factor (SCF) receptor causes rapid heart failure. Implantation of mesenchymal stem cells (MSC) slows progression to heart failure after MI. We hypothesized that MSC engineered to over-express SCF would restore cardiac function better than unmodified MSC. MSC from C57Bl/6 mice bone marrow were transfected with SCF cDNA cloned into pcDNA-3, and a stable transfectant was selected in G418 medium. C57Bl/6 mice (N) underwent coronary ligation and received medium (M), or 3×105 MSC (C) or MSC transfected with SCF (CS) into the anterior left ventricle. Cardiac function was assessed by pressure-volume (PV) loops on day 28 (n=10 per group). Hearts were perfusion-fixed in situ and cut into transverse sections for computerized planimetry (n = 5 per group). When measured in vivo, myocardial SCF increased 2.9-fold in M, 5.5-fold in C, and 17.3-fold in SC groups compared to the N group (P<0.05). All analysis showed significant improvement of post MI ventricular size and function in cell groups compared to M group. PV loop analysis showed a 1.9-fold increase (P < 0.001) in the slope of preload-recruitable stroke work in CS group compared with C group. The improvement in end-systolic elastance was similar to the improvement in preload-recruitable stroke work. In addition, the left ventricular volume was 0.6-fold smaller (P<.01) in the CS group compared with the C group. However 4 out of 20 survivors in CS group developed fibrosarcomas infiltrating into the cell transplanted area within 3 months of cell implantation. Use of genetically modified MSC over-expressing SCF better prevented ventricular dilation and restored cardiac function following MI. Unfortunately, this approach was associated with tumorogenesis and our data strongly suggests that malignant transformation was caused by the specific combination of techniques that we used to generate our cell line.

Differential Effect of Imatinib and Synergism of Combination Treatment with Chemotherapeutic Agents in Malignant Glioma Cells

Imatinib mesylate (STI571, Gleevec) is a signal transduction inhibitor and novel anti-cancer agent. It selectively inhibits aberrantly activated tyrosine kinases in malignant cells, for example, bcr-abl in leukaemia, platelet-derived growth factor receptor and stem cell factor receptor (c-Kit) in solid cancers including malignant glioma. However, recently published clinical studies with imatinib monotherapy in patients with malignant glioma demonstrated only very modest anti-tumour activity. The aim of this study was to investigate the biological activity of imatinib, its cellular mechanisms of action and its synergism with other chemotherapeutic agents in human malignant glioma cells in culture. Expression of PDGF/R and c-Kit was analyzed by RT-PCR. Proliferation was measured by MTT assays and drug synergy was assessed by the Chou-Talalay method. Cell cycle and apoptosis were analyzed by flow cytometry and migration by monolayer migration assays. Multi-immunoblot was performed on imatinib-treated and control malignant glioma cells. Results indicate that imatinib is more effective in inhibiting cell colony formation and migration rather than proliferation. Imatinib treatment caused cell cycle arrest of glioma cells in G0-G1 or G2/M, with significant elevation of a few cyclin-dependent kinases. Furthermore, imatinib acted synergistically with chemotherapy agents, such as the DNA alkylating agent, temozolomide, and riboneucleotide reductase inhibitors, for example, hydroxyurea at varied effective dose levels. In conclusion, imatinib exerts varied biological effects on malignant glioma cells in culture. Synergistic interaction of imatinib with chemotherapy agents may be related to cell cycle control mechanisms and could be potentially important in a clinical setting.

MicroRNA-221 regulates high glucose-induced endothelial dysfunction

Persistent hyperglycemia in diabetes causes endothelial cell dysfunction. Exposure to high levels of glucose, which mimics hyperglycemia, induced expression of microRNA 221 (miR-221) but reduced expression of c-kit, the receptor for stem cell factor in human umbilical vein endothelial cells (HUVECs). In addition, high glucose treatment impaired endothelial cell migration. Incubation with the antisense miR-221 oligonucleotide AMO-221 reduced expression of miR-221 and restored c-kit protein expression in HUVECs treated with high levels of glucose. Furthermore, AMO-221 treatment abolished the inhibitory effect of high glucose exposure on HUVECs transmigration. Thus, under hyperglycemic conditions, miR-221 is induced in HUVECs, which consequently triggers inhibition of c-kit and impairment of HUVECs migration. These findings suggest that manipulation of the miR-221-c-kit pathway may offer a novel strategy for treatment of vascular dysfunction in diabetic patients.

Green fluorescent protein transgene driven by Kit regulatory sequences is expressed in hematopoietic stem cells

The transcriptional regulation of stem cell genes is still poorly understood. Kit, encoding the stem cell factor receptor, is a pivotal molecule for multiple types of stem/progenitor cells. We previously generated mouse lines expressing transgenic green fluorescent protein under the control of Kit promoter/first intron regulatory elements, and we demonstrated expression in hematopoietic progenitors. In the present work we investigated whether the transgene is also expressed in hematopoietic stem cells of adult bone marrow and fetal liver. To this purpose, we tested, in long-term repopulating assays, cell fractions expressing different levels of green fluorescent protein within Kit-positive or SLAM-selected populations. The experiments demonstrated transgene expression in both fetal and adult hematopoietic stem cells and indicated that the transgene is transcribed at distinctly lower levels in hematopoietic stem cells than in pluripotent and committed progenitors. These results, together with previous data, show that a limited subset of DNA sequences drives gene expression in number of stem cell types (hematopoietic stem cells, primordial germ cells, cardiac stem cells). Additionally, our results might help to further improve high level purification of hematopoietic stem cells for experimental purposes. Finally, as the Kit/green fluorescent protein transgene is expressed in multiple stem cell types, our transgenic model provides powerful in vivo system to track these cells during development and tissue regeneration.

The stem cell factor–c-KIT pathway must be inhibited to enable apoptosis induced by BCR–ABL inhibitors in chronic myelogenous leukemia cells

Imatinib is an effective first-line therapy for chronic myelogenous leukemia (CML) that acts by targeting the tyrosine kinase activity of BCR-ABL. To overcome resistance, second-generation inhibitors of BCR-ABL have been developed. Among these, nilotinib is more potent against BCR-ABL than imatinib, and is effective against many imatinib-resistant BCR-ABL mutants. In this study, an in vitro flow cytometry assay to analyze imatinib- and nilotinib-induced apoptosis in CML cells has been developed. Both the drugs induced significant apoptosis in CD34+ cells from 36 CML bone marrow samples (P<10(-4)), whereas CD34+ cells from BCR-ABL negative samples were unaffected. When the experiments were carried out in the presence of a cocktail of cytokines, nilotinib- but not imatinib-induced apoptosis was inhibited. This differential inhibition was confirmed on K562 cells. A blocking anti-CD117 antibody alleviated the antiapoptotic effect of cytokines against nilotinib. Moreover, using short hairpin RNA against BCR-ABL, we showed that K562 cells were not dependent on BCR-ABL signaling as long as the stem cell factor (SCF) receptor pathway was activated. We conclude that the c-KIT pathway may substitute for BCR-ABL tyrosine kinase to activate survival signals, and that c-KIT must be inhibited besides Bcr-Abl to allow apoptosis of CML cells.

NAD(P)H oxidase isoform Nox2 plays a prosurvival role in human leukaemia cells

The mechanism involved in the prosurvival effect of interleukin-3 on the human acute myeloid leukaemia cell line M07e is investigated. A decrease in intracellular reactive oxygen species (ROS) content, glucose transport activity and cell survival was observed in the presence of inhibitors of plasma membrane ROS sources, such as diphenylene iodonium and apocynin, and by small interference RNA for Nox2. Moreover, IL-3 incubation stimulated the synthesis of Nox2 cytosolic sub-unit p47phox and glucose transporter Glut1. Thus, the inhibition of ROS generation by Nox inhibitors stimulated apoptosis showing that ROS production, induced by IL-3 via Nox2, protects leukaemic cells from cell death. Also incubation with receptor tyrosine kinase inhibitors, such as anti-leukaemic drugs blocking the stem cell factor receptor (c-kit), showed similar effects, hinting that IL-3 transmodulates c-kit phosphorylation. These mechanisms may play an important role in acute myeloid leukaemia treatment, representing a novel therapeutic target.

Phenotypic Characterization of Human CD34+ Cells Selected from G-CSF Mobilized Leukopheresis Using Baxter's Isolex™ 300i Magnetic Cell Selection System (v2.5)

Baxter's Isolex™ 300i Magnetic Cell Selection System (v2.5) is used to select CD34+ cells from G-CSF mobilized leukopheresis units. This study focused on evaluating the cellular composition of the post-Isolex product as well as producing a detailed characterization of the CD34+ cells. A multi-color flow cytometry assay was developed to characterize the Isolex product with a detailed focus on the CD34+ cells. A hematopoietic colony-forming assay was used to determine the clonal potential of the selected cells. Cell function was evaluated by measuring the migration potential of the CD34+ cells toward SDF-1. The post-Isolex cell suspension product contained an average of 96.44% ± 0.84% CD34+ cells. The majority of the remaining cells present were T and B cell lymphocytes with an average 1.74% ± 0.78% CD3 positive T-Cells and 1.61% ± 0.23% CD19 positive B-Cells. The platelets comprised 1.92% ± 0.92% of the product and 99% of this population were co-aggregated to the CD34+ cells. Non-CD34, immature white blood cells averaged less than 0.5% of the total post-Isolex population. A majority of the CD34+ stem cells are lineage-committed HPCs as determined by KDR-CD34+ (94.90% ± 1.99%) and CD38+CD34+ (93.87% ± 2.76%). Commitment to a myeloid cell line (CD33+CD34+) was determined to be 34.75% ± 23.10%. Migration potential of the stem cells toward SDF-1 as defined by dual positivity for CXCR4 and CD34 averaged 1.95% ± 2.49%. While the CXCR4 receptor positivity appears low, the migratory response of the CD34+ cells toward SDF-1 was robust. An examination of VEGF populations in the post-Isolex product averaged 0.32% ± 0.17% for VEGF R2 (KDR) and 34.25% ± 12.74% for VEGF R1. Several adhesion markers were assayed and the positivity was equal to or less than 1% for CD146, CD144, CD73, and CD29. Other adhesion markers, such as CD105 (endoglin) averaged 34.04% ± 20.64% and CD99 averaged 66.33% ± 13.25%. CD117, which binds to the receptor for stem cell factor (SCF), averaged 69.63% ± 6.38%. CD133, which is considered an early HPC marker, averaged 73.90% ± 5.97%. CD90, which is useful for defining a population of highly proliferative cells capable of long-term culture, averaged 60.50% ± 8.50%. The EPC phenotypic profile combination, KDR+CD34+CD133+, averaged 0.30% ± 0.22% in our post-Isolex population. In the hematopoietic colony-forming assay, the Isolex-selected CD34+ cells were able to produce colonies with an efficiency of 20% with CFU-GM forming a majority of the colonies. By utilizing multi-color flow cytometry, we were able to account for the cell populations found in the Isolex product and to further characterize the CD34+ selected cell population. The hematopoietic colony-forming assay gave insight into the efficiency and growth potential of the CD34+ selected cells. The functional migration assay determined that the CD34+ selected cells exhibited a robust migratory response toward SDF-1.

Stem cell factor receptor KIT (CD117) in aseptic hip prosthesis loosening.

This study aimed to investigate specific inflammatory pathomechanisms, i.e. the expression of the stem cell factor receptor KIT (CD117) in tissue specimens from patients with aseptic hip prosthesis loosening (AHPL) with special emphasis on colocalization with the mast cell specific marker tryptase. Immunohistochemical analysis of CD117 was performed in tissue specimens from 10 patients with aseptically loosened acetabular components of failed non-cement total hip replacements and compared to control samples obtained at primary hip surgery (n=4). The CD117 expressing cells were characterized further with mast cell tryptase (MCT) by serial section analysis and a double staining method. CD117 and MCT expression was examined by semi-quantitative analysis. Additionally, double labeling of the CD117 or MCT expression by immunohistochemistry and of polyethylene (PE) particles by Oil Red reaction was performed. In AHPL, CD117 was almost exclusively detected in MCT positive cells. Co-expression tended to be highly correlated (r=0.86, p<0.01). CD117 was found mainly in two regions: first, in perivascular lymphocyte-rich areas and; secondly, near macrophages and multinucleated giant cells (MGC). PE particles were not detected in CD117 and MCT positive cells. In control samples, CD117/MCT positive cells were less frequent. This is the first report on CD117 expression in AHPL. CD117 is almost exclusively expressed in a distinct mast cell subgroup. As an important growth factor receptor, CD117 could play a major role in recruitment and activation of mast cells in AHPL. Furthermore, mast cells do not contain significant amounts of PE particles. However, it remains to be investigated whether this cell population could influence phagocytosis of PE particles. (Journal of Applied Biomaterials and Biome-chanics 2005; 3: 11-7).