Stem Cell Factor C-kit Research Articles

0130 : Is the stem cell antigen 1 involved in the brain natriuretic peptide effect on cardiac precursor cells?

Cardiac cellular therapy can be intended by stimulation of “endogenous” cardiac precusor cells (CPCs), identified by several markers such as the Stem Cell Antigen 1 (Sca-1) or the stem cell factor c-kit. CPCs are able to proliferate and differentiate into beating cardiomyocytes in vivo and in vitro after brain natriuretic peptide (BNP) treatment. BNP is a cardiac hormone, whose role in the heart remains to be clarified. Interestingly, BNP treatments, in vivo and in vitro , induced an upregulation of mRNA coding for Sca-1 and a 2 fold increase of the number of CPCs. In previous work, we showed that Sca-1 KO mice had two times less CPCs, and two times less BNP mRNA expression compared to Wild Type mice. The aim of this project is to determine whether the effect of BNP on CPCs is linked to Sca-1 expression. Sca-1 KO mice developed a dilated cardiomyopathy with ageing. To determine whether reduced amount of BNP is responsible for the development of this cardiac disease, neonatal Sca-1 KO mice were treated with BNP for 2 weeks. Mice were sacrificed 13 weeks later, and cardiac parameters were measured by echocardiography. The thickness of the left ventricular posterior wall and the ejection fraction were increased in BNP-treated Sca-1 deficient mice (+15% and + 28%, respectively) compared to untreated Sca-1 KO mice. To explore the cellular mechanisms, in vitro experiments were performed. Non Myocytes cells (NMCs), containing CPCs, have been isolated from the heart of Sca-1 KO mice and cultured with or without BNP in media favouring either cell proliferation or differentiation into cardiomyocytes. BNP is not able to induce Sca-1KO cell proliferation, whereas CPC differentiation into cardiomyocytes is maintained even in absence of Sca-1 expression. In conclusion, BNP modulates the cardiomyopathy developed by Sca-1 KO mice. BNP effect on CPC proliferation is linked to Sca-1 expression, but its effect on differentiation is Sca-1 independent.

Transmyocardial Revascularization Enhances Bone Marrow Stem Cell Engraftment in Infarcted Hearts Through SCF-C-kit and SDF-1-CXCR4 Signaling Axes.

We investigated the roles of stem cell factor (SCF)-c-kit and stromal derived factor-1 (SDF-1)-CXCR4 signaling axes in transmyocardial revascularization (TMR)-enhanced engraftment of transplanted bone marrow stem cells (BMSCs) in infarcted hearts. 3weeks after LAD ligation, female Lewis rats underwent 10-channel needle-TMR, followed by daily IV injections of 1 million male donor BMSC for 5days, either wild type (WT) or with knockdown (K/D) of c-kit or CXCR4, accomplished via a shRNA + plasmid in a lentiviral vector (N = 6/group). In our rat infarct model, 3days after last BMSC injection, the number of BMSCs that homed into infarct was affected by both TMR and donor cell type, with greater BMSC engraftment with TMR and with WT BMSC (TMR, cell type, and interaction, P < 0.05). At 1week, these differences persisted (TMR and cell type, P < 0.05). At 3days, TMR significantly upregulated transcription of c-kit (TMR, p < 0.05), SCF (TMR and cell type, P < 0.05), CXCR4 (TMR and cell type, p < 0.05), and SDF-1 (TMR and cell type, P < 0.05). At 1week, we saw similar declines in expression of c-kit (cell type, P < 0.05), SCF (TMR, P < 0.05), CXCR4 (TMR and cell type, P < 0.05), and SDF-1 (TMR, P < 0.05). At 1week, TMR improved LV ejection fraction (LVEF) (N = 5) when WT BMSCs were infused, but knockdown of either c-kit or CXCR4 completely abrogated this TMR-mediated augmentation of BMSC reparative effect (TMR and cell type, P < 0.05). Downregulation of either c-kit or CXCR4 in BMSC decreased engraftment of circulating BMSC and inhibited reparative effects of TMR.

Transmyocardial Revascularization Enhances Mesenchymal Stem Cell Engraftment in Infarcted Hearts through SCF–c-Kit and SDF-1–CXCR4 Signaling Axes

Purpose We investigated the roles of stem cell factor(SCF)–c-kit and stromal derived factor-1(SDF-1)–chemokine receptor type 4(CXCR4) stem cell signaling axes in transmyocardial revascularization (TMR)-enhanced engraftment of transplanted mesenchymal stem cells (MSC) in infarcted hearts. Methods and Materials 3 weeks after LAD ligation, female Lewis rats underwent 10-channel needle TMR, followed by daily IV injections of 1 million male donor MSC for 5 days, either wild type (WT) or with knockdown (K/D) of c-kit or CXCR4, accomplished via a shRNA+plasmid in a lentiviral vector. Experimental groups included: WT MSC with or without TMR, c-kit K/D MSC with or without TMR, and CXCR4 K/D MSC with or without TMR (N=6/group). Results In vitro cell surface expression of c-kit (N=3) was reduced from 14±0.7% of WT MSC to 1.6±0.4% c-kit K/D MSC, and CXCR4 (N=3) was reduced from 39±10% WT MSCs to 3.7±0.7% CXCR4 K/D MSC after 1 week (p Conclusions Downregulation of either c-kit or CXCR4 in MSC decreased engraftment of circulating MSC and inhibited the reparative effects of TMR. Hence, both SCF–c-kit and SDF-1–CXCR4 signaling axes are required for TMR-augmented repair of the infarcted heart.

Elimination of Human Lung Cancer Stem Cells through Targeting of the Stem Cell Factor–c-kit Autocrine Signaling Loop

Cancer stem cells (CSC) are thought to be responsible for tumor initiation and tumor regeneration after chemotherapy. Previously, we showed that chemotherapy of non-small cell lung cancer (NSCLC) cells lines can select for outgrowth of highly tumorigenic and metastatic CSCs. The high malignancy of lung CSCs was associated with an efficient cytokine network. In this study, we provide evidence that blocking stem cell factor (SCF)-c-kit signaling is sufficient to inhibit CSC proliferation and survival promoted by chemotherapy. CSCs were isolated from NSCLC cell lines as tumor spheres under CSC-selective conditions and their stem properties were confirmed. In contrast to other tumor cells, CSCs expressed c-kit receptors and produced SCF. Proliferation of CSCs was inhibited by SCF-neutralizing antibodies or by imatinib (Gleevec), an inhibitor of c-kit. Although cisplatin treatment eliminated the majority of tumor cells, it did not eliminate CSCs, whereas imatinib or anti-SCF antibody destroyed CSCs. Significantly, combining cisplatin with imatinib or anti-SCF antibody prevented the growth of both tumor cell subpopulations. Our findings reveal an important role for the SCF-c-kit signaling axis in self-renewal and proliferation of lung CSCs, and they suggest that SCF-c-kit signaling blockade could improve the antitumor efficacy of chemotherapy of human NSCLC.

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.

The Stem Cell Factor-cKIT Pathway Must Be Inhibited to Enable Apoptosis Induced by Bcr-Abl Inhibitors in Chronic Myelogenous Leukaemia Cells

Imatinib is an effective first line therapy for early chronic phase chronic myelogenous leukaemia (CML) that acts by targeting the tyrosine kinase activity of BCR-ABL. However, mutations occurred that procure imatinib resistance and, to overcome this, second generation inhibitors of BCR-ABL have been developed. Among these, nilotinib is significantly more potent against BCR-ABL than imatinib but is used in the same order of concentration as imatinib in vivo. Moreover, nilotinib is more specific towards BCR-ABL than imatinib which, at efficient concentration, also inhibits c-KIT. In the current study an in vitro flow cytometry assay to analyze imatinib- and nilotinib-induced apoptosis in CML cells has been developed. Both drugs induced significant apoptosis in CD34 positive cells from 36 CML bone marrow samples collected at diagnosis while CD34 positive cells from 12 other myeloproliferative disorders samples were unaffected, confirming the high specificity of both inhibitors on BCR-ABL positive cells (p<10−9). Nilotinib was confirmed to be 100 times more potent than imatinib to induce apoptosis of CD34 positive cells. When the experiments were performed in the presence of a cocktail of cytokines, inhibitions of 26% for spontaneous apoptosis, 22% for imatinib-induced apoptosis (n=20, p<0.005) and 71% for nilotinib-induced apoptosis (p<10−6) were observed. The inhibition essentially occurred in CD117+/CD34+ cells. This differential inhibition by cytokines was confirmed on K562 cells (34% for imatinib and 71% for nilotinib). A blocking anti-CD117 antibody alleviated the anti-apoptotic effect of cytokines against nilotinib. Moreover, using shRNA against BCR-ABL, we demonstrated that K562 cells were not dependant of BCR-ABL expression as long as the 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 beside BCR-ABL to allow apoptosis of CML cells.

Clinicopathological study on cholangiolocellular carcinoma suggesting hepatic progenitor cell origin

Cholangiolocellular carcinoma (CLC), a subtype of cholangiocellular carcinoma (CC), is thought to originate from the ductules/canals of Hering, where hepatic progenitor cells (HPCs) are located. We investigated the clinicopathological features of 30 CLCs and their relationship to HPCs. We evaluated the expression of hepatocytic markers (hepatocyte paraffin-1, canalicular polyclonal carcinoembryonic antigen, and CD10), biliary/HPC markers (keratin [K]7, K19, and neural cell adhesion molecule), the adenosine triphosphate binding cassette transporters: multidrug resistance protein 1, multidrug resistance-associated protein (MRP)1, MRP3, and breast cancer resistance protein, using immunohistochemistry and electron microscopy. In addition, gene expression profiling of CLC was performed and compared with the profile of hepatocellular carcinoma (HCC) with or without HPC features (K19 expression). In surrounding nontumoral tissue, K7-positive and K19-positive HPCs/ductular reaction were observed. More than 90% of the tumor was composed of CLC areas that showed small monotonous and/or anastomosing glands, strongly positive for K7 and K19. Especially at the tumor boundary, all cases showed a HCC-like trabecular area characterized by canalicular CD10/polyclonal carcinoembryonic antigen expression, and submembranous K7 expression, similar to intermediate hepatocytes. K7-positive/K19-positive HPCs were also seen. Out of 30 cases, 19 showed papillary and/or clear glandular formation with mucin production, representing CC areas. These three different areas showed transitional zones with each other. We observed an increased expression of MRP1, MRP3, and breast cancer resistance protein in the tumor. Electron microscopy findings in HCC-like trabecular areas confirmed the presence of HPCs and intermediate hepatocytes. HPC markers, K7, K19, prominin-1, receptor for stem cell factor c-kit, octamer-4 transcription factor, and leukemia inhibitory factor were upregulated (P < 0.05), while albumin was downregulated in CLC (P = 0.007) toward K19-negative HCCs. Comparison of CLC with K19-positive HCCs indicated a high homology. All these findings highly suggest a progenitor cell origin of CLC.

Melanocyte fate in neural crest is triggered by Myb proteins through activation of c-kit.

The c-myb proto-oncogene and its oncogenic derivative v-mybAMV encode transcriptional regulators engaged in the commitment of hematopoietic cells. While the c-Myb protein is important for the formation and differentiation of various progenitors, the v-MybAMV oncoprotein induces in chicks a progression and transformation of the single (monoblastic) cell lineage. Here we present the first evidence of cell fate-directing abilities of c-Myb and v-MybAMV proteins in avian neural crest (NC), where both proteins determine melanocytogenesis. The increased concentration of c-Myb induces progression into dendritic melanocytes and differentiation. The v-myb oncogene converts essentially all NC cells into melanocytes and causes their transformation. Both Myb proteins activate in NC cells expression of the c-kit gene and stem cell factor c-Kit signaling--one of the essential pathways in melanocyte development. These observations suggest that the c-myb-c-kit pathway represents a common regulatory scheme for both hematopoietic and neural progenitors and establishes a novel experimental model for studies of melanocytogenesis and melanocyte transformation.

Susceptibility to Basal Cell Carcinoma is Associated with High Dermal Mast Cell Prevalence in Non-sun-exposed Skin for an Australian Population¶

In a Danish population, basal cell carcinoma (BCC) patients have a higher dermal mast cell prevalence in buttock skin than controls. This finding was supported by a functional link in mice between histamine-staining dermal mast cells and the extent of susceptibility to UV-B–induced systemic immunomodulation. It was important to confirm that this association was maintained in an Australian population with very different ancestry and sun exposure patterns. Australian BCC patients (n = 26) had significantly higher densities of mast cells in the dermis of buttock skin than control subjects (n = 25) (P= 0.0003, Mann–Whitney U-test). However, this correlation was lost at the sun-exposed site of the hand (P= 0.547, Mann–Whitney U-test). To further evaluate whether a relationship exists between dermal mast cell prevalence in sun-exposed skin and incidence of BCC in a larger study, biopsies of dorsal hand skin were obtained from an age-stratified random sample of 166 Queensland subjects, together with the 51 South Australian subjects, and dermal mast cell prevalence was quantified. Older subjects (over the median age of 42 years) had a greater incidence of BCC development (P= 0.0001, chi-square test) and significantly higher mast cell densities in hand skin (P= 0.0001, chi-square test) than younger subjects. However, mast cell density in sun-exposed hand skin was not significantly associated with BCC incidence. Finally, cellular expression of c-kit correlated with mast cell prevalence in non–sun-exposed skin, thereby implicating the stem cell factor–c-kit axis in the intrinsic mechanisms that regulate prevalence. These results show that high prevalence of dermal mast cells in buttock skin but not hand is associated with BCC development in an Australian population.

Acute Renal Failure Secondary to Imatinib Mesylate Treatment in Prostate Cancer

To report a case of acute renal failure associated with the administration of imatinib mesylate. A 64-year-old man diagnosed with prostate cancer was enrolled in a Phase I trial of imatinib mesylate plus taxotere on a protocol that required a run-in period of imatinib mesylate alone. During therapy with imatinib mesylate, the patient developed acute renal failure, requiring hemodialysis. A renal biopsy revealed tubular vacuolization. Renal failure resolved with cessation of imatinib mesylate. Imatinib mesylate is a protein tyrosine kinase inhibitor that inhibits the BCR-ABL tyrosine kinase, the receptor tyrosine kinases for platelet-derived growth factor, and stem cell factor c-kit. Prostate cancer has been identified as a target for therapy with imatinib mesylate. This patient had no other confounding factors for the cause of the renal failure. An objective causality assessment determined that imatinib mesylate was the probable cause of the acute renal failure. The presence of a primary glomerular disease was excluded by biopsy. Imatinib mesylate-induced acute renal failure has now been linked to toxic effects on renal tubular cells in 3 cases. Renal function should be closely monitored during imatinib mesylate therapy.

Expression of neuronal nitric oxide synthase (nNOS) and nitric-oxide-induced changes in cGMP in the urothelial layer of the guinea pig bladder

The urothelium plays a sensory role responding to deformation of the bladder wall; this involves the release of adenosine trisphosphate (ATP) and nitric oxide (NO), which affect afferent nerve discharge and bladder sensation. The urothelial cells responsible for producing ATP and NO and the cellular targets, other than afferent nerves, for ATP and NO remain largely unexplored. Sub-urothelial interstitial cells (SU-ICs) lie immediately below the urothelium and respond to NO with a rise in cGMP. To determine which cells might target SU-ICs by producing NO, areas of dome, lateral wall and base wall were treated with isobutyl-methyl-xanthine, exposed to the NO donor diethylamino NONOate and then fixed for immunohistochemistry. Surface urothelial cells (SUCs) in the base and dome expressed neuronal nitric oxide synthase (nNOS), whereas those in the lateral wall did not. Distinct populations of SUCs were present in the bladder base. SUCs with significant amounts of nNOS lay adjacent to cells with low levels of nNOS. In specific base regions, the few SUCs present contained nNOS within discrete intracellular particles. In the basal urothelial cell (BUC) layer of the lateral wall, nNOS-positive (NOS(+)) BUCs neither showed an elevation in cGMP in response to NO, nor expressed the beta1 sub-unit of soluble guanylate cyclase, protein kinase I or protein kinase II. Thus, they produced but did not respond to NO. The BUC layer also stained for the stem cell factor c-Kit suggesting its involvement in urothelial cell development. No NOS(+) BUCs were present in the SUC-sparse region in the bladder base. Exogenous NO produced an elevation in cGMP in SUCs and SU-ICs. The distribution and proportion of these target cells varied between the dome, lateral wall and base. cGMP(+) SU-ICs were present as a dense layer in the bladder base but were rarely seen in the lateral wall, which contained nNOS(+) BUCs. No nNOS(+) BUCs and cGMP(+) SU-ICs were apparent in the dome. The degree of complexity in nNOS distribution and NO target cells is therefore greater than has previously been described and may reflect distinct physiological functions that have yet to be identified.

Increment of murine spermatogonial cell number by gonadotropin-releasing hormone analogue is independent of stem cell factor c-kit signal.

Recent studies have demonstrated that GnRH-analogues can stimulate regeneration of spermatogenesis of rats when administered after testicular damages. Although the mechanism of this phenomenon has not been elucidated yet, stem cell factor (SCF) produced by Sertoli cells was proposed to mediate the effects of GnRH-analogues on spermatogonial proliferation and/or survival. In the present study, we quantitatively evaluated the proliferation of spermatogonia and addressed whether SCF mediates the effect of GnRH-analogue on spermatogonial proliferation, using a novel approach combining spermatogonial transplantation and laser confocal microscopic observation. In the first experiment, using wild-type mice as recipients for spermatogonial transplantation, the number of donor spermatogonia per 100 Sertoli cells in each spermatogenic colony was significantly higher in the experimental group of mice treated with leuprorelin, a GnRH-agonist, than that of the control group at 4 and 5 wk after transplantation. In the second experiment, Steel/Steeldickie (Sl/Sld) mutant mice, which lack expression of membrane bound form SCF, were used as recipients. As seen in the first experiment, the number of undifferentiated spermatogonia was significantly higher in leuprorelin-treated than in the control group. Since undifferentiated spermatogonia do not express the receptor of SCF, the present study clearly demonstrates that neither membrane-bound nor secreted forms of SCF are involved in the mechanism of GnRH-analogue's effect on spermatogonial proliferation and/or survival.

The Stem Cell Factor–c-kit System and Mast Cells in Human Pancreatic Cancer

Stem cell factor (SCF) and its receptor c-kit take part in the regulation of developmental processes of mast cells, hematopoietic stem cells, and melanocytes, as well as in the growth control of human malignancies. To explore the possible role of the SCF–c-kit system and of mast cells in pancreatic cancer, the concomitant expression and distribution of the two molecules were examined in 17 normal and 26 cancerous human pancreatic tissues and in 6 cultured pancreatic cancer cell lines. Mast cell distribution was also evaluated in the same tissue samples. In addition, the effects of SCF and of the c-kit tyrosine-kinase inhibitor STI571 on the growth of the cancer cell lines and of the normal pancreatic ductal cell line TAKA-1 were assessed. SCF immunoreactivity was absent in acinar, ductal, and islet cells of the normal pancreas and faint in pancreatic cancer tissues and cell lines. In contrast, c-kit was clearly present in some normal and hyperplastic ducts of the normal pancreas, in the cancer cells of 73% of the tumor samples, and in all the cell lines tested. Mast cells, identified by tryptase and chymase immunostaining on consecutive tissue sections, showed immunoreactivity for SCF and c-kit in both normal and cancerous specimens and their number was significantly increased (p = 0.03) in pancreatic cancer compared with the normal pancreas. SCF showed a dose-dependent growth inhibitory effect on TAKA-1 cells (p < 0.001), whereas pancreatic cancer cells were resistant to the SCF-induced growth inhibition. Nonetheless, the growth of TAKA-1 cells and pancreatic cancer cells was inhibited by the c-kit tyrosine kinase inhibitor STI571. In conclusion, the SCF–c-kit system, possibly with the contribution of mast cells, may have a growth-regulating role in the normal pancreas, which is altered during malignant transformation.

Zinc-finger transcription factor Slug contributes to the function of the stem cell factor c-kit signaling pathway

The stem cell factor c-kit signaling pathway (SCF/c-kit) has been previously implicated in normal hematopoiesis, melanogenesis, and gametogenesis through the formation and migration of c-kit(+) cells. These biologic functions are also determinants in epithelial-mesenchymal transitions during embryonic development governed by the Snail family of transcription factors. Here we show that the activation of c-kit by SCF specifically induces the expression of Slug, a Snail family member. Slug mutant mice have a cell-intrinsic defect with pigment deficiency, gonadal defect, and impairment of hematopoiesis. Kit(+) cells derived from Slug mutant mice exhibit migratory defects similar to those of c-kit(+) cells derived from SCF and c-kit mutant mice. Endogenous Slug is expressed in migratory c-kit(+) cells purified from control mice but is not present in c-kit(+) cells derived from SCF mutant mice or in bone marrow cells from W/W(v) mice, though Slug is present in spleen c-kit(+) cells of W/W(v) (mutants expressing c-kit with reduced surface expression and activity). SCF-induced migration was affected in primary c-kit(+) cells purified from Slug-/- mice, providing evidence for a role of Slug in the acquisition of c-kit(+) cells with ability to migrate. Slug may thus be considered a molecular target that contributes to the biologic specificity to the SCF/c-kit signaling pathway, opening up new avenues for stem cell mobilization.