A case of undifferentiated carcinoma of the gallbladder with anomalous arrangement of the pancreaticobiliary ductal system.

Anomalous junction of the pancreaticobiliary duct (AJPBD) is a congenital anomaly associated with gallbladder carcinoma. Especially patients with noncystic dilatation and without dilatation of the biliary tract are at risk of gallbladder carcinoma. A 56-year-old woman with advanced gallbladder cancer associated with AJPBD but without dilatation of the biliary tract was treated at our hospital. Although histologically cancer cells remained in the layer of the proprial mucosa, extensive metastases to lymph nodes including the paraaorta and peripancreas were detected. According to the TNM classification this case was of Stage IVB. The cancer consisted of medullary round cells, and was diagnosed as undifferentiated carcinoma. After surgery poor prognosis was expected, but three years have elapsed with no recurrence. The case is of interest because of two points of discrepancy: the primary cancer did not show deep invasion but demonstrated extensive lymph node metastases; the cancer was histologically malignant but prognosis was relatively good.

[All-trans retinoic acid combined with low-dose cytosine arabinoside treatment for acute myelogenous leukemia with trilineage myelodysplasia--a case report].

AML with trilineage myelodysplasia (AML/TMDS) is recognized as having of poor prognosis due to its resistance to chemotherapy in comparison with de novo AML. An AML/TMDS patient who failed to respond to ordinary induction therapy achieved complete remission with combination therapy consisting of all-trans retinoic acid (ATRA) and low-dose Ara-C. No serious toxicity was observed. ATRA combined with low-dose Ara-C could be an alternative treatment for this kind of patient.

Tumor-like splenic extramedullary hematopoiesis in a patient with myelofibrosis.

A 61-year-old woman, who was diagnosed in 1982 as having polycythemia vera, was admitted to our hospital in July 1998 because of a splenic tumor in an enlarged spleen due to myelofibrosis. As it was difficult to identify the etiology of the splenic tumor, partial splenectomy was carried out. The resected tumor proved to be an extremely proliferative lesion as the result of extramedullary hematopoiesis. Since it is difficult to diagnose the etiology of splenic tumor, the collection and analysis of reports of relevant cases may well facilitate diagnosis.

Diurnal changes in intraepithelial lymphocytes (IELs) in the small intestine of mice.

Diurnal changes in small intestinal intraepithelial lymphocytes (IELs) were examined in 8- to 10-week-old BALB/cA male mice. The ratio of T cell subsets expressing CD8 alpha alpha homodimer/CD8 alpha beta heterodimer was found to be higher in the dark period than that in the light period. Increased expression of Thy-1.2 on gamma delta T cells was also observed in the light period. No significant changes were found in other subsets. This is the first report to document diurnal changes in the small intestinal IELs in mice.

EGF-induced activation of 70-kDa S6 kinase in CHO cells expressing human EGF receptors.

We investigated epidermal growth factor (EGF)-induced activation of 85-kDa/110-kDa phosphatidylinositol (PI)-3-kinase and 70-kDa S6 kinase in Chinese hamster ovary cells expressing the human EGF receptor. EGF-induced activation of p70 S6 kinase was comparable to that induced by insulin, whereas that of PI-3-kinase in anti-phosphotyrosine immunoprecipitates was very small compared with insulin. Wortmannin, a p85/p110 PI-3-kinase inhibitor, inhibited EGF-induced activation of p70 S6 kinase in a dose-dependent manner. Given that several proteins homologous to catalytic subunit of p85/p110 PI-3-kinase have been identified and that wortmannin inhibits distinct form of PI-3-kinase, the present results suggest that wortmannin-sensitive kinases that resemble catalytic subunit of p85/p110 PI-3-kinase may participate in the signaling pathway from EGF receptors to p70 S6 kinase.

Grb2/Ash binds directly to tyrosines 1068 and 1086 and indirectly to tyrosine 1148 of activated human epidermal growth factor receptors in intact cells.

The activation of receptor tyrosine kinases generates tyrosine-phosphorylated recognition motifs for the binding of signaling proteins containing Src homology 2 domains. We determined the binding sites of Grb2/Ash, an Src homology 2 domain-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing human EGF receptor mutants in which one of the autophosphorylation sites was retained. In intact cells, the amount of Grb2/Ash coimmunoprecipitated with mutant receptors retaining tyrosines 992, 1068, 1086, 1148, or 1173 was approximately 10, 85, 55, 50, or 20% of wild-type levels, respectively. The association of Grb2/Ash with in vitro autophosphorylated EGF receptor mutants was detectable in those retaining either tyrosines 1068 or 1086 but not in other mutants including those retaining tyrosine 1148. In peptide inhibition assay, phosphorylated peptides representing tyrosines 1068 and 1086 inhibited the binding of Grb2/Ash to in vitro autophosphorylated wild-type EGF receptors, whereas the other peptides representing tyrosines 992, 1148, and 1173 failed to inhibit the binding. Given that tyrosine 1148 of the activated EGF receptor is a major binding site of Shc (Okabayashi, Y., Kido, Y., Okutani, T., Sugimoto, Y., Sakaguchi, K., and Kasuga, M. (1994) J. Biol. Chem. 269, 18674-18678), these results indicate that tyrosines 1068 and 1086 of activated human EGF receptors are direct high affinity binding sites of Grb2/Ash and that tyrosine 1148 is an indirect binding site through Shc in intact cells.

Tyrosines 1148 and 1173 of activated human epidermal growth factor receptors are binding sites of Shc in intact cells.

Autophosphorylation of receptor tyrosine kinases provides binding sites for signaling proteins containing Src homology 2 (SH2) domains. We determined the binding sites of Shc, SH2-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing EGF receptor mutants in which autophosphorylation sites, either alone or in combination, were replaced by phenylalanine. Binding of Shc to EGF receptor mutants lacking single tyrosine residues at 1148 or 1173 decreased by approximately 60 or approximately 15%, respectively, whereas other single point mutants bound the wild-type level of Shc. Binding of Shc markedly decreased in mutants lacking both tyrosine residues at 1148 and 1173. In peptide inhibition assay, phosphorylated nonameric peptide representing tyrosine 1148, DNPDpYQQDF, but not pentameric peptide, pYQQDF, inhibited the binding of glutathione S-transferase-Shc SH2 domain fusion protein to in vitro autophosphorylated EGF receptors, suggesting that N-terminal sequences adjacent to phosphotyrosine are necessary for the association of Shc. Based on results of peptide inhibition assays in which phosphorylated peptides representing tyrosines 992, 1148, and 1173 inhibited Shc binding to the receptor, we constructed another EGF receptor mutant in which one of these tyrosine residues was retained. The amount of Shc bound to mutant receptors retaining tyrosines 1148, 1173, or 992 was approximately 80, approximately 40, or approximately 10% of wild-type level, respectively. These results indicate that tyrosine 1148 of activated human EGF receptors is a major binding site of Shc and tyrosine 1173 is a secondary binding site in intact cells.

In vitro inhibitory effect of somatostatin on secretin action in exocrine pancreas of rats.

BACKGROUND: Exocrine pancreatic function is influenced by pancreatic islet hormones. Although the existence of somatostatin receptors has been shown on pancreatic acinar cells, the in vitro effect of somatostatin on exocrine secretory function has not been established. METHODS: Using isolated rat pancreatic acini, the effect of somatostatin analog SMS 201-995 (SMS) and somatostatin 14 (S-14) on amylase release, cyclic adenosine monophosphate (cAMP) production, and hormone binding were determined. RESULTS: SMS inhibited the potentiating effect of secretin on amylase response to cholecystokinin octapeptide (CCK-8) in a concentration-dependent manner. The inhibitory effects of SMS and S-14 were similar on a molar basis and were observed when vasoactive intestinal polypeptide (VIP) but not 8bromoadenosine 3':5' cyclic monophosphate was used instead of secretin and when carbachol, bombesin, A23187, and 12-O-tetradecanoylphorbol 13-acetate were used instead of CCK-8. SMS inhibited secretin-induced cAMP production, and the dose-inhibition curve for cAMP was similar to that for amylase release. SMS had no influence on 125I-secretin and 125I-VIP binding. CONCLUSIONS: Somatostatin acts directly on acinar cells and inhibits secretin potentiation of secretory response in part by inhibiting secretin-induced cAMP production.

[Effect of cholecystokinin and secretin on insulin binding to rat pancreatic acini and pancreatic cancer cell line AR42J cells].

In order to clarify the interaction of hormones which exert various effects on the exocrine pancreas, we investigated the effect of cholecystokinin (CCK) and secretin on subsequent insulin binding to pancreatic acini and cultured AR42J cells derived from azaserine-induced acinar cell carcinoma of the pancreas. CCK at concentrations of 100pM-10nM inhibited subsequent 125I-insulin binding to pancreatic acini. 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited 125I-insulin binding whereas A23187 had little effect, suggesting that the inhibitory effect of CCK is mediated by protein kinase C. On the other hand, 100pM-10nM secretin had no effect on subsequent 125I-insulin binding to pancreatic acini, although higher concentrations of forskolin and 8 bromoadenosine 3', 5'-cyclic monophosphate inhibited 125I-insulin binding. In addition, secretin exerted no potentiating effect on the inhibitory effect of CCK on 125I-insulin binding to pancreatic acini. Based on these results, we further investigated the effect of CCK and TPA on subsequent 125I-insulin binding to AR42J cells. In this carcinoma cell line, inhibitory effect of CCK and TPA on insulin binding was completely abolished. The present results suggest, therefore, that hormonal interaction may play an important role in the regulation of exocrine pancreatic function including acinar cell growth.

[Clinical evaluation of intra-arterial methotrexate, vinblastine, doxorubicin and cis-platinum for bladder cancer].

Chemotherapy regimens including Methotrexate, Vinblastine. Doxorubicin and Cis-platinum (M-VAC) have shown objective responses in more than 50% of uroepithelial tumors. And intra-arterial chemotherapy can theoretically increase the drug concentration delivered to the tumor with maximum efficacy and less toxicity. In this report we review our experience at Matsuyama Red Cross Hospital between September 1987 and February 1991 using intra-arterial M-VAC in 21 evaluable patients with bladder cancer. Response was observed in 62% of the treated patients, two with complete remission and eleven with partial remission. WBC nadir and platelet nadir were mild to moderate. Other adverse effects such as mucositis, hair loss and creatinine elevation were seen in a small number of patients. Intra-arterial M-VAC can be one of the most useful forms of chemotherapy for patients with bladder cancer.

[Inhibitory effect of somatostatin analog, SMS 201-995, on exocrine secretion from isolated rat pancreatic acini].

In vitro effect of somatostatin analog, SMS 201-995 (SMS), on pancreatic exocrine secretion was investigated using isolated rat pancreatic acini. SMS had no effect on basal, cholecystokinin octapeptide (CCK-8)- or secretin-stimulated amylase release. SMS inhibited pancreatic amylase release in response to simultaneous stimulation with secretin and CCK-8 in a dose-dependent manner. Significant inhibition was observed with 10 nM SMS and maximal inhibition with 0.1-1 microM SMS. Amylase release in response to the combination of 100 pM CCK-8, 1 nM secretin and 0.1-1 microM SMS was similar to that to 100 pM CCK-8 alone. Secretin significantly increased acinar cell cAMP content. SMS partially inhibited an increase in cAMP content induced by secretin. The present study has demonstrated, therefore, that SMS directly inhibits the potentiating effect of secretin on exocrine secretion in part by inhibiting an increase in secretin-induced cAMP accumulation in rat pancreatic acinar cells.

Rat prostatic growth factors: purification and characterization of high and low molecular weight epidermal growth factors from rat dorsolateral prostate.

Growth factors which possibly participate in androgen-induced proliferation of rat prostate epithelial cells have been purified and characterized. Four distinct forms of growth factor were found in the extract of rat dorsolateral prostate. One of the factors was a member of heparin-binding growth factor (HBGF) family judging from its high affinity for heparin-Sepharose. The other three factors were capable of competing with [125I]epidermal growth factor (EGF) for the cell surface receptor, and recognized by anti-rat EGF antiserum. These EGF-like factors (EGF1-EGF3) were purified by ion-exchange chromatography, gel filtration and reverse phase HPLC. EGF1 showed microheterogeneity on chromatographic and electrophoretic separation and N-terminal sequence analysis. EGF1 showed an average molecular weight of about 35,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. These results indicated that EGF1 was a mixture of high molecular weight forms of EGF. The molecular weights of EGF2 and EGF3 were similar to that of rat submaxillary gland EGF (Mr = 5400). The amino acid sequence of EGF2 was identical with that of rat EGF except for the N- and C-terminal amino acids: aspartic acid instead of asparagine was found at the N-terminal position and C-terminal arginine was missing in EGF2. Although the N-terminal sequence of EGF3 (1-19) was identical with that of EGF2, the two factors were completely separated by gel filtration indicating a difference in the C-terminal structure. EGF1, EGF2 and EGF3 but not HBGF stimulated proliferation of primary cultured rat dorsolateral prostate epithelial cells.

Role of cyclic AMP and polypeptide growth regulators in growth inhibition by interferon in PC-3 cells.

Participation of growth factors and intracellular cAMP in direct antiproliferative action of interferon alpha (IFN-alpha) was investigated in PC-3 human prostate carcinoma cell line. IFN-alpha inhibited proliferation of PC-3 cells in a dose-dependent manner in vitro, and the effect was reversible. Fibroblast growth factor, epidermal growth factor and platelet-derived growth factor, when added to the culture medium, showed no effect on growth of PC-3 cells in presence or absence of IFN-alpha. Transforming growth factor beta (TGF-beta) significantly inhibited PC-3 cell growth, and the effect was additived to that of IFN-alpha. TGF-beta content in conditioned medium of PC-3 cells was not affected by treatment with IFN-alpha. On the other hand, IFN-alpha increased intracellular cAMP concentration about 20-fold. Dibutyryl cAMP and reagents which elevated intracellular cAMP level also inhibited PC-3 cell growth. These indicated that direct antiproliferative effect of IFN-alpha on PC-3 cells was at least partly mediated by cAMP, and that neither growth factors nor a growth inhibitor participated in the action of IFN-alpha.

Differences in growth requirements between epithelial and stromal cells derived from rat ventral prostate in serum-free primary culture.

Different procedures of enzymatic digestion of rat prostatic tissue and unique sets of mitogenic factors made it possible to culture practically pure populations of epithelial and stromal cells without previous separation of the two types of cells. Keratin-positive epithelial cells dissociated by trypsin and collagenase from adult rat ventral prostate proliferated in medium WAJC 404 supplemented with epidermal growth factor, insulin, cholera toxin, and bovine pituitary extract. Proliferation of epithelial cells was completely inhibited by dexamethasone as low as 30 nM. On the other hand, fibroblast-like stromal cells released by trypsin digestion required a plastic substratum coated with calf serum or fibronectin, and proliferated in Eagle's minimum essential medium supplemented with cholera toxin, bovine pituitary extract, dexamethasone, and bovine serum albumin. Epidermal growth factor and insulin had negligible effect on proliferation of stromal cells. Physiological concentrations of dihydrotestosterone and estradiol showed no effect on proliferation of both types of cells.

Synthesis and antitumor activity of new amphiphilic alkylglycerolipids substituted with a polar head group, 2-(2-trimethylammonioethoxy)ethyl or a congeneric oligo(ethyleneoxy)ethyl group.

A new series of amphiphilic 1-octadecyl glycerolipids (eleven compounds, 1a-k) were designed and synthesized, in which the 3-phosphocholine portion of platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) was replaced by the 2-(2-trimethylammonioethoxy)ethyl group and congeneric groups having oligo(ethyleneoxy)ethyl bridges of various lengths at position 3, together with modification at position 2 (lower alkyl, acetonyl, acetoacetyl, carboxymethyl and pyrimidin-2-yl groups). These ether lipids, characterized by a nonphosphorus lysoglycerolipid structure, showed potent antitumor activity in vitro (human promyelocytic leukemia cells, HL-60, and human epidermoid carcinoma cells, KB) and in vivo (mouse sarcoma S180 and mouse mammary carcinoma MM46). Maximal in vitro potency was obtained with 1-O-octadecyl-2-O-(2-pyrimidinyl)-3-O-[2-(2-trimethylammonioethoxy )ethyl] glycerol (1g; IC50 values for both HL-60 and KB were 0.32 microgram/ml, indicating a higher activity than alkyl-lysophospholipid, ET18-OMe). Several appropriately 2-substituted 1-octadecylglycerolipids with the 3-[2-(2-trimethylammonioethoxy)ethyl] group (e.g., methyl, 1b; butyl, 1f; 2,2,2-trifluoroethyl, 1j; and acetonyl, 1k) showed a potent life-span-prolonging effect on mice with ascites sarcoma S180 and on those with mammary carcinoma MM46, when administered intraperitoneally at 16.5 and 12.5 mg/kg/d, respectively. Compounds 1b and 1k showed definite tumor growth inhibition against solid sarcoma S180 in mice, whether given p.o. or i.v. at 16.5 mg/kg/d. Studies on the structure-activity relationships indicate that the metabolic stability to phospholipase C or related enzymes is at least partly responsible for the potent antitumor activity of this series of ether lipids.

[Vincristine, adriamycin, mitomycin-C and UFT (VAM-UFT) therapy in progressive or recurrent breast cancer].

Since June 1984, 23 cases of progressive or recurrent breast cancers were treated with combination chemotherapy of VAM-UFT consisting of vincristine, adriamycin, mitomycin C and UFT. Clinical effects of VAM-UFT therapy were 3 CR, 12 PR, and the response rate was 65.2%. Its effective interval was 3 months. But the patients treated with over 4 cycles of VAM-UFT therapy showed an 85% response rate, with a 5-month effective interval. In each patient's background, a shorter disease free interval tended to be more highly effective, but other factors were not significant. Scirrhous carcinoma of pathology evidenced slightly high response rate. Compared with the survival time of patients treated with under 3 cycles and over 4 cycles of this therapy, the latter was significantly longer. Toxicity involved leukocytopenia (74%), thrombocytopenia (22%), anemia (30%), alopecia (91%), nausea and vomiting (87%) and stomatitis (35%), but cases in which the treatment was stopped were not observed. Therefore VAM-UFT therapy had a highly therapeutic effect, reflected in an 85% response rate, for progressive or recurrent breast cancers.

Synthesis and antitumor activity of new alkylphospholipids containing modifications of the phosphocholine moiety.

New antitumor alkylglycerophospholipids, in which primarily the phosphocholine moiety of the platelet activating factor (PAF) molecule was modified, were synthesized from 1-alkyl-2-substituted glycerols by introducing polar head phosphoryl groups having methylene bridges of various lengths (from 2 to 14 carbons). They were tested for PAF agonistic activity and antitumor properties. In a series of 1-octadecyl-2-acetoacetylglycerophospholipids (1a-f), an increase in the length of the methylene bridge separating the phosphate and trimethylammonio group in the polar head side chain at position 3 of the glycerol backbone resulted in a progressive decrease in PAF agonistic activity and a characteristic change in antitumor activity against human promyelocytic leukemia cells (HL-60). Maximal potency was obtained with the compound having a decamethylene bridge (1e, IC50 value = 1.5 microgram/ml). Thus, alkylphospholipids possessing a decamethylene bridge and a variety of substituents at position 2 (1g-n) were synthesized. They showed potent inhibitory activity with IC50 values ranging from 0.4 to 1.9 micrograms/ml, depending on the nature of the 2-substituent in the phospholipid molecule. In in vivo tests of the present series of alkylglycerophospholipids (1a--n), using mice bearing sarcoma 180 and mice with mammary carcinoma MM46 (both cells and compounds were given i.p.), 1-octadecyl-2-acetoacetyl-3-glyceryl omega-trimethylammoniodecyl phosphate (1e) showed the most potent life-prolonging effect. The structure-activity relationships are discussed.