C-kit Protein Expression Research Articles

The function of hyperpolarization-activated cyclic nucleotide-gated channel in diabetic cystopathy.

We aimed at investigating changes in the expression and physiological function of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in interstitial cells of Cajal (ICC) in diabetic state. Twenty adult female Sprague-Dawley (SD) rats were randomly assigned to control and Zucker diabetic fatty (ZDF) group. The protein and mRNA expression of HCN isoforms and C-kit in the rat bladders were detected using Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The bladder contraction was evaluated using a bladder smooth muscle strip test. Whole cell patch-clamp techniques were used to detect the activity of HCN channels. Immunofluorescent staining was used to the positive expression of HCN and C-kit in ICC. cAMP, as HCN channel-specific stimulant, could increase the frequency and amplitude of spontaneous contractions in both group, while cAMP inducing contraction of ZDF rats, was still significantly lower compared with the control group. Acute bladder ICCs were isolated by collagenase digestion. Classic Ih current pattern was recorded on ICCs while Ih current amplitude of ICCs from ZDF diabetic rats was significantly lower than the control group. The expression and mRNA of HCN1-4 isoforms in ZDF diabetic rats were both significantly lower compared with the control group. Meanwhile, the number of c-kit positive cells in ZDF diabetic rats showed no significant differences compared with controls. The morphological structure of ICC in the bladder of ZDF rats was relatively loose and the number of their cell process was apparently decreased. The structure of ICCs in ZDF rats was relatively loose, their connection to each other was also diminished. The expression of HCN was down-regulated and its response to cAMP was also decreased. HCN channels in bladder ICCs might regulate detrusor contraction. Changes in HCN expression and activity in bladder ICCs might be one of the most important mechanisms of diabetic cystopathy.

Loss of c-KIT expression in breast cancer correlates with malignant transformation of breast epithelium and is mediated by KIT gene promoter DNA hypermethylation

KIT Proto-Oncogene Receptor Tyrosine Kinase (KIT) is a transmembrane receptor tyrosine kinase which plays an important role in regulation of cell proliferation, survival and migration. Interestingly, the role of c-KIT in malignant transformation seems to be highly tissue-specific and it can act either as an oncogene or tumor suppressor gene. Here we analyzed the expression of c-KIT in normal breast tissues and tissues from different stages encompassing major steps of breast tumor development. Our study showed, that the c-KIT protein expression is gradually lost during the process of breast tissue transformation. The analysis of previously published datasets revealed that c-KIT expression in breast malignancies was downregulated at mRNA level. Because sequencing studies did not identify any recurrent mutations or copy number alterations, we proposed a potential epigenetic mechanism for the downregulation of c-KIT expression. In-silico analysis of the KIT promoter revealed the presence of CpG islands, therefore we performed bisulfite sequencing of normal breast epithelial tissues as well as breast tumor samples. We found, that KIT promoter is hypermethylated in breast tumors compared to normal breast tissues. Furthermore, treatment of breast cancer cell lines, that lack the expression of c-KIT, with methyltransferase inhibitor 5-Azacytidine (5Aza-2dC) resulted in increased expression of c-KIT mRNA. Collectively, our studies demonstrate that c-KIT expression is epigenetically downregulated during breast epithelium transformation and cancer development via KIT promoter hypermethylation.

A naphthalene diimide G-quadruplex ligand inhibits cell growth and down-regulates BCL-2 expression in an imatinib-resistant gastrointestinal cancer cell line

Gastro-intestinal tumours (GISTs) are driven by aberrant expression of the c-KIT oncoprotein. They can be effectively treated by the kinase inhibitor imatinib, which locks the c-KIT kinase domain into an inactive conformation. However resistance to imatinib, driven by active-site mutations, is a recurrent clinical challenge, which has been only partly met by the subsequent development of second and third-generation c-KIT inhibitors.It is reported here that a tetra-substituted naphthalene diimide derivative, which is a micromolar inhibitor of cell growth in a wild-type patient-derived GIST cell line, has a sub-micromolar activity in two distinct patient-derived imatinib-resistant cell lines. The compound has been previously shown to down-regulate expression of the c-KIT protein in a wild-type GIST cell line. It does not affect c-KIT protein expression in a resistant cell line to the same extent, whereas it profoundly down-regulates the expression of the anti-apoptopic protein BCL-2. It is proposed that the mechanism of action involves targeting quadruplex nucleic acid structures, and in particular those in the BCL-2 gene and its RNA transcript. The BCL-2 protein is up-regulated in the GIST-resistant cell line, and is strongly down-regulated after treatment. The compound strongly stabilises a range of G-quadruplexes including a DNA one from the BCL-2 promoter and an RNA quadruplex from its 5′-UTR region. A reporter assay construct incorporating the 5′-UTR quadruplex sequence demonstrates down-regulation of BCL-2 expression.

Combination of Luteolin and Solifenacin Improves Urinary Dysfunction Induced by Diabetic Cystopathy in Rats.

BackgroundThe purpose of the present study was to assess the effect of luteolin and solifenacin on diabetic cystopathy (DCP) and to investigate the mechanism of action. A novel link between the overexpression of c-Kit in the bladder and voiding dysfunction was identified in rats with DCP.Material/MethodsA rat model of DCP was successfully established by intraperitoneal injection of streptozotocin and a diet high in glucose and lipids, and animals were treated with luteolin and solifenacin. The effect of luteolin and solifenacin on urinary dysfunction in DCP rats was investigated by assessing bladder pressure and performing a volume test. The protein levels of c-Kit, stem cell factor (SCF), p110, and phosphorylated p110 in the bladder were detected by Western blot analysis and immunohistochemical staining.ResultsIn DCP rats, the protein levels of c-Kit, SCF and phosphorylated p110 in the bladder were significantly increased. However, oral treatment of DCP rats with luteolin combined with solifenacin resulted in effective improvement of overactive bladder and reduced the protein expression of c-Kit, SCF, and phosphorylated p110. Moreover, the effect of luteolin combined with solifenacin on maximum voiding pressure and residual urine volume was improved compared to that of luteolin alone.ConclusionsLuteolin improved overactive bladder in DCP rats, which may be due to SCF/c-kit inhibition, as well as the downregulation of the phosphoinositide-3 kinase signaling pathway. Moreover, solifenacin enhanced the potential pharmacological effect of luteolin in the treatment of DCP.

Signaling Cascades Activated by UVB in Human Melanocytes Lead to the Increased Expression of Melanocyte Receptors, Endothelin B Receptor and c-KIT.

A single exposure of normal human melanocytes (NHMs) to ultraviolet B (UVB) radiation induces a distinct increase in the expression of c-KIT and endothelin B receptor (EDNRB) and upregulates the expression of microphthalmia-associated transcription factor (MITF). In this review, we clarify the signaling mechanisms by which UVB stimulates the expression of MITF in NHMs, thus leading to upregulation of those two important melanogenic receptors. The increased expression of MITF in UVB-exposed NHMs is accompanied by a markedly stimulated and prolonged phosphorylation of p38/CREB. The UVB-stimulated expression of c-KIT and EDNRB could be completely abolished by a p38 inhibitor concomitant with a reduced phosphorylation of CREB and a downregulation of MITF expression. The UVB exposure of NHMs stimulates the phosphorylation of p38 and c-jun N-terminal kinase, but not ERK, followed by the increased phosphorylation of MSK1 and subsequently CREB. Postirradiation treatment with the MSK1 inhibitor H89 significantly downregulates the increased mRNA and protein expression of MITF, EDNRB and c-KIT in UVB-exposed NHMs. Our findings indicate for the first time that the increased expression of MITF that leads to the upregulation of melanocyte-specific proteins in UVB-exposed NHMs is mediated via activation of the p38/MSK1/CREB axis but not the ERK/RSK/CREB axis.

Acute Cholecystitis Reduces Interstitial Cells of Cajal in Porcine Gallbladder Through Decreased mRNA Synthesis

Background/Aims: Acute cholecystitis is a common gastrointestinal disorder, often characterized by acute cholecystitis with gallbladder motility disorder. Interstitial cells of Cajal (ICCs) are the pacemaker cells of gut motility in the gastrointestinal tract. Disruption of ICC function is related to motility disorders. The aim of this study was to explore the cellular and molecular mechanisms of ICCs in acute cholecystitis and after the resolution of acute inflammation. Materials and Methods: Fifty adult guinea pigs were randomly divided into five groups: a sham-administered group (control group); two groups that were intraperitoneally administered an anti-polyclonal neutrophil (PMN) antibody 24 h before common bile duct ligation (CBDL); and two groups of guinea pigs that were subjected to CBDL without receiving the PMN antibody. Guinea pigs that underwent CBDL were held for 24 h or 48 h after surgery before being subjected to laparotomy and cholecystectomy. Immunohistochemistry, TUNEL assays, western blotting, and real-time PCR were performed to determine ICC morphology and density, to detect ICC apoptosis, and to examine stem cell factor (SCF) and c-kit protein expression and SCF and c-kit mRNA levels, respectively. Results: Both hematoxylin-eosin staining and histological inflammation scores in the PMN groups were lower than those in the control groups (P < 0.01). No differences were observed in ICC morphology between groups. During acute cholecystitis, ICCs numbers were reduced. Conversely, the density of ICCs increased after inflammation was relieved (P < 0.01). In addition, SCF and c-kit protein and mRNA expression levels decreased during acute cholecystitis (P < 0.05) and increased after inflammation was relieved (P < 0.05). Furthermore, ICC apoptosis increased during acute cholecystitis and decreased after resolution of acute cholecystitis (P < 0.01). Conclusions: In acute cholecystitis, ICC injury may be related to gallbladder motility disorder.

Effect of bisacodyl on rats with slow transit constipation.

The effect of bisacodyl on the treatment of rats with slow transit constipation (STC) was studied. Forty-five female Wister rats were divided into control group, STC group, and STC bisacodyl group. The immunohistochemical method was used to determine interstitial cells of Cajal (ICC) and the expression of c-Kit protein. Body mass and the number of defecations were significantly decreased in the STC group compared with the control group on the 100th day after diphenoxylate administration, while dry weight of feces was significantly increased and the intestinal transit time was prolonged. There were significant differences in the number of defecations, dry weight of feces, and intestinal transit time among the three groups. The number of defecations was higher, dry weight of feces was lower, and intestinal transit time was shorter in the STC bisacodyl group compared to the STC group. In addition, ICC basement membrane dissolution occurred in the colon wall of the STC group. The connection between ICC and surrounding cells was destroyed, and the nucleus shrunken to different degrees. Moreover, c-Kit expression in the STC group was significantly lower than the control group. The connection between ICC and surrounding cells in the STC bisacodyl group was significantly stronger than the STC group, and the number of ICC and the expression of c-Kit were increased. Bisacodyl could reduce the severity of STC in rats by increasing the number of ICC and the expression of c-Kit.

Stage-specific expression of DDX4 and c-kit at different developmental stages of the porcine testis

Spermatogenesis begins with spermatogonial stem cells (SSCs), which are located in the basement membrane of the adult testes. Previous studies have described specific biomarkers for undifferentiated porcine spermatogonia or SSCs; however, these markers are not sufficient to understand spermatogenesis at different developmental stages. The objective of this study was characterize the expression of DEAD-Box polypeptide 4 (DDX4, also known as VASA) and tyrosine-protein kinase kit (c-kit), as potential markers of male germ cells in the porcine testis. In porcine testis tissue at prepubertal stages (5, 30, and 60 days), DDX4 and c-kit protein expression was detected in the most undifferentiated spermatogonia, which also express protein gene product 9.5 (PGP9.5). However, in porcine testis tissues from pubertal and postpubertal stages (90, 120, and 150 days), DDX4 and c-kit were not detected in PGP9.5-positive undifferentiated spermatogonia. The DDX4 expression pattern was similar to that of c-kit in the porcine testis. In adult porcine testes, DDX4-expressing cells were located on the lumenal side, compared to synaptonemal complex protein 3-positive primary spermatocytes, but DDX-4 was not co-expressed with acrosin, a known acrosome marker. In addition, DDX4 was detected in PGP9.5-expressing porcine SSCs in culture. Based on our results, we suggest that DDX4 and c-kit are putative markers of undifferentiated spermatogonia in the prepubertal porcine testis. While in the postpubertal porcine testis, they are markers of differentiated spermatocytes. These findings may facilitate future studies of porcine spermatogenesis.

Investigation of c-KIT and Ki67 expression in normal, preneoplastic and neoplastic canine prostate

Backgroundc-KIT expression has been related to bone metastasis in human prostate cancer, but whether c-KIT expression can be similarly classified in canine prostatic tissue is unknown. This study assessed c-KIT and Ki67 expression in canine prostate cancer (PC). c-KIT gene and protein expression and Ki67 expression were evaluated in forty-four canine prostatic tissues by immunohistochemistry, RT-qPCR and western blot. Additionally, we have investigated c-KIT protein expression by immunoblotting in two primary canine prostate cancer cell lines.ResultsEleven normal prostates, 12 proliferative inflammatory atrophy (PIA) prostates, 18 PC, 3 metastatic lesions and two prostate cancer cell cultures (PC1 and PC2) were analysed. The prostatic tissue exhibited varying degrees of membranous, cytoplasmic or membranous/cytoplasmic c-KIT staining. Four normal prostates, 4 PIA and 5 prostatic carcinomas showed positive c-KIT expression. No c-KIT immunoexpression was observed in metastases. Canine prostate cancer and PIA samples contained a higher number of Ki67-positive cells compared to normal samples. The median relative quantification (RQ) for c-KIT expression in normal, PIA and prostate cancer and metastatic samples were 0.6 (0.1-2.5), 0.7 (0.09-2.1), 0.7 (0.09-5.1) and 0.1 (0.07-0.6), respectively. A positive correlation between the number of Ki67-positive cells and c-KIT transcript levels was observed in prostate cancer samples. In the cell line, PC1 was negative for c-KIT protein expression, while PC2 was weakly positive.ConclusionThe present study identified a strong correlation between c-KIT expression and proliferative index, suggesting that c-KIT may influence cell proliferation. Therefore, c-KIT heterogeneous protein expression among the samples (five positive and thirteen negative prostate cancer samples) indicates a personalized approach for canine prostate cancer.

Changes in the interstitial cells of Cajal and neuronal nitric oxide synthase positive neuronal cells with aging in the esophagus of F344 rats.

The aging-associated cellular and molecular changes in esophagus have not been established, yet. Thus we evaluated histological structure, interstitial cells of Cajal (ICCs), neuronal nitric oxide synthase (nNOS)-positive cells, and contractility in the esophagus of Fischer 344 rat at different ages (6-, 31-, 74-weeks, and 2-years). The lamina propria thickness and endomysial area were calculated. The immunoreactivity of c-Kit, nNOS and protein gene product (PGP) 9.5 was counted after immunohistochemistry. Expression of c-Kit, stem cell factor (SCF), nNOS and PGP 9.5 mRNA was measured by real-time PCR, and expression of c-Kit and nNOS protein was detected by Western blot. Isovolumetric contractile force measurement and electrical field stimulation (EFS) were conducted. The lamina propria thickness increased (6 week vs 2 year, P = 0.005) and the endomysial area of longitudinal muscle decreased with aging (6 week vs 2 year, P<0.001), while endomysial area of circular muscle did not significantly decrease. The proportions of NOS-immunoreactive cells and c-Kit-immunoreactive areas declined with aging (6 week vs 2 year; P<0.001 and P = 0.004, respectively), but there was no significant change of PGP 9.5-immunopositiviy. The expressions of nNOS, c-Kit and SCF mRNA also reduced with aging (6 week vs 2 year; P = 0.006, P = 0.001 and P = 0.006, respectively), while the change of PGP 9.5 mRNA expression was not significant. Western blot showed the significant decreases of nNOS and c-Kit protein expression with aging (6 week vs 2 year; P = 0.008 and P = 0.012, respectively). The EFS-induced esophageal contractions significantly decreased in 2-yr-old rat compared with 6-wk-old rats, however, L-NG-Nitroarginine methylester did not significantly increase the spontaneous and EFS-induced contractions in the 6-wk- and 2-yr-old rat esophagus. In conclusion, an increase of lamina propria thickness, a decrease of endomysial area, c-Kit, SCF and NOS expression with preserved total enteric neurons, and contractility in aged rat esophagus may explain the aging-associated esophageal dysmotility.

Abstract 463: MicroRNA-7 suppresses RB1 expression leading to chromosomal instability in leukemia cells harboring c-Kit mutation

Abstract C-Kit mutation D816V is a well-known indicator of poor prognosis in acute myeloid leukemia (AML) harboring t(8;21) chromosomal translocations. However, the mechanism the D816V mutation promotes therapeutic resistance is still under investigation. C-Kit V814 mutation is a murine counterpart of the human D816V. We utilized a murine IL-3 dependent cell line Ba/F3 with or without the c-Kit V814 mutation to investigate its downstream signaling pathway and the effect on the expression of bcl-2 family members and critical granulocytic transcription factors. The c-Kit V814 and wild type (WT) c-Kit were retrovirally transduced into Ba/F3 cells. Unexpectedly, Gata1 mRNA was significantly downregulated to one-tenth in V814+Ba/F3 cells (p &amp;lt; 0.01), contrary to the previous reports that WT c-Kit signaling induces Gata1 expression. When V814+Ba/F3 cells were treated with several c-Kit signaling pathway inhibitors, mRNA and protein expression levels of Gata1 recovered to normal levels only with MEK 1/2 inhibitor (PD325901) treatment. We then hypothesized that miRNAs might control Gata1 transcription. We used an array to identify differences in miRNA expression between Ba/F3 cells and V814+Ba/F3 cells, and between V814+Ba/F3 cells treated with or without PD325901. Only miR-7a-5p and miR-706 were significantly upregulated in V814+Ba/F3 cells compared with Ba/F3 without the V814 mutation, and downregulated (&amp;gt; 2-fold) in cells treated with PD325901 compared with controls. We focused our further analysis on miR-7 as it is highly conserved in vertebrates. The differences in miR-7 expression were confirmed using qRT-PCR (10.5-fold change, p &amp;lt; 0.01). In silico analysis implicated Rb1 as a candidate miR-7 target that may regulate Gata1 transcription. Luciferase reporter constructs containing murine and human Rb1 miR-7 target sequence exhibited 0.51 and 0.49 times lower luciferase activity than the controls, respectively (p &amp;lt; 0.01). To further determine the biological significance of the c-Kit mutation that would transform the cells to more aggressive phenotype, we focused on DNA and chromosomal instability caused by RB1 deterioration. We exposed WT c-Kit Ba/F3 and V814+Ba/F3 cells to 4 Gy irradiation and then used immunohistochemistry to analyze the frequency of γH2AX foci in the cells. The percentage of cells with more than 10 γH2AX foci was significantly higher in V814+Ba/F3 cells than WT Ba/F3 cells (27% vs. 14%, p &amp;lt; 0.05), Here, we clearly show that a single mutation of c-Kit is sufficient to regulate miR-7 expression leading to RB1 translational suppression. Since c-Kit mutation requires only a single step to inhibit RB1, this mechanism is more likely to facilitate oncogenesis than bi-allelic RB1 chromosomal and/or gene alterations in leukemia cells. Taken together, we identified miR-7 as a suppressor of RB1 in V814 mutation-positive cells, which might reflect the responsibility of D816V to refractory feature in AML. Citation Format: Keiji Kurata, Shinichiro Kawamoto, Ryota Masutani, Kimikazu Yakushijin, Katsuya Yamamoto, Hiroshi Matsuoka, Takayuki Takubo, Hironobu Minami. MicroRNA-7 suppresses RB1 expression leading to chromosomal instability in leukemia cells harboring c-Kit mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 463. doi:10.1158/1538-7445.AM2017-463

The Dome Wall of Bladder Acts as a Pacemaker Site in Detrusor Instability in Rats.

BackgroundThe aim of this study was to confirm that the interstitial cells of Cajal (ICCs) in the dome wall of the bladder are pacemaker cells, and that the dome wall of the bladder acts as a pacemaker site in the detrusor instability (DI) rat model.Material/MethodsThe model of DI in Wistar rats was established and urodynamic studies measuring the bladder volume and pressure were performed. The detrusor excitability was investigated using the amplitude and frequency of phasic contraction of strips. The localization and quantity of ICCs was identified by immunohistochemistry and c-KIT protein expression in the rat bladder. PCR assay and Western blot were used to assess the expression of HCN2 and Cx43.ResultsThe bladder capacity, residual volume, voiding volume, and maximum voiding pressure were significantly increased in the DI group. The contraction frequency and amplitude of the strips from the dome of the bladder in the DI group were higher than the triangle, body, and base parts. Both the concentration of c-KIT positive ICCs cells and expression of the c-KIT protein in the dome wall were higher than in other parts of the bladder. The expression of HCN2 and Cx43 in each part of the DI rat group were obviously higher than each part in the control group. Compared to the body, base, and triangle parts, the expression of HCN2 and Cx43 in the dome wall were obviously higher in the DI group.ConclusionsThe quantity of ICCs was higher in the dome wall and the dome wall of bladder acts as a pacemaker site in the DI rat model.

Effect of acupuncture at points selected from different regions on SCF-kit signaling pathway in diabetic gastroparesis rats

To observe the effect of acupuncture at points selected from different regions on the positive expression of interstitial cells of Cajal (ICC) and the stem cell factor (SCF) in gastric antrum tissues in diabetic gastroparesis (DGP) rats, and to explore the influence of region-based point selection on the acupoint combination efficacy. Sixty Sprague-Dawley (SD) rats were randomly divided into a normal group (group A), a model group (group B), a group of Zusanli (ST 36) plus Zhongwan (CV 12) (group C), a group of Zusanli (ST 36) plus Neiguan (PC 6) (group D), and a group of Zusanli (ST 36) plus non-meridian non-acupoint points (group E), based on the random number table (n=12). DGP rat model was established by single intraperitoneal injection of 2% streptozotocin and common diet. After successful modeling, the rats were treated once a day for 4 weeks. Positive ICC and SCF expressions were measured by immunohistochemistry. Compared with group A, the gastrointestinal propulsion rate of group B showed a statistically significant decrease (P<0.05). Compared with group B, the gastrointestinal propulsion rate and the expression of ICC in the gastric antrum were significantly higher in group C, group D and group E, and the between-group differences were statistically significant (P<0.05); the expression of C-kit protein in group C was statistically significantly higher than that in group D and group E (P<0.05). The expression of SCF protein was significantly increased in group C than in group B, and the difference was statistically significant (P<0.05). Acupuncture can improve the symptoms of delayed gastric emptying in DGP model rats, and regulate the expression of ICC and SCF in gastric antrum tissues. The effect of Zusanli (ST 36) plus Zhongwan (CV 12) in the gastric region is superior to that of the Zusanli (ST 36) plus distal Neiguan (PC 6) or non-meridian non-acupoint point, indicating that region-based point selection is the key factor affecting the effect of acupoint combination.

Change in the Interstitial Cells of Cajal and nNOS Positive Neuronal Cells with Aging in the Stomach of F344 Rats

The gastric accommodation reflex is an important mechanism in gastric physiology. However, the aging-associated structural and functional changes in gastric relaxation have not yet been established. Thus, we evaluated the molecular changes of interstitial cell of Cajal (ICC) and neuronal nitric oxide synthase (nNOS) and the function changes in the corpus of F344 rats at different ages (6-, 31-, 74-wk and 2-yr). The proportion of the c-Kit-positive area in the submucosal border (SMB) and myenteric plexus (MP) layer was significantly lower in the older rats, as indicated by immunohistochemistry. The density of the nNOS-positive immunoreactive area also decreased with age in the SMB, circular muscle (CM), and MP. Similarly, the percent of nNOS-positive neuronal cells per total neuronal cells and the proportion of nNOS immunoreactive area of MP also decreased in aged rats. In addition, the mRNA and protein expression of c-Kit and nNOS significantly decreased with age. Expression of stem cell factor (SCF) and the pan-neuronal marker PGP 9.5 mRNA was significantly lower in the older rats than in the younger rats. Barostat studies showed no difference depending on age. Instead, the change of volume was significantly decreased by L-NG63-nitroarginine methyl ester in the 2-yr-old rats compared with the 6-wk-old rats (P = 0.003). Taken together, the quantitative and molecular nNOS changes in the stomach might play a role in the decrease of gastric accommodation with age.

Diagnostic utility of C-kit protein (CD117) expression in differentiating adenoid cystic carcinoma and polymorphous low grade Adenocarcinoma.

Background & Objective:To evaluate usefulness of immunohistochemical marker C-kit (CD117) in differentiating Adenoid cystic carcinoma (AdCC) from Polymorphous low-grade adenocarcinoma (PLGA) in patients of salivary gland carcinomas. AdCC is a malignant salivary gland neoplasm with poor prognosis. PLGA is a salivary gland malignancy with indolent growth pattern. Differentiating between the two entities is a diagnostic challenge. We evaluated the role of C-kit in differentiating the two.Methods:This is a Cross sectional study. Samples of 19 tumors including 12 AdCC and 4 PLGA was evaluated at Department of Histopathology, Armed Forces Institute of Pathology, Rawalpindi from December 2015 to August 2016. Immunohistochemical techniques were used to analyze the level of c-kit expression in AdCC (n = 12), polymorphous low-grade adenocarcinoma (PLGA) (n = 6). Samples were stained using monoclonal antibody against C-kit. Statistical analysis of the data was done using SPSS version 21.Results:Strong diffuse cytoplasmic reactivity was observed in more than 50% of the tumor cells of AdCC whereas less than 20% of cells showed negative to weak positivity in PLGA. Hence, the difference in the expression of c-kit between AdCC and PLGA was statistically significant (p value <0.002).Conclusions:CD117 expression itself can be used as a marker in differential diagnosis of salivary gland neoplasms. However, the percentage of the CD117 immunoreactive cells and the staining intensities appeared to be important factors in distinguishing AdCC from PLGA.

Immunohistochemical profiling of receptor tyrosine kinases, MED12, and TGF-βRII of surgically resected small cell lung cancer, and the potential of c-kit as a prognostic marker.

The limited number of available treatments for patients with small-cell lung cancer (SCLC) has prompted us to further investigate the biology of SCLC by molecular profiling. We collected formalin-fixed paraffin-embedded tumor samples from 127 patients with SCLC, who had undergone surgery at 16 institutions between January 2003 and January 2013, and analyzed the association between disease-specific survival and protein expression of c-kit, c-Met, epidermal growth factor receptor, human EGFR-related 2, vascular endothelial growth factor receptor II, anaplastic lymphoma kinase, mediator complex subunit 12 (MED12), and transforming growth factor beta receptor II (TGF-βRII) by immunohistochemistry (IHC). Of the 125 evaluable samples, all tumors expressed MED12, and 123 samples (98.4%) expressed TGF-βRII. MED12 was highly expressed in the nucleus in 92% of the positive samples while TGF-βRII was highly expressed in the cytoplasm in 55% of the positive samples. High c-kit expression was an independent favorable prognostic marker confirmed by multivariate analysis (hazard ratio: 0.543, 95% confidence interval: 0.310–0.953, p = 0.033). Both the relapse free-survival and overall survival of patients who underwent adjuvant chemotherapy were statistically longer in those with high c-kit expression (n = 38) than those with intermediate, low, or no c-kit expression (n = 19) (not reached vs 11.6 months, p = 0.021; not reached vs 25.9 months, p = 0.028). IHC for c-kit may offer a prognostic marker for early-stage SCLC, and the results for MED12 and TGF-βRII may suggest the biological characteristics of SCLC. Further investigation of the roles of their related molecules in early stage SCLC is required.

Experimental study of Bisacodyl in the treatment of slow transit constipation model rats

To explore the efficacy and the mechanism of Bisacodyl in treating slow transit constipation model rats. A total of 30 healthy rats were enrolled. Twenty rats received intragastric diphenoxylate to develop slow transit constipation (STC) model, and 10 untreated rats were set as blank control. STC rats were subdivided into two groups: STC Bisacodyl group (fed with Bisacodyl) and STC control group (common feed). Body weight, number and dry weight of faeces, and intestinal transit time were compared among 3 groups. Interstitial cells of Cajal(ICC) and c-Kit protein expression were measured by immunohistochemical staining. Restults Compared to blank control rats, at 100-day of receiving intragastric diphenoxylate, above 20 rats presented the decrease of body weight and feces number, the increase of dry weight of faeces, and the delay of intestinal transit time, indicating the successful establishment of STC rat model. One month after feeding, compared to STC control group, STC Bisacodyl grap had an increased feces number[(36.6±6.8) pill/day vs. (26.8±6.0) pill/day], decreased dry weight of feces [(150.6±10.5) mg/pill vs. (171.6±16.3) mg/pill] and shortened intestinal transit time [(416.9±50.6) minutes vs. (495.3±66.8) minutes], and the differences were statistically significant(all P<0.05). Dissolution of ICC basement membrane, damage of connection between ICC and surrounding cells, and atrophy of ICC nucleus structure were found in STC control rats. ICC (8.20±1.92 per field] and c-Kit expression (12.68%±2.59% ) in STC control rats were significantly lower than those in blank control rats(36.00±6.25 per field and 71.50 %±8.27%) (P=0.000). Compared to STC control group, the connection between ICC and surrounding cells enhanced obviously, ICC (18.80±3.70 per field) and c-Kit expression (45.91%±6.80%) were significantly higher in STC Bisacodyl group (all P=0.000). Bisacodyl treatment can relieve STC symptoms, which may be associated with increased ICC number and c-Kit protein expression.

Relationship between colonic function change and interstitial cells of Cajal in rats with spinal cord injury

Objective To investigate the correlation of colonic function change with interstitial cells of Cajal (ICC) in rats with spinal cord injury and further evaluate the mechanism of intestinal dysfunction after spinal cord injury. Methods Using the modified Allen’s device, experimental spinal cord injury was induced by dropping a 10 g weight from the height of 25 cm on the T10 segment. A total of 10 male adult SD rats were divided into normal group (n=20), sham-operated group(n=20) and injury group (n=60), according to the random number table. At postoperative 1, 2 and 4 weeks, proximal colon tissues in each group were collected for morphological study using HE staining, bioelectricity study, ICC count and neuron absorbance value using double-labeling immunofluorescence staining, and expression of c-kit protein in ICC using Western blot method. Results Proximal colon epithelial cells arranged neatly in normal and sham-operated groups, while the lodging and shedding of villus epithelial cells and increased thickness of mucous layer and muscular layer were seen in injury group. Bioelectricity in injury group disordered with large amplitude variation and unstable waveform, compared to the regular biological activity in normal and sham-operated groups. Intramuscular ICC cell count in injury group was 57.38±4.69 at 1 week, 32.26±3.62 at 2 weeks and 25.07±3.05 at 4 weeks, significantly less than that in normal and sham-operated groups(P<0.05). At 2 weeks, mean absorbance value of the neuron in injury group (71.46±4.69) was less than that in normal and the sham-operated groups (114.30±24.44, 108.11±26.46)(P<0.05). Expression of c-kit protein in ICC cells was 0.745±0.017 at 1 week, 0.492±0.480 at 2 weeks and 0.409±0.032 at 4 weeks, significantly less than that in normal and sham-operated groups (P<0.05). Conclusion Colonic function in rats presents significant changes after spinal cord injury, and the changes may relate to the decrease of intramuscular ICC cell count and c-kit level in the colon. Key words: Intestines; Interstitial cells of Cajal; Spinal cord injures

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells.

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies.

Hiding inside? Intracellular expression of non-glycosylated c-kit protein in cardiac progenitor cells

Cardiac progenitor cells including c-kit+ cells and cardiosphere-derived cells (CDCs) play important roles in cardiac repair and regeneration. CDCs were reported to contain only small subpopulations of c-kit+ cells and recent publications suggested that depletion of the c-kit+ subpopulation of cells has no effect on regenerative properties of CDCs. However, our current study showed that the vast majority of CDCs from murine heart actually express c-kit, albeit, in an intracellular and non-glycosylated form. Immunostaining and flow cytometry showed that the fluorescent signal indicative of c-kit immunostaining significantly increased when cell membranes were permeabilized. Western blots further demonstrated that glycosylation of c-kit was increased during endothelial differentiation in a time dependent manner. Glycosylation inhibition by 1-deoxymannojirimycin hydrochloride (1-DMM) blocked c-kit glycosylation and reduced expression of endothelial cell markers such as Flk-1 and CD31 during differentiation. Pretreatment of these cells with a c-kit kinase inhibitor (imatinib mesylate) also attenuated Flk-1 and CD31 expression. These results suggest that c-kit glycosylation and its kinase activity are likely needed for these cells to differentiate into an endothelial lineage. In vivo, we found that intracellular c-kit expressing cells are located in the wall of cardiac blood vessels in mice subjected to myocardial infarction. In summary, our work demonstrated for the first time that c-kit is not only expressed in CDCs but may also directly participate in CDC differentiation into an endothelial lineage.