Increased expression of glial cell line-derived neurotrophic factor and neurturin in a case of colon adenocarcinoma associated with diffuse ganglioneuromatosis.

Intestinal ganglioneuromatosis (GN) is an uncommon disease of the enteric nervous system (ENS) and its pathogenesis remains unclear. Here we describe a unique case of diffuse GN of the intestinal wall associated with colon adenocarcinoma occurring in a 38-year-old female. Because it is well-known that glial cell line-derived neurotrophic factor (GDNF) and its receptor components, GDNF family receptor-alpha 1 (GFR-alpha 1) and RET receptor tyrosine kinase, play a crucial role in the development of ENS, their expression was analyzed by immunohistochemistry. Interestingly, GDNF as well as a related neurotrophic factor, neurturin (NTN), were expressed at high levels in adenocarcinoma cells whereas expression of GFR alpha 1 and RET was undetectable in them. In contrast, GFR-alpha 1 showed positive staining in both proliferating ganglion cells and glial cells, and RET immunoreactivity was found mainly in ganglion cell bodies. These findings suggested that GDNF and NTN expression in adenocarcinoma cells may play an important role in the pathogenesis of GN.

Human thymine-DNA glycosylase maps at chromosome 12q22-q24.1: a region of high loss of heterozygosity in gastric cancer.

Spontaneous hydrolytic deamination of 5-methylcytosine leads to T:G mismatches in double-stranded DNA and comprises a major threat for the integrity of both the DNA primary sequence as well as the epigenetic information stored in the DNA methylation pattern. Failure of the cellular DNA repair machinery to recognize and repair such mismatched nucleotides can lead to a mutator phenotype and subsequent carcinogenesis. A thymine-DNA glycosylase (TDG) has been described that initiates T:G mismatch repair by specifically excising the mismatched T. We have studied the TDG genomic locus and the expression of this enzyme to evaluate its role in cancer development. TDG is highly expressed in thymus and is expressed at lower levels in all human tissues analyzed. The TDG gene has 10 exons covering a region of >25 kb and is located on chromosome 12q22-q24.1. Because gastric tumors have been shown to contain a high percentage of C-->T mutations at CpG sites, we used a microsatellite found in intron 8 of the TDG locus to screen gastric tumor samples for loss of heterozygosity. Although our analysis showed loss of heterozygosity in 10 of 24 samples (42%), none of those tumor samples revealed a mutation in the coding sequence of the remaining TDG allele as analyzed by single-strand conformational polymorphism. Expression of the TDG was not determined because of the limited availability of RNA in these primary tumor samples. At present, we have found no evidence that TDG is central to the development of gastric cancer, limiting the importance of TDG in T:G mismatch repair and subsequent carcinogenesis.

Characterization of the human homologue of RAD54: a gene located on chromosome 1p32 at a region of high loss of heterozygosity in breast tumors.

A search of the Human Genome Sciences database of expressed sequence-tagged DNA fragments, for sequences containing homology to known yeast DNA recombination and repair genes, yielded a cDNA fragment with high homology to RAD54. Here we describe the complete cDNA sequence and the characterization of the genomic locus coding for the human homologue of the yeast RAD54 gene (hRAD54). The yeast RAD54 belongs to the RAD52 epistasis group and appears to be involved in both DNA recombination and repair. The hRAD54 gene maps to chromosome 1p32 in a region of frequent loss of heterozygosity in breast tumors and encodes a protein of M(r) 93,000 that displays 52% identity to the yeast RAD54 protein. The hRAD54 protein sequence additionally contains all seven of the consensus segments of a superfamily of proteins with presumed or proven DNA helicase activity. Mutations in genes with consensus helicase homology have been found in cancer-prone syndromes such as xeroderma pigmentosum and Bloom syndrome as well as Werner's syndrome, in which patients age prematurely, and the X-linked mental retardation with alpha-thalassemia syndrome, ATR-X. We have examined the hRAD54 gene in several breast tumors and breast tumor cell lines and, although the gene region appears to be deleted in several tumors, at present we have found no coding sequence mutations.

Characterization of intracellular signals via tyrosine 1062 in RET activated by glial cell line-derived neurotrophic factor.

Glial cell line derived neurotrophic factor (GDNF) signals through a multicomponent receptor complex consisting of RET receptor tyrosine kinase and a member of GDNF family receptor alpha (GFRalpha). Recently, it was shown that tyrosine 1062 in RET represents a binding site for SHC adaptor proteins and is crucial for both RAS/mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/AKT signaling pathways. In the present study, we characterized how these two pathways diverge from tyrosine 1062, using human neuroblastoma and primitive neuroectodermal tumor cell lines expressing RET at high levels. In response to GDNF stimulation, SHC bound to GAB1 and GRB2 adaptor proteins as well as RET, and SHC and GAB1 were highly phosphorylated on tyrosine. The complex formation consisting of SHC, GAB1 and GRB2 was almost abolished by replacement of tyrosine 1062 in RET with phenylalanine. Tyrosine-phosphorylated GAB1 was also associated with p85 subunit of PI3-K, resulting in PI3-K and AKT activation, whereas SHC-GRB2-SOS complex was responsible for the RAS/ERK signaling pathway. These results suggested that the RAS and PI3-K pathways activated by GDNF bifurcate mainly through SHC bound to tyrosine 1062 in RET. Furthermore, using luciferase reporter-gene assays, we found that the RAS/ERK and PI3-K signaling pathways are important for activation of CREB and NF-kappaB in GDNF-treated cells, respectively. Oncogene (2000) 19, 4469 - 4475.

Fez1/lzts1 alterations in gastric carcinoma.

PURPOSE: Loss of heterozygosity (LOH) involving the short arm of chromosome 8 (8p) is a common feature of the malignant progression of human tumors, including gastric cancer. We have cloned and mapped a candidate tumor suppressor gene, FEZ1/LZTS1, to 8p22. Here we have analyzed whether FEZ1/LZTS1 alterations play a role in the development and progression of gastric carcinoma. EXPERIMENTAL DESIGN: We examined Fez1/Lzts1 expression in 8 gastric carcinoma cell lines by Western blot, and in 88 primary gastric carcinomas by immunohistochemistry. Twenty-six of these 88 primary gastric carcinomas were also microdissected and tested for LOH at the FEZ1/LZTS1 locus and for mutation of the FEZ1/LZTS1 gene. Furthermore, we studied the FEZ1/LZTS1 gene regulation and transcriptional control and the methylation status of the 5' region of the gene in all 8 gastric carcinoma cell lines. RESULTS: Fez1/Lzts1 protein was barely detectable in all of the gastric cancer cell lines tested and was absent or significantly reduced in 39 of the 88 (44.3%) gastric carcinomas analyzed by immunohistochemistry, with a significant correlation (P < 0.001) to diffuse histotype. DNA allelotyping analysis showed allelic loss in 3 of 17 (18%) and microsatellite instability in 4 of 17 (23.5%) cases informative for D8S261 at the FEZ1/LZTS1 locus. When we compared the presence of LOH with Fez1/Lzts1 expression, we found loss of protein expression in all three of the tumors with allelic imbalance at D8S261. A missense mutation was detected in one case that did not express Fez1/Lzts1. Hypermethylation of the CpG island flanking the Fez1/Lzts1 promoter was evident in six of the eight cell lines examined as well as in the normal control. CONCLUSIONS: Our findings support FEZ1/LZTS1 as a candidate tumor suppressor gene at 8p in a subtype of gastric cancer and suggest that its inactivation is attributable to several factors including genomic deletion and methylation.

Identification of human SEP1 as a glial cell line-derived neurotrophic factor-inducible protein and its expression in the nervous system.

Glial cell line-derived neurotrophic factor (GDNF) signals through multisubunit receptor complex consisting of RET tyrosine kinase and a glycosylphosphatidylinositol-anchored coreceptor called GDNF family receptor alpha1 (GFRalpha1). In the current study, we cloned a human SEP1 gene as a GDNF-inducible gene using human neuroblastoma cells that express RET and GFRalpha1. The induction of the SEP1 gene showed two peaks at 0.5-2 h and 24-48 h after GDNF stimulation by Northern blotting and quantitative real-time reverse transcriptase polymerase chain reaction. The late induction was also confirmed at protein levels by Western blotting with anti-SEP1 antibody. Immunostaining revealed that the expression of the SEP1 protein was detected in cell body, elongated neurites and growth cone-like structure of neuroblastoma cells treated with GDNF. In addition, we found a high level of SEP1 expression in neurons of the dorsal root and superior cervical ganglia and motor neurons of the spinal cord of mice in which RET is also expressed. SEP1 was co-immunoprecipitated with alpha- and beta-tubulins from the lysate of mouse brain. These results thus suggested that SEP1 is a GDNF-inducible and microtubule-associated protein that may play a role in the nervous system.

Substance P inhibits catecholamine biosynthesis stimulated by carbamylcholine in cultured adrenal chromaffin cells.

The effect of substance P on catecholamine biosynthesis was examined using cultured bovine adrenal chromaffin cells as a model for the sympathoadrenergic system. Substance P markedly inhibited the formation of [14C]catecholamines from L-[14C]tyrosine stimulated by cholinergic agonist, but caused no significant effect on the biosynthesis stimulated by depolarizing agent. In addition, this inhibitory action was completely prevented by the addition of substance P antagonists. Under the conditions in which the inhibition of catecholamine biosynthesis was observed, substance P also inhibited the influx of extracellular 45Ca2+ into these cells, and this inhibitory action on Ca2+ influx was almost identical to that on the biosynthesis. These results provide evidence for a possible role of substance P as a putative neuromodulator in the sympathoadrenergic system.

Muscarinic receptor-mediated calcium efflux from cultured bovine adrenal chromaffin cells.

The effect of stimulation of the muscarinic receptor on Ca2+ mobilization in cultured bovine adrenal chromaffin cells was examined. Acetylcholine (ACh) increased the uptake of 45Ca2+ and [Ca2+]i whose levels decreased with time after reaching peaks. It also enhanced the efflux of 45Ca2+ from the cells. Its effect was inhibited by the specific muscarinic receptor antagonist atropine (Atr), but not by the nicotinic receptor antagonist hexamethonium (C6). The increase in muscarine (Mus)-stimulated 45Ca2+ efflux was reduced concentration-dependently by deprivation of extracellular Na+. These results suggest that muscarinic stimulation of the ACh receptor stimulates Na+/Ca2+ exchange in cultured bovine adrenal chromaffin cells.

Calcium efflux from cultured bovine adrenal chromaffin cells induced by bradykinin.

The effect of bradykinin on Ca2+ efflux from cultured bovine adrenal chromaffin cells was examined. Bradykinin enhanced the efflux of 45Ca2+ from the cells in a concentration dependent manner (10(-9)-10(-6) M). This effect was inhibited by a specific bradykinin B2-receptor antagonist, but not by a B1-receptor antagonist. Nifedipine, Co2+ and Cd2+ did not inhibit the bradykinin-stimulated 45Ca2+ efflux from the cells. 12-O-Tetradecanoyl phorbol 13-acetate, an activator of protein kinase C, also had no effect on the efflux of 45Ca2+ from the cells. The increase in bradykinin-stimulated 45Ca2+ efflux was reduced by removal of extracellular Na+. These results suggest that bradykinin stimulates Na+/Ca2+ exchange in cultured bovine adrenal chromaffin cells.

cDNA sequencing and expression of rat mast cell tryptase.

A cDNA encoding rat mast cell tryptase (rMCT) was successfully cloned, and sequenced, from peritoneal cells of Lewis rats infected with Nippostrongylus brasiliensis by the reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods. The cDNA was 1,097 base-pairs long, and included 822 base-pairs of an open reading frame. As judged from the deduced amino acid sequence, rMCT is highly homologous to mouse mast cell protease-6, and is considered to be translated as a prepro-enzyme with a 19-amino acid signal peptide, a 10-amino acid activation peptide, and a 245-amino acid mature enzyme. The rMCT mRNA was not detected in peritoneal cells of mast cell-deficient Ws/Ws rats, though it was strongly detected in ones of littermate +/+ and Lewis rats. In addition to in peritoneal mast cells, the rMCT mRNA was detected in the tongue. However, mRNA signals were not detected in the small intestine regardless of N. brasiliensis infection. Nor were mRNA signals detected in RBL2H3 rat basophilic leukemia cells. In the lung, the rMCT mRNA was strongly detected after infection with N. brasiliensis, though it was only faintly detected before infection. These results suggest that the rMCT is basically specific for connective tissue mast cells, but not for mucosal mast cells and that it is up-regulated in the lung during the inflammatory process of a parasitic infection.

Palytoxin: a potent stimulator of catecholamine release from cultured bovine adrenal chromaffin cells.

The effect of palytoxin (PTX), a potent marine toxin, on catecholamine release from cultured bovine adrenal chromaffin cells was examined. PTX at concentrations of over 10(-9) M induced catecholamine release dose-dependently. About 40-50% of the total cellular catecholamine was released during 20-min incubation with 3 x 10(-8) M PTX. PTX-induced catecholamine release was dependent on both extracellular Na+ and Ca2+, and was inhibited by organic and inorganic Ca2+ channel blockers, but not by tetrodotoxin. PTX-induced increase in 45Ca2+ influx into the cells, which was associated with catecholamine release, was also inhibited by these Ca2+ channel blockers. These results indicated that PTX-induced catecholamine release was mediated by activation of Na(+)-dependent, tetrodotoxin (TTX) insensitive voltage-dependent Ca2+ channels.

Potentiation by ouabain of bradykinin-induced catecholamine secretion and calcium influx into cultured bovine adrenal chromaffin cells: evidence for involvement of Na+ influx associated with the bradykinin B2-receptor.

The effect of bradykinin (BK), in the presence of ouabain, an inhibitor of Na(+)-K+ ATPase, on catecholamine (CA) secretion was studied in cultured bovine adrenal chromaffin cells, to determine whether Na+, as well as Ca2+, is involved in BK-receptor mediated CA secretion. BK (10(-8)-10(-5) M)-induced CA secretion was markedly potentiated by addition of ouabain (10(-5) M), was blocked by a BK-B2 receptor antagonist, and was decreased in Ca(2+)-free medium. BK-induced increase in 45Ca2+ influx was also potentiated by addition of ouabain. The cultured cells were first incubated with BK for 30 min in Ca(2+)-free medium in the presence or absence of ouabain and then stimulated for 15 min with Ca(2+)-medium without BK or ouabain. Prior stimulation of the cells, BK induced 22Na+ influx and increased Ca(2+)-induced CA secretion and these stimulatory effects of BK were potentiated by added ouabain. When the cells were stimulated with BK and ouabain in Na(+)-free sucrose medium, the Ca(2+)-induced CA secretion was greatly reduced. These results indicated that activation of the BK-B2 receptor and inhibition of the Na+ pump both increase the intracellular Na+ level, resulting in increase in Ca2+ influx and CA secretion.

Effect of orally administered heat-killed Enterococcus faecalis FK-23 preparation on leukopenia in mice treated with cyclophosphamide.

Cyclophosphamide (CY) treated mice were orally administered heat-killed Enterococcus faecalis FK-23 preparation (FK-23) to define the effect of FK-23 on leukopenia associated with chemotherapy. FK-23 had no inhibiting effect on CY induced leukopenia, whereas it augmented leukocyte reconstitution in CY-treated mice. The percentage of neutrophils increased in the mice orally given FK-23. Furthermore, an increase in a myeloid/erythroid ratio and neutrophilic lineages was found in bone marrow of FK-23 treated mice. These findings suggest that FK-23 may have a potent effect on the augmentation of leukocyte reconstituting capacity in patients receiving chemotherapy.

Processing of CD109 by furin and its role in the regulation of TGF-beta signaling.

CD109 is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, whose expression is upregulated in squamous cell carcinomas of the lung, esophagus, uterus and oral cavity. CD109 negatively regulates transforming growth factor (TGF)-beta signaling in keratinocytes by directly modulating receptor activity. In this study, we further characterized CD109 regulation of TGF-beta signaling and cell proliferation. We found that CD109 is produced as a 205 kDa glycoprotein, which is then processed in the Golgi apparatus into 180 kDa and 25 kDa proteins by furin (furinase). 180 kDa CD109 associated with GPI-anchored 25 kDa CD109 on the cell surface and was also secreted into the culture medium. To investigate whether furinase cleavage of CD109 is necessary for its biological activity, we mutated arginine 1273 in the CD109 furinase cleavage motif (amino acid 1270-RRRR-1273) to serine (R1273S). Interestingly, CD109 R1273S neither significantly impaired TGF-beta signaling nor affected TGF-beta-mediated suppression of cell growth, although it was expressed on the cell surface as a 205 kDa protein. Consistent with this finding, the 180 kDa and 25 kDa CD109 complex, but not CD109 R1273S, associated with the type I TGF-beta receptor. These findings indicate that processing of CD109 into 180 kDa and 25 kDa proteins by furin, followed by complex formation with the type I TGF-beta receptor is required for the regulation of TGF-beta signaling in cancer cells and keratinocytes.

Roles of induced expression of MAPK phosphatase-2 in tumor development in RET-MEN2A transgenic mice.

Germline mutations in the RET tyrosine kinase gene are responsible for the development of multiple endocrine neoplasia 2A and 2B (MEN2A and MEN2B). However, knowledge of the fundamental principles that determine the mutant RET-mediated signaling remains elusive. Here, we report increased expression of mitogen-activated protein kinase phosphatase-2 (MKP-2) in carcinomas developed in transgenic mice carrying RET with the MEN2A mutation (RET-MEN2A). The expression of MKP-2 was not only induced by RET-MEN2A or RET-MEN2B mutant proteins but also by the activation of endogenous RET by its ligand, glial cell line-derived neurotrophic factor (GDNF). MKP-2 expression was also evident in the MKK-f cell line, which was established from a mammary tumor developed in a RET-MEN2A transgenic mouse. Inhibition of MKP-2 attenuated the in vitro and in vivo proliferation of MKK-f cells, which was mediated by the suppression of cyclin B1 expression. Furthermore, we found that MKP-2 is highly expressed in medullary thyroid carcinomas derived from MEN2A patients. These findings suggest that the increased expression of MKP-2 may play a crucial role in oncogenic signaling downstream of mutant RET, leading to deregulation of cell cycle.

Fine mapping of thymus enlargement gene 1 (Ten1) in BUF/Mna rats.

Two polymeric autosomal loci, Ten1 and Ten2, regulate thymus enlargement in BUF/Mna (B) rats. Previously, we mapped Ten1 on chromosome (Chr) 1 to a 20 cM region between Myl2 and D1Mgh11, and Ten2 on Chr 13. To further characterize the precise position of Ten1, 34 and 37 microsatellite markers, that have a polymorphism between the B and WYK (W) and between the B and MITE (M) strains, were used for linkage analysis of thymus enlargement in 105 (WBF1 x B) blackcross (BC) and 78 (B x BMF1) BC rats, respectively. Our data showed that the D1Rat168, D1Rat112, D1Rat323, D1Got186, D1Got187 and D1Got188 markers each gave a peak logarithm of odds (LOD) score of 10.68 for linkage to the thymus ratio in (WBF1 x B) BC rats, and that the D1Rat168, D1Rat197, D1Got184, D1Got186 and D1Got188 markers each gave a peak LOD score of 7.82 in (B x BMF1) BC rats. The two LOD score peaks are coincident in the position of the rat genetic map. All of the markers mentioned above are located in the region between Igf2 and D1Mgh11, in which synteny is conserved with human 11q15.5 and the distal end of mouse Chr 7 or with human 11q13 and the proximal end of mouse Chr 19. Genes existing in these regions are discussed as candidate genes for Ten1.

Chromosomal mapping of genetic locus associated with thymus-size enlargement in BUF/Mna rats.

The thymoma-prone rat of the BUF/Mna strain is a useful model for human thymoma. In this strain thymoma development is regulated by a single autosomal susceptible gene, Tsr-1. At pre-thymoma age, BUF/Mna rats have extremely large thymuses, when compared to those of other strains of rats. Genetic studies in crosses between BUF/Mna rats with large thymuses and WKY/NCrj rats with small thymuses suggested the presence of a major autosomal gene, Ten-1, which contributes to thymus enlargement in a backcross population. Linkage studies between Ten-1 and microsatellite markers in backcross rats of (WKY/NCrj x BUF/Mna)F1 x BUF/Mna have led to the localization of Ten-1 in chromosome 1. This result may provide an approach to clone Tsr-1, which could be allelic to Ten-1.

Inhibitory action of hydralazine on catecholamine secretion from cultured bovine adrenal chromaffin cells.

Hydralazine caused a concentration-dependent inhibition of the secretion of catecholamines induced by carbamylcholine or high K+ from cultured bovine adrenal chromaffin cells, and also caused the significant inhibition of radioactive calcium uptake induced by carbamylcholine into the cells. However, hydralazine failed to inhibit the secretion of catecholamines evoked by the calcium-ionophore, A23187. The inhibitory action of hydralazine on catecholamine secretion induced by carbamylcholine was not affected by increasing the concentration of calcium ion in the reaction mixture. These observations therefore seem to indicate that the inhibitory action of hydralazine is not due to either the blocking of receptors for carbamylcholine or the disruption of the secretory machinery, and suggest that the drug may cause the inhibition of catecholamine secretion through its blocking action on calcium influx into the cells.