C-kit mRNA Research Articles

CD1d expressed in mast cell surface enhances IgE production in B cells by up-regulating CD40L expression and mediator release in allergic asthma in mice

Mast cells play important roles via FcεRI-mediated activation in allergic asthma. A nonpolymorphic MHC I-like molecule CD1d, which is mainly expressed in APCs, presents glycolipid Ag to iTCR on iNKT cells and modulates allergic responses. This study aimed to investigate the role of CD1d on IgE production and mast cell activation related to allergic asthma. Bone marrow-derived mast cells (BMMCs) from C57BL/6 Wild type (WT) or KO (CD1d−/−) mice were activated with Ag/Ab (refer to WT-act-BMMCs and KO-act-BMMCs, respectively) or α-Galactosylceramide (WT-αGal-BMMCs, KO-αGal-BMMCs) in the presence of iNKT cells. WT, KO or BMMC-transferred KO mice were sensitized and/or challenged by OVA or α-Gal to induce asthma. KO-act-BMMCs reduced intracellular Ca2+ levels, expression of signaling molecules (Ras, Rac1/2, PLA2, COX-2, NF-κB/AP-1), mediator release (histamines, leukotrienes and cytokines/chemokines), and total IgE levels versus the corresponding WT-BMMCs. KO mice reduced total and OVA-specific serum IgE levels, number of mast cells, recruiting molecules (CCR2/CCL2, VCAM-1, PECAM-1), expression of tryptase, c-kit, CD40L and cytokine mRNA, co-localization of c-kit and CD1d or iNKT cells in BAL cells or lung tissues, and PCA responses, compared with the corresponding WT mice. BMMC-transferred KO-both mice showed the restoration of all allergic responses versus KO-both mice (Ag/Ab reaction plus α-Gal). KO-αGal-BMMCs or KO-αGal mice did not show any responses. Our data suggest that CD1d-expressed mast cells may function as APC cells for iNKT cells and exacerbate airway inflammation and remodeling through up-regulating IgE production via B cell Ig class switching and mediator release in mast cells of OVA-challenged mice.

Antagonistic Effects Of Ozone (O3) Exposure and Glucocorticoid Treatment On Airway Hyperresponsiveness (AHR) and Surfactant Protein D (SP-D) Production In Mice

S U N D A Y 559 Bronchial Mast Cell Markers and Clinical Asthma Severity In Steroid Refractory Asthmatics Dr. Mandeep Hundal, MD, Dr. Katherine N. Cahill, MD, Robert Pedicini, Allison Crosby-Thompson, Stefanie Dutile, Usha Govindarajulu, PhD, Dr. Kartik Shenoy, MD, Dr. Mark Dransfield, Dr. Emily DiMango, Dr. Serpil C. Erzurum, MD, Dr. Mario Castro, MD, MPH, Dr. Nizar N. Jarjour, MD, Dr. Howard Katz, PhD, Dr. Joshua A. Boyce, MD, FAAAAI, Dr. Elliot Israel, MD, FAAAAI; Brigham and Women’s Hospital, Boston, MA, Brigham and Women’s Hospital, Division of Rheumatology, Immunology and Allergy, Boston, MA, Temple University School of Medicine, University of Alabama School of Medicine, AL, Columbia University, New York, NY, Department of Pathobiology, Cleveland Clinic, Washington University School of Medicine, St. Louis, MO, University of Wisconsin School of Medicine and Public Health, Madison, WI. RATIONALE: Mast cells are thought to play a significant role in asthma and are resistant to corticosteroid effects. Bronchial mast cells express chymase, tryptase and the c-kit receptor. We investigated the relationship between these mast cell markers and clinical asthma severity measurements in patients with steroid refractory asthma. METHODS: We recruited adult asthmatics with severe asthma symptoms (ACQ>1.5) on continuous treatment with high-dose inhaled corticosteroids (ICS) and at least one additional controller therapy. All subjects had confirmed airway hyperresponsiveness with methacholine challenge (PC20< 10 mg/ml). Endobronchial biopsies were obtained from these subjects (n527) andmast cells were immunostained for tryptase and chymase. Bronchoalveolar lavage (BAL) fluid (n528) was processed and c-kit mRNAwas measured in the BAL cell pellet using qPCR. RESULTS: Intramucosal chymase positive mast cell numbers show a positive correlation with log PC20 (r50.45, p50.02); suggesting decreasing methacholine hyperresponsiveness with increasing chymase positive mast cells. Intramucosal tryptase positive mast cell counts did not show any significant association with log PC20. FEV1/FVC ratio had a negative correlation with c-kit mRNA in the BAL cell pellet (r5-0.37, p50.05). CONCLUSIONS: Increased c-kit mRNA in BAL is related to worsening airway obstruction suggestingmast cell activity contributes to poor asthma control in steroid unresponsive asthmatics. It has been previously reported that chymase positive mast cells in severe asthma correlate with better lung function. The negative correlation between chymase positive mast cells and airway hyperresponsiveness in our study supports a unique pathobiological role for this mast cell phenotype in severe asthmatics.

Effects of lipopolysaccharide-induced inflammation on the interstitial cells of Cajal

Interstitial cells of Cajal (ICC) have recently been found to display phenotypic changes. The present study is designed to determine whether phenotypic changes occur in ICC associated with an inflammatory microenvironment and whether the ICC phenotype could be recovered after the discontinuation of inflammatory stimuli. Immunohistochemistry studies revealed that the functional ICC marker, c-kit, was markedly reduced in patients with Hirschsprung's disease (n = 34) compared with controls (n = 12), whereas another marker of ICC, CD34, was not altered significantly. Compared with the vehicle group (n = 6), intraperitoneal injection of lipopolysaccharide (LPS; 1.5 mg/kg) in mice (n = 6) significantly induced plasma tumor necrosis factor-alpha (TNF-α) levels as determined by enzyme-linked immunosorbent assay. Western blot and real-time polymerase chain reaction assessment further showed that LPS injection markedly suppressed intestinal c-kit protein and mRNA expression, which could be blocked by Toll-like receptor 4 (TLR4) deficiency (n = 6) rather than TLR2 deficiency (n = 6) and had no effects on CD34. Compared with the vehicle group (n = 6), intraperitoneal TNF-α (30 μg/kg) administration (n = 6) also significantly reduced intestinal c-kit protein and mRNA levels but not CD34 levels. However, the reduction of c-kit induced by TNF-α injection was not suppressed by TLR4 deficiency (n = 6). Intestinal c-kit protein and mRNA levels were markedly restored after the discontinuation of TNF-α administration for 7 days. Moreover, immunofluorescence analysis of primary ICC further confirmed that exposure to TNF-α for 24 h suppressed c-kit expression, which could be restored after discontinuation of TNF-α exposure. CD34 expression was not altered upon exposure to TNF-α. Thus, phenotypic changes in ICC occur in an inflammatory microenvironment in the gut and LPS, TLR4 and TNFα are crucial to this process.

Development of an in vitro model of acquired resistance to toceranib phosphate (Palladia®) in canine mast cell tumor

BackgroundMast cell tumors (MCTs) are the most common skin tumors in dogs and exhibit variable biologic behavior. Mutations in the c-kit proto-oncogene are associated with the tumorigenesis of MCTs, resulting in growth factor-independent and constitutive phosphorylation of the KIT receptor tyrosine kinase (RTK). Toceranib (TOC) phosphate (Palladia®) is a KIT RTK inhibitor that has biological activity against MCTs. Despite these benefits, patients ultimately develop resistance to TOC. Therefore, there is a need to identify distinguishing clinical and molecular features of resistance in this population.ResultsThe canine C2 mastocytoma cell line contains an activating mutation in c-kit. Three TOC-resistant C2 sublines (TR1, TR2, TR3) were established over seven months by growing cells in increasing concentrations of TOC. TOC inhibited KIT phosphorylation and cell proliferation in a dose-dependent manner in the treatment-naïve, parental C2 line (IC50 < 10 nM). In contrast, the three sublines were resistant to growth inhibition by TOC (IC50 > 1,000 nM) and phosphorylation of the KIT receptor was less inhibited compared to the TOC-sensitive C2 cells. Interestingly, sensitivity to three structurally distinct KIT RTK inhibitors was variable among the sublines, and all 3 sublines retained sensitivity to the cytotoxic agents vinblastine and lomustine. Sequencing of c-kit revealed secondary mutations in the juxtamembrane and tyrosine kinase domains of the resistant sublines. These included point mutations in TR1 (Q574R, M835T), TR2 (K724R), and TR3 (K580R, R584G, A620S). Additionally, chronic TOC exposure resulted in c-kit mRNA and KIT protein overexpression in the TOC-resistant sublines compared to the parental line. C2, TR1, TR2, and TR3 cells demonstrated minimal P-glycoprotein (P-gp) activity and no functional P-gp.ConclusionsThis study demonstrates the development of an in vitro model of acquired resistance to targeted therapy in canine MCTs harboring a c-kit-activating mutation. This model may be used to investigate the molecular basis of and strategies to overcome TOC resistance.

Ano1 is a better marker than c-Kit for transcript analysis of single interstitial cells of Cajal in culture.

The interstitial cells of Cajal (ICC) drive the slow wave-associated contractions in the small intestine. A commonly used marker for these cells is c-Kit, but another marker named Ano1 was recently described. This study uses single-cell RT-PCR, qPCR and immunohistochemistry to determine if Ano1 could be reliably used as a molecular marker for ICC in single-cell mRNA analysis. Here, we report on the relationship between the expression of c-Kit and Ano1 in single ICC in culture. We observed that Ano1 is expressed in more than 60% of the collected cells, whereas c-Kit is found only in 22% of the cells (n = 18). When we stained ICC primary cultures for c-KIT and ANO1 protein, we found complete co-localization in all the preparations. We propose that this difference is due to the regulation of c-Kit mRNA in culture. This regulation gives rise to low levels of its transcript, while Ano1 is expressed more prominently in culture on day 4. We also propose that Ano1 is more suitable for single-cell expression analysis as a marker for cell identity than c-Kit at the mRNA level. We hope this evidence will help to validate and increase the success of future studies characterizing single ICC expression patterns.

Role and mechanism of tranilast preventing the progression of tubulointerstilial fibrosis in diabetic kidney diseases

To determine the role and mechanism of tranilast preventing the progression of tubulointerstilial fibrosis in diabetic kidney disease (DKD). Sprague-Dawley rats were randomly divided into a control group (n=6), DKD model group (n=8), low dose tranilast group [200 mg/(kg.d), n=8], and high dose tranilast group [400 mg/(kg.d), n=8]. Tranilast was administered daily after the model was built. Rats were sacrificed at day 56, 24 hour urine was collected to measure 24-hour urine albumin excretion, and blood was collected to determine the renal function and serum albumin. Then the kidneys were harvested and subjected to studies. The expression of C3aR, E-cadherin, α-SMA, fibronectin(FN), collagen I (Col I), stem cell factor (SCF) and c-kit were detected by immunohistochemical staining respectively. The expression of E-cadherin, α-SMA, FN, Col I, SCF and c-kit protein was analyzed by Western blot, and the expression of FN, Col I, SCF and c-kit mRNA was examined by RT-PCR. Tranilast can inhibit the infiltration of mast cells in the kidneys of DKD rats. The expression of α-SMA in the kidneys of DKD rats inereased significantly (P<0.05), while the expression of E-cadherin decreased (P<0.05). Tranilast increased the expression of E-cadherin and decreased the expression of α-SMA in the prophase of DKD dose dependently. The expressions of FN and Col I were increased in the tubulointerstitial fields in DKD model rats (P<0.05). After the tranilast treatment, these changes were relieved to a certein degree (P<0.05). The expression of SCF and c-kit in the tubular and interstitial tissue was slight. The increased expressions of SCF and c-kit protein and mRNA in DKD model rats were downregulated by tranilat (P<0.05). The expressions of SCF and c-kit were positively correlated with the infiltration degree of mast cells and the expressions of FN, Col I. Mast cells participate in and aggravate the renal tubulointerstitial fibrosis in DKD rats. Tranilast can reverse the EMT of renal tubular cells and inhibit the tubulointersitial fibrosis of DKD by blocking the infiltration of mast cells induced by SCF/c-kit pathway.

Effects of different concentrations of concanavalin A and follicle stimulating hormone on goat primordial follicles activation, survival and gene expression

This study aims to investigate the effects of concanavalin A (Con A) and follicle stimulating hormone (FSH) on the survival and activation of primordial follicles, as well as on gene expression in cultured goat ovarian tissue. Ovarian tissues were cultured for one or six days in medium supplemented with different concentrations of Con A (0, 5, 10, 20 and 40μg/mL – experiment 1). Non-cultured (control) and ovarian fragments cultured for one or six days were processed for histological evaluation. After choosing the best concentration of Con A (10μg/mL), ovarian tissues were cultured for 6 days in medium supplemented with Con A (10μg/mL), FSH (50ng/mL) or both (experiment 2). Then, the ovarian fragments were processed for histology, or stored to evaluate the expression of the mRNAs for PCNA, c-kit, KL, GDF-9 and BMP-15 by PCR in real time. The results showed that after 6 days of culture, the presence of Con A (10 or 40μg/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. The addition of FSH (50ng/mL) alone or in combination with Con A (10μg/mL) was able to induce the primordial follicles activation, but no positive interaction was observed. However, after culture, FSH increased the expression of PCNA mRNA, but this effect was blocked by Con A. Con A increased the levels of c-KIT mRNA in relation to uncultured control, but Con-A or both Con A and FSH reduced the levels of KL mRNA. In addition, a reduction of BMP-15 mRNA expression was found in follicles cultured in control medium, but not in media containing FSH. Furthermore, follicles cultured in presence of Con A had higher levels of mRNA for GDF-9 than those cultured in control medium. In conclusion, Con A and FSH are able to improve goat primordial follicles activation and regulate gene expression in ovarian cortical tissue that was cultured for 6 days.

Changes in mast cells and hepatic expression of c-kit and stem cell factor in the rat model of chronic hepatitis

To study the potential role of mast cells and the related molecular mechanism in chronic hepatitis (CH) using a rat model system. Thirty Wistar rats (15 males, 15 females; weight range: 230-290 g) were randomly divided into the normal contrast (NC) group and experimental CH group. The CH group received subcutaneous injection of CCl4 and a diet high in cholesterol and alcohol content and low in protein and choline content. Throughout the 4-week modeling period, aseptic blood samples were taken to test plasma tryptase (TS) and hyaluronic acid (HA) levels. The rats were euthanized to assess the changes in liver mast cells by histology and morphology analyses and the changes in liver expression of c-kit and stem cell factor (SCF) proteins by immunohistochemistry and mRNAs by RT-PCR. Compared to the NC group, the CH group had higher plasma and liver concentration of HA (78.09 +/- 38.55 vs. 145.14 +/- 52.54 ng/ml, 51.58 +/- 20.45 vs. 106.59 +/- 43.15 ng/100 mg; t = 2.457 and 2.825 respectively, both P less than 0.05) and TS (0.416 +/- 0.143 vs 0.753 +/- 0.210 mg/ml; t = 4.165, P less than 0.05). The CH group also showed fatty degeneration and fibrosis with many degranulating and degranulated mast cells filled with purple granula located around the liver blood vessels and in fiber-intervals. The CH livers also showed a significantly higher number of mast cells (2.167 +/- 0.924 vs. NC: 10.92 +/- 1.575; t = 7.633, P less than 0.05) and stronger intensity of c-kit staining (2.783 +/- 0.577 vs. 12.86 +/- 3.126; t = 9.511, P less than 0.05) and SCF staining (3.383 +/- 1.583 vs. 15.58 +/- 6.431; t = 9.625, P less than 0.05). The expressions of c-kit and SCF were positively correlated with HA level (r = 0.478 and 0.556 respectively, both P less than 0.05). The c-kit and SCF mRNA expression levels were also significantly higher in the CH liver tissues. Mast cell degranulation and histamine release is significantly increased under conditions of chronic hepatitis, and the related mechanism may involve up-regulation of the membrane receptor c-kit and its ligand SCF.

Effect of flutamide on folliculogenesis in the fetal porcine ovary – Regulation by Kit ligand/c-Kit and IGF1/IGF1R systems

In pigs, primordial to primary follicle transition occur in the late pregnancy. The interactions between Kit ligand (KL) and its receptor (c-Kit), as well as insulin-like growth factor 1 (IGF1) and cognate receptor (IGF1R) are crucial for the primordial follicle activation. It is well established that hormonal disruption induces abnormalities in the developing reproductive system. Hence, this study investigated the influence of antiandrogen, flutamide, on genes involved in the primordial to primary follicle transition. Pregnant gilts were injected with flutamide (50mg/kg bw, seven times, every day) or corn oil (control groups) starting on gestation days 83 (GD90) or 101 (GD108). Fetal ovaries were excised on days 90 and 108 of gestation. The proportion of primordial and primary follicles was determined, and immunohistochemistry for c-Kit and IGF1R was conducted. To assess KL, c-Kit, IGF1 and IGF1R mRNA expression real-time PCR was performed. Ovaries from both GD90 and GD108 animals exhibited a greater proportion of primordial to primary follicles when compared to respective control groups. C-Kit and IGF1R were immunolocalized in the oocytes of primordial and primary follicles. Both c-Kit mRNA and protein levels and KL mRNA expression were diminished in GD90 group. IGF1R expression decreased at mRNA and protein levels, whereas IGF1 mRNA expression was increased in GD90 and GD108 groups. In summary, our findings may indicate that the interactions between KL and c-Kit as well as IGF1 and IGF1R are relevant to the initiation of follicular transition from primordial into primary follicles and can be affected by AR signaling.

Demethylation effect of inhibitor As2O3 on expression of SHP-1 and C-kit genes in leukemia HL-60 cells

This study was aimed to investigate the expression level of SHP-1 and C-kit genes in acute leukemia HL-60 cells and effect of inhibitor As2O3 demethylation on SHP-1 and C-kit genes expression. RT-PCR was used to detect the expression level of SHP-1 and C-kit mRNA in drug-treated cell group and control group. The methylation specific PCR (MSP) was applied to measure the methylation status of SHP-1 gene in HL-60 cells. The results showed that after being treated with As2O3 the recovery of SHP-1 gene expression was observed in HL-60 cells in which SHP-1 mRNA originally did not expressed, meanwhile the expression level of C-kit mRNA in HL-60 cells with high expression decreased. When HL-60 cells were treated with As2O3 of 1.0, 2.5, 5.0 µmol/L, the demethylation effects was enhanced, the expression of SHP-1 mRNA displayed an ascending tendency, and expression of C-kit mRNA showed an descending tendency in dose-dependent manner (P < 0.05). It is concluded that the absence of SHP-1 mRNA expression in HL-60 cells and recovery of expression after treatment with As2O3 suggest the hypermethylation of SHP-1 gene related with pathogenesis of leukemia, and the abnormal increase of C-kit mRNA expression maybe exist in formation of leukemia. The effect of As2O3 on expression of SHP-1 and C-kit shows dose-dependency, the higher the As2O3 concentration, the higher the SHP-1 expression and the lower the C-kit expression, moreover, the effect of As2O3 shows time-dependency in specific concentration. The SHP-1 mRNA expression negatively relates with C-kit mRNA expression, suggesting that the decrease or absence of SHP-1 expression in leukemia cells weakens the negative regulation on C-kit signaling pathway, thus plays a role in the formation of leukemia.

Evaluation of tyrosine-kinase receptor c-kit mutations, mRNA and protein expression in canine lymphoma: Might c-kit represent a therapeutic target?

c-kit plays an important role in proliferation, survival and differentiation of hematopoietic progenitor cells. In human hematopoietic malignancies, c-kit is mostly expressed by progenitor cell neoplasms and seldom by mature cell neoplasms. Aim of this study was to evaluate c-kit expression in canine lymphoma. Twenty-five B-cell lymphomas and 21 T-cell lymphomas were enrolled in the study. c-kit mRNA and protein expression was measured in lymph node fine needle aspirates by quantitative real-time RT-PCR, flow cytometry and immunocytochemistry, while the occurrence of KIT mutations on exons 8–11 and 17 was investigated by direct cDNA sequencing. KIT mRNA was amplifiable but below the limit of quantification in 76% of B-cell lymphomas and 33% of T-cell lymphomas. Remaining samples showed a very low expression of KIT, except for some high grade (HG) T-cell lymphomas where a comparatively higher mRNA amount was observed. Transcriptional data were confirmed at the protein level. No gain-of-function mutations were observed. Among canine lymphomas, T-cell lymphoma typically shows an aggressive biological behavior, partly being attributable to the lack of efficacious treatment options, and the evidence of c-kit expression in HG T-cell lymphomas might represent the rationale for its routinely diagnostic evaluation and the use of tyrosine kinase inhibitors in future clinical trials.

Evaluation of tyrosine-kinase receptor c-KIT (c-KIT) mutations, mRNA and protein expression in canine leukemia: Might c-KIT represent a therapeutic target?

The tyrosine-kinase receptor c-KIT (c-KIT) plays an important role in proliferation, survival and differentiation of progenitor cells in normal hematopoietic cells. In human hematological malignancies, c-KIT is mostly expressed by progenitor cell neoplasia and seldom by those involving mature cells. Tyrosine kinase inhibitors (TKIs) are actually licensed for the first- and second-line treatment of human hematologic disorders. Aim of the present study was to evaluate c-KIT mRNA and protein expression and complementary DNA (cDNA) mutations in canine leukemia. Eleven acute lymphoblastic leukemia (ALL) and acute undifferentiated leukemia (AUL) and 12 chronic lymphocytic leukemia (CLL) were enrolled in this study. The amounts of c-KIT mRNA and protein were determined, in peripheral blood samples, by using quantitative real time RT-PCR, flow cytometry and immunocytochemistry, respectively. The presence of mutations on c-KIT exons 8–11 and 17 were investigated by cDNA sequencing. Higher amounts of c-KIT mRNA were found in ALL/AUL compared to CLL, and this latter showed a lower pattern of gene expression. Transcriptional data were confirmed at the protein level. No significant gain-of-function mutations were ever observed in both ALL/AUL and CLL. Among canine hematological malignancies, ALL/AUL typically show a very aggressive biological behavior, partly being attributable to the lack of efficacious therapeutic options. The high level of c-KIT expression found in canine ALL/AUL might represent the rationale for using TKIs in future clinical trials.

Effect of flutamide on folliculogenesis in the fetal porcine ovary – regulation by Kit Ligand/c-Kit and IGF-1/IGF-1R systems

a b s t r a c t In pigs, primordial to primary follicle transition occur in the late pregnancy. The interac- tions between Kit ligand (KL) and its receptor (c-Kit), as well as insulin-like growth factor 1 (IGF1) and cognate receptor (IGF1R) are crucial for the primordial follicle activation. It is well established that hormonal disruption induces abnormalities in the developing repro- ductive system. Hence, this study investigated the influence of antiandrogen, flutamide, on genes involved in the primordial to primary follicle transition. Pregnant gilts were injected with flutamide (50 mg/kg bw, seven times, every day) or corn oil (control groups) start- ing on gestation days 83 (GD90) or 101 (GD108). Fetal ovaries were excised on days 90 and 108 of gestation. The proportion of primordial and primary follicles was determined, and immunohistochemistry for c-Kit and IGF1R was conducted. To assess KL, c-Kit, IGF1 and IGF1R mRNA expression real-time PCR was performed. Ovaries from both GD90 and GD108 animals exhibited a greater proportion of primordial to primary follicles when compared to respective control groups. C-Kit and IGF1R were immunolocalized in the oocytes of pri- mordial and primary follicles. Both c-Kit mRNA and protein levels and KL mRNA expression were diminished in GD90 group. IGF1R expression decreased at mRNA and protein levels, whereas IGF1 mRNA expression was increased in GD90 and GD108 groups. In summary, our findings may indicate that the interactions between KL and c-Kit as well as IGF1 and IGF1R are relevant to the initiation of follicular transition from primordial into primary follicles and can be affected by AR signaling.

Involvement of interstitial cells of Cajal in experimental severe acute pancreatitis in rats

To observe the changes in interstitial cells of Cajal (ICC) in rats with experimental severe acute pancreatitis (SAP). A total of twenty-four SD rats were randomly divided into two groups (n = 12), namely the sham (S) group and the SAP group; the SAP rat model was established by retrograde injection of 5% sodium taurocholate (1.0 mL/kg) into the pancreatic duct. Twenty-four hours later intestinal motility was assessed by testing small intestinal propulsion rate, and then the rats were sacrificed. The pancreas and jejunum were resected and underwent routine pathologic examination. Immunohistochemical staining was used to detect c-kit-positive cells in the jejunum. Expression of c-kit mRNA was detected by real-time polymerase chain reaction, and the expression of c-kit protein was evaluated by Western blotting. Ultrastructure of ICC was evaluated by transmission electron microscopy. There was bleeding, necrosis and a large amount of inflammatory cell infiltration in pancreatic tissue in the SAP group, while in jejunal tissue we observed a markedly denuded mucosal layer, loss of villous tissue and a slightly dilated muscular layer. The small intestinal propulsion rate was 68.66% ± 2.66% in the S group and 41.55% ± 3.85% in the SAP group. Compared with the S group, the rate of the SAP group decreased sharply. The density of c-kit-positive cells in the SAP group was significantly lower than in the S group; the respective mean densities were 88.47 ± 10.49 in the S group and 56.11 ± 7.09 in the SAP group. The levels of c-kit protein and mRNA were 0.36 ± 0.04 and 1.29 ± 0.91 in the SAP group, respectively, which were significantly lower than those in the S group (0.53 ± 0.06, 0.64 ± 0.33, respectively). In the SAP group, ICC profiles showed the same change tendency, such as vacuolation of mitochondria, irregular vacuoles and loosened desmosome-like junctions. Decreased c-kit-positive cells and ultrastructural changes in ICC resulting from blockade of the c-kit signaling pathway are involved in the intestinal dysmotility associated with SAP.

51 Platinum(II) phenanthroimidazoles with “clicked” side chains as selective G-quadruplex DNA binders

G-quadruplexes (noncanonical secondary structures), have gained recognition as viable targets for chemotherapeutic drug design based on their ability to interfere with cancer cell proliferation. These DNA structures, held together by Hoogsteen hydrogen bonds, result from the folding of guanine (G)-rich DNA sequences in the presence of potassium or sodium cations.(Lane et al., 2008) G-quadruplex (G4)-forming sequences have been identified throughout the human genome, and depending on the sequence, different monomeric topologies prevail. These varying topologies present a means of specific targeting of one polymorph rather than all polymorphs. Two regions that have been highly investigated as G4 targets for small molecule drugs are the telomeres and the promoters of oncogenes. To date, both organic and inorganic compounds with varying degrees of efficacy in their targeting of G4 structures have been reported.(Georgiades et al., 2010; Monchaud & Teulade-Fichou, 2008) We have previously shown that Pt(II) phenanthroimidazole binders are good stabilizers of both intermolecular and intramolecular human-telomere-derived G4 motifs.(Castor et al., 2012; Kieltyka et al., 2008) Now, we focus on broadening our target to include those G4s derived from the c-myc and c-kit oncogene promoters due to their apparent ability in controlling gene expression through regulating transcription and translation. We hypothesized that the addition of side chains to our existing phenanthroimidazole core will enable our complexes to discriminate between different groove environments presented by topologically distinct G4s. Thus, we have taken our core and have appended “clicked”-amine containing side chains from the phenyl moiety of the phenylphenanthroimidazole. Through a high-throughput fluorescence intercalator displacement assay, circular dichroism, surface plasmon resonance, and molecular modeling, we have evaluated three complexes with G4s derived from human telomere, c-myc, and c-kit sequences and have shown enhanced binding of the complexes with the c-kit sequence and an unprecedented kinetic profile from SPR. We hope that this kinetic profile results in a long residence time while interacting with the c-kit promoter G-quadruplex, resulting in lower levels of c-kit mRNA in human glioblastoma cells and subsequent down-regulation of the gene expression.

The identification of c-Kit-positive cells in the intestine of chicken

The ultrastructure of the interstitial cells of Cajal (ICC) has been examined in birds, but the distribution of these cells remains obscure because a suitable marker is lacking. In the present study, the identification and expression of c-Kit-positive cells in the chicken intestine were demonstrated by means of in situ hybridization histochemistry and the expression of the c-Kit gene by real-time quantitative PCR. Two types of cells stained positive for c-Kit mRNA. The first group consisted of spindle-shaped or bipolar cells identified as ICC. The ICC were found at a variety of locations: at the level of the myenteric plexus between the circular and longitudinal muscle and intermingled with smooth muscle cells within muscle bundles in the circular and longitudinal muscle layers. The ICC were also identified along the submucosal layer. The second group was composed of round-shaped cells, which resembled mast cells. Mast cells were mainly found in the lamina propria region as well as in the submucosal layer. The expression of the c-Kit gene by real-time quantitative PCR revealed the expression of c-Kit mRNA throughout the lamina muscularis and mucosa of the intestine; however, the quantitation was variable in different regions. This study reveals conclusively for the first time the distribution of ICC, quantifies the expression of c-Kit mRNA in the intestine of adult chicken, and also compares the c-Kit-positive cell types morphologically.

1679P - Heat Shock Protein 90 Inhibition Downregulates C-Kit Expression in Gist Cells Through Diminishing Transcription and Enhancing Protein Degradation via both Autophagy and Proteasome Pathways

ABSTRACT Background Gastrointestinal stromal tumor (GIST) is a mutant oncoprotein c-Kit droved cancer. Patients suffered drug resistance after prolonged standard treatment of tyrosine kinase inhibitor (TKI), imatinib mesylate (IM). Inhibition of heat-shock protein 90 (Hsp90), a chaperone responsible for protein maturation and stability, causing its client proteins degradation, as c-Kit, is a developing therapy for cancer. However, the mechanisms of Hsp90 inhibition-induced c-Kit downregulation in post-translational and transcriptional levels have rarely been investigated. Methods and results Our data showed that NVP-AUY922 (AUY922), a new class of Hsp90 inhibitor, inhibited the growth of GIST882 and GIST48 cells with mutant c-Kit protein expression that was accompanied with reduction of both total and phosphor-c-Kit protein and induction of apoptosis. Pharmacological inhibition on either autophagy or proteasome degradation pathway could partially reverse endogenous c-Kit turnover and AUY922-induced c-Kit reduction. These findings were further confirmed by the co-localization of c-Kit with either LC-3B or acridine orange-labeled autophagosome in confocal microscopy, and delay of c-Kit degradation by silencing autophagosome essential Beclin-1 protein. In addition, AUY922 could also reduce c-Kit mRNA without affecting its degradation. Our preliminary data showed that activities and nuclear levels of several transcription factors in GIST cells were diminished after AUY922 treatment. DNA affinity precipitation assay and chromatin immunoprecipitation (ChIP) will be performed to validate the binding of the candidate transcript factors on the c-Kit promoter. Conclusions This is the first report showing the involvement of autophagy in endogenous as well as Hsp90 inhibitor-induced c-Kit degradation. Moreover, AUY922 treatment resulted in downregulation of c-Kit through protein degradation and transcriptional mRNA regulation in GIST cells. These findings address the mechanisms of AUY922 on c-Kit protein downregulation and highlight the potential use of AUY922 in TKI-refractory, c-Kit-expressing GIST. Disclosure All authors have declared no conflicts of interest.

Prognostic significance of c-KIT in vulvar cancer: bringing this molecular marker from bench to bedside

BackgroundVulvar carcinomas are rare tumors, and there is limited data regarding molecular alterations. To our knowledge there are no published studies on c-KIT and squamous cell carcinomas of the vulva (VSCC). Although there are a significant number of other tumor types which express c-KIT, there remains controversy as to its relationship to patient outcome. Thus, we wished to investigate such controversial findings to determine the prognostic importance of c-KIT by evaluating its protein and mRNA expression in VSCCs, correlating these findings with clinicopathological features and Human Papillomavirus (HPV) infection.Methodsc-KIT expression was scored by immunohistochemistry (IHC) as positive or negative in 139 formalin-fixed paraffin-embedded (FFPE) cases of vulvar carcinomas arrayed in a tissue microarray (TMA) using the DAKO® A4502 rabbit polyclonal c-KIT antibody (diluted 1:100). c-KIT mRNA was evaluated by qRT-PCR in 34 frozen samples from AC Camargo Hospital Biobank (17 tumoral and 17 non-tumoral samples) using TaqMan probes–Applied Biosystems [Hs00174029_m1]. HPV genotyping was assessed in 103 samples using Linear Array® HPV Genotyping Test kit (Roche Molecular Diagnostics, Basel, Switzerland). All results obtained were correlated with clinical and pathological data of the patients.Resultsc-KIT protein was positive by immunohistochemistry in 70.5% of the cases and this was associated with a higher global survival (p = 0.007), a higher recurrence-free survival (p < 0.0001), an absence of associated lesions (p = 0.001), lymph node metastasis (p = 0.0053), and HPV infection (p = 0.034). Furthermore, c-KIT mRNA quantitation revealed higher levels of transcripts in normal samples compared to tumor samples (p = 0,0009).ConclusionsOur findings indicate that those vulvar tumors staining positively for c-KIT present better prognosis. Thus, positivity of c-KIT as evaluated by IHC may be a good predictor for use of more conservative surgery techniques and lymph node dissection in vulvar cancer. So part of the essence of our study is to see the possibility of translating our current results from the bench to the bedside. This will help provide patients a more appropriate, less mutilating treatment, in order to keep the maximum physical and psychic quality as possible to these women.

Panax ginseng Modulates Cytokines in Bone Marrow Toxicity and Myelopoiesis: Ginsenoside Rg1 Partially Supports Myelopoiesis

In this study, we have demonstrated that Korean Panax ginseng (KG) significantly enhances myelopoiesis in vitro and reconstitutes bone marrow after 5-flurouracil-induced (5FU) myelosuppression in mice. KG promoted total white blood cell, lymphocyte, neutrophil and platelet counts and improved body weight, spleen weight, and thymus weight. The number of CFU-GM in bone marrow cells of mice and serum levels of IL-3 and GM-CSF were significantly improved after KG treatment. KG induced significant c-Kit, SCF and IL-1 mRNA expression in spleen. Moreover, treatment with KG led to marked improvements in 5FU-induced histopathological changes in bone marrow and spleen, and partial suppression of thymus damage. The levels of IL-3 and GM-CSF in cultured bone marrow cells after 24 h stimulation with KG were considerably increased. The mechanism underlying promotion of myelopoiesis by KG was assessed by monitoring gene expression at two time-points of 4 and 8 h. Treatment with Rg1 (0.5, 1 and 1.5 µmol) specifically enhanced c-Kit, IL-6 and TNF-α mRNA expression in cultured bone marrow cells. Our results collectively suggest that the anti-myelotoxicity activity and promotion of myelopoiesis by KG are mediated through cytokines. Moreover, the ginsenoside, Rg1, supports the role of KG in myelopoiesis to some extent.

Electroacupuncture improves behavioral recovery and increases SCF/c-kit expression in a rat model of focal cerebral ischemia/reperfusion

This study aims to investigate the mechanism of electroacupuncture (EA) in promoting behavioral recovery after focal cerebral ischemia/reperfusion. The SD rats received filament occlusion of the right middle cerebral artery for 2 h followed by reperfusion for 1, 3, 7, 14, and 21 days, respectively. Rats were randomly divided into sham group, model group and EA group. After 2 h of the reperfusion, EA was given at bilateral "Hegu" point (LI 4) in the EA group. Neurobehavioral evaluation, the expression of stem cell factor (SCF), its receptor c-kit and matrix metalloproteinase-9 (MMP-9) protein and mRNA in the cortical ischemic region were measured. EA treatment can improve behavioral recovery after ischemia/reperfusion. Compared with the sham group, the positive cells and mRNA expression of SCF, c-kit, MMP-9, the protein expression of SCF were increased significantly in the model and EA groups (P < 0.001). Compared with the model group, the positive cells, protein and mRNA expression of SCF were increased significantly in EA groups (P < 0.01). The positive cells and mRNA expression of c-kit were increased in EA groups beginning at 3 day and remained significantly high thereafter. The expression of MMP-9 positive cells and mRNA were deceased significantly in the 1 day subgroup in EA (P < 0.01), but increased significantly in the 3, 7 days subgroups (P < 0.01). We conclude that EA treatment up-regulates the positive cells and mRNA expression of SCF, c-kit and MMP-9 after cerebral ischemia/reperfusion. EA may promote neurobehavioral recovery by increasing the protein and mRNA expression of SCF, c-kit and MMP-9 after cerebral ischemia/reperfusion.