Search

Pancreatic adenocarcinoma is mostly fatal and generally resistant to conventional treatments, such that effective therapies with tolerable side effects are desperately needed. Ion channels including the transient receptor potential (TRP) channels have been implicated in human malignancies, but their roles in pancreatic cancer were mostly unknown. Recent identification of the melastatin-subfamily members of the TRP family of ion channels, and their functions in pancreatic epithelia and adenocarcinoma, is expected to provide a new perspective to understanding the mechanism underlying pancreatic tumorigenesis. In this report, we present the clinical and pathological features of a mini-series of patients with pancreatic adenocarcinoma, which aberrantly exhibits immunoreactivity against the TRPM8 channel. We have recently demonstrated the proliferative role of TRPM8 channel in pancreatic cancer cells. Here, we present evidence that RNA interference-mediated silencing of TRPM8 induces replicative senescence in pancreatic adenocarcinoma cells. This suggests that the aberrantly expressed TRPM8 channel may contribute to pancreatic tumorigenesis by preventing oncogene-induced senescence, and targeted inhibition of TRPM8 may enhance tumor sensitivity to therapeutics. Based on these observations, we hypothesize that the TRPM8 ion channel plays a crucial role in the growth and progression of pancreatic neoplasia during tumorigenesis. We propose that TRPM8 can be exploited as a clinical biomarker and as a therapeutic target for developing personalized therapy in pancreatic adenocarcinoma.

The transient receptor potential TRPM7 ion channel is required for cellular proliferation in pancreatic epithelia and adenocarcinoma. To elucidate the mechanism that mediates the function of TRPM7, we examined its role in survival of pancreatic cancer cells. RNA interference-mediated silencing of TRPM7 did not induce apoptotic cell death. TRPM7-deficient cells underwent replicative senescence with up-regulation of p16(CDKN2A) and WRN mRNA. The combination of anti-TRPM7 siRNA and gemcitabine produced enhanced cytotoxicity as compared to gemcitabine alone. Thus, TRPM7 is required for preventing senescence, and modulation of TRPM7 expression may help improve treatment response of pancreatic cancer by combining with apoptosis-inducing agents., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Histone deacetylases (HDACs) and RNA polymerase III (POLR3) play vital roles in fundamental cellular processes, and deregulation of these enzymes has been implicated in malignant transformation. Hdacs and Polr3 are required for exocrine pancreatic epithelial proliferation during morphogenesis in zebrafish. We aim to test the hypothesis that Hdacs and Polr3 cooperatively control exocrine pancreatic growth, and combined inhibition of HDACs and POLR3 produces enhanced growth suppression in pancreatic cancer. In zebrafish larvae, combination of a Hdac inhibitor (Trichostatin A) and an inhibitor of Polr3 (ML-60218) synergistically prohibited the expansion of exocrine pancreas. In human pancreatic adenocarcinoma cells, combination of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and ML-60218 produced augmented suppression of colony formation and proliferation, and induction of cell cycle arrest and apoptotic cell death. The enhanced cytotoxicity was associated with supra-additive upregulation of the pro-apoptotic regulator BAX and the cyclin-dependent kinase inhibitor p21(CDKN1A). tRNAs have been shown to have pro-proliferative and anti-apoptotic roles, and SAHA-stimulated expression of tRNAs was reversed by ML-60218. These findings demonstrate that chemically targeting developmental regulators of exocrine pancreas can be translated into an approach with potential impact on therapeutic response in pancreatic cancer, and suggest that counteracting the pro-malignant side effect of HDAC inhibitors can enhance their anti-tumor activity.

Transient receptor potential (TRP) ion channels are essential for normal functions and health by acting as molecular sensors and transducing various stimuli into cellular and physiological responses. Growing evidence has revealed that TRP ion channels play important roles in a wide range of human diseases, including malignancies. In light of recent discoveries, it has been found that TRP melastatin-subfamily members, TRPM7 and TRPM8, are required for normal and cancerous development of exocrine pancreas. We are currently investigating the mechanisms which mediate the functional roles of TRPM7 and TRPM8 and attempting to develop these ion channels as clinical biomarkers and therapeutic targets for achieving the goal of personalized therapy in pancreatic cancer.

Histone deacetylases (HDACs) play important roles in the epigenetic control of development, and aberrant expression of HDACs has been implicated in human diseases including cancer. Among the mammalian HDACs, HDAC1 has been extensively studied, but its role in exocrine pancreatic morphogenesis and cancer is still poorly understood. The goal of this study is to determine the functional role of HDAC1 in normal development of exocrine pancreas using zebrafish as the model organism as well as in human pancreatic adenocarcinoma. The zebrafish germline loss-of-function mutation hdac1(hi1618) caused impaired cell cycle progression in pancreatic epithelia, resulting in growth arrest and dysmorphogenesis of exocrine pancreas. In human pancreatic adenocarcinoma tissues and cell lines, HDAC1 was expressed at variably elevated levels. RNA interference-induced silencing of HDAC1 diminished proliferation of the cancer cells and cell cycle progression. The proliferative arrest in the developing exocrine pancreas and pancreatic cancer cells was associated with up-regulated expression of the cyclin-dependent kinase inhibitors and the sonic hedgehog signaling components. This study indicates that HDAC1 is required for pancreatic epithelial proliferation in development and cancer. We hypothesize that aberrant expression of HDAC1 modulates the developmental and signaling pathways in exocrine pancreatic epithelia and consequently the genes required for cellular proliferation during development and progression of pancreatic neoplasia.

Genetic analysis of pancreatic development has provided new insights into the mechanisms underlying the formation of exocrine pancreatic neoplasia. Zebrafish sweetbread (swd) mutants develop hypoplastic acini and dysmorphic ducts in the exocrine pancreas, with impeded progression of cell division cycle and of epithelial growth. Positional cloning and allelic complementation have revealed that the swd mutations affect the transient receptor potential melastatin-subfamily member 7 (trpm7) gene, which encodes a divalent cation-permeable channel with kinase activity. Supplementary Mg(2+) partially rescued the exocrine pancreatic defects of the trpm7 mutants by improving cell-cycle progression and growth and repressing the suppressor of cytokine signaling 3a (socs3a) gene. The role of Socs3a in Trpm7-mediated signaling is supported by the findings that socs3a mRNA level is elevated in the trpm7 mutants, and antisense inhibition of socs3a expression improved their exocrine pancreatic growth. TRPM7 is generally overexpressed in human pancreatic adenocarcinoma. TRPM7-deficient cells are impaired in proliferation and arrested in the G0-G1 phases of the cell division cycle. Supplementary Mg(2+) rescued the proliferative defect of the TRPM7-deficient cells. Results of this study indicate that Trpm7 regulates exocrine pancreatic development via the Mg(2+)-sensitive Socs3a pathway, and suggest that aberrant TRPM7-mediated signaling contributes to pancreatic carcinogenesis.

The roles of transient receptor potential (TRP) cation channels in pancreatic tumorigenesis are essentially unknown. Here, we focus on the TRP melastatin-subfamily (TRPM) members. Expression of the thermally regulated transmembrane Ca(2+)-permeable channel TRPM8 is consistently up-regulated in human pancreatic adenocarcinoma cell lines and tissues. TRPM8-deficient pancreatic cancer cells have reduced ability of proliferation and cell cycle progression with elevated levels of cyclin-dependent kinase inhibitors. These results indicate that TRPM8 is aberrantly over-expressed in pancreatic adenocarcinoma and required for cellular proliferation, and they support further investigation of the potential of TRPM8 as a clinical biomarker and therapeutic target in pancreatic adenocarcinoma., (2010 Elsevier Ireland Ltd. All rights reserved.)

Advanced age is considered a risk factor for pancreatic cancer, but this relationship at the molecular and genetic level remains unclear. We present a clinical case series focusing on an association between pancreatic adenocarcinoma and Werner syndrome (WS) that is an autosomal recessive genetic disorder characterized by accelerated aging and cancer predisposition, and is caused by loss-of-function mutations in the WS RecQ helicase gene (WRN). Although pancreatic adenocarcinoma mostly occurs in a sporadic fashion, a minority of cases occurs in the context of susceptible individuals with hereditary syndromes. While WS has not been previously recognized as a risk factor for developing malignant tumors of the exocrine pancreas, the clinicopathologic features of three reported patients suggest a contributory role of WRN deficiency in pancreatic carcinogenesis. Molecular genetic analyses support the role of WRN as a tumor suppressor gene, although recent evidence reveals that WRN can alternatively promote oncogenicity depending on the molecular context. Based upon the clinico-pathologic features of these patients and the role of WRN in experimental models, we propose that its loss-of-function predisposes the development of pancreatic adenocarcinoma through epigenetic silencing or loss-of-heterozygosity of WRN. To test this hypothesis, we are investigating the mechanistic role of WRN in pancreatic cancer models including a pancreatic adenocarcinoma cell line generated from a human with WS. These studies are expected to provide new insight into the relationship between aging and pancreatic tumorigenesis, and facilitate development of novel strategies for patient-tailored interventions in this deadly malignancy.

Combined targeting of distinct cellular signaling mechanisms may improve the efficacy and reduce the toxicity of therapy in pancreatic cancer. Histone deacetylases (HDACs) control cellular functions through epigenetic modulation, and HDACs inhibitors suppress cell growth in pancreatic adenocarcinoma. The Hedgehog (Hh) pathway regulates the development of the pancreas, and aberrant Hh signaling promotes the initiation and progression of pancreatic neoplasia. We hypothesize that HDACs and the Hh pathway cooperatively interact to regulate cellular proliferation of the exocrine pancreas. A combination of the HDAC inhibitor SAHA and the Smoothened antagonist SANT-1 was evaluated for their ability to suppress growth of the Gemcitabine-resistant pancreatic adenocarcinoma cell lines Panc-1 and BxPC-3. The combination of SAHA and SANT-1 supra-additively suppressed cellular proliferation and colony formation. Flow cytometric and immunohistochemical analyses indicated that enhanced induction of apoptotic cell death, cell cycle arrest in G(0)/G(1) phase, and ductal epithelial differentiation are involved. Cell death was associated with nuclear localization of survivin, increased bax expression, and activation of caspases 3 and 7. Consistent with the cell cycle arrest and cytodifferentiation, the cyclin-dependent kinase inhibitors p21(waf) and p27(kip1) were upregulated, and cyclin D1 downregulated. The potentiated anti-proliferative effect by the combination of SAHA and SANT-1 may involve cooperative suppression of the Hh pathway activity, as shown by the upregulation of HHIP by SAHA, and enhanced repression of of Ptc-1 mRNA expression. In summary, we have developed a molecular target-based therapeutic approach that overcomes chemoresistance in pancreatic cancer cells by chemically inhibiting HDACs and Hh signaling in combination.

The role of RNA polymerase III (Pol III) in developing vertebrates has not been examined. Here, we identify a causative mutation of the second largest Pol III subunit, polr3b, that disrupts digestive organ development in zebrafish slim jim (slj) mutants. The slj mutation is a splice-site substitution that causes deletion of a conserved tract of 41 amino acids in the Polr3b protein. Structural considerations predict that the slj Pol3rb deletion might impair its interaction with Polr3k, the ortholog of an essential yeast Pol III subunit, Rpc11, which promotes RNA cleavage and Pol III recycling. We engineered Schizosaccharomyces pombe to carry an Rpc2 deletion comparable to the slj mutation and found that the Pol III recovered from this rpc2-delta yeast had markedly reduced levels of Rpc11p. Remarkably, overexpression of cDNA encoding the zebrafish rpc11 ortholog, polr3k, rescued the exocrine defects in slj mutants, indicating that the slj phenotype is due to deficiency of Rpc11. These data show that functional interactions between Pol III subunits have been conserved during eukaryotic evolution and support the utility of zebrafish as a model vertebrate for analysis of Pol III function.

Although many of the genes that regulate development of the endocrine pancreas have been identified, comparatively little is known about how the exocrine pancreas forms. Previous studies have shown that exocrine pancreas development may be modeled in zebrafish. However, the timing and mechanism of acinar and ductal differentiation and morphogenesis have not been described. Here, we characterize zebrafish exocrine pancreas development in wild type and mutant larvae using histological, immunohistochemical and ultrastructural analyses. These data allow us to identify two stages of zebrafish exocrine development. During the first stage, the exocrine anlage forms from rostral endodermal cells. During the second stage, proto-differentiated progenitor cells undergo terminal differentiation followed by acinar gland and duct morphogenesis. Immunohistochemical analyses support a model in which the intrapancreatic ductal system develops from progenitors that join to form a contiguous network rather than by branching morphogenesis of the pancreatic epithelium, as described for mammals. Contemporaneous appearance of acinar glands and ducts in developing larvae and their disruption in pancreatic mutants suggest that common molecular pathways may regulate gland and duct morphogenesis and differentiation of their constituent cells. By contrast, analyses of mind bomb mutants and jagged morpholino-injected larvae suggest that Notch signaling principally regulates ductal differentiation of bipotential exocrine progenitors.

Elucidation of basic mechanisms that regulate pancreatic organogenesis may help define molecular pathways involved in the development of exocrine pancreas cancer. The zebrafish has emerged as a powerful model for genetic dissection of the mechanisms underlying vertebrate organogenesis including formation of the pancreas. Unique properties of zebrafish enable genetic and embryological analyses not feasible using other vertebrate model organisms. The optical clarity of the zebrafish embryos allows visual detection of markers for pancreatic morphogenesis and cytodifferentiation by whole mount immunohistochemistry and RNA in situ hybridization. This feature, coupled with the accessibility of the externally fertilized zebrafish embryo and the small size and fecundity of adult zebrafish, facilitates large-scale forward genetic screens using chemical or insertional mutagenesis techniques. Furthermore, these properties allow high throughput studies that target functions of known genes via antisense or enforced expression studies. Together, such studies are predicted to identify novel genes, or known genes essential for pancreas development. Work in zebrafish is predicted to complement research performed using other vertebrate model organisms, and may help identify markers that define early stages of pancreatic tumorigenesis as well as potential targets for therapy.

Mammalian studies have implicated important roles for the basic helix-loop-helix transcription factor Ptf1a-p48 in the development of both exocrine and endocrine pancreas. We have cloned the Ptf1a-p48 ortholog in Danio rerio. Early zebrafish ptf1a expression is observed in developing hindbrain and in endodermal pancreatic precursors. Analysis of ptf1a and insulin expression reveals a population of exocrine precursors that, throughout early development, are temporally and spatially segregated from endocrine elements. Morpholino-mediated knockdown of ptf1a confirms early divergence of these endocrine and exocrine lineages. Ptf1a morphants lack differentiated exocrine pancreas, but maintain normal differentiation and organization of the principal islet. In addition to the exocrine phenotype, ptf1a knockdown also reduces the prevalence of a small population of anterior endocrine cells normally found outside the principal islet. Together, these findings suggest the presence of distinct ptf1a-dependent and ptf1a-independent precursor populations in developing zebrafish pancreas.

Pancreatic cancer is increasingly prevalent and almost uniformly fatal. Studies of the molecular genetics of sporadic and hereditary cases of pancreatic cancer as well as the molecular biology of pancreatic development may advance our understanding of the mechanism underlying pathogenesis of this malignancy. Based on a case of pancreatic adenocarcinoma in a patient with Peutz-Jeghers syndrome (PJS), the clinicopathologic features and molecular genetics of pancreatic tumors associated with this hereditary cancer syndrome are reviewed. Particular emphasis is placed on the developmental roles and biochemical functions of STK11/LKB1, the gene mainly responsible for PJS. Modeling pancreatic cancer in animal models such as the mouse and zebrafish will further our understanding of the pathogenesis of this important disease, and the studies derived from these model organisms can be potentially applied for developing novel preventive and therapeutic strategies.

In bone marrow-derived mast cells (BMMCs), the Kit receptor tyrosine kinase mediates diverse responses including proliferation, survival, chemotaxis, migration, differentiation, and adhesion to extracellular matrix. In connective tissue mast cells, a role for Kit in the secretion of inflammatory mediators has been demonstrated as well. We recently demonstrated a role for phosphatidylinositide-3' (PI 3)-kinase in Kit-ligand (KL)-induced adhesion of BMMCs to fibronectin. Herein, we investigated the mechanism by which Kit mediates enhancement of Fc epsilon RI-mediated degranulation, cytoskeletal rearrangements, and adhesion in BMMCs. Wsh/Wsh BMMCs lacking endogenous Kit expression, were transduced to express normal and mutant Kit receptors containing Tyr-->Phe substitution at residues 719 and 821. Although the normal Kit receptor fully restored KL-induced responses in Wsh/Wsh BMMCs, Kit gamma 719F, which fails to bind and activate PI 3-kinase, failed to potentiate degranulation and is impaired in mediating membrane ruffling and actin assembly. Inhibition of PI 3-kinase with wortmannin or LY294002 also inhibited secretory enhancement and cytoskeletal rearrangements mediated by Kit. In contrast, secretory enhancement and adhesion stimulated directly through protein kinase C (PKC) do not require PI 3-kinase. Calphostin C, an inhibitor of PKC, blocked Kit-mediated adhesion to fibronectin, secretory enhancement, membrane ruffling, and filamentous actin assembly. Although cytochalasin D inhibited Kit-mediated filamentous actin assembly and membrane ruffling, secretory enhancement and adhesion to fibronectin were not affected by this drug. Therefore, Kit-mediated cytoskeletal rearrangements that are dependent on actin polymerization can be uncoupled from the Kit-mediated secretory and adhesive responses. Our results implicate receptor-proximal PI 3-kinase activation and activation of a PKC isoform in Kit-mediated secretory enhancement, adhesion, and cytoskeletal reorganization.

The pleiotropic effects of the Kit receptor system are mediated by Kit-Ligand (KL) induced receptor autophosphorylation and its association with and activation of distinct second messengers, including phosphatidylinositol 3'-kinase (PI3-kinase), p21ras and mitogen-activated protein kinase (MAPK). To define the role of PI3-kinase, p21ras and MAPK in Kit-mediated cell proliferation, survival and adhesion in bone marrow-derived mast cells (BMMC), mutant Kit receptors were expressed in Wsh/Wsh BMMC lacking endogenous c-kit expression. The introduction of both murine Kit(S) and KitL (isoform containing a four amino acid insert) into Wsh/Wsh BMMC restored KL-induced proliferation, survival and adhesion to fibronectin, as well as activation of PI3-kinase, p21ras and MAPK, and induced expression of c-fos, junB, c-myc and c-myb mRNA. Substitution of tyrosine 719 in the kinase insert with phenylalanine (Y719F) abolished PI3-kinase activation, diminished c-fos and junB induction, and impaired KL-induced adhesion of BMMC to fibronectin. In addition, the Y719F mutation had partial effects on p21ras activation, cell proliferation and survival, while MAP kinase activation was not affected. On the other hand, Y821F substitution impaired proliferation and survival without affecting PI3-kinase, p21ras and MAPK activation, and induction of c-myc, c-myb, c-fos and c-jun mRNA, while KL-induced cell adhesion to fibronectin remained intact. In agreement with a role for PI3-kinase in Kit-mediated cell adhesion, wortmannin blocked Kit-mediated cell adhesion at concentrations known to specifically inhibit PI3-kinase. We conclude, that association of Kit with p85PI3-K, and thus with PI3-kinase activity, is necessary for a full mitogenic as well as adhesive response in mast cells. In contrast, tyrosine 821 is essential for Kit-mediated mitogenesis and survival, but not cell adhesion.

The receptor tyrosine kinase Kit and Kit ligand (KL), encoded at the murine white spotting (W) and steel (Sl) loci, respectively, function in hematopoiesis, melanogenesis, and gametogenesis. To understand the mechanism of turnover of Kit in mast cells, mutant receptors generated in vitro were heterologously expressed in Wsb/Wsh mast cells lacking endogenous c-kit expression, and the effects of mutations on KL-induced internalization and ubiquitination/degradation of Kit were studied. Upon binding of KL, KL.Kit receptor complexes were rapidly internalized, and the turnover was accelerated by ubiquitin-mediated degradation. Inactivation of the Kit kinase resulted in a reduced rate of internalization of KL.Kit complexes, degradation of kinase-inactive receptor complexes was relatively slow, and receptor ubiquitination was absent. But abolishment of KL-induced receptor association and activation of phosphatidylinositol 3'-kinase and of tyrosine 821 autophosphorylation did not affect KL-induced internalization and ubiquitination/degradation of Kit. Furthermore, Kit receptors can be down-regulated by proteolytic cleavage induced by either activation of protein kinase C or by isopropyl alcohol. In summary, KL-induced internalization of KL.Kit complexes and ubiquitination/degradation require an active kinase. By contrast, proteolytic cleavage of Kit mediated by protein kinase C activation is independent of kinase activity.

The receptor tyrosine kinase Kit and its cognate ligand KL/steel factor are encoded at the white spotting (W) and Steel (Sl) loci of the mouse, respectively. Mutations at both the W and the Sl loci affect hematopoiesis including the stem cell hierarchy, erythropoiesis, and mast cells, as well as gametogenesis and melanogenesis. In addition, mutant mice display an increased sensitivity to lethal doses of irradiation. The role of KL/c-kit in cell proliferation and survival under conditions of growth factor-deprivation and gamma-irradiation was studied by using bone marrow-derived mast cells (BMMC) as a model. Whereas apoptosis induced by growth factor deprivation in BMMC is a stochastic process and follows zero order kinetics, gamma-irradiation-induced apoptosis is an inductive process and follows higher order kinetics. In agreement with these results, gamma-irradiation-induced apoptosis in BMMC was shown to be dependent on p53 whereas apoptosis induced by deprivation is partly dependent on p53, implying that there are other mechanisms mediating apoptosis in KL-deprived BMMC. In the presence and in the absence of serum, KL stimulated proliferation by promoting cell cycle progression. The presence of KL was required only during the early part of the G1 phase for entry into the S phase. At concentrations lower than those required for proliferation, KL suppressed apoptosis induced by both growth factor-deprivation and gamma-irradiation, and internucleosomal DNA fragmentation characteristic of apoptosis. The ability of KL to suppress apoptosis was independent of the phase of the cell cycle in which the cells were irradiated and suppression of apoptosis was a prerequisite for subsequent cell cycle progression. Moreover, addition of KL to gamma-irradiated and growth factor-deprived cells could be delayed for up to 1 h after irradiation or removal of growth factors when cells became irreversibly committed to apoptosis. KL and IL-3 induce suppression of apoptosis in mast cells by different mechanisms based on the observations of induction of bcl-2 gene expression by IL-3 but not by KL. It is proposed that the increased sensitivity of W and Sl mutant mice to lethal irradiation results from paucity of the apoptosis suppressing and proliferative effects of KL.

The proto-oncogene c-kit is allelic with the white spotting locus (W) on mouse chromosome 5 and it encodes a transmembrane protein tyrosine kinase which belongs to the platelet-derived growth factor and macrophage-colony stimulating factor (CSF-1) receptor subfamily. In an effort to study the function of the c-kit receptor, specifically the physiological mechanism of controlling the signal induced by the ligand, the effect and mechanism of down-regulation of the c-kit receptor by the kit ligand (KL) was investigated in mast cells. Following preincubation with KL, the capacity of mast cells to bind kit antibody was reduced and binding of radiolabeled KL to mast cells decreased with similar kinetics, suggesting that KL stimulates the loss of c-kit receptor from the cell surface. After binding to the c-kit receptor, KL was rapidly internalized, and degradation of the receptor was accelerated. The c-kit receptor was transmodulated by the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and by the calcium ionophore ionomycin. TPA- and ionomycin-induced down-regulation of the c-kit receptor was accompanied by release of the extracellular domain of the receptor, presumably by proteolytic cleavage near the transmembrane domain. Release of the extracellular domain of the c-kit receptor occurred also in untreated cells but at a slow rate. In addition, ionomycin induced shedding of the intact c-kit receptor. In mast cells depleted of protein kinase C, the c-kit receptor remained sensitive to down-regulation induced by KL and ionomycin, but not by treatment with TPA. Therefore, the down-regulation of the c-kit receptor induced by KL, activated protein kinase C, and an increased level of intracellular calcium is mediated through independent mechanisms.

The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor that is allelic with the murine white-spotting locus (W). W mutations affect melanogenesis, gametogenesis, and hematopoiesis during development and adult life, and they result from the partial or complete loss of c-kit function. The W42 allele is a W mutation with severe effects in both the homozygous and the heterozygous states. Previous analysis of the W42 allele identified a missense mutation in an essential amino acid of the c-kitW42 kinase domain that abolishes the in vitro kinase activity of the c-kitW42 protein but does not affect its normal expression. These results suggested that the c-kitW42 allele was a dominant negative mutation within the context of c-kit-mediated signal transduction. To further explore the dominant negative characteristics of the W42 mutation, we have generated transgenic mice in which ectopic expression is driven by the human beta-actin promoter (hAP). Two mouse lines carrying the hAP-c-kitW42 transgene show an effect on pigmentation and the number of tissue mast cells. The patchy coat color pattern of the line 695 mice may reflect variable expression of the transgene in melanoblast progenitors and their descendants and, consequently, is indicative of a function for c-kit in early melanoblasts. Germ cell development and erythropoiesis, however, do not appear to be affected by the transgene. Mice expressing the c-kitW42 transgene therefore recapitulate some of the phenotypes of mice with W mutations. These results are therefore in agreement with the molecular basis of the W42 mutation and the dominant-negative characteristics of the c-kitW42 protein product.

This study compared the effect of single equimolar oral doses of cimetidine (100 mg/kg) or ranitidine (139 mg/kg) on rat hepatic mixed function oxidases. Cimetidine significantly (p less than 0.05) increased hexobarbital sleeping time and prolonged aminopyrine and theophylline elimination. In contrast, ranitidine did not significantly affect hexobarbital sleeping time and theophylline elimination but significantly (p less than 0.025) increased aminopyrine elimination. Aminopyrine N-demethylase activity in vitro was significantly (p less than 0.05) inhibited by cimetidine pretreatment but significantly (p less than 0.025) increased by ranitidine pretreatment. The direct addition of cimetidine or SKF 525A to the 10,000g supernatant fraction from controlled liver homogenates decreased aminopyrine N-demethylase activity, whereas the direct addition of ranitidine tended to increase aminopyrine N-demethylase activity. A significant correlation (r = 0.65, p less than or equal to 0.005) was observed between hexobarbital sleeping time in vivo and aminopyrine N-demethylase activity in vitro in the same rat. The results of this study showed that cimetidine inhibited mixed function oxidases, whereas ranitidine had no effect or tended to stimulate mixed function oxidases.