C-kit Protein Research Articles

Co-regulated Intestinal c-kit by Interleukin-17 and Interleukin-10 on Post-infectious Irritable Bowel Syndrome in Mice

Purpose The aim of this study was to investigate the probable immune mechanisms of PI-IBS. Methods C57L/B6 mice, which have been infected by 500 Trichinella larvae for 56 days, were used to PI-IBS model mice, the levels of the intestinal mRNAs and proteins of c-kit, interleukin-10 (IL-10), and IL-17 were measured by RTPCR and western blotting or ELISA. The locations of c-kit were detected using immunohistochemical staining. Results Levels of the c-kit protein and mRNA were up-regulated in the whole PI-IBS mice intestine. Compared to the control bowel segments, IL-17 levels were significantly higher in the duodenum and ileum, and IL-10 levels were lower in jejunum, ileum, and colon. By immunohistochemical staining, the increased signals of the c-kit were mainly detected in the submucosa and myenteron. Conclusions These results suggested that increased levels of the c-kit resulted in the changes of the intestinal motility and visceral sensitivity in PI-IBS mouse model induced by Trichinella Spiralis infection, and which may be coregulated by IL-17 and IL-10 in pathophysiology of PI-IBS.

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.

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.

Effects of electronically stimulating Tianshu (ST 25) and Dachangshu (BL 25), Quchi (LI 11) and Shangjuxu (ST 37) on the expressions of jejunum c-kit protein and c-kit mRNA in rats with functional diarrhea

OBJECTIVETo investigate the effects of electronically stimulating Tianshu (ST 25) and Dachangshu (BL 25), Quchi (LI 11) and Shangjuxu (ST 37) on the jejunum c-kit protein and c-kit mRNA in rats with functional diarrhea (FD). METHODSFD models were established through intragastric administration with folium sennae. Experimental rats were then divided into 4 groups: blank group, model group, electroacupuncture group I [Tianshu (ST 25) and Dachangshu (BL 25) of both sides] and electroacupuncture group II [Quchi (Li 11) and Shangjuxu (ST 37) of both sides], 10 in each. After treatment with electroacupuncture for 10 days, The expressions of jejunum c-kit protein and c-kit mRNA in each group were detected with Western blot and Real-Time quantitative real-time polymerase chain reaction (PCR). RESULTSThe expressions of c-kit protein and c-kit mRNA in the model group increased significantly compared to those in the blank group (P < 0.01); the expressions in electroacupuncture group I significantly decreased compared to those in the model group (P < 0.01). CONCLUSIONOur findings suggest that electronically stimulating both Tianshu (ST 25) and Dachangshu (BL 25) significantly increased the expressions of jejunum c-kit protein and c-kit mRNA in FD rats, which means the treatment might have better therapeutic effects on FD.

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.

Vestibular Mast Cell Density in Vulvodynia: A Case-Controlled Study.

To identify whether mast cell densities in vulvar biopsies from the vestibule are associated with vulvodynia. We enrolled 100 women aged 19 to 59 years with confirmed vulvodynia cases, 100 racially matched controls, and 100 black control women. All had vulvar biopsies performed at the 7 o'clock position of the vestibule, which were then immunostained to detect c-KIT protein. The numbers of c-KIT positive mast cells per ×400 magnification field were manually counted, and t tests and logistic regression were used to assess the association with case-control status. Of the biopsies, 235 were adequate samples for c-KIT testing for mast cells. The mast cell density was substantially lower in black control women (13.9 ± 10.9) in comparison to white control women (22.5 ± 13.2 p < 0.001): hence the analysis was confined to white cases and racially matched control women. Compared with racially matched controls, cases were younger, more likely to be married, and reported a higher household income. The average number of mast cells per ×400 magnification field overall was 19.1 ± 13.2 (range, 0-62). There was no difference in the mast cell count between racially matched cases (22.4 ± 13.9 per ×400 field) and controls (22.5 ± 13.2) in either the univariate or multivariable analyses. Within the group of cases, there was no difference in mast cell density based on the presence or absence of a variety of urogenital symptoms. No difference in mast cell density in biopsies of the vestibule was found between white cases and racially matched controls. Black control women have a lower mast cell density compared with white control women.

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.

Detection of novel polymorphisms in the ckit gene of canine patients with lymphoma, melanoma, haemangiosarcoma, and osteosarcoma.

Tyrosine kinase inhibitors (TKIs) that specifically target cKIT represent a therapeutic approach for non-resectable canine mast cell tumours (MCTs) grade II/III. The therapeutic benefit of TKIs has been investigated in other tumours based on clinical response rates and identification of gain-of-function mutations. In the present study, cKIT expression in 14 dogs with osteosarcoma, melanoma, haemangiosarcoma, lymphoma, and fibrosarcoma was analysed. Tissue samples were used for cKIT sequencing to (I) detect the cKIT transcript and to (II) identify gain-of-function mutations. The cKIT transcript was detected in ten patients. Four novel amino acid substitutions and five silent polymorphisms were identified. Furthermore, an insertion mutation (GNSK) was discovered in the tissue, but not in the blood sample of one dog. CKIT expression was identified in a variety of canine tumours and, therefore, TKIs might have a broader therapeutic indication apart from treatment of MCTs. Further investigations will be necessary to localize the cKIT protein in the respective tumours and to evaluate the functional consequence of the cKIT variants identified in the present study.

Screening of candidate G-quadruplex ligands for the human c-KIT promotorial region and their effects in multiple in-vitro models.

Stabilization of G-quadruplex (G4) structures in promoters is a novel promising strategy to regulate gene expression at transcriptional and translational levels. c-KIT proto-oncogene encodes for a tyrosine kinase receptor. It is involved in several physiological processes, but it is also dysregulated in many diseases, including cancer. Two G-rich sequences able to fold into G4, have been identified in c-KIT proximal promoter, thus representing suitable targets for anticancer intervention. Herein, we screened an “in house” library of compounds for the recognition of these G4 elements and we identified three promising ligands. Their G4-binding properties were analyzed and related to their antiproliferative, transcriptional and post-transcriptional effects in MCF7 and HGC27 cell lines. Besides c-KIT, the transcriptional analysis covered a panel of oncogenes known to possess G4 in their promoters.From these studies, an anthraquinone derivative (AQ1) was found to efficiently downregulate c-KIT mRNA and protein in both cell lines. The targeted activity of AQ1 was confirmed using c-KIT–dependent cell lines that present either c-KIT mutations or promoter engineered (i.e., α155, HMC1.2 and ROSA cells).Present results indicate AQ1 as a promising compound for the target therapy of c-KIT-dependent tumors, worth of further and in depth molecular investigations.

Up-regulation of the Ang II/AT1 receptor may compensate for the loss of gastric antrum ICC via the PI3k/Akt signaling pathway in STZ-induced diabetic mice

The classic renin–angiotensin system (RAS) is a complex system in which angiotensin II (Ang II) has been identified as an important endogenous regulator that influences both smooth muscle contraction and cell growth. Although a local RAS is known to exist in the gastrointestinal tract, it is unclear whether Ang II is involved in the loss of gastric interstitial cells of Cajal (ICC) in diabetic mice. The present study was designed to investigate the effect of Ang II on ICC survival in streptozotocin (STZ)-induced diabetic mice. Western blot, immunofluorescence, isometric muscle recording, enzyme-linked immunosorbent assay (ELISA) and a cell counting kit-8 were used in this research. Our results demonstrate that the c-Kit and membrane-bound stem cell factor (mSCF) protein expression levels in gastric smooth muscle were decreased in STZ-induced diabetic mice. However, the angiotensin receptor type 1 (AT1R) expression levels in gastric smooth muscle and angiotensin-converting enzyme (ACE) expression levels in gastric mucosa were increased. The effect of Ang II on the tonic contraction of gastric smooth muscle was potentiated in diabetic mice, and the plasma Ang II level was enhanced. Ang II increased mSCF expression, cell proliferation, and Akt-Ser473 phosphorylation in cultured gastric smooth muscle cells (GSMCs). These effects were reduced by specific inhibitors ZD7155 (an AT1R antagonist) and LY294002 (a PI3-kinase inhibitor). Our results suggest that Ang II increases mSCF expression and cell proliferation in cultured GSMCs in a PI3K/Akt signaling-dependent manner. ACE and AT1R up-regulation in the stomach may help compensate for ICC loss in STZ-induced diabetic mice.

The Role of Interstitial Cells of Cajal in Acute Cholecystitis in Guinea Pig Gallbladder

Background/Aims: Acute cholecystitis is common in gallbladder motility disorder. Interstitial cells of Cajal (ICCs) in the gallbladder are involved in the regulation of gallbladder motility. The aim of this study was to explore the change of gallbladder ICCs in acute cholecystitis. Methods: Thirty adult guinea pigs were randomly divided into 3 groups: a sham-operated group (healthy controls) and 2 study groups. The animals in the study group were subjected to bile duct ligation and then to laparotomy and cholecystectomy at 24 and 48 hours after surgery. Immunohistochemistry, immunohistofluorescence, and laser confocal microscopy were performed to observe the shape, size, morphology, and density of gallbladder ICCs. Western blot and real-time PCR were performed to detect stem cell factor and c-kit protein and mRNA expression, respectively. Results: There were no differences in the shape, size, and morphology of the gallbladder ICCs in the control and the two acute cholecystitis groups. Density of gallbladder ICCs, SCF level, and c-kit protein and mRNA expression all decreased in the acute cholecystitis groups. Further, SCF level and c-kit protein and mRNA expression decreased with progress of acute cholecystitis (all P < 0.05). Conclusion: Acute cholecystitis can decrease ICCs through repression of SCF and c-kit expression and that ICCs loss play a role in acute cholecystitis.

Effect of Neutrophils on Gallbladder Interstitial Cajal-Like Cells in Guinea Pig Model of Acute Cholecystitis

Background: Acute cholecystitis is a common condition in gallbladder motility disorder. Interstitial Cajal-like cells (ICLCs) in the gallbladder are known as one of the players in the complex motility mechanisms affecting gallbladder motility. Aim: This study explored morphological symptoms and molecular mechanisms underlying gallbladder ICLC changes induced by acute cholecystitis. Materials and Methods: Fifteen adult guinea pigs were randomly divided into 3 groups: sham-operated group (healthy controls) and 2 experimental groups wherein these guinea pigs were subjected to common bile duct ligation to induce acute cholecystitis. Neutrophils were isolated from the peripheral blood of sham-operated animals and from the experimental animals at 24 and 48 h after surgery, and co-cultured with gallbladder ICLCs. The morphology of gallbladder ICLCs was examined by laser confocal immunofluorescence microscopy, TUNEL assay was used to detect apoptosis, and western blot and real-time PCR were performed to detect stem cell factor (SCF) and c-kit protein and mRNA expression, respectively. Results: No morphological differences in the gallbladder ICLCs were observed between single-culture and co-culture with healthy control neutrophil groups. However, the ICLCs in all co-culture groups with acute inflammation were impaired. In the co-culture groups, the rate of ICLC apoptosis was significantly higher than that in the single-culture group. SCF and c-kit protein and mRNA expression levels decreased in all co-culture groups as well. Conclusion: We demonstrated that the neutrophils are involved in gallbladder ICLC injury in acute cholecystitis cases and associated with gallbladder motility disorder.

Effects of sacral nerve stimulation with acupuncture on gut transit time and c-kit expression in colon of rats with slow transit constipation.

Sacral nerve stimulation (SNS) is an alternative surgical approach to alleviate fecal incontinence and constipation. This study aimed to explore the effects and underlying mechanisms of SNS with acupuncture on gut transit time and colon c-kit protein expression in rats with slow transit constipation (STC). Fifty Sprague-Dawley rats were randomly divided into five groups: blank control, SNS, Mosapride, sham SNS, and STC model control group. The STC model was established by subcutaneous injection of morphine. Each group was treated over a 15-day period. Gut transit time was measured 1 day before the treatment started and after 5, 10, and 15 days of treatment. After the 15-day treatment, animals were sacrificed and colonic tissues were collected for analysis of c-kit protein expression, using western blot analysis. We found significant differences in gut transit time in the SNS group compared with the Mosapride group after 5 (P = 0.001) and 10 (P = 0.004) days of treatment. After 15 days of treatment, there were no differences in gut transit time among the SNS, Mosapride, and blank control groups. However, significant differences were observed when comparing the SNS and Mosapride groups with the STC model and sham SNS groups. A decreased c-kit protein expression was observed in the STC model control, sham SNS, and Mosapride groups, compared with the SNS group (P = 0.001). Our data indicate that SNS can decrease gut transit time and increase the expression of c-kit protein in rats with STC to improve colon transit function.

Management of gastrointestinal stromal tumors: a prospective and retrospective study

Background Gastrointestinal stromal tumors (GISTs) are a group of unusual neoplasms arising from the interstitial cells of Cajal. GISTs are the most common mesenchymal tumors of the gastrointestinal (GI) tract. Diagnosis always requires immunohistochemical staining for the expression of c-KIT protein (CD-117). Purpose The aim of this study was to present the prospective and retrospective experience of the Gastrointestinal Surgery Department, Alexandria Faculty of Medicine, in the management of GISTs. Materials and methods This study was carried out on 102 patients: a prospective study on 22 patients from April 2013 to April 2015 and a retrospective study on 80 patients between January 2009 and March 2013. All patient data, the different clinical presentations, the impact of surgical treatment, complications, follow-up, and survival data were collected and analyzed. Results This study included 102 patients (63 men and 39 women) who presented with GISTs on clinical, radiological, and/or endoscopic aspects. Their mean age at diagnosis was 49.18 ± 14.58 years. The most frequent presenting symptom was GI bleeding, seen in 42 patients (41.18%). Twenty-five patients (24.51%) presented with abdominal swelling and pain. Twenty-four patients (23.53%) presented with anemia for investigation. Eight patients (7.84%) presented with repeated attacks of abdominal pain only. The tumors were located in the stomach in 54 patients (52.9%). Upper gastrointestinal endoscopy was performed in 64 patients (62.75%). Upper gastrointestinal endoscopy revealed the presence of a gastric lesion in 46 patients and a duodenal lesion in six patients and was completely free in 12 patients. Complete resection was achieved in 92 patients (92%), whereas eight patients (8%) had incomplete resection. Conclusion This study concludes that GISTs can occur anywhere in the GI tract but most commonly in the stomach. The prognosis is strictly related to the size of the tumor, number of mitoses, and completeness of surgical resection.

C-Kit immunoexpression patterns differ in melanotic and amelanotic canine oral melanomas

&lt;p&gt;Melanomas são as mais frequentes neoplasias malignas da cavidade bucal de cães. Sabe-se que a proliferação de células e expressão de conexina diferem em melanomas melanóticos e amelanóticos da cavidade bucal de cães. Este estudo analisou a expressão da proteína c-Kit em melanomas melanóticos e amelanóticos da cavidade bucal canina. Um total de 34 melanomas bucais caninos (19 melanóticos e 15 amelanóticos) foram coletados. Os melanomas amelanóticos apresentaram evolução mais rápida e maior incidência de metástase. Foi constatado um número significativamente maior de células positivas para c-Kit em neoplasias amelanóticas. Além disso, a intensidade de imunomarcação de c-Kit foi predominantemente mais forte em melanomas amelanóticos. Estes resultados confirmam um papel potencial para c-Kit em melanomas orais caninos, com diferenças claras em padrões de expressão entre os dois tipos histológicos de tumor, melanóticos e amelanóticos. Este trabalho destaca a importância de um estudo detalhado das mutações c-Kit em melanomas orais caninos para ser possível a melhor compreensão dos mecanismos moleculares envolvidos no desenvolvimento da doença.&lt;/p&gt;

SATB1 Expression Marks Lymphoid-Lineage-Biased Hematopoietic Stem Cells in Mouse Bone Marrow

Background: Lifelong hematopoiesis is maintained by cell differentiation in which signaling pathways and transcription factors coordinately induce step-wise maturation of hematopoietic stem cells (HSCs) toward downstream effector cells. In addition, the organization of chromatin structure that creates accessible sites of target genes is also essential so as to ensure temporally and spatially adequate control of internal gene expression. Murine HSCs can be isolated with high efficiency using surface molecules including lineage-related markers, c-Kit, Sca-1, Flt3 and SLAM family proteins. However, even the highly enriched HSC fraction is still heterogeneous regarding differentiation potential, and how the HSC diversity reflects the heterogeneity of intrinsic gene-expression in HSCs is as-yet-unknown. We previously identified Special AT-rich Sequence Binding protein 1 (SATB1), a global chromatin regulator, as a lymphoid-related gene in the HSC differentiation (Satoh and Yokota et al. Immunity 2013). Indeed, SATB1 overexpression strongly enhanced both T and B lymphopoietic potential of murine HSCs whereas SATB1 deficiency caused malfunctions of HSCs in the lymphopoietic activity. Furthermore, another report showed that SATB1-deficient HSCs were less quiescent in transplanted recipients and more prone to differentiate preferentially to myeloid-erythroid lineages (Will et al. Nat Immunol 2013). These results suggested that SATB1 is likely indispensable not only for the lymphopoietic potential but also for the integrity of HSCs. Here, to better understand the mechanism how SATB1 influences homeostatic HSC functions in adult bone marrow (BM), we have developed a new mouse model in which SATB1 expression can be precisely monitored along the HSC differentiation.Methods: The Tomato gene, coding a red fluorescent protein, was knock-in to the coding region of endogenous Satb1 gene. The heterozygous SATB1/Tomato knock-in mice in which one Satb1 allele was replaced with the Tomato were used to sort HSCs in adult BM. The sorted cells were evaluated for the differentiation potential with methylcellulose colony assays and co-cultures with MS5 stromal cells. Further, the long-term reconstitution ability was evaluated by transplantation to lethally irradiated mice. To obtain transcriptome information, total RNA was isolated from SATB1/Tomato- and SATB1/Tomato+ HSCs, and then next-generation sequencing was performed. The data were analyzed with the Ingenuity Pathway Analysis software.Results: We defined Lin- Sca1+ c-KitHi (LSK) CD150+ Flt3- cells as HSCs, especially adopting FLT3- to exclude FLT3+ lymphoid-primed multipotent progenitors from our functional analyses. We found that the LSK CD150+ Flt3- fraction contains substantial number of SATB1/Tomato+ cells. While both SATB1/Tomato- and SATB1/Tomato+ HSCs produced numerous CFU-Mix and CFU-GM/G/M colonies, the latter were less potent to produce BFU-E. In co-culture with MS5 stromal cells that support B and myeloid lineages, the output of B lineage cells from SATB1+ HSCs was more robust than that of SATB1- HSCs. Upon transplantation, enhanced B-lineage engraftment was observed in the SATB1+ HSC-transplanted recipients. Interestingly, while the two types of HSCs showed obvious difference in the differentiation potential toward lymphoid or myeloid lineage, both HSCs reconstituted the LSK CD150+ Flt3- fraction that similarly contained SATB1/Tomato- and SATB1/Tomato+ cells. With the RNA-sequencing data of SATB1- and SATB1+ HSCs, biological pathway analyses revealed that the "Hematological System Development and Function" pathway was remarkably up-regulated in the SATB1+ HSCs. Among subcategories of the "Hematological System Development and Function" pathway, the "quantity of lymphocytes" pathway was increased whereas "quantity of myeloid cells" and "quantity of granulocytes" pathways were decreased.Conclusion: We have developed a new mouse system that can be used to identify and isolate viable lymphoid-biased HSCs in the most primitive hematopoietic cell fraction of adult BM. While the SATB1- and SATB1+ HSCs differ genetically and functionally, both subtypes have displayed a self-renewal activity with mutual interconversion in transplanted recipients. These findings suggest that functional heterogeneity and variability within the HSC population is, at least in part, a manifestation of SATB1 expression. DisclosuresYokota:SHIONOGI & CO., LTD.: Research Funding.

Prognostic Value of C-kit and VEGF Expression in Small Cell Lung Cancer

SESSION TITLE: Lung Cancer Treatment Posters SESSION TYPE: Original Investigation Poster PRESENTED ON: Wednesday, October 28, 2015 at 01:30 PM - 02:30 PM PURPOSE: Lung cancer is the leading cause of cancer related death in the world. Small cell lung cancer (SCLC) is an aggressive form of lung cancer with poor clinical course. Despite using new technologies and chemotherapeutic agents, little progress has been made in prolonging survival in SCLC. Performans status (PS), stage, age, gender, number of metastatic sites, weight loss and biochemical variables, such as serum albumin, sodium, alkaline phosphatase, lactat dehidrogenase levels are used to define prognosis in SCLC. However investigations are going on for finding a beter prognostic marker. The C-kit protein, which has type 3 receptor tyrosine kinase activity has been implicated in the pathophysiology of many tumors, including SCLC. Vascular endothelial growth factor (VEGF) expression was also corrolated to an increased risk of metastatic disease and a worse survival due to the role of angiogenesis in SCLC. METHODS: In our study we aimed to evaluate VEGF and C-kit expression and relation between survival and clinical factors affecting the prognosis of 53 patients who were diagnosed with SCLC between January 2005- August 2010. The expressions of VEGF and C-kit were studied immonuhistochemically. RESULTS: The expression of VEGF was detected in 64,2% and the expression of C-kit was detected in 60,4% of the patients. The results showed that VEGF and C-kit expressions were not associated with prognostic factors and survival. A multivariate analysis was perfomed to evaluate independent prognostic role of age, weight loss, PS, serum albumin level and stage. This analysis indicated that PS (p=0.001) and serum albumin (p=0.032) level had independent prognostic value on overall survival. CONCLUSIONS: Although our results did not show any correlations we recommend that the prognostic value of VEGF and C-kit expressions, must be further investigated in prospective and large studies due to their role in pathogenesis. CLINICAL IMPLICATIONS: The role of VEGF and C-kit expressions as an important event in the pathogenesis of SCLC are still debated, and remain to be clarified whether this expressions are relevant for tumor growth and survival. Based on the available data reported in the literature, C-kit tyrosine kinase inhibitors and VEGF-targeting monoclonal antibody bevacizumab have substantial clinical benefits. As a result, to understand the prognostic value of VEGF and C-kit expressions new studies and large series needed. DISCLOSURE: The following authors have nothing to disclose: Gulen Topaloglu, Aydin Yilmaz, Funda Demirag, Yurdanur Erdogan, Sezgi Sahin Duyar, Ulkü Yilmaz No Product/Research Disclosure Information

Resveratrol and curcumin ameliorate di-(2-ethylhexyl) phthalate induced testicular injury in rats

The present study aimed to evaluate the protective role of resveratrol and curcumin on oxidative testicular damage induced by di-(2-ethylhexyl) phthalate (DEHP). Male Wistar rats were divided into six groups; three groups received oral daily doses of DEHP (2g/kgBW) for 45days to induce testicular injury. Two of these groups received either resveratrol (80mg/kgBW) or curcumin (200mg/kgBW) orally for 30days before and 45days after DEHP administration. A vehicle-treated control group was also included. Another two groups of rats received either resveratrol or curcumin alone. Oxidative damage was observed by decreased levels of total antioxidant capacity (TAC) and glutathione (GSH) and increased malondialdehyde (MDA) level in the testes of DEHP-administered rats. Serum testosterone level as well as testicular marker enzymes activities; acid and alkaline phosphatases (ACP and ALP) and lactate dehydrogenase (LDH) showed severe declines. DEHP administration caused significant increases in the testicular gene expression levels of Nrf2, HO-1, HSP60, HSP70 and HSP90 as well as a significant decrease in c-Kit protein when compared with the control group. Histopathological observations provided evidence for the biochemical and molecular analysis. These DEHP-induced pathological alterations were attenuated by pretreatment with resveratrol and curcumin. We conclude that DEHP-induced injuries in biochemical, molecular and histological structure of testis were recovered by pretreatment with resveratrol and curcumin. The chemoprotective effects of these compounds may be due to their intrinsic antioxidant properties along with boosting Nrf2, HSP 60, HSP 70 and HSP 90 gene expression levels and as such may be useful potential tools in combating DEHP-induced testicular dysfunction.

Analysis of POU5F1, c-Kit, PLAP, AP2γ and SALL4 in gonocytes of patients with cryptorchidism

Cryptorchidism is a risk factor for the development of testicular germ cell tumors (TGCTs). The most common type of TGCT in cryptorchidism is seminoma. The intratubular germ cell neoplasia unclassified (ITGCNU) is a histological pattern preceding the development of seminomas and non-seminomas. It was suggested that in patients with cryptorchidism, the gonocytes remained undifferentiated with pluripotent abilities expressing proteins like POU domain class 5 transcription factor 1 (POU5F1), tyrosine kinase receptor c-Kit, placental-like alkaline phosphatase (PLAP), the transcription factor AP2γ and sal-like protein 4 (SALL4) that confer to the gonocytes this ability and therefore make them susceptible to develop ITGCNU. The aim of the present study was to determine if the gonocytes of patients with cryptorchidism express POU5F1, c-Kit, PLAP, AP2γ and SALL4 proteins after their differentiation period. Based on this, we evaluated samples of testicular tissue from newborns to 16-year old subjects with or without cryptorchidism in search of POU5F1, c-Kit, PLAP, AP2γ and SALL4 using immunocytochemical method, the results of which were validated by RT-PCR. The results showed that control subjects witnessed a down-regulation in the expression of these five proteins in the first year of life, which eventually disappeared. On the other hand, it was determined that 21.6% (8/37) of the patients with cryptorchidism continued to express, at least, one of the proteins analyzed in this study after the second year of life. And only 5.4% (2/37) of the patients were positive to the five markers. These data sustain the proposed hypothesis that in cryptorchid patients, ITGCNU arises from gonocytes that fail in their differentiation process to spermatogonia with conservation of the proteins (POU5F1, c-Kit, PLAP, AP2γ and SALL4) that maintain pluripotency and undifferentiated characteristics and which are responsible for making the gonocytes susceptible to malignancy. However, we cannot guarantee that these patients present neoplastic transformation.