Osamu Nakano

Missense mutations and a deletion of the p53 gene in human gastric cancer.

To investigate the molecular pathogenesis of human gastric cancers the p53 gene, a suppressor oncogene, was analyzed in 12 human gastric cell lines. Southern blot and Northern blot analysis revealed a total deletion of p53 gene in KATO-III cells but no major abnormality of p53 gene in other cell lines. By the use of the reverse-transcriptase polymerase chain reaction and direct sequencing 7 cell lines showed point mutations of p53 gene resulting in amino-acid substitutions. Most of them were rare mutations which had not been observed in other types of cancers. One of these mutations was also detected through the use of PCR and oligomer-specific hybridization. Six out of 7 cell lines with mutations of p53 gene also lost one allele of chromosome 17p. Immunoblotting of cell lysates with an antibody specific to p53 demonstrated the absence of p53 protein in KATO-III cell. By contrast, the high levels of the p53 protein were observed in 5 cell lines all of which contained mutations of p53 gene. These results further suggest that the inactivation of p53 gene may play an important role in the transformation of gastric cells to the malignant phenotype. KATO-III cells might be a good model for studying the significance of the loss of p53 gene in cellular transformation.

Prostaglandin Protects Isolated Guinea Pig Chief Cells against Ethanol Injury via an Increase in Diacylglycerol

We studied cellular processes activated by prostaglandins (PG) that are involved in the protection of gastric chief cell injury estimated in terms of dye exclusion test, release of lactate dehydrogenase (LDH), or 51Cr from prelabeled chief cells. Pretreatment of chief cells with 3 x 10(-6) M PGE2 or PGE1 at 37 degrees C and pH 7.4 for 15 min maximally reduced not only ethanol- but also taurocholic acid-caused LDH release from chief cells. PGs equipotently stimulated increases in the accumulation of diacylglycerol and cyclic AMP without elevating intracellular Ca2+ concentrations in gastric chief cells. The rank order of the potency was equal to that of PGs to reduce the injury. Pretreatment of chief cells with synthetic 1-oleoyl-2-acetyl-sn-glycerol (OAG) or 12-o-tetradecanoyl phorbol 13-acetate (TPA) reduced the injury of chief cells, while 4 alpha-phorbol 12,13-didecanoate, an inactive phorbol ester, failed to reduce the injury and 1-(5-isouinolinylsulfonyl)-2-methylpiperazine (H7) blocked the protective action of PGE2. On the other hand, forskolin and dbcAMP had no effect on ethanol-caused LDH release and diacylglycerol formation in chief cells. These results suggest that PGE2 and PGE1 possess the direct protective action against ethanol- or taurocholic acid-caused injury in chief cells, presumably through the activation of the diacylglycerol/protein kinase C signaling pathway.

Induction of cyclooxygenase protein and stimulation of prostaglandin E2 release by epidermal growth factor in cultured guinea pig gastric mucous cells

The present study was undertaken to investigate whether epidermal growth factor (EGF) could stimulate prostaglandin E2 release, and if so, by what mechanism EGF would exert such an effect in gastric mucosal cells. In cultured guinea pig gastric mucous cells, EGF dosedependently stimulated prostaglandin E2 release, with maximal stimulation observed at 10 ng/ml. EGF stimulated an increase in cyclooxygenase activity, which was reduced by protein synthesis inhibitor, actinomycin D, and cycloheximide. EGF also stimulated the enzyme protein synthesis estimated by Western blot analysis, whereas EGF did not stimulate phospholipase A2 activity. These results suggest that such an effect of EGF onde novo synthesis of cyclooxygenase protein and prostaglandin E2 release may be involved at least in part in the mechanism of EGF-induced local regulation of gastric mucosal integrity.

Expression of two types of neurofibromatosis type 1 gene transcripts in gastric cancers and comparison of GAP activities.

To understand the molecular mechanism of gastric tumorigenesis, the status of neurofibromatosis type 1 (NF1) gene was analyzed in human gastric cancer cell lines. Although the sequencing of the GTPase activating protein (GAP)-related region of NF1 (NF1-GRD) revealed no apparent mutation, the NF1-GRD transcript (type I) and that containing an additional 63 bp insert in the center of NF1-GRD (type II) were equally expressed in most gastric cancer cells. By contrast, type II was predominantly expressed in normal stomach mucosa. When these two types of NF1-GRD were bacterially expressed and their GAP activities were tested, both types of NF1-GRD similarly stimulated ras GTPase activity. However, arachidonic acid inhibited GAP activities of two types of NF1-GRD to different extents. These results suggest that the increased expression of type I NF1 protein may modulate ras-related signal transduction and it may be related to the control of the gastric cellular proliferation.

ACUTE PROMYELOCYTIC LEUKEMIA IN CROHN'S DISEASE : CASE REPORT AND REVIEW OF THE LITERATURE

A 19-year-old man with a documented 2-year history of Crohns disease abruptly developed leukopenia and thrombocytopenia. A diagnosis of acute promyelocytic leukemia was established by bone marrow cytology. Chromosomal analysis of bone marrow aspirate revealed aberrations of no. 8 trisomy and translocation between no. 15 and no. 17 [46,XY,t(15q+,17q-)/47,XY,+8,t(15q+,17q-)]. Nine cases of Crohns disease complicated by leukemia have been reported, including the present one; once again, a relationship between Crohns disease and leukemia is suggested.

Effects of antiulcer drugs on phosphatidylcholine synthesis in isolated guinea pig gastric glands

To better understand phosphatidylcholine synthesis in the stomach, we isolated guinea pig gastric glands and examined their [3H] choline incorporation into phosphatidylcholine in response to either antiulcer drugs such as geranylgeranylacetone (GGA) and H2-receptor antagonists or agents that cause phosphatidylcholine synthesis in other tissues. [3H]Choline incorporation was stimulated by GGA, palmitate, and 12-O-tetradecanoylphorbol-13-acetate (TPA). Dibutyryl cyclic-AMP had no effect. By contrast with GGA, famotidine, ranitidine, and cimetidine equipotently inhibited [3H] choline incorporation into phosphatidylcholine. GGA, palmitate, and TPA increased phosphatidyl-[3H] choline and decreased phosphoryl-[3H] choline as compared with control in tissues that had been pulsed with [3H] choline. On the other hand, no more decrease in [3H] choline incorporation at chase periods was observed in pulse-labeled glands in response to each H2-receptor antagonist. The particulate fraction of glands that had been incubated with GGA or palmitate had more CTP-phosphocholine cytidylyltransferase activity than that of glands incubated without agents. A decrease in choline kinase activity was not observed in the cytosolic fraction of glands that had been incubated with cimetidine. These results suggest that GGA and palmitate stimulate phosphatidylcholine synthesis by activating cytidylyltransferase, and H2-receptor antagonists may affect phosphatidylcholine synthesis by inhibiting choline uptake in the gastric glands.

Difference in effects of sodium fluoride and cholecystokinin on diacylglycerol accumulation and calcium increase in guinea pig gastric chief cells.

In isolated guinea pig gastric chief cells, sodium fluoride (NaF) stimulated a monophasic increase in diacylglycerol accumulation, while cholecystokinin (CCK) strongly stimulated its biphasic accumulation. NaF evoked an increase in initial Ca2+ influx rate with a slow increase in intracellular free Ca2+ concentration [( Ca2+]i), while CCK stimulated a rapid increase in [Ca2+]i followed by a late sustained phase of the [Ca2+]i increase. Lanthanum chloride (La3+) effectively blocked NaF-stimulated increase in [Ca2+]i, but it blocked only CCK-stimulated late sustained phase of [Ca2+]i increase. The effect of NaF on pepsinogen secretion was enhanced in the presence of 100 microM AlCl3. Furthermore, pertussis toxin did not affect NaF-evoked diacylglycerol accumulation at all. These results suggest that NaF may activate a pertussis-toxin insensitive guanine nucleotide regulatory protein (G protein) coupled to a signal transducing mechanism which seems to be distinct from that activated by CCK, thereby inducing increases in diacylglycerol accumulation, Ca2+ influx and pepsinogen secretion in guinea pig gastric chief cells.

Idiopathic chronic calcifying pancreatitis with diabetes mellitus. Analysis of pancreatic stone protein gene

SummaryWe present a case of a 27-year-old female suffering from chronic calcifying pancreatitis with diabetes mellitus. Radiographic examinations and exocrine pancreatic function tests revealed considerable dilatation of pancreatic ducts with large intraductal calculi and exocrine pancreatic insufficiency, respectively. Recent literature indicates that a decrease in the activity of pancreatic stone protein (PSP), which inhibits CaCO3 crystal formation in pancreatic juice, is closely related to the development of chronic calcifying pancreatitis. The patient had no apparent cause or family history of pancreatitis. We therefore investigated the possibility that alterations in the PSP gene might explain the chronic pancreatitis seen in this patient. Six exons of the PSP gene amplified by polymerase chain reaction were directly sequenced, but there was no apparent base mutation observed. Furthermore, Southern blot analysis revealed neither rearrangement nor deletion of the PSP gene in the genomic DNA of this case. However, this genetic approach will be useful for future study of the etiology of hereditary pancreatitis.

Characterization of cholecystokinin receptors on guinea pig gastric chief cell membranes

The binding of cholecystokinin (CCK) to its receptors on guinea pig gastric chief cell membranes were characterized by the use of 125I-CCK-octapeptide (CCK8). At 30 degrees C optimal binding was obtained at acidic pH in the presence of Mg2+, while Na+ reduced the binding. In contrast to reports on pancreatic and brain CCK receptors, scatchard analysis of CCK binding to chief cell membranes revealed two classes of binding sites. Whereas, in the presence of a non-hydrolyzable GTP analog, GTP gamma S, only a low affinity site of CCK binding was observed. Chief cell receptors recognized CCK analogs, with an order of potency of: CCK8 greater than gastrin-I greater than CCK4. Although all CCK receptor antagonists tested (dibutyryl cyclic GMP, L-364718 and CR1409) inhibited labeled CCK binding to chief cell membranes, the relative potencies of these antagonists in terms of inhibiting labeled CCK binding were different from those observed in either pancreatic membranes or brain membranes. The results indicate, therefore, that on gastric chief cell membranes there exist specific CCK receptors, which are coupled to G protein. Furthermore, chief cell CCK receptors may be distinct from pancreatic or brain type CCK receptors.

PGE2 protects isolated cells against injury through multiple mechanisms

SummaryIn order to clarify how PGE2 regulates gastric mucosal integrity, we examined the effects of PGE2 on ethanol-caused injury of isolated gastric chief cells, cultured gastric mucous cells from guinea pigs and Balb/c 3T3 fibroblasts. Pretreatment of these cells with PGE2 reduced ethanol-caused injury of the cells. Furthermore, pretreatment of gastric mucous cells with indomethacin enhanced ethanol-caused injury, suggesting that endogenous PGE2 may be involved in the cell protection. PGE2 stimulated an increase in diacylglycerol (DG) accumulation in chief cells and treatment of chief cells with synthetic DG reduced the injury of the cells. However, DG accumulation was not observed in gastric mucous cells treated with PGE2. Therefore PGE2 may protect the cells from injury through a variety of mechanisms. In addition, PGE2 enhanced the survival of the quiescent fibroblasts cultured in the absence of serum, while PGE2 had no survival enhancing effect on gastric mucous cells. These results sugest that the mechanism by which PGE2 preserves the cell viability may depend on not only cell types used but also how the cells are injured.