C-kit Protein Expression Research Articles

Enhanced Gastric Ghrelin-Induced c-Kit Protein Expression in Rats with Gastric Outlet Obstruction

Enhanced Gastric Ghrelin-Induced c-Kit Protein Expression in Rats with Gastric Outlet Obstruction

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.

Patient profile modulates cardiac c-kit+ progenitor cell availability and amplification potential

Human c-kit(+) cardiac progenitor cells (CPCs) are multipotent and may be used for cardiac repair. The effect of cardiovascular risk factors and medications on CPCs isolation efficiency, c-kit stem cell marker expression, and exvivo proliferative potential is unknown and was examined in the present work. Cells from human right atrial appendages (n = 50) were expanded in culture; after ∼16 days (T0), it was established the percentage of CPCs and c-kit protein mean fluorescence intensity (MFI) by fluorescence activated cell sorting (FACS). Thereafter, CPCs were isolated by high throughput sorting; after culturing for 4 passages CPCs-derived cells were re-analyzed to assess c-kit(+) cell percentage and enrichment vs T0. The association between 19 demographic and clinical variables to CPCs number and MFI at T0, and CPCs enrichment at P4, was determined by multiple linear regression analysis with stepwise selection procedure. The results revealed that (1) at T0, the number of isolated CPCs directly correlated to β-blocker treatment; (2) at T0, c-kit protein expression directly correlated to pulmonary hypertension (PH); (3) at P4, CPC's enrichment inversely correlated to smoke, atrial fibrillation (AF), a history of myocardial infarction, whereas it directly correlated to PH and statins. Patient clinical profile and medications differently modulate CPCs isolation and amplification potential exvivo. These results may provide new insights for the understanding of cardiac homeostasis and suggest both limitations and possible enhancing strategies for the therapeutic use of cardiac-resident progenitor cells.

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.

Expression of c-kit protein in cancer versus normal breast tissue

Aim of the studyThere are at least four main signal transduction pathways within human cells which are activated by interaction of an extracellular ligand with its corresponding receptors. One of them is activation of protein kinase. Actually, any of these proteins at any level of the signaling cascade in a human cell can undergo mutation and cause irregular cellular proliferation and finally result in cancer. C-kit is alternatively called stem cell factor receptor (SCFR) or CD117. It appears that lack of c-kit expression accompanies progression of some tumors, e.g. lung, breast, GIST. The aim of this study was to evaluate C-kit protein expression level within cancer cases.Material and methodsSixty specimens of breast cancer and 60 non-cancerous breast tissue specimens were evaluated by IHC for C-kit presentation. We used positive GIST slides as controls. Epi-info ver 6.04 (CDC, WHO) was used for analysis.ResultsC-kit was negative in all breast cancer specimens. C-kit was negative in 47 (78%) of 60 non-cancerous breast tissue specimens, but was positive in 13 (22%) of them (p < 0.0001).ConclusionsThere is a reduction in C-kit expression with malignant transformation of breast epithelium. C-kit is believed to play a role in breast carcinogenesis. However, we should follow patients with normal or benign breast tissue to indicate any correlation between C-kit presentation and breast cancer development.

P3-01-11: Increased Gene Copy Number of c-KIT and VEGFR2 at 4q12 in Primary Breast Cancer Is Related to an Aggressive Phenotype and Impaired Prognosis.

Abstract Introduction: Triple-negative breast cancer (TNBC) accounts for approximately 15% of all female breast cancer and is associated with aggressive clinical behaviour. No targeted treatments are available for TNBC. Drugs inhibiting tyrosine kinases, such as vascular endothelial growth factor receptor 2 (VEGFR2) and c-KIT, have however shown some promising results for patients with TNBC. The aim of the present study was to investigate whether gains and/or amplifications of VEGFR2 and c-KIT occur in TNBC. These genes may constitute novel candidate biomarkers for selecting TNBC patients for treatment with tyrosine kinase inhibitors. Material &amp; Methods: Fluorescence in situ hybridization (FISH) was used to quantify gene copy numbers of VEGFR2 and c-KIT in 83 primary human breast cancers, of which 31 were classified as TNBC. Gains were defined as ≥4 gene copies in more than 40% of the cancer cells, while amplification was defined as gene copy/CEP ratio &amp;gt;2 in more than 10% of the cancer cells. A tumour was considered FISH positive for c-KIT and/or VEGFR2 if it displayed copy number gain and/or amplification. Immunohistochemical (IHC) staining was performed for assessment of c-KIT protein expression. Results: Ten (32%) of the TNBCs were VEGFR2 FISH positive and nine (29%) were c-KIT FISH positive, whereas non-TNBCs were FISH positive for VEGFR2 and c-KIT in nine (18%) cases for both genes. No significant difference in frequency between TNBCs and non-TNBCs was found. There was a correlation between FISH positivity for VEGFR2 and c-KIT (c2 test, P&amp;lt;0.001), and VEGFR2 and c-KIT FISH positivity correlated to ER/PgR negativity and high Nottingham histological grade (NHG). A significantly worse breast cancer specific survival (BCSS) was seen for FISH positive cases in the whole cohort as well as among untreated patients and non-TNBCs, but not among TNBC cases. Discussion: The high correlation between VEGFR2 and c-KIT FISH positivity suggests that the genes are co-amplified. Increased copy number of both genes was related to aggressive disease and a worse prognosis, and thus has the potential of functioning as a novel predictive biomarker for selected targeted therapy particularly in the difficult-to-treat TNBC patient category. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-11.

C-KIT messenger RNA and protein expression and mutations in canine cutaneous mast cell tumors

Cutaneous mast cell tumors (MCTs) are among the most common neoplasms in dogs and show a highly variable biologic behavior. Histological grading, cell proliferation markers, and KIT immunohistochemistry are typically used to predict post-surgical prognosis. In the present study, c-KIT messenger RNA (mRNA) expression was measured in canine MCTs and its relationship with tumor grade, immunohistochemical staining pattern, post-surgical prognosis, and mutations was investigated. A significant increase of c-KIT mRNA was observed in MCTs versus healthy skin and surgical margins. Mutations were observed in 8.3% of cases. The KIT staining pattern was investigated for both grading systems. In particular, staining pattern III was associated with grade II (G2) and G3 MCTs, while staining patterns I and II were associated with G1 and G2 MCTs. Considering the 2-tier histological grading, the high grade was mainly associated with pattern III (71%) while the low grade was associated with patterns II (70%) and I (28%). A weak association between the KIT staining pattern and outcome was also observed. The results obtained suggest that c-KIT mRNA is overexpressed in canine MCT, although the fold variations were not associated with the protein localization or complementary DNA mutations. These observations suggested that the 3 events were independent. The histological grading and the KIT staining pattern have prognostic value as previously published. Staining pattern I could be especially helpful in predicting a good prognosis of G2 MCTs. Sequence mutations were not necessarily suggestive of a worse prognosis, but might be useful in choosing a chemotherapy protocol.

Octreotide Ameliorates Intestinal Dysmotility by Interstitial Cells of Cajal Protection in a Rat Acute Necrotizing Pancreatitis Model

Intestinal motility is impaired in acute necrotizing pancreatitis (ANP). The aim of present study was to investigate the effects of octreotide on the small intestinal motor function during experimentally induced ANP. L-Ornithine was intraperitoneally injected to induce ANP. Octreotide was administrated subcutaneously every 8 hours. The small intestine migrating myoelectrical complexes and slow waves in vivo were recorded before and after (24, 48, and 72 hours) ANP induction. The morphological alterations of interstitial cells of Cajal (ICCs) in deep muscular plexus were evaluated by immunohistochemistry and Western blots. Disturbed migrating myoelectrical complex cycle length and decreased dominant frequency of slow waves exacerbated gradually with time. The bolus applications of octreotide per 8 hours attenuated these functional abnormalities. The result of morphological study suggested that octreotide might ameliorate the damage of ICCs at 48 and 72 hours after ANP induction. Decreased expression of c-Kit protein at 72 hours was also attenuated by octreotide. The pathogenesis of the ileus in ANP may be related to the sustained deficiencies in ICCs. Octreotide may ameliorate the severity of ileus by minimizing the injury of ICCs.

Targeted Therapy in Lung Cancer; Lessons Learned from Past Experiences

Targeted Therapy in Lung Cancer; Lessons Learned from Past Experiences

C-kit and bcl-2 Are Not Useful Markers in Differentiating Adenoid Cystic Carcinoma from Polymorphous Low-Grade Adenocarcinoma

Aims. To evaluate the immunohistochemical expression of bcl-2 and c-kit proteins in the salivary gland tumors, adenoid cystic carcinoma, and polymorphous low-grade adenocarcinoma with a view to tumor differentiation. Methods. Immunohistochemical studies were performed on 29 adenoid cystic carcinoma and 23 polymorphous low-grade adenocarcinoma archival cases with bcl-2 and c-kit antibodies using standard procedures, and the data were statistically analyzed. Results. The difference in bcl-2 expression in adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma was not statistically significant. Whilst the difference in c-kit expression was statistically significant (), the difference proved small so as to be of no practical or diagnostic importance. Moderate to strong immunostaining was noted in adenoid cystic carcinoma with c-kit, the pattern and intensity varying within different histological subtypes, being greater the more aggressive the tumor subtype. Conclusions. Bcl-2 and c-kit are not useful as immunohistochemical markers in differentiating adenoid cystic carcinoma from polymorphous low-grade adenocarcinoma.

A comprehensive profiling of colorectal cancer patients through multiplexed functional pathway signature and somatic mutation analysis for effective therapy selection.

e14103 Background: Colorectal cancer (CRC) is a leading cause of death in the developed nations. The development of effective forms of cytotoxic chemotherapy for colorectal cancer has been paralleled by the emergence of monoclonal antibodies against proteins that are thought to be important in the proliferation of malignant cells. Recent clinical studies showed that EGFR-targeting agent improved response rate when added to chemotherapy, however patients with KRAS mutations did not benefit from such treatment. Here we report a comprehensive profiling of pathwy signal proteins for their level of expression/activation and presence of somatic mutations in tumor tissues obtained from 136 CRC patients. Methods: The collaborative enzyme enhanced reactive immunoassay (CEER) is a multiplexed protein microarray platform requiring co-localization of two detector enzyme-conjugated-antibodies. Once target proteins are captured on the microarray-surface, channeling events between two detector enzymes in proximity enable the profiling of the target proteins with high sensitivity. In this study, tumor lysates were prepared from frozen tissues obtained from 136 CRC patients. In addition to a multiplexed analysis for panel of mutations in KRAS and BRAF genes, the level of expression and activation of HER1, HER2, HER3, cMET, cKIT, IGF-1R, PI3K, AKT, ERK and other pathway proteins were determined using CEER technology. Results: KRAS and BRAF mutations were found in ~40% (54/136) and ~5% (6/136) respectively in this cohort. The most frequent mutant alleles were G12D (14%) and G13D (13%). Varying levels of RTKs and associated activation patterns were observed. Levels of phosphorylated ERK and AKT correlated to expression/activation patterns of HER1, cMET, HER3 and prevalence of each biomarker along with pathway redundancy will be presented. Conclusions: A comprehensive profiling of multiplexed pathway/ somatic mutation analysis provides critical information needed to select the most effective targeted agents to overcome pathway redundancy /activating mutation related drug resistance through treatment combination or sequencing.

Selection of targeted agent(s) based on matching differential protein expression and mutational profile in NSCLC patients.

e21085 Background: Treatment of nonsmall-cell lung cancer (NSCLC) with tyrosine kinase inhibitors (TKIs) shows initial response, but most tumors develop acquired resistance to TKIs due to secondary resistance mutations (T790M) and amplification of other RTKs. Patients stratified for mono therapy treatment based on genotype alone is not sufficient as other kinases contribute to survival and necessitate inhibition of multiple kinases. Methods: The Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) is a multiplexed protein microarray platform requiring co-localization of two detector enzyme-conjugated-antibodies. CEER is capable of high detection sensitivity from FNA/CTC viable samples (23-35 gage needle). Lysates were prepared from frozen tissues obtained surgically from 71 Caucasian and 29 Asian patients with NSCLC. Expression and activation of EGFR, ErbB2, ErbB3, cMET, IGF1R, cKIT, Shc, PI3K, AKT, ERK, VEGFR2, Src, FAK, Stat5, JAK2, CrKL, and other pathway proteins were profiled. Genotyping on panel of mutations in KRAS, EGFR, and BRAF were performed on all samples. Results: We have observed differential expression patterns between Asian and Caucasian patients. KRAS is mutated in 30% of all the patients. In Asians, EGFR over expression is higher (27%) compared to the Caucasians (3%). Caucasian patients expressed high Her3 (58%), cMET (25%), and co expression of both cMET and HER3 (27%). VEGFR2 is highly expressed in 17% of all patients. Patients over expressing cMET, HER3, and HER2 will benefit from combination of pan-HER and cMET inhibitors. Those over expressing cMET and EGFR will benefit from cMET and EGFR inhibitors. Lastly, patients over expressing VEGFR2 and cMET will benefit from cMET and VEGFR inhibitors. A comprehensive differential biomarker prevalence for each NSCLC sub-populations for all tested RTKs and signaling proteins will be presented. Conclusions: Mono therapy and patient selection based on genotype is not an effective criteria for treatment options. Instead, a comprehensive pathway profile using FNA/CTC should guide selection of appropriate therapy (combined or sequenced), and shifts in pathway profile should be monitored for appropriate response.

Abstract 2310: Down-regulating BCL-2 gene expression in a resistant GIST cell line by a RNA G-quadruplex interacting small molecule ligand

Abstract We have previously shown that KIT protein expression in the gastrointestinal tumour (GIST) cell line GIST882 can be down-regulated using a small molecule approach that targets expression at the transcriptional level. The small-molecule naphthalene diimide derivative ND1 developed as a G-quadruplex telomere targeting agent can inhibit telomerase activity and can also reduce c-kit mRNA levels, consistent with a dual G-quadruplex mechanism of action in this cell line. An Imatinib (Gleevec) resistant patient-derived cell line GIST48 expresses high levels of the anti-apoptotic protein bcl-2. The compound ND1 down-regulates the expression of bcl-2 in this resistant cell line, while having no discernable effect on c-kit protein expression levels or telomerase activity, both associated with DNA quadruplex stabilization. Instead we observe that translation of the apoptosis-related protein is impeded, while mRNA levels remain unaffected. The identification of a G-rich quadruplex-forming sequence in the 5’-UTR region of the bcl-2 gene, upstream of the translation start site, lead us to the development of a dual luciferase reporter assay. Utilizing a psiCHECK-2 vector we inserted the G-rich 67 base pair bcl-2 5’-UTR gene sequence upstream of the Renilla luciferase start codon. In vitro experiments have shown that the G-rich sequence can form a stable RNA G-quadruplex and interfere with translation. Our in vivo transfection experiments with ND1 in a GIST cell line shows concentration-dependent inhibition of bcl-2 protein expression. RNA G-quadruplex formation and ligand stabilization may thus be a novel anti-cancer route for the down-regulation of the anti-apoptotic protein bcl-2 in chemo-resistant cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2310. doi:10.1158/1538-7445.AM2011-2310

Influence of Shenqing Recipe on Morphology and Quantity of Colonic Interstitial Cells of Cajal in Trinitrobenzene Sulfonic Acid Induced Rat Colitis

To observe the influence of Shenqing Recipe (SQR), a kind of Traditional Chinese Medicine, on the morphology and quantity of colonic interstitial cells of Cajal (ICC) in trinitrobenzene sulfonic acid (TNBS)-induced rat colitis, and to investigate the possible mechanism of SQR in regulating intestinal dynamics. Sixty rats were randomly divided into normal control, model 1, model 2, mesalazine, and high-dose, and low-dose SQR groups with 10 rats in each group. TNBS (10 mg) dissolved in 50% ethanol was instilled into the lumen of the rat colon of the latter five groups to induce colitis. On the 4th day after administration of TNBS, each treatment group was administered one of the following formulations by enteroclysis gavage once a day for 7 days: 600 mg•kg⁻¹•d⁻¹ mesalazine, 2.4 g•kg⁻¹•d⁻¹ SQR, and 1.2 g•kg⁻¹•d⁻¹ SQR. Model 2 rats received normal saline solution. After 7 days colonic samples were collected. While the colonic samples of model 1 group were collected on the 3rd day after TNBS administered. Ultrastructure of ICC in the damaged colonic tissues was observed with transmission electron microscope. Expression of c-kit protein in colonic tissue was determined by immunohistochemical staining and Western blot. The ultrastructure of colonic ICC in the rat model of TNBS-induced colitis showed a severe injury, and administration of SQR or mesalazine reduced the severity of injury. Similarly, the expression of c-kit protein of TNBS-induced colitis rat model was significantly decreased compared with the normal control group (P < 0.05). Treatment with SQR or mesalazine significantly increased the expression of c-kit protein compared with the administration of control formulations (P < 0.05), especially the high-dose SQR group. SQR could alleviate and repair the injured ICC, and improve its quantity, which might be involved in regulating intestinal motility.

Effect of inhibition of heat-shock protein 90 on mutant c-Kit protein as modulated by mRNA expression and protein degradation through both proteasome and autophagy-mediated pathways.

54 Background: c-Kit is a known client protein of heat-shock protein 90 (Hsp90). Inhibition of Hsp90 can reduce the expression of c-Kit protein in human mastocytoma and gastrointestinal stromal tumor (GIST) cells, which is conceived through the proteasome degradation pathway. Methods: COS-1 cells transfected with vectors containing CMV promoter-driven various c-Kit mutants (imatinib- sensitive or imatinib-resistant) and human GIST882 cells (with endogenous exon 13 mutant c-Kit) were used to investigate the efficacy NVP-AUY922, a new class of Hsp90 inhibitor, on suppressing the constitutive activation of mutated c-Kit. Detail mechanisms of protein regulation, as RNA transcription, RNA stability, and protein degradation were studied. Results: NVP-AUY922 is more potent than 17-AAG to inhibit the proliferation of imatinib-sensitive GIST882 cells. Further study showed that NVP-AUY922 down-regulated both total and phosphorylated c-Kit proteins in dose- and time-dependent manners in both GIST882 cells and mutant c-Kit (including imatinib-resistant exon11/17 double mutants) expressed in COS-1 cells. Surprisingly, the NVP-AUY922-induced c-Kit degradation in both cell models could be reversed by proteasome-degradation inhibitor and autophagy inhibitor. The involvement of autophagy in NVP-AUY922-induced c-Kit protein degradation was further confirmed by co-localization of c-Kit and autophagosome by immuno-staining. In addition, NVP-AUY922 treatment resulted in a reduction of c-Kit mRNA level in GIST882 cells but not c-Kit expressing COS-1 cells, which could be explained by the use of CMV promoter in artificial COS-1 cell model. Conclusions: Taken together, these findings provide the first evidence that down-regulation of mutant c-Kit protein by Hsp90 inhibitor involved both transcription and post-translation levels. Of the latter, autophagy- as well as proteasome-mediated degradation pathways were involved. These observations highlight the use of NVP-AUY922 either alone or in combination with autophagy modulators in imatinib-refractory, c-Kit-expressing GIST. Supported by 99D1-TC-CADOH03. [Table: see text]

Anti-apoptotic and growth-promoting markers in adult medulloblastomas

the aim of this study was to investigate the pathologic features, proliferation potential and expression of some anti-apoptotic and growth-promoting markers in adult medulloblastomas. we analyzed the immunohistochemical expression of survivin, c-KIT, Bcl-2, fascin, p-53 and Ki-67 in 18 adult medulloblastomas (> 16 years of age). study included 14 males and 4 females (mean age: 22.9 ± 8.2). 14 cases were classical, 2 desmoplastic/nodular and 2 large cell medulloblastomas. Moderate-to-high nuclear survivin expression was observed with high percentages (55 - 100%) in all medulloblastomas while Bcl-2 was mildly positive in only 1 case. Interestingly, mild-to-moderate cytoplasmic c-KIT expression was demonstrated in 16 cases (89%) without membranous accentuation. Fascin expression was observed in 13 medulloblastomas (72%), 9 of which showing moderate to high immunoreactivity. Mild p53 expression was present in only 4 cases (22%). Mean Ki-67 index was 20.6% (range 8 - 55%). frequent nuclear survivin expression implies the predominance of anti-apoptotic factors in pathogenesis of adult medulloblastomas. It may also be a potential therapeutic target for adult medulloblastomas. Although Blc-2 immunoreactivity was previously reported in approximately 30% in medulloblastomas, we have observed that it is rarely expressed in the present series of adult medulloblastomas. To our knowledge, this is the first study evaluating fascin expression in medulloblastomas. Its presence may be related to the neuronal differentiation. Mild-to-moderate cytoplasmic c-KIT immunoreactivity without membranous staining in adult medulloblastomas may support the previous studies reporting low level of c-KIT protein expression with lack of activating mutations in medulloblastomas. It seems p53 is rarely involved in the course of develepment of adult medulloblastomas.

39 REGULATION OF THE SEMINIFEROUS TUBULES MOTILITY IN PATIENTS WITH SECONDARY SCO: PRESENCE OF C-KIT + CELLS

Introduction: Sertoli-cell-only (SCO) syndrome, is also called germ cell aplasia. The histological diagnosis of Sertoli-cell-only (SCO) syndrome is confirmed when the examined tissue reveals that all seminiferous tubules contain only Sertoli cells. Theoretically the SCO is caused by a prenatal defect in migration of germ cells into the seminiferous tubules, resulting in sterility. Conversely, secondary SCO is a result of postnatal damage to healthy testicular tissue that may result only in a focal histological SCO pattern. Primary and secondary SCO differ substantially in tubular wall histology, in morphology and function of Sertoli cells, and in the appearance of the interstitial tissue. However, it is possible that they also differ in the motility of the seminiferous tubules. Objective: In this research we aim to identify and quantify the c-kit + cells in human testes with histological confirmation of SCO secondary. Material and Methods: Seven samples with confirmed diagnosis of secondary SCO were taken from the Laboratory of Reproduction, Faculty of Medicine of the University of Chile were obtained, and the appropriate authorizations for bioethics. Paraffin sections were mounted on silanized slides to conduct immunohistochemistry protocols HRP / DAB to identify cells expressing c-kit protein with the primary antibody anti c-kit (CD177, clone YR145, BSB 5322). Optical microscopy to quantify the number of positive cells per unit area present in the interstitial compartment of the testis (using Micrometrics SE Premium software, 2009). Then the means and standard deviations between patients were compared by t test with p 0.05. Results: The biopsies with a diagnosis of secondary SCO is possible to recognize the presence of c-kit + cells through immunohistochemistry. Phenotypically these cells in the interstitium were observed with a core comatina rounded and dense, with abundant perinuclear cytoplasm in an approximate ratio of 3/1 to the core. Equally distributed in the interstitium itself also in the vicinity of the seminiferous tubules. Discussion: C-kit cells are involved in contractile activity in a wide variety of organs, and usually associated with contractile cells function properly. These cells operate independently of the general nervous system. Normally in a healthy testicle cells are scarce and rarely seen their cytoplasm, which is occupying the proprietary extensions that contact with the effector cells. Therefore, the diagnosis of secondary SCO observed changes in the quantity (more, p 0.05) and distribution of cytoplasm (mainly perinuclear) taking lead to modifications in the motility of the seminiferous tubules and runoff of its content, which together lead to changes in male fertility (Grant Fondecyt No. 1101046, 2010). 40 THE EFFECTS OF PROGESTERONE AND EXOGENOUS GONADOTROPIN ON THE INCIDENCE OF APOPTOSIS IN MOUSE BLASTOCYSTS M. Salehnia1, F. Panahi1, M.F. Moghadam2. 1Department of Anatomy, Tarbiat Modares University, Tehran, Iran, 2Department of Biotechnology, Tarbiat Modares University, Tehran, Iran

The role of c‐kit‐positive interstitial cells in mediating phasic contractions of bladder strips from streptozotocin‐induced diabetic rats

• To investigate the role of c-kit-positive interstitial cells (ICCs) in mediating muscarinic receptor-induced phasic contractions of isolated bladder strips from streptozotocin(STZ)-induced diabetic rats and to confirm the expression and location of ICCs in the rat bladder. • Bladders were removed from STZ-induced diabetic rats at 1, 4 and 12 weeks after induction of diabetes and from age-matched controls. • To investigate the functional role of ICCs in mediating phasic contractions, bladder strips were isolated from control and diabetic rats and mounted in tissue baths. • Strips were stimulated with low concentrations of the muscarinic receptor agonist carbachol (CCH; 0.1 µm) to induce phasic contractions and the effect of increasing concentrations (1-50 µm) of imatinib (Glivec® or Gleevec®, formerly STI571), a c-kit tyrosine kinase inhibitor, was then investigated. • For molecular studies, to detect expression of the c-kit tyrosine kinase receptor (c-kit), total cellular RNA was extracted from rat bladders and reverse-transcribed to obtain complementary DNA (cDNA). • Reverse transcription-polymerase chain reaction (RT-PCR) was then performed using primers specific to the c-kit sequence and amplified products separated by agarose gel electrophoresis. • Amplified PCR products were excised from the gel, sequenced and compared with the known c-kit sequence to confirm their identity. • For immunohistochemical detection, whole mount preparations of control rat bladders were fixed in acetone and labelled using antibodies directed to the ICC marker c-kit. • In functional studies, CCH induced phasic contractions in bladder strips from control and diabetic rats. Bladder strips from 1-week diabetic rats showed CCH-induced phasic contractions, which were greater in amplitude, but had lower frequency, than the controls, whilst no such differences were apparent at later time points of diabetes. • Imatinib decreased the amplitude and the frequency of the CCH-induced phasic contractions in both control and diabetic tissues in a concentration-dependent manner, although in diabetic tissues this effect was only seen at the higher concentrations of imatinib. RT-PCR of bladder cDNA yielded a single amplicon of 480 bp. • The sequence of this amplicon showed a 98% homology with the published c-kit sequence, thus confirming c-kit mRNA expression in both control and 1-week diabetic rat bladder. • Expression of c-kit protein was also detected in a network of cells on the edge of and between smooth muscle bundles of control rat bladders by positive immunoreactivity to c-kit specific antibodies. • These data show the presence of c-kit-positive ICCs in rat urinary bladder and their importance in mediating muscarinic receptor-induced phasic contractions of bladder strips from control and diabetic rats. The role of these ICCs does not seem to be significantly altered by the diabetic state.

Effects of glial cell line-derived neurotrophic factor on the proliferation and differentiation of PLZF and c-Kit of spermatogonial stem cells of rats in vitro

Objective To investigate the effects of glial cell line-derived neurotrophic factor(GDNF)on the gene transcriptions and expressions of promyelocytic leukaemia zinc finger(PLZF)and c-Kit in spermatogonial stem cells(SSC).Methods SSC was cultured in DMEM plus different concentrations of GDNF(0,10,50,100 ng/ml).Quantitative reverse transcription PCR was used to detect the mRNA of PLZF and c-Kit.The expressions of PLZF and c-Kit protein were detected by Western blot assay.Results With the control group and 10ng/ml of GDNF group cell growth has no obvious influence,but with 50 ng/ml of GDNF group and 100 ng/ml of GDNF group cell growth was enhanced significantly.In the control group,the PLZF mRNA was 0.28±0.13 and c-Kit mRNA was 0.65±0.21.In the 10 ng/ml of GDNF group,PLZF mRNA was 0.27±0.14 and c-Kit tuRNA was 0.62±0.19.Compared with the control group,10 ng/ml of GDNF group had not influence on PLZF and c-Kit mRNA.In the 50 ng/ml of GDNF group PLZF mRNA was 0.64±0.28 and c-Kit mRNA was 0.34±0.15.Compared with the control group,50 ng/ml of GDNF enhanced the transcription of PLZF mRNA(P＜0.05),decreased the transeription of c-Kit mRNA (P＜0.05).In the 100 ng/ml of GDNF gmup,PLZF mRNA was 0.68±0.27 and c-Kit mRNA was 0.28±0.18.In the 100 ng/ml of GDNF group,there was no difference compared with 50 ng/ml of GDNF.In the control group,PLZF protein was 0.34±0.13 and c-Kit protein was 0.72±0.27.In 10 ng/ml of GDNF group,PLZF protein was 0.38±0.18 and c-Kit protein was 0.69±0.26.Compared with the control group,10 ng/ml of GDNF had no influence on PLZF and c-Kit protein.In 50 ng/ml of GDNF group,PLZF protein was 0.68±0.26 and c-Kit protein was 0.35±0.15.50 ng/ml of GDNF enhanced the expression of PLZF protein(P＜0.05),decreased the expression of c-Kit protein(P＜0.05).In 100 ng/ml of GDNF group PLZF protein was 0.70±0.27 and c-Kit protein Was 0.32±0.11,100 ng/ml of GDNF had no influence on PLZF and c-Kit protein compared with 50 ng/ml of GDNF.Conclusions Higher concentration of GDNF could enhance the transcription and expression of PLZF,and decrease the transcription and expression of c-Kit in SSC.GDNF has possible role to regulate and control proliferation and differentiation of SSC. Key words: Spermatogonia; Glial cell line-derived neurotrophic factor; Promyelocytic leukaemia zinc finger; Proto-oncogene proteins; c-kit

Exogenous stem cell factor improves interstitial cells of Cajal restoration after blockade of c-kit signaling pathway

Objective. Interstitial cells of Cajal (ICC) have been endowed with considerable intrinsic plasticity. Blockade of the c-kit signaling pathway results in the shift of ICC towards a smooth muscle-like phenotype. Little is known about stem cell factor (SCF), the ligand of c-kit, and the role it plays in the process of restoration. The aim of this study was to determine whether exogenous SCF can promote ICC replenishment following the blockade of c-kit signaling. Material and methods. Neutralizing anti-c-kit monoclonal antibody (ACK2) was administered to mice for 8 days after birth. Jejunal muscle strips were cultured up to 7 days. Electrical rhythmic changes were monitored and ICC were examined by immunohistochemistry. Expression of c-kit mRNA was detected by reverse transcriptase-polymerase chain reaction, and expression of Kit protein was detected by Western blot. Results. When c-kit receptors were blocked, ICC nearly disappeared from the jejunum accompanied by the loss of electrical slow waves. By day 7, after in vitro culture with SCF (100 ng/ml), the amplitude of muscle strip slow waves was restored to 0.19 ± 0.07 mV (p < 0.05), whereas the frequency recovered to 13.7 ± 3.32/min (p < 0.01). Furthermore, labeling for c-kit+ cells in the myenteric plexus increased and c-kit mRNA and protein expression were up-regulated compared to that of non-treatment with SCF. Conclusions. The c-kit signaling pathway, activated by SCF, is the critical pathway associated with the control of ICC survival and proliferation. The restoration of ICC number and jejunal electrical rhythm, resulting from blockade of the c-kit signaling pathway, could be facilitated by local SCF administration.