C-kit mRNA Expression Research Articles

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.

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.

생약복합조성물(HemoHIM)의 사람 비만세포주 활성 억제 효과

방사선에 대한 방호와 면역기능 조절을 목적으로 새로운 생약복합조성물인 HemoHIM을 개발하였다. 식품 원료로 사용 가능한 생약재 3종 당귀, 천궁, 백작약의 에탄올 분획을 열수추출물에 첨가하여 HemoHIM을 제조하였다. HemoHIM의 항알레르기 효과를 검증하기 위하여 사람 비만세포주 HMC-1을 사용해 compound 48/80으로 유도되는 히스타민 분비량과 PMA/A23187로 유도되는 염증성 사이토카인의 분비량을 측정하였다. 히스타민의 양은 형광분석법으로, 염증성 사이토카인 IL-6, IL-8, TNF-<TEX>$\alpha$</TEX>, GM-CSF의 양은 효소결합 면역측정법으로 측정하였다. 저농도의 HemoHIM에 의해 히스타민 분비량이 억제되었고, 모든 농도에서 IL-6, TNF-<TEX>$\alpha$</TEX>, GM-CSF의 분비량은 억제되었지만 IL-8은 고농도에서만 억제되었다. 사이토카인의 mRNA 발현량은 HemoHIM의 농도 의존적으로 억제되었다. 그리고 c-kit와 Fc<TEX>$\varepsilon$</TEX>RI의 mRNA 발현량도 모든 농도에서 억제되었지만, tryptase의 mRNA 발현량은 저 농도에서만 억제되었다. 이상의 결과로 HemoHIM이 비만세포의 활성을 억제하는 효과가 있다는 것을 알 수 있었으며, 상대적으로 독성이 적은 항알레르기 제재로 개발할 가능성을 제시한 것으로 생각된다. In our previous study, a new herbal preparation (HemoHIM) was developed as a functional food for the radioprotection and immunomodulatory agents. In order elucidate the mechanism involved, we examined the effect of HemoHIM on the compound 48/80-induced histamine release, and on the phorbol 12-myristate 13-acetate (PMA)/calcium ionophore (A23187)-induced inflammatory cytokine secretion in HMC-1. The cell culture supernatants were harvested, and the cytokines (IL-4, IL-6, IL-8, TNF-<TEX>$\alpha$</TEX>, GM-CSF) in the supernatants were measured by enzyme-linked immunosorbent assay. The total RNA of the cells was extracted, and the cytokines or c-kit/tryptase/Fc<TEX>$\varepsilon$</TEX>RI's messenger RNA expressions were examined using reverse transcriptase polymerase chain reaction. Under low concentrations, HemoHIM inhibited histamine release in HMC-1 stimulated compound 48/80. Furthermore HemoHIM inhibited PMA/A23187-induced inflammatory cytokines' secreation or mRNA expression in a dose-dependent manner. But IL-8 secretion was not inhibited by low concentrayion of HemoHIM, respectively. The mRNA expression of c-kit and Fc<TEX>$\varepsilon$</TEX>RI were also inhibited in a dose-dependent manner. Tryptase mRNA expression was only inhibited by low concentration of HemoHIM. These results indicated that HemoHIM might be an useful agent for protection against allergy as well as immune modulation, especially since it is a relatively nontoxic natural product.

MRNA Expression of Platelet-Derived Growth Factor Receptor-β and C-KIT: Correlation With Pathologic Response to Cetuximab-Based Chemoradiotherapy in Patients With Rectal Cancer

Deviant expression of platelet-derived growth factor receptor-beta (PDGFRbeta) and c-kit was shown in patients with colorectal cancer. In the present study, mRNA expression of PDGFRbeta and c-kit in 33 patients with locally advanced rectal cancer undergoing preoperative chemoradiotherapy with cetuximab/capecitabine/irinotecan in correlation with the tumor regression rate was investigated. Pretherapeutic biopsy cores and tumor material from the resected specimens were collected in parallel with normal rectal mucosa. The expression levels of PDGFRbeta and c-kit were measured by quantitative polymerase chain reaction. Tumors were classified as good responders (tumor regression grade [TRG], 2-3) or poor responders (TRG, 0-1). The TRG evaluation of the resected specimen was TRG 0-1 in 11 and TRG 2-3 in 22. The median normalized ratios in the pretreatment mucosa vs. tumor biopsy cores was as follows: PDGFRbeta ratio of 15.2 vs. 49.5 (p <0.0001) and c-kit ratio of 0.94 vs. 0.67 (p = 0.014). The same tendency was observed for the median PDGFRbeta ratios after chemoradiotherapy completion: 34.2 vs. 170.0 (p <0.0001). The PDGFRbeta and c-kit mRNA expression values in the pretreatment tumor biopsy cores were lower than the values in the resected specimens: PDGFRbeta ratio 49.5 vs. 170.0 (p = 0.0002) and c-kit ratio 0.67 vs. 1.1 (p = 0.0003). Nevertheless, no correlation was seen between the pretherapeutic PDGFRbeta and c-kit mRNA expression and the pathologic regression rate. Cetuximab-based chemoradiotherapy increased PDGFRbeta levels even further compared with the pretreatment samples and deserves further investigation.

Expression of C-kit Messenger Ribonucleic Acid and C-kit Protein in the Gallbladders in Guinea Pigs of High Cholesterol Diet

The c-kit protooncogene receptor and its ligand-stem cell factor regulating the proliferation and survival of interstitial cells of Cajal (ICCs) have been described. The aim of this study was to determine the expression of c-kit mRNA and c-kit protein in the gallbladders in guinea pigs of high cholesterol diet (HCD). The gallbladder samples from 16 guinea pigs of HCD and from 16 guinea pigs of standard diet (StD) were used for this study. Expression of c-kit mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR), and expression of c-kit protein was detected by Western blot analysis. Serum total cholesterol (TC) (39 +/- 6 vs. 109 +/- 20 mg/dl), low density lipoprotein (LDL) cholesterol (24 +/- 4 vs. 71 +/- 10 mg/dl), high density lipoprotein (HDL) cholesterol (2.4 +/- 0.4 vs. 7.0 +/- 1.6 mg/dl), and triglyceride (TG) (58 +/- 8 vs. 118 +/- 23 mg/dl) concentrations were significantly higher in the HCD group than in the StD group of guinea pigs (P < 0.001, respectively). Decreased expression of c-kit mRNA was demonstrated in the HCD group compared with the StD group. The ratio of c-kit mRNA and GAPDH was 0.56 +/- 0.09 in controls and 0.50 +/- 0.07 in the HCD group (P = 0.033). C-kit protein expression significantly declined in the HCD group. The mean value of optical density was 129 +/- 25 in the StD group and 103 +/- 19 in the HCD group (P = 0.0009). The data indicate that the expression of c-kit mRNA and c-kit protein significantly decreased in the gallbladders in guinea pigs of HCD.

The roles of pericystic cells and Sertoli cells in spermatogonial proliferation stimulated by some growth factors in organ culture of newt ( Cynops pyrrhogaster) testis

We have previously shown FSH promotes spermatogonial proliferation and their differentiation into primary spermatocytes in organ culture of newt testicular fragments. Several growth factors identified in newt testis, such as stem cell factor (SCF), insulin-like growth factor (IGF)-I, and neuregulin (NRG)1, also stimulate spermatogonial proliferation in the organ culture. However, any growth factor added in vitro might not work on spermatogonia directly, but act on somatic cells such as Sertoli cells or pericystic cells, because size-selective barrier exists around a cyst which is enclosed by Sertoli cells. In order to determine the target somatic cells of the growth factors as well as the role of pericystic cells in spermatogonial proliferation and differentiation, we searched for agents that kill pericystic cells selectively. We found that treatment of the testicular fragments with trypan blue (TB) caused cell death of only pericystic cells and significant abolishment of the activity of SCF to stimulate spermatogonial proliferation, while the activities of neither IGF-I nor NRG1 were affected. In addition, the potency of neither IGF-I nor FSH to stimulate the differentiation of spermatogonia into primary spermatocytes was abolished by TB treatment. Consistent with these results, only the mRNA expression of c-kit was reduced by TB treatment, whereas those of FSH receptor, SCF, IGF-I, IGF-I receptor, Immunoglobulin-like domain-containing NRG1, ErbB2, and ErbB4 were unaffected. These results indicate that SCF stimulates pericystic cells, while IGF-I and NRG1, as well as FSH, activate Sertoli cells, resulting in stimulation of spermatogonial proliferation in organ culture of testicular fragments.

Production of the Soluble Form of KIT, s-KIT, Abolishes Stem Cell Factor-Induced Melanogenesis in Human Melanocytes

The signaling of stem cell factor (SCF) and its receptor KIT (membrane-bound KIT; m-KIT) plays an important role in melanocyte development, survival, proliferation, and melanogenesis. It has been demonstrated in other systems that a soluble form of m-KIT released from the cell surface (s-KIT) regulates SCF signaling, although there have been no reports pertaining to the existence and the biological role of s-KIT in melanocytes. In this study, we therefore examined the involvement of s-KIT in melanogenesis. Western blotting analysis revealed that treatment with phorbol 12-myristate-13-acetate (PMA) or 4-aminophenylmercuric acetate (APMA) induced s-KIT production in cultured human melanocytes. Inhibitors of tumor necrosis factor-alpha-converting enzyme (TACE) and metalloproteinases (MMPs) muted this release of s-KIT into the media. Human recombinant s-KIT added to melanocytes inhibited SCF-induced phosphorylation of m-KIT, resulting in suppression of SCF-induced melanogenesis. Additionally, APMA-induced s-KIT production abolished SCF-induced melanogenesis as effectively as a KIT-neutralizing antibody. Concomitantly, APMA and TACE inhibitors significantly decreased and increased melanin synthesis, respectively, in an in vitro skin model. Taken together, these findings provided an insight into the elaborate mechanism of SCF/m-KIT signaling in human melanocytes and suggested that production of s-KIT contributes to the regulation of human skin pigmentation. Journal of Investigative Dermatology (2008) 128, 1763-1772; doi:10.1038/jid.2008.9; published online 31 January 2008.

SiRNA-Mediated Silencing of c-kit in Mouse Primary Spermatogonial Cells Induces Cell Cycle Arrest

Several genes/gene products are known to act in a concert to regulate the process of spermatogenesis. One such gene is c-kit, a transmembrane tyrosine kinase receptor which plays an indispensable role in the maturation and differentiation of spermatogonial germ cells (SGCs). In the present study, siRNA approach was used to assess the role of c-kit in survival and proliferation of murine primary SGCs. The effect of different concentrations of anti-c-kit siRNA-1 and siRNA-2 (0.15, 0.315, 0.625, 1.25, 2.50, 5, and 10 nM) on c-kit protein and mRNA expression at post-transfection time (0, 6, 12, 24, 48, and 72 hours) was assessed using an array of techniques such as flow cytometry, ELISA, Western blot, and RT-PCR. Transfection of cells with anti-c-kit siRNAs (0.15-10 nM) at various time points after (0-72 hours) showed significant knockdown c-kit mRNA and protein expression. MTT, Alamar blue assays, and RT-PCR were used to investigate the effects of c-kit silencing on survival, proliferation, distribution, and apoptosis of cells. Experiments were also conducted to determine the effects of c-kit knockdown on cell cycle distribution, DNA laddering, and apoptosis. The results indicated that the transfection with anti-c-kit siRNA induces DNA fragmentation and cell cycle arrest at G(2)/M phase leading to significant reduction in cell viability and proliferation. In addition, enhanced suppression of c-kit protein in P815 cells was observed after transfection as compared to ES-E14TG2alpha cells, suggesting early onset of c-kit protein repression in P815 cells leading to prolongation in cell doubling time. In conclusion, our data provide the first evidence of specific knockdown of c-kit expression in mouse primary SGCs, which emphasizes the critical role played by c-kit in germ cell survival, proliferation, and apoptosis.

Stem cell factor and c-kit are involved in hepatic recovery after acetaminophen-induced liver injury in mice

Stem cell factor (SCF) and its receptor c-kit are important in hematopoiesis and cellular proliferation. c-kit has also been identified as a cell surface marker for progenitor cells. We have previously shown that there is a large reservoir of hepatic SCF, and this molecule plays a significant role in liver regeneration after 70% hepatectomy. In the current study, we further examined the expression of SCF and c-kit in acetaminophen (APAP)-induced liver injury in C57BL/6J mice or SCF-deficient sl-sld mice and their appropriate wild-type controls. Following APAP-induced liver injury, c-kit mRNA expression increased, with peak levels detected 48 h postinjury. Hepatic SCF mRNA levels after APAP injury were also increased, with peak levels seen 16 h post-APAP. The mortality rate in SCF-deficient mice treated with APAP was significantly higher than that of wild-type mice; furthermore, administration of exogenous SCF significantly reduced the mortality of APAP-treated wild-type mice. Bromodeoxyuridine incorporation experiments showed that SCF significantly increased hepatocyte proliferation at 48 and 72 h in APAP-treated mice. SCF inhibited APAP-induced hepatocyte apoptosis and increased Bcl-2 and Bcl-xL expression, suggesting that this decrease in hepatocyte apoptosis is mediated through Bcl-2 and Bcl-xL. In summary, SCF and c-kit expression was increased after APAP-induced liver injury. Administration of exogenous SCF reduces mortality in APAP-treated mice, increases hepatocyte proliferation, and prevents hepatocyte apoptosis induced by APAP, suggesting that these molecules are important in the liver's recovery from these injuries.

Effects of an ARB on Endothelial Progenitor Cell Function and Cardiovascular Oxidation in Hypertension

Angiotensin II (Ang II) receptor blocker (ARB) has been reported to have protective effects on the cardiovascular system independent of blood pressure reduction. Endothelial progenitor cells (EPCs) play a significant role in neovascularization of ischemic tissue. The average lifespan of EPCs was recently reported to be shortened by oxidative stress and regulated by anti-oxidative mechanisms. It has been reported that EPCs are present in peripheral blood and have the ability to repair cardiovascular damage. We investigated the effects of an ARB, candesartan, on EPC function and cardiovascular oxidation in salt-loaded, stroke-prone, spontaneously hypertensive rats (SHR-SP) in vivo. Salt-loaded SHR-SP were treated with candesartan (1 mg/kg/day), a diuretic (trichlormethiazide, TCM, 1.6 mg/kg/day), or an antioxidant (tempol, 5 mg/kg/day) for 2 weeks. Peripheral blood mononuclear cells (MNCs) were isolated and cultured to assay EPC colony formation and migration. Oxidative stress in EPCs was evaluated by thiobarbituric acid reactive substance (TBARS) assay. We evaluated messenger RNA (mRNA) expression of c-kit in the heart, the renin-angiotensin system (RAS) in EPC colonies, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit in cardiovascular organs. Candesartan and tempol, but not TCM, markedly increased EPC colony number in SHR-SP and reduced TBARS. Candesartan also significantly decreased mRNA expression of NADPH oxidase subunits in cardiovascular organs and increased cardiac c-kit mRNA expression. EPCs expressed mRNAs of renin, cathepsin D, chymase, and Ang II type 1 and type 2 receptors. Candesartan, an ARB, improves EPC dysfunction and increases cardiac c-kit expression through the anti-oxidative mechanism in hypertension. The local RAS induces oxidative stress and regulates the EPC functions.

249. Regulated expression of KIT protein in juvenile and adult germ cells of the rodent testis

KIT receptor is an established marker of differentiating spermatogonia and its activation is required to trigger spermatogonial maturation. KIT mRNA, however, can be detected in undifferentiated spermatogonia in the absence of protein expression, as previously established by us in the irradiated adult rat testes [1]. This differential regulation of mRNA and protein is presumably modulated by either local hormone action or by cues from the adult testicular microenvironment. Endogenous regulatory factors known to stimulate KIT synthesis in juvenile male germ cells in vitro are bone morphogenetic protein 4 (BMP4) and retinoic acid (RA), while factors known to suppress KIT at the onset of spermatogenesis have not yet been identified. Activin A, implicated in KIT downregulation in a murine erythroleukemia cell line [2], is produced within the juvenile mammalian testis and influences activities of spermatogonia and Sertoli cells. We hypothesised that activin acts to repress KIT expression in spermatogonia and therefore modulate spermatogonial behaviour. Evidence for this was first derived from Sertoli and germ cell co-cultures of day 8 wild type mouse testes in which exogenous activin addition caused a dose-dependent reduction of KIT mRNA. Whole testes mRNA analyses of two activin transgenic mouse models, the newborn Inhba−/− (lacking activin A) and postnatal InhbaBK/BK (decreased bioactive activin), revealed a significant elevation in KIT expression relative to wild type littermates. In the postnatal day 7 InhbaBK/BK testes, an elevated proportion of differentiated spermatogonia, increased cell surface KIT protein levels, enhanced mRNA levels of a known downstream target of KIT signalling pathway, cyclind3 and a meiotic marker, Sycp3, were observed. These data provide the first comprehensive evidence for activin modulation of KIT expression at spermatogenesis onset, in germ cells of the juvenile testis. This finding is of fundamental importance to other KIT-dependent processes. (1) Prabhu, S.M., et al. Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis. Reproduction, 2006. 131(3): p. 489–99. (2) Hino, M., et al. Down-modulation of c-kit mRNA and protein expression by erythroid differentiation factor/activin A. FEBS Lett, 1995. 374(1): p. 69–71.

Adipogenic, osteogenic and myofibrogenic differentiations of a rat malignant fibrous histiocytoma (MFH)-derived cell line, and a relationship of MFH cells with embryonal mesenchymal, perivascular and bone marrow stem cells

Malignant fibrous histiocytoma (MFH) is regarded as an undifferentiated pleomorphic sarcoma with unproven histogenesis. We investigated pathobiological characteristics of a rat MFH cell line (MT-9). Immunocytochemically, MT-9 cells and MT-9-induced tumours reacted to vimentin, A3 (rat MFH cell-specific antibody), macrophage markers and α-SMA (myofibroblastic marker), indicating that MT-9 showed both histiocytic and (myo)fibroblastic features. Adipogenic supplement-added MT-9 showed increased accumulation of lipid droplets. Addition of BMP-2 or osteogenic supplement to MT-9 enhanced osteoblastic markers (ALP activity, osteocalcin mRNA expression and calcification). TGF-β1-treated MT-9 revealed increased numbers of α-SMA-immunopositive cells, and enhanced protein levels of α-SMA and fibronectin, indicating myofibrogenesis. In rat tissues, A3 labelled with immature mesenchymal and perivascular cells in foetuses and neonates, and with marrow stem cells in adults. c-kit mRNA expression was seen in bone marrows and MT-9. Collectively, progenitors of MFH should be sought in lineage of marrow stem cells capable of differentiating into mesenchymal cells.

Myoelectric activity and motility of the Roux limb after cut or uncut Roux-en-Y gastrojejunostomy

To explore the mechanisms of uncut Roux-en-Y gastrojejunostomy, which is used to decrease the occurrence of Roux stasis syndrome. The changes of myoelectric activity, mechanic motility and interstitial cells of Cajal (ICC) of the Roux limb after cut or uncut Roux-en-Y gastrojejunostomy were observed. When compared with the cut group, the amplitude (1.15 +/- 0.15 mV vs 0.48 +/- 0.06 mV, P < 0.05) and frequency (14.4 +/- 1.9 cpm vs 9.5 +/- 1.1 cpm, P < 0.01) of slow waves and the incidence (98.2% +/- 10.4% vs 56.6% +/- 6.4%, P < 0.05) and amplitude (0.58 +/- 0.08 mV vs 0.23 +/- 0.06 mV, P < 0.01) of spike potential of the Roux limb in the uncut group were significantly higher. The migrating myoelectric complexes (MMC) phase III duration in the uncut group was significantly prolonged (6.5 +/- 1.1 min vs 4.4 +/- 0.8 min, P < 0.05), while the MMC cycle obviously shortened (42.5 +/- 6.8 vs 55.3 +/- 8.2 min, P < 0.05). Both gastric emptying rate (65.5% +/- 7.9% vs 49.3% +/- 6.8%, P < 0.01) and intestinal impelling ratio (53.4% +/- 7.4% vs 32.2% +/- 5.4%, P < 0.01) in the uncut group were significantly increased. The contractile force index of the isolated jejunal segment in the uncut group was significantly higher (36.8 +/- 5.1 vs 15.3 +/- 2.2, P < 0.01), and the expression of c-kit mRNA was significantly increased in the uncut group (0.82 +/- 0.11 vs 0.35 +/- 0.06, P < 0.01). Uncut Roux-en-Y gastrojejunostomy may lessen the effects of operation on myoelectric activity such as slow waves, spike potential, and MMC, decrease the impairment of gastrointestinal motility, and remarkably increase the expression of c-kit mRNA.

Effects of STI571 on the Expression of c-kit Receptor in Mononuclear Cells from the Bone Marrow of Patients with Acute Non-Lymphocytic Leukemia.

Objective: To explore the effects of STI571 on the expression of c-kit gene of the bone marrow mononuclear cells from patients with acute non-lymphocytic leukemia (ANLL) and to study the gene difference expression in original leukemia cells from the bone marrow of ANLL patients with CD117 overexpress treated by STI571, so as to explore initially the mechanism of the restraint of proliferation caused by STI571.Methods: The expression of CD117 and C-kit mRNA level were assayed by FACS and RT-PCR before and after different concentration STI571 cultivatio. The changes of gene expression were examined with the cDNA array in original leukemia cells after treated by STI571 and control.Results: After STI571 treatment, the expression of CD117 and the mRNA expression of c-kit were significantly lower than before (P<0.05). There were over 730 different genes in each pair of chips, in which 156 different genes expressed together. In all different genes, 66.67% (104/156) were up-regulated, 33.33% (52/156) were down-regulated. Cellular skeleton accounted to 4.49% (7/156), oncogenes and tumor supressors 6.41% (10/156), cells receptor 7.05% (11/156), extracellular signal transduction proteins 23.07% (36/156), cyclin proteins7.05% (11/156), apoptosis correlation proteins 1.92% (3/156) and other genes accounted to50% (78/156). Genes with over ten times relative difference amounted to 26.92% (42/156).Conclusion: The restraint of c-kit on ANLL fresh cells was related to concentration of STI571. The mechanism of the restraint of proliferation caused by STI571 may relate to three ways: 1. STI571 realizes its biological function by affecting different signal conduction pathways through protein tyrosine kinase, guanine-nucleotide-binding protein and protein kinase C; 2. STI571 restrains the infinite differentiation of cells through affecting of tumor cellular mitosis; 3. Destroy cellular skeleton.

Restoration of hepatic mast cells and expression of a different mast cell protease phenotype in regenerating rat liver after 70%‐hepatectomy

Mast cells (MC) can undergo significant changes in number and phenotype; these alterations result in the differential expression of growth factors and cytokines. Kit ligand (KL; stem cell factor) is produced by mesenchymal cells, and in the liver by biliary epithelial cells. Recent studies suggest that KL, and its receptor c-kit, may be involved in liver regeneration after loss of liver mass. However, KL is also the major growth, differentiating, chemotactic, and activating factor for MC. The aim of our study was to elucidate the dynamics and phenotype of hepatic MC and KL/c-kit expression during liver regeneration after partial (70%) hepatectomy in the rat. Regenerating livers were harvested after 1, 3, 7, and 14 days, respectively (n = 6 each day). MC were stained for naphthol-AS/D-chloroacetate esterase and counted as MC per bile ductule. MC phenotype was assessed by rat MC protease (RMCP)-1 and -2 immunofluorescence staining, in order to distinguish RMCP-1 positive connective tissue MC (CTMC) from RMCP-2 positive mucosa MC (MMC). mRNA expression of RMCP, c-kit, and the differentially spliced variants of KL was quantified by RT-PCR. MC counts per bile ductule decreased in regenerating rat liver tissue at day 3, compared with native livers, and became normal thereafter. Hepatic MC were predominantly of a CTMC phenotype expressing RMCP-1, as previously published; after hepatectomy, between 76 and 99% of all MC double-expressed RMCP-1 and -2, compatible with an MMC phenotype. The ratio of the two alternatively spliced mRNAs for KL (KL-1 : KL-2), and c-kit mRNA expression did not differ significantly between regenerating livers and the livers of sham operated animals. These results suggest that hepatic mast cells are restored during liver regeneration after partial hepatectomy in the rat. Restored MC express an MMC phenotype, suggesting migration from outside into the regenerating liver. Alternative splicing of KL is affected by the surgical procedure in general, and, together with its receptor c-kit, doesn't seem to be involved in liver regeneration after partial hepatectomy in the rat. Further functional studies, and studies in regenerating human livers might offer the possibility of elucidating the role of the hepatic mast cell, and its different protease phenotypes during liver regeneration after surgical loss of liver mass.

Expression of c-kit messenger ribonucleic acid and c-kit protein in sigmoid colon of patients with slow transit constipation

The c-kit protooncogene receptor and its ligand stem cell factor regulate the proliferation and survival of germ cells as well as interstitial cells of Cajal (ICCs). Decreased numbers of ICCs and defects in its networks have been reported in the colon of patients with slow transit constipation (STC). However, little information about the c-kit messenger ribonucleic acid (mRNA) and protein expression in the constipated colon is available. The aim of this study was to determine whether the expression of c-kit mRNA and c-kit protein declined in the colon in STC. The sigmoid colonic samples from 12 patients with STC and from eight age-matched patients with non-obstructed colorectal cancer were used for this study. Expression of c-kit mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), and expression of c-kit protein was detected by Western blot analysis. Decreased expression of c-kit mRNA was demonstrated in the STC group compared with the control group. The ratio of c-kit and beta-actin was 1.26+/-0.32 in controls and 1.17+/-0.41 in the STC group (U=0.500, P=0.029). c-kit protein expression significantly declined in the STC group. The mean value of optical density was 162.97+/-5.43 in the control group and 96.64+/-8.80 in the STC group (U=0.000, P=0.021). The data indicate that the expression of c-kit mRNA and c-kit protein significantly decreased in the colon of STC, suggesting that the c-kit signal pathway may play an important role in ICC reduction in STC.

Role of stem cell factor and its receptor in the pathogenesis of pediatric aplastic anemia.

In order to investigate the levels of stem cell factor (SCF) and its receptor c-kit protein and mRNA in pediatric aplastic anemia (AA) and their relevance to the pathogenesis, immunocytochemical and in situ hybridization were utilized to detect the expression of SCF and its receptor c-kit gene protein and mRNA, respectively in 59 children with AA and 51 normal controls. The relationship between SCF and c-kit and the pathogenesis of AA was analyzed subsequently. The results showed that the positive rate of SCF protein and mRNA expression in children with AA was significantly lower than that in healthy controls (P < 0.05). However, there was no significant difference in the positive rate of c-kit protein and mRNA expression between children with AA and control group (P > 0.05). It was concluded that the expression of SCF is significantly decreased in children with AA, which may be closely associated with the pathogenesis of the AA. c-kit may be unrelated to the development of pediatric AA. Therefore, AA in children may have abnormalities at SCF/c-kit signal transduction levels.

216. c-kit Expression study: timing of onset in rodent testis and irradiated rat testis model

Primordial germ cell and spermatogonial cell function is essential for normal male fertility. These cells require Sertoli–germ cell interactions, specifically somatic cell-derived stem cell factor (SCF) that acts through the c-kit receptor to govern primordial germ cell migration in the foetus, spermatogonial differentiation during puberty and adulthood, and Leydig cell steroidogenesis. We performed a comprehensive study of the c-kit mRNA expression profile in the pre- and post-pubertal mouse and rat testes by in situ hybridisation. Expression of c-kit mRNA was first visualised in germ cells after birth, with the levels concordant with the number and appearance of the differentiated spermatogonial subtypes in both the rat and the mouse. We also studied c-kit expression in the irradiated adult rat testis, in which only undifferentiated spermatogonia are present. After treatment with Cetrorelix, GnRH antagonist (3 days, 1, 2 and 4 weeks) germ cell maturation is re-initiated. Expression of c-kit messenger RNA was observed in the undifferentiated spermatogonia in both untreated and treated testes sections. In contrast, c-kit protein expression was undetectable until 4 weeks of hormone treatment. This suggests that c-kit mRNA and protein expression are differentially regulated and that protein expression relates to somatic cell function.

In vivo efficacy of STI571 in xenografted human small cell lung cancer alone or combined with chemotherapy

STI571, or imatinib, selectively inhibits BCR/ABL, PDGFR and c-kit kinase activity. It has been reported that a large proportion of small cell lung cancer (SCLC) cell lines and tumors express c-kit and that STI571 inhibits tumor cell growth. We therefore investigated the therapeutic efficacy of STI571, alone or combined with chemotherapy, in human SCLC cells or tumors xenografted into nude mice. The level of c-kit mRNA expression was variable in SCLC tumors (positive for 2 of 4 xenografts), and c-kit protein was not detected by immunohistochemistry. On the 4 xenografted tumors, PDGFRalpha and PDGFRbeta were not detected by immunohistochemistry. STI571 induced inhibition of proliferation of the SCLC6 cell line without inducing apoptosis; in contrast, in combination with etoposide or topotecan, the growth inhibition of SCLC6 cells induced by STI571 was increased, with apoptotic DNA fragmentation. Four human SCLC xenografts (SCLC6, SCLC61, SCLC74 and SCLC108) were transplanted into mice. After intraperitoneal injection of STI571, we observed 80%, 40% and 78% growth inhibition of SCLC6, SCLC61 and SCLC108 tumors, respectively, without any significant inhibition of SCLC74 tumor growth. In mice bearing responsive SCLC tumors, we observed an increase of growth inhibition induced by chemotherapy (etoposide + ifosfamide or topotecan) by concomitant and continuous administration of STI571, associated with an increase of toxic deaths. In SCLC6-bearing mice receiving sequential treatments, we observed a reduction of toxic deaths but a decrease of synergistic antitumor efficacy. In conclusion, the efficacy of STI571 alone in SCLC xenografted tumors was variable and did not depend on c-kit expression. Moreover, a significant increase of chemotherapy-induced growth inhibition was obtained by concomitant administration of STI571 that should be carefully investigated in SCLC patients.

The expression patterns of mRNA-encoding stem cell factor, internal stem cell factor and c-kit in the prepubertal and adult porcine ovary.

The receptor, c-kit, and its ligand, stem cell factor (SCF), are important regulators of ovarian follicle growth and development. The aim of this study was to identify the sites of expression of mRNA for c-kit and SCF in prepubertal and mature (pregnant and non-pregnant) animals. Ovaries were recovered from prepubertal animals, non-pregnant sows and five sows at approximately 3 months of gestation. Ovine SCF and c-kit DNA were cloned into plasmid vectors to produce RNA probes. Expression of mRNA encoding SCF and c-kit were detected via in situ hybridization. Both mRNA were detected throughout ovaries from all animals. This study provides evidence that the growth-factor complex is required throughout follicle development, and also for continued maintenance of the corpus luteum (CL) in the mature animal. SCF mRNA was localized to the granulosa cell layer and was also extensively expressed in endothelial tissue and throughout the CL. c-kit mRNA was detected in the theca layer, oocytes and also in CL. In conclusion, expression of SCF and c-kit mRNA in granulosa and theca cells, respectively, indicate an important interaction between somatic cells throughout follicle development and that in the mature animal, SCF and c-kit potentially have a role in maintaining progesterone secretion by the CL. The observations of continued expression of SCF and c-kit throughout development suggest that there may be differences in the role of this receptor-ligand complex between large mono- vs. poly ovulatory species, such as the pig.