C-kit Ligand Research Articles

The orphan nuclear receptor Ear-2 (Nr2f6) is a novel negative regulator of T cell development

We describe a novel role for the orphan nuclear receptor Ear-2 in regulating T cell development. Retrovirus-mediated overexpression of Ear-2 (EAR-2++) in a bone marrow (BM) transplantation assay resulted in limited T cell development and a greater than tenfold decrease in thymus size and cellularity relative to controls. Ear-2-transduced murine BM hematopoietic stem cells (HSCs) in OP9-DL1 cultures showed a proliferation deficit during days 1-5 after induction of differentiation, which corresponded to increased expression of the cell cycle regulators p21 (cdkn1a) and p27 (cdkn1b), as well as increased expression of Hes1, Notch3, Egr1, and Scl (Tal1) and decreased expression of Gli1, Gfi-1, HoxA9, PU.1, Nrarp, and Tcf1. In addition, there was a block in differentiation at the DN4 to double-positive (DP) transition accompanied by an increase in apoptosis, similar to the deficit seen in the RORγt null mouse. Gene expression profiling revealed that, like the RORγt-deficient mouse, EAR-2++ DP cells had decreased expression of BclXL and increased expression of the proapoptosis gene Bad. In addition, EAR-2++ DP cells had decreased expression of Bcl11b, PU.1, and HoxA9, and increased expression of Id2. Based on these findings, we conclude that EAR-2++ cells were able to migrate to, but not fully repopulate, the thymus because of a cell-intrinsic defect in the proliferation of DN1 cells followed by a block in differentiation from the DN4 to DP stage of T cell development. We conclude that Ear-2 is a novel negative regulator of T-cell development and that downregulation of Ear-2 is indispensable for the proliferation of DN1 cells and the survival of DN4-DP cells.

Histamine synthesis is required for granule maturation in murine mast cells

Mast cells are the major sources of histamine, which is released in response to immunological stimulations. The synthesis of histamine is catalyzed by histidine decarboxylase (HDC). Previous studies have shown that Hdc(-/-) mast cells exhibit aberrant granule morphology with severely decreased granule content. Here, we investigated whether the histamine synthesized in mast cells regulates the granule maturation of murine mast cells. Several genes, including those encoding granule proteases and enzymes involved in heparin biosynthesis, were downregulated in Hdc(-/-) peritoneal mast cells. Impaired granule maturation was also found in Hdc(-/-) BM-derived cultured mast cells when they were cocultured with fibroblasts in the presence of c-kit ligand. Exogenous application of histamine and several H4 receptor agonists restored the granule maturation of Hdc(-/-) cultured mast cells. However, the maturation of granules was largely normal in Hrh4(-/-) peritoneal mast cells. Depletion of cellular histamine with tetrabenazine, an inhibitor of vesicular monoamine transporter-2, did not affect granule maturation. In vivo experiments with mast cell deficient Kit(W) /Kit(W-v) mice indicated that the expression of the Hdc gene in mast cells is required for granule maturation. These results suggest that histamine promotes granule maturation in mast cells and acts as an proinflammatory mediator.

Development of an Efficient Targeted Cell-SELEX Procedure for DNA Aptamer Reagents

BackgroundDNA aptamers generated by cell-SELEX offer an attractive alternative to antibodies, but generating aptamers to specific, known membrane protein targets has proven challenging, and has severely limited the use of aptamers as affinity reagents for cell identification and purification.MethodologyWe modified the BJAB lymphoblastoma cell line to over-express the murine c-kit cell surface receptor. After six rounds of cell-SELEX, high-throughput sequencing and bioinformatics analysis, we identified aptamers that bound BJAB cells expressing c-kit but not wild-type BJAB controls. One of these aptamers also recognizes c-kit endogenously expressed by a mast cell line or hematopoietic progenitor cells, and specifically blocks binding of the c-kit ligand stem cell factor (SCF). This aptamer enables better separation by fluorescence-activated cell sorting (FACS) of c-kit+ hematopoietic progenitor cells from mixed bone marrow populations than a commercially available antibody, suggesting that this approach may be broadly useful for rapid isolation of affinity reagents suitable for purification of other specific cell types.Conclusions/SignificanceHere we describe a novel procedure for the efficient generation of DNA aptamers that bind to specific cell membrane proteins and can be used as high affinity reagents. We have named the procedure STACS (Specific TArget Cell-SELEX).

C-Kit/c-Kit ligand interaction of bone marrow endothelial progenitor cells is influenced in a cigarette smoke extract-induced emphysema model

ABSTRACTBackground: Smoking causes lung endothelial cell apoptosis and emphysema. Derived from bone marrow, circulating endothelial progenitor cells (EPCs) maintain vascular integrity by replacing and repairing damaged endothelial cells. Smoking influences the number of circulating EPCs. Recruitment of EPCs from bone marrow to peripheral blood depends on the interaction of c-Kit/soluble c-Kit ligand (sKitL). We hypothesized that smoking might influence c-Kit(+) EPCs/sKitL interaction in bone marrow in the development of smoking-related emphysema. In this study, we used a cigarette smoke extract (CSE)-induced emphysema model. Methods: Mice were injected intraperitoneally with PBS/CSE and sacrificed at day 28. Lung function and pathology of lung tissue were measured to characterize the model. Expressions of c-Kit in the lung tissue were assayed. Bone marrow cells were isolated by red blood cell lysis. EPCs/c-Kit(+) EPCs in nonred blood cells were analyzed by flow cytometry. Expressions of KitL and MMP-9, and activity MMP-9 in bone marrow were measured. Results: Our data demonstrated that gene and protein expressions of c-Kit were decreased in the lung tissue in this model. Compared with the control group, the number of bone marrow nonred blood cells was unchanged following CSE treatment, while the depletion of bone marrow EPCs/c-Kit(+) EPCs was significant. The level of sKitL was reduced in the bone marrow in the model. The reduction of sKitL was associated with deregulated KitL expression and decreased MMP-9 activity. Conclusions: The interaction between c-Kit and sKitL in bone marrow EPCs, a critical step in endothelial repair, is negatively affected in a CSE-induced emphysema model.

C-Kit SCF receptor (CD117) expression andKITgene mutation in conjunctival pigmented lesions

To investigate the presence of KIT gene mutations and immunoreactivity in 85 conjunctival melanocytic tumours and to clarify the role of KIT as a potential therapeutic target in this group of patients. Eighty-five conjunctival pigmented tumours [27 melanomas, 12 primary acquired melanosis (PAMs) and 46 nevi] were immunostained for KIT. Intensity and pattern of expression were evaluated. Molecular analysis to identify KIT mutations was performed in 15 selected cases (tumour-rich areas >50%). KIT immunostaining score and pattern were statistically related to patients' age, sex, diagnostic category, presence of relapse, disease-free survival, presence of metastases, metastasis-free survival, limbal versus nonlimbal tumour location and thickness of melanomas. KIT stains were documented in 48% of melanomas, 50% of PAMs and 24% of nevi. The mean score of KIT staining in the melanomas/PAMs group was significantly different from nevi (p = 0.0076). No statistically significant differences were detected between either c-kit immunostaining score or pattern and each of the other clinico-pathologic parameters considered. No KIT gene mutations were detected in melanomas and nevi. A silent mutation/polymorphism in KIT exon 13 was found in one PAM. Despite the high level of KIT immunostains in PAMs and melanomas, this parameter seems not to be a good predictor of the presence of molecular mutations. KIT-activating mutations should be considered an uncommon event in this tumour.

FIH-1/c-Kit Signaling: A Novel Contributor to Corneal Epithelial Glycogen Metabolism

Corneal epithelial cells have large stores of glycogen, which serve as their primary energy source. Recently, we demonstrated that factor-inhibiting hypoxia-inducible factor 1 (FIH-1) diminished glycogen stores in vitro and in vivo, working through the Akt/Glycogen Synthase Kinase (GSK)-3β pathway. In this study we investigated the relationship between FIH-1 and c-kit as it pertains to limbal and corneal epithelial glycogen stores. Limbal and corneal epithelia from wild-type FIH-1(-/-) and Kit(W/Wv) mice were stained with periodic acid Schiff (PAS) to detect glycogen. RNA samples prepared from laser-capture microdissected populations of limbal epithelium were subjected to real-time quantitative PCR to determine c-kit ligand expression. Submerged cultures of primary human corneal epithelial keratinocytes (HCEKs) transduced with FIH-1 were treated with c-kit ligand to establish further a FIH-1/c-kit interaction via Western analysis. Akt phosphorylation was assessed by Western blotting. The limbal epithelial cells of FIH-1 null mice had an increase in glycogen levels as well as increased c-kit ligand mRNA compared with wild-type controls. Consistent with a FIH-1/c-kit association, the diminished Akt signaling observed in FIH-1-overexpressing HCEKs could be restored by the addition of c-kit ligand. Interestingly, Akt signaling and glycogen content of the corneal epithelium were significantly decreased in c-kit mutant mice. c-Kit signaling has been shown to affect glucose metabolism via the Akt/GSK-3β pathway. An inverse relationship between FIH-1 and c-kit signaling pathways accounts, in part, for differences in glycogen content between corneal and limbal epithelial cells.

Arthritis augments breast cancer metastasis: role of mast cells and SCF/c-Kit signaling

IntroductionBreast cancer remains the second leading cause of cancer-related deaths for women in the United States. Metastasis is regulated not only by intrinsic genetic changes in malignant cells, but also by the microenvironment, especially those associated with chronic inflammation. We recently reported that mice with autoimmune arthritis have significantly increased incidence of bone and lung metastasis and decreased survival associated with breast cancer. In this study, we evaluated the mechanism underlying the increased metastasis.MethodsWe used two mouse models; one that develops spontaneous autoimmune arthritis (SKG mice) injected with metastatic breast cancer cells (4T1), and another that develops spontaneous breast cancer (MMTV-PyV MT mice) injected with type II collagen to induce autoimmune arthritis. Mast cell levels and metastasis were monitored.ResultsFirst, we confirmed that breast tumor-bearing arthritic mice have a significantly higher incidence of bone and lung metastasis than do their nonarthritic counterparts. Next, we showed increased recruitment of mast cells within the primary tumor of arthritic mice, which facilitates metastasis. Next, we report that arthritic mice without any tumors have higher numbers of mast cells in the bones and lungs, which may be the underlying cause for the enhanced lung and bone metastases observed in the arthritic mice. Next, we showed that once the tumor cells populate the metastatic niches (bones and lungs), they further increase the mast cell population within the niche and assist in enhancing metastasis. This may primarily be due to the interaction of c-Kit receptor present on mast cells and stem cell factor (SCF, the ligand for ckit) expressed on tumor cells. Finally, we showed that targeting the SCF/cKit interaction with an anti-ckit antibody reduces the differentiation of mast cells and consequently reduces metastasis.ConclusionThis is the first report to show that mast cells may play a critical role in remodeling not only the tumor microenvironment but also the metastatic niche to facilitate efficient metastasis through SCF/cKit interaction in breast cancer with arthritis.

Maternal undernutrition does not alter Sertoli cell numbers or the expression of key developmental markers in the mid-gestation ovine fetal testis

BackgroundThe aim of this study was to determine the effects of maternal undernutrition on ovine fetal testis morphology and expression of relevant histological indicators. Maternal undernutrition, in sheep, has been reported, previously, to alter fetal ovary development, as indicated by delayed folliculogenesis and the altered expression of ovarian apoptosis-regulating gene products, at day 110 of gestation. It is not known whether or not maternal undernutrition alters the same gene products in the day 110 fetal testis.Design and methodsMature Scottish Blackface ewes were fed either 100% (Control; C) or 50% (low; L) of estimated metabolisable energy requirements of a pregnant ewe, from mating to day 110 of gestation. All pregnant ewes were euthanized at day 110 and a sub-set of male fetuses was randomly selected (6 C and 9 L) for histology studies designed to address the effect of nutritional state on several indices of testis development. Sertoli cell numbers were measured using a stereological method and Ki67 (cell proliferation index), Bax (pro-apoptosis), Mcl-1 (anti-apoptosis), SCF and c-kit ligand (development and apoptosis) gene expression was measured in Bouins-fixed fetal testis using immunohistochemistry.ResultsNo significant differences were observed in numbers of Sertoli cells or testicular Ki67 positive cells. The latter were localised to the testicular cords and interstitium. Bax and Mcl-1 were localised specifically to the germ cells whereas c-kit was localised to both the cords and interstitium. SCF staining was very sparse. No treatment effects were observed for any of the markers examined.ConclusionsThese data suggest that, unlike in the fetal ovary, maternal undernutrition for the first 110 days of gestation affects neither the morphology of the fetal testis nor the expression of gene products which regulate apoptosis. It is postulated that the effects of fetal undernutrition on testis function may be expressed through hypothalamic-pituitary changes.

Testis tissue explantation cures spermatogenic failure in c-Kit ligand mutant mice

Male infertility is most commonly caused by spermatogenic defects or insufficiencies, the majority of which are as yet cureless. Recently, we succeeded in cultivating mouse testicular tissues for producing fertile sperm from spermatogonial stem cells. Here, we show that one of the most severe types of spermatogenic defect mutant can be treated by the culture method without any genetic manipulations. The Sl/Sl(d) mouse is used as a model of such male infertility. The testis of the Sl/Sl(d) mouse has only primitive spermatogonia as germ cells, lacking any sign of spermatogenesis owing to mutations of the c-kit ligand (KITL) gene that cause the loss of membrane-bound-type KITL from the surface of Sertoli cells. To compensate for the deficit, we cultured testis tissues of Sl/Sl(d) mice with a medium containing recombinant KITL and found that it induced the differentiation of spermatogonia up to the end of meiosis. We further discovered that colony stimulating factor-1 (CSF-1) enhances the effect of KITL and promotes spermatogenesis up to the production of sperm. Microinsemination of haploid cells resulted in delivery of healthy offspring. This study demonstrated that spermatogenic impairments can be treated in vitro with the supplementation of certain factors or substances that are insufficient in the original testes.

Analysis of Genetic Interactions Involving Maternal and Offspring Genotypes at Different Loci: Power Simulation and Application to Testicular Cancer

The analyses of genetic interaction between maternal and offspring genotypes are usually conducted considering a single locus. Here, we propose testing maternal × offspring (M×O) and maternal × maternal (M×M) genotype interactions involving two unlinked loci. We reformulate the log-linear approach of analyzing cases and their parents (family trios) to accommodate two loci, fit fuller models to avoid confounding in a first analysis step and propose that the model be reduced to the most prominent effects in a second step. We conduct extensive simulations to assess the validity and power of this approach under various model assumptions. We show that the approach is valid and has good power to detect M×O and M×M interactions. For example, the power to detect a dominant interaction relative risk of 1.5 (both M×O and M×M) is 70% with 300 trios and approaches 100% with 1,000 trios. Unlike the main effects, M×O and M×M interactions are conditionally independent of mating types, and consequently, their power is not affected by missing paternal genotypes. When applied to single-locus M×O interaction, our method is as powerful as other existing methods. Applying the method to testicular cancer, we found a nominally significant M×M interaction between single nucleotide polymorphisms from C-Kit Ligand (KITLG) and Sex Hormone Binding Globulin (SHBG) using 210 families (relative risk 2.2, P = 0.03). This finding supports a role of maternal hormones in offspring testicular cancer and warrants confirmation in a larger dataset.

Abstract 4273: C-kit expression promotes prostate cancer cell migration and invasion through Brca2 downregulation

Abstract In many human cancers, expression or activity of the receptor tyrosine kinase (RTK) c-kit is often associated with increased malignancy. In prostate cancer (PCa), we reported an association between expression of c-kit and clinical progression, with the highest expression seen in castrate-resistant metastatic bone specimens. We also found de novo c-kit expression in experimental intraosseous tumors formed by otherwise c-kit negative PCa cells. However, the biological consequences of c-kit expression and activation in PCa cells remain unknown. Here, we show that c-kit overexpression in PC3 and C4-2B PCa cells significantly enhances their migratory and invasive ability in vitro. Consistent with this result, we found that expression and activation of c-kit in PCa cells decreased the expression of E-cadherin and increased the expression of vimentin, suggesting an epithelial mesenchymal transition-like phenomenon. Furthermore, activation by c-kit ligand in PC3 and C4-2B cells transfected with c-kit lead to phosphorylation of Akt. This contributes to the enhanced invasiveness of c-kit-expressing PCa cells, as demonstrated by an inhibitory effect by PI3K/Akt inhibitors LY294002 and Wortmannin. In an attempt to identify genes differentially expressed in c-kit-expressing and -non-expressing PCa cells, we used a microarray containing 84 genes, representing growth factor receptors, transcription factors, cell cycle, and DNA repair genes. The analysis of the array demonstrated a consistent down-regulation of Brca2 in both c-kit-expressing PC3 and C4-2B cells, further validated by qRT-PCR and immunoblotting. Re-introduction of Brca2 into c-kit-expressing PCa cells suppressed c-kit-enhanced invasiveness to levels similar to those shown by c-kit-non-expressing cells, and led to reduced phosphorylation of Akt. Our results provide evidence that c-kit expression in PCa cells, an event which is induced upon interaction with the bone microenvironment, promotes tumor migration and invasion through an Akt-dependent mechanism regulated by Brca2 down-regulation. The identification of this novel signaling axis and its importance in PCa migration and invasion reveal potential therapeutic targets for regulating the expansion of PCa metastatic lesions in bone. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4273. doi:1538-7445.AM2012-4273

499 URINARY STEM CELL FACTOR AS A NOVEL DIAGNOSTIC AND THERAPEUTIC BIOMARKER FOR OVERACTIVE BLADDER

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology II1 Apr 2012499 URINARY STEM CELL FACTOR AS A NOVEL DIAGNOSTIC AND THERAPEUTIC BIOMARKER FOR OVERACTIVE BLADDER Yasue Kubota, Yoshiyuki Kojima, Takashi Hamakawa, Makoto Imura, Yasuhiro Shibata, Shoichi Sasaki, and Kenjiro Kohri Yasue KubotaYasue Kubota Nagoya, Japan More articles by this author , Yoshiyuki KojimaYoshiyuki Kojima Nagoya, Japan More articles by this author , Takashi HamakawaTakashi Hamakawa Nagoya, Japan More articles by this author , Makoto ImuraMakoto Imura Nagoya, Japan More articles by this author , Yasuhiro ShibataYasuhiro Shibata Nagoya, Japan More articles by this author , Shoichi SasakiShoichi Sasaki Nagoya, Japan More articles by this author , and Kenjiro KohriKenjiro Kohri Nagoya, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.569AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent reports have demonstrated that c-kit acts as not only a marker of interstitial cells of Cajal, but also plays a significant role in the control of bladder spontaneous activity, and could be a target for the clinical treatment of overactive bladder (OAB). We previously demonstrated that c-kit ligand, stem cell factor (SCF), was expressed in the urothelium of human bladder and secreted by the urothelium. The objective of this study was to investigate whether SCF could be a potential biomarker for diagnosis and therapeutic efficacy of OAB. METHODS One hundred ninety eight normal healthy volunteers (male:female=98:98) without OAB, lower urinary tract symptoms and other urinary tract-related diseases, were enrolled as a control to determine the reference range of urinary SCF level. In addition, 280 patients with untreated OAB were also enrolled. OAB was diagnosed on the basis of the overactive bladder symptom score (OABSS). Urinalysis was performed to rule out urinary tract infection and microscopic hematuria. Urinary SCF levels were measured by the enzyme-linked immunosorbent assay (ELISA). ELISA was performed by one laboratory assistant who was blind to the clinical classification of the patients. At the same time, 3 ml urine was taken to measure the urinary creatinine (Cre) level. All experiments were carried out in triplicate. The total urinary SCF levels were normalized to the concentration of urinary Cre. The study was approved by the ethics committee of our institution. RESULTS The reference range of SCF/Cre was obtained by measuring the values in healthy volunteers. The 95% reference range was calculated as (mean-1.96 standard deviations; SD) to (mean+1.96SD), and provided that the data were normally distributed. According to this calculation, the normal range of urinary SCF was determined under 3.6x10-4. There was no significant difference in urinary SCF/Cr level between male and female. The average urinary SCF level was significantly higher in OAB patients (12.7 ± 11.9 x10-4) than in the healthy volunteers group (1.7 ± 0.9 x10-4; p <0.00001). The urinary SCF level showed a significant positive correlation with OABSS (r=0.67, p=0.001). It was significantly decreased after 12-week administration of anticholinergic agents (p<0.05), and showed a significant positive correlation with improvement of OABSS (p<0.004). CONCLUSIONS The urinary SCF could be a novel diagnostic and therapeutic biomarker for overactive bladder, because it showed significant correlation with severity of OAB and efficacy of anti-cholinergic agents. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e205 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yasue Kubota Nagoya, Japan More articles by this author Yoshiyuki Kojima Nagoya, Japan More articles by this author Takashi Hamakawa Nagoya, Japan More articles by this author Makoto Imura Nagoya, Japan More articles by this author Yasuhiro Shibata Nagoya, Japan More articles by this author Shoichi Sasaki Nagoya, Japan More articles by this author Kenjiro Kohri Nagoya, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The canonical Wnt pathway shapes niches supportive of hematopoietic stem/progenitor cells

AbstractConsiderable information has accumulated about components of BM that regulate the survival, self-renewal, and differentiation of hematopoietic cells. In the present study, we investigated Wnt signaling and assessed its influence on human and murine hematopoiesis. Hematopoietic stem/progenitor cells (HSPCs) were placed on Wnt3a-transduced OP9 stromal cells. The proliferation and production of B cells, natural killer cells, and plasmacytoid dendritic cells were blocked. In addition, some HSPC characteristics were maintained or re-acquired along with different lineage generation potentials. These responses did not result from direct effects of Wnt3a on HSPCs, but also required alterations in the OP9 cells. Microarray, PCR, and flow cytometric experiments revealed that OP9 cells acquired osteoblastic characteristics while down-regulating some features associated with mesenchymal stem cells, including the expression of angiopoietin 1, the c-Kit ligand, and VCAM-1. In contrast, the production of decorin, tenascins, and fibromodulin markedly increased. We found that at least 1 of these extracellular matrix components, decorin, is a regulator of hematopoiesis: upon addition of this proteoglycan to OP9 cocultures, decorin caused changes similar to those caused by Wnt3a. Furthermore, hematopoietic stem cell numbers in the BM and spleen were elevated in decorin-knockout mice. These findings define one mechanism through which canonical Wnt signaling could shape niches supportive of hematopoiesis.

Identification of a cKit+ Colonic Crypt Base Secretory Cell That Supports Lgr5+ Stem Cells in Mice

Paneth cells contribute to the small intestinal niche of Lgr5(+) stem cells. Although the colon also contains Lgr5(+) stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5(+) stem cells. We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types. Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117(+) crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit(+) goblet cells were interdigitated with Lgr5(+) stem cells. In vivo, this colonic cKit(+) population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit(+) cells. When isolated from mouse colon, cKit(+) cells promoted formation of organoids from Lgr5(+) stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit(+) cells using a toxin-conjugated antibody, organoid formation decreased. cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5(+) stem cells.

Variants in KITLG predispose to testicular germ cell cancer independently from spermatogenic function

Epidemiological data suggest an association and a common pathogenetic link between male infertility and testicular germ cell tumor (TGCT) development. Genome-wide studies identified that TGCT susceptibility is associated with KITLG (c-KIT ligand), which regulates the formation of primordial germ cells, from which TGCT is believed to arise and spermatogenesis develops. In this study, we analyzed the link between KITLG, TGCT, and spermatogenic disruption by performing an association study between the KITLG markers rs995030 and rs4471514 and 426 TGCT cases and 614 controls with normal and abnormal sperm count. We found that TGCT risk was increased more than twofold per copy of the major G allele and A allele in KITLG rs995030 and rs4471514 (odds ratio (OR)=2.38, 95% confidence interval (95% CI)=1.81-3.12; OR=2.43, 95% CI=1.86-3.17 respectively), and homozygotes for the risk allele had a sevenfold increased risk of TGCT. KITLG markers were strongly associated with seminoma subtype (per allele risk increased more than threefold, homozygote risk increased by 13- to 16-fold) and weakly with nonseminoma. KITLG markers were not associated with sperm production, as no difference was observed in men with normozoospermia and azoo-oligozoospermia, both in controls and in TGCT cases. In conclusion, this study provides evidence that KITLG variants are involved in TGCT development and they represent an independent and strong specific risk factor for TGCT independently from spermatogenic function. A shared genetic cause and a common pathogenetic link between TGCT development and impairment of spermatogenesis are not evident from this study.

Quantitative and semiquantitative immunoassay of growth factors and cytokines in the conditioned medium of STO and CF-1 mouse feeder cells

Feeder cells of irradiated mouse fibroblasts are commonly used for, and are generally necessary for, the in vitro maintenance and growth of many fastidious cell types, particularly embryonic stem cells or induced pluripotent stem cells. Quantitative and semiquantitative immunoassays of conditioned media were performed to identify some of the soluble cytokines, chemokines, protein hormones, and cell matrix/adhesion molecules that are elaborated from two commonly used feeder cells, STO and CF-1. Among those quantitatively assayed, the most abundant cytokine proteins expressed by the feeder cells were activin A, hepatocyte growth factor (HGF), insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein (IGFBP)-6, macrophage colony-stimulating factor (a.k.a. CSF-1), and pigment epithelium-derived factor (a.k.a. serine protease inhibitor, clade F, member 1). CF-1 cells expressed ten times more activin A than STO cells and also produced larger amounts of interleukin-6 and IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5. Conversely, STO cell produced almost ten times more HGF and five times more stem cell factor (a.k.a. c-kit ligand) than CF-1 cells. Assayed semiquantitatively, relatively large amounts of chemokines were produced by both feeder cells including fractalkine (CX3CL1), interferon-inducible protein 10 (a.k.a. CXCL10 and cytokine-responsive gene-2, CRG-2), monocyte chemotactic protein (MCP)-1 (a.k.a. CCL2 and junctional epithelium chemokine (JE), MCP-5/CCL12), keratinocyte-derived chemokine (a.k.a. CXCL1 and growth-related oncogene alpha, GROα), nephroblastoma overexpressed gene (CCN3, IGFBP-9), stromal cell-derived factor 1 (CXCL12), and serpin E1 (PAI-1). In contrast to one another, STO produced more CXCL16 than CF-1 cells, and CF-1 cell produced more MCP-5 (CCL12), macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), pentraxin-3 (TSG-14), and platelet factor-4 (CXCL4) than STO cells. Soluble adhesion molecule, sICAM (ICAM-1, CD54), was expressed by CF-1 cells, but not STO cells, and similarly, the cell matrix-associated molecules endocan (endothelial cell-specific molecule 1), endostatin (collagen XVIII), and matrix metalloproteinase 3 were expressed more by CF-1 cells. Tissue inhibitor of metalloproteinases 1 was robustly expressed by both feeder cells. Other proteins primarily detected from CF-1 cells included retinol-binding protein 4 and FGF21, while STO cells secreted more interferon gamma. Both feeder cells produced no or low amounts of LIF, tumor necrosis factor alpha, vascular endothelial growth factor (VEGF), VEGF-B, prolactin, various interleukins, fibroblast growth factor (FGF)-1, FGF-2, FGF-7, EGF, HB-EGF, and amphiregulin. The results may explain some of the cell growth and maintenance responses by various types of cells co-cultured on STO or CF-1 feeder cells.

In Vitro Culture Reveals Microenvironment-Dependent Growth, Heterogeneity and Hierarchical Structure of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells

Abstract 1483In Vitro culture reveals microenvironment-dependent growth, heterogeneity and hierarchical structure of primary human pediatric pre-B cell acute lymphoblastic leukemia (pre-B ALL) cells.J.-H. Shieh1, P. Steinherz2, J Shieh3 and M. A.S. Moore1. 1Moore Laboratory. Cell Biology Program and 2Leukemia and Lymphoma Studies, Department of Pediatrics, Memorial-Sloan Kettering Cancer Center, New York, NY. 3Department of Biology, Brandeis Univ., Waltham, MAPre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children. Although this pediatric pre-B ALLs are treatable, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. To address this issue, we systemically evaluated the effects of various tissue culture parameters to the growth of primary pre-B ALL cells. A serum-free MS-5 cells (a murine bone marrow stromal cell line) co-culture system is capable of expanding the pre-B ALL CD34+CD19+ cells and supporting their differentiation to CD34−CD19+ B cells. This expansion requires a contact between the stromal cells and the pre-B ALL cells, and is inhibited by fetal bovine serum and IL-6 in a dose-dependent manner. c-Kit ligand and Flt3 ligand can reverse the IL-6 inhibition. Expansion of individual CD34+CD19+ cells revealed a hierarchical structure with respect to CD34 antigen expression and an heterogeneity in cell proliferation. When the pre-B ALL cells were sorted into CD34dim and CD34bright populations, the CD34dim cells were capable of a faster proliferation but gradually lost their CD34 antigen. In contrast, the CD34bright cells were more slowly proliferating and retained their CD34 antigen. We transduced the B-ALL cells with a fusion gene expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL). These GFP-Lu-pre-B ALL cells display the similar in vitro characteristics and in vivo xenograftment to NOD/SCID IL2R gamma null (NSG) mice as the non-transduced pre-B ALL cells. One hundred, 103, 104 or 105 GFP-LU-pre-B ALL CD34+ cells were i.v. transplanted to NSG mice. Both 104 and 105 cells resulted in the engraftment of the leukemia cells in limbs and cranium as judged by imaging after 6 weeks, and 103 cells engrafted after 13 weeks. When the 105 cells-transplanted mice were sacrificed after 14 weeks, the harvested peripheral blood, spleen (3–4×108cells/spleen) and bone marrow (5−10×106 cells/femur) displayed 2–3%, 51–55% and 75–81% of human CD34+CD19+ cells, respectively. Human CD34−CD19+ cells were 1–2%, 12–13% and 15–21%, respectively. Therefore, our stromal culture system supports leukemic stem cell/leukemia initiating cell proliferation and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo, and reveals the heterogeneity and hierarchical structure of human pre-B ALL cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides powerful tools to dissect the pathophysiology of human pre-B ALL, and to screen new drugs for pre-B ALL therapy. Disclosures:No relevant conflicts of interest to declare.

Mechanism of activation of human c-KIT kinase by internal tandem duplications of the juxtamembrane domain and point mutations at aspartic acid 816

The proto-oncogene c-KIT receptor has been implicated as an essential component in the activation of leukemic cells. The internal tandem duplication (ITD) of c-KIT has also been identified as a predominant cause of acute myeloid leukemia (AML), although its role in the activation process is still unclear. To investigate the biological mechanisms of c-KIT activation, we generated a c-KIT receptor bearing two different immunological tags, HA and Flag tags. In this study, we demonstrated that the mutant (Mt)-ITD and Asp816 (D816Y) c-KIT receptors spontaneously formed dimers and that these Mt-ITD forms of c-KIT displayed high levels of phosphorylation and increased cellular tyrosine phosphorylation. The amount of wild-type homodimers increased following the addition of the c-KIT ligand, while the level of mutant homodimers was less affected by the addition of the c-KIT ligand. Furthermore, we demonstrated that Mt-ITD and activating point mutations of D816Y induced constitutive activation of c-KIT kinase in the absence of ligand in COS-1 cells. These data suggest a novel mechanism for the regulation of cell growth autonomy. Overall, our study suggests that c-KIT activation might have significant effects on hematopoietic cells and might help to improve our understanding of the pathogenesis of systemic mast cell disease, gastrointestinal stromal tumors and AML and potentially lead to the development of novel therapeutic approaches.

Cytokine and angiogenic factor (CAF) profiling for identification of markers associated with response to cediranib in metastatic renal cell carcinoma (mRCC).

362 Background: Cediranib is a highly potent and selective signaling inhibitor of vascular endothelial growth factor (VEGF) receptors−1, −2, −3 and c-Kit. In a phase II trial, patients (pts) with mRCC were randomized 3:1 to treatment with cediranib or placebo to primarily assess the difference in change in tumor size at 12 weeks. Cediranib resulted in a significant reduction in tumor size vs. placebo (Mulders et al. ESMO. 2009). Here we investigated soluble biomarkers in serum and concentration changes during treatment. Methods: Pts received cediranib 45 mg/day PO or placebo for the first 12 weeks on treatment. At that time (or upon progression if earlier), treatment was unblinded and pts on placebo were given the option of receiving cediranib. Serum was collected from 61 pts at baseline (BL; cediranib 46, placebo 15), from 50 pts on day (D) 28, and from 45 pts on D84. Multiplex bead suspension arrays and ELISA were used to measure CAF concentrations including VEGF, placental growth factor (PlGF), sVEGFR-2, PDGFbb, HGF, MMP-9, multiple chemokines and interleukins (IL). The objectives of this exploratory analysis were to assess whether baseline CAFs were associated with changes in tumor size after 12 weeks of treatment and whether CAF levels changed while on treatment. Results: Of 39 CAFs available at BL, lower than median (‘low') concentrations of IL-10, VEGF, PlGF, stem cell factor (SCF), and monokine-induced by interferon-gamma (MIG) were associated with larger decreases in tumor size than high concentrations (independent of treatment arm), whereas the opposite trend was observed for IL-5 and TRAIL. Consistent with inhibition of VEGF signaling, cediranib treatment resulted in changes over time in VEGF (increase), sVEGFR-2 (decrease) and PlGF (increase) concentrations. In addition, SCF, a c-Kit ligand and M-CSF decreased, while VCAM-1 and TRAIL increased during cediranib treatment. Conclusions: Candidate CAFs associated with response to cediranib in mRCC such as IL-10 and VEGF were identified. Patients treated with cediranib showed distinct CAF changes compared with placebo. These results require independent validation in a larger trial. [Table: see text]

Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner

Stem cell factor (SCF) known as the c-kit ligand, plays important roles in spermatogenesis, melanogenesis and early stages of hematopoiesis. As for the latter, SCF is essential for growth and expansion of hematopoietic stem and progenitor cells. We herein describe the production of recombinant murine SCF from Escherichia coli as soluble thioredoxin-fusion protein. The formation of insoluble and inactive inclusion bodies, usually observed when SCF is expressed in E. coli, was almost entirely prevented. After purification based on membrane adsorber technology, the fusion protein was subsequently cleaved by TEV protease in order to release mature mSCF. Following dialysis and a final purification step, the target protein was isolated in high purity. Bioactivity of mSCF was proven by different tests (MTT analogous assay, long-term proliferation assay) applying a human megakaryocytic cell line. Furthermore, the biological activity of the uncleaved fusion protein was tested as well. We observed a significant activity, even though it was less than the activity displayed by the purified mSCF. In summary, avoiding inclusion body formation we present an efficient production procedure for mSCF, one of the most important stem cell cytokines.