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Pancreatic cancer is a devastating malignant tumor in humans and the development of new modalities of treatment is needed. We studied the mechanism of the growth-inhibitory effect of cisplatin (CDDP) on human pancreatic cancer cells in connection with the status of the p53 gene and expression of the bcl-2 family. COLO-357 cells with wild-type p53 gene and T3M4, Panc-1 and AsPC-1 cells with mutant-p53 gene were used. Growth of these cells was inhibited by CDDP in a dose-dependent manner in both serum-deprived and serum-supplemented conditions. CDDP induced apoptosis of COLO-357 and T3M4 cells in the serum-supplemented condition, whereas necrosis of these cells was induced by CDDP at high concentrations in the serum-deprived condition. Although expression of bax mRNA and its protein product were enhanced, while bcl-2 protein was decreased by CDDP in COLO-357 cells, expression of mRNA of the bcl-2 family and protein product were not influenced by CDDP in T3M4 cells. Increased expression of bax and reduced expression of bcl-2 are involved in the growth-inhibitory effect of CDDP on pancreatic cancer cells with wild-type p53 gene.

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We investigated the effects of hepatocyte growth factor (HGF) and transforming growth factor alpha (TGF alpha) on cell growth in four human pancreatic cancer cell lines. Changes in the expression of mRNAs of HGF, c-met, TGF alpha, and epidermal growth factor receptor (EGFR) by treatment with HGF and TGF alpha were observed. Cell growth with growth factors was assessed with the MTT assay and compared with basal growth without growth factors. Although HGF stimulated cell growth in AsPC-1, COLO-357, and T3M4 cells, Panc-1 cells showed no response to HGF. TGF alpha stimulated the growth of all the above cells. The expression of c-met mRNA under nonstimulated conditions was detected with Northern blotting in all cells. Treatment with HGF slightly enhanced the expression of c-met mRNA only in COLO-357 cells. The intensity of EGFR expression was consistent, and HGF mRNA was not detected during induction experiments in any cell type. Concomitant treatment with HGF and TGF alpha exerted an effect that was additive or less on the growth of all cells. Expression of TGF alpha was enhanced by HGF treatment only in AsPC-1 cells. These results suggested that HGF and TGF alpha stimulated cell growth through a final common pathway of signal transduction.

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Human pancreatic cancers overexpress the epidermal growth factor (EGF) receptor (EGFR) and all 5 ligands that bind to this receptor, including amphiregulin. It is not known, however, whether amphiregulin contributes in an autocrine manner to enhance pancreatic cancer cell growth. Therefore, we used an amphiregulin antisense oligonucleotide (AR-AS) to suppress amphiregulin expression in T3M4 human pancreatic cancer cells. These cells express high levels of EGFR and amphiregulin. AR-AS abolished amphiregulin immunoreactivity in T3M4 cells, decreased amphiregulin release into the medium and inhibited cell growth in a dose-dependent manner. Exogenous amphiregulin reversed AR-AS-mediated growth inhibition. A random oligonucleotide (AR-R) did not alter either cell growth or cellular amphiregulin immunoreactivity. AR-AS also increased cellular EGFR protein levels and enhanced the growth-inhibitory actions of TP40, a chimeric protein consisting of transforming growth factor-alpha coupled to Pseudomonas exotoxin that internalizes into cells via EGFR. These findings indicate that there is an important EGFR/ amphiregulin autocrine loop in T3M4 cells and raise the possibility that modalities aimed at abrogating amphiregulin action may prove useful in pancreatic cancer, especially when used in conjunction with EGFR-targeted therapy.

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Insulin receptor substrate-1 (IRS-1) is a multisite docking protein implicated in mitogenic signaling following activation of the insulin and insulin-like growth factor I receptors. In the present study we characterized IRS-1 expression in human pancreatic cancer. Northern blot analysis revealed high levels of IRS-1 mRNA transcripts in ASPC-1 and MIA PaCa-2 human pancreatic cancer cell lines, and lower levels in COLO-357, PANC-1, and T3M4 cells. Immunoblotting with anti-IRS-1 antibodies indicated that IRS-1 protein levels paralleled IRS-1 mRNA levels. Analysis of RNA isolated from normal and cancerous human pancreatic tissues indicated that 7 of 16 pancreatic cancer samples overexpressed IRS-1 mRNA transcripts by comparison with the normal pancreas and that insulin mRNA levels were abundant in many tumors. These data suggest that IRS-1 contributes to the signaling pathways that lead to excessive growth stimulation in human pancreatic cancer.

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Insulin-like growth factor-II (IGF-II) action and expression were examined in 3 human pancreatic cancer cell lines. IGF-II expression was also studied in 17 normal and 12 malignant pancreatic tissues. IGF-II enhanced the growth of all 3 cell lines. In COLO-357 and PANC-1 cells, one-half maximal stimulation occurred at 0.3 and 0.4 nM IGF-II, respectively. In ASPC-1 cells, one-half maximal stimulation occurred at 0.9 nM IGF-II. A monoclonal antibody (alpha IR3) that blocks ligand binding to the insulin-like growth factor I (IGF-I) receptor (IGF-IR) inhibited IGF-II-mediated growth stimulation, and IGF-II enhanced insulin-receptor substrate 1 (IRS-1) phosphorylation. IGF-II mRNA transcripts were present in COLO-357 cells, in 4 of 17 normal human pancreatic tissues, and in 8 of the 12 cancer samples. By immunohistochemistry, IGF-II was present in the islets of both normal and malignant pancreatic tissues, and occasionally in the cancer cells within the tumor mass. These findings indicate that IGF-II acts via IGF-IR to enhance mitogenic signaling in pancreatic cancer cells and suggest that islet-derived IGF-II may contribute to pancreatic cancer cell growth in vivo.

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The epidermal growth factor (EGF) receptor is activated by EGF and other EGF-like growth factors, including heparin-binding epidermal growth factor-like growth factor (HB-EGF). We characterized the biological actions of HB-EGF in PANC-1 and COLO-357 human pancreatic cancer cell lines, and determined whether the presence of HB-EGF in human pancreatic carcinomas correlates with patient survival. HB-EGF enhanced the growth of both cell lines in a dose-dependent manner, with a potency that was generally similar to that of EGF and transforming growth factor-alpha (TGF-alpha). HB-EGF also readily induced tyrosine phosphorylation of the EGF receptor in these cells. Immunohistochemical analysis of 47 pancreatic cancer tissues revealed the presence of HB-EGF immunoreactivity in the cancer cells in 50% of the tumors. However, the presence of HB-EGF was not associated with a statistically significant decrease in the post-operative survival period. Furthermore, coexpression of HB-EGF and the EGF receptor was not associated with shorter patient survival. These findings suggest that HB-EGF activates the EGF receptor in human pancreatic cancer cells, but that it is not involved in enhancing the biological aggressiveness of this malignancy in vivo.

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Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) group of heparin-binding polypeptides. In the present study we sought to determine whether KGF is expressed in human pancreatic cancers. Using reverse transcriptase polymerase chain reaction (RT-PCR), a cDNA fragment of KGF was cloned and used to analyze Northern blots of RNA isolated from normal and cancerous human pancreatic tissues. Seven of 16 (44%) pancreatic cancer samples revealed significant overexpression of the 2.4 kilobase KGF mRNA transcript by comparison with the normal pancreas. Northern blot analysis failed to reveal the KGF transcript in several cultured human pancreatic cancer cell lines. However, by PCR analysis, some of the cell lines expressed KGF mRNA. Furthermore, 5 of 7 tested cell lines expressed the KGF receptor, and the growth of one cell line was enhanced by human recombinant KGF. These results suggest that KGF may participate in aberrant paracrine and autocrine pathways in human pancreatic cancer.

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Insulin and glucagon are thought to play important roles as hepatotrophic factors in acute viral hepatitis (AVH); however, few reports have investigated the responses and relationships of each of these hormones to liver damage in detail. We studied insulin and glucagon responses during the acute and recovery phases of AVH. We performed a glucose tolerance test (GTT) and an insulin sensitivity test (IST) in each phase in 11 patients with AVH. In 8 additional patients in the acute phase (total n = 19), were compared immunoreactive insulin (IRI) and C-peptide immunoreactivity (CPR) levels with transaminase levels. In the acute phase, IRI concentrations were normal from fasting to 60 min, despite an increased CPR level. In the recovery phase, IRI and CPR levels increased significantly. Immunoreactive glucagon levels in both phases did not differ significantly from those in controls. During the IST, the insulin sensitivity index in both phases was significantly lower than that in the controls. Fasting IRI and sigma IRI showed significant negative correlations with transaminase levels. We found enhanced insulin secretion and a decrease in plasma insulin in the acute phase of AVH. The discrepancy between IRI and CPR responses in the acute phase suggests an increase in the degradation or consumption of insulin in the liver.

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We assessed the potential roles of insulin-like growth factor-I (IGF-I) and the IGF-I receptor (IGF-IR) in human pancreatic cancer. IGF-I enhanced the growth of ASPC-1 and COLO-357 human pancreatic cancer cells, and this effect was significantly inhibited by a highly specific monoclonal anti-IGF-IR antibody (alpha IR3). Both cell lines expressed mRNA transcripts for IGF-IR, and basal cell growth was significantly reduced by an IGF-IR antisense oligodeoxynucleotide. IGF-I mRNA transcripts were not detected in either cell line or in two additional pancreatic cancer cell lines. In contrast, analysis of 12 pancreatic cancers revealed a 32-fold increase (P < 0.01) in IGF-I mRNA levels by comparison with the low levels observed in the normal pancreas. By in situ hybridization, IGF-I mRNA grains were present in both the cancer cells and in the surrounding connective tissue. Six of the cancers exhibited a 4.4-fold increase in IGF-IR mRNA levels. These findings suggest that IGF-I may participate in aberrant autocrine and paracrine activation of IGF-IR in pancreatic cancer in vivo.

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Human pancreatic ductal adenocarcinomas overexpress the epidermal growth factor (EGF) receptor. Betacellulin is a mitogenic polypeptide that binds and activates this receptor. To determine whether betacellulin has a role in human pancreatic cancer, we studied its expression in cultured human pancreatic cancer cell lines and in normal and cancerous pancreatic tissues. Five of 6 pancreatic cancer cell lines expressed the 3 kb betacellulin mRNA moiety, T3M4, MiaPaCa-2 and COLO-357 cells exhibiting the highest expression levels. EGF, heparin-binding EGF-like growth factor (HB-EGF), and basic fibroblast growth factor (bFGF) increased betacelullin mRNA levels. Only 2 of 15 normal samples and 1 of 10 cancer samples failed to exhibit the betacellulin transcript. Densitometric analysis of the autoradiographs revealed a 7.5-fold increase in betacellulin mRNA levels in the cancer tissues by comparison with the normal tissues. By in situ hybridization, the duct-like cancer cells exhibited many betacellulin mRNA in situ hybridization grains. These findings indicate that human pancreatic cancer cells express betacellulin in culture and in vivo, and suggest that this EGF-like ligand may participate in aberrant autocrine and paracrine activation of the EGF receptor in human pancreatic cancer.

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The epidermal growth factor (EGF) receptor (EGFR) is activated by EGF and other EGF-like growth factors, including amphiregulin (AR). We characterized the localization and mitogenic action of AR in T3M4 and COLO-357 human pancreatic cancer cell lines and determined whether the presence of AR in human pancreatic cancers correlates with patient survival. Both T3M4 and COLO-357 cells were found to be extremely sensitive to AR, one-half maximal stimulation occurring at a concentration of 70 and 50 pM, respectively. The magnitude of the stimulatory effect was greater with AR than with EGF. Both cell lines exhibited AR immunostaining, which was present in a variable manner in the cytoplasm, nucleus and nucleoli. Immunohistochemical analysis of 62 pancreatic cancer tissues revealed the presence of nuclear and/or cytoplasmic AR immunoreactivity in the cancer cells. Cytoplasmic, but not nuclear localization of AR in the pancreatic cancer cells was associated with a statistically significant decrease in the post-operative survival period. The presence of EGFR alone in the cancer cells did not correlate with decreased survival, whereas coexpression of cytoplasmic AR and EGFR was associated with shorter survival. Distant metastases did not always exhibit cytoplasmic AR immunoreactivity. These findings point to the existence of an EGFR: AR autocrine loop in human pancreatic cancer which may contribute to its biological aggressiveness.

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Heparin-binding EGF-like growth factor (HB-EGF) is a polypeptide with an apparent molecular weight of 22 kilodalton that is related to epidermal growth factor (EGF) and that binds and activates the EGF receptor. We examined HB-EGF biological action and expression in human pancreatic cancer cell lines, and compared HB-EGF expression in normal and cancerous pancreatic tissues. HB-EGF enhanced the growth of human pancreatic cancer cells in a dose-dependent manner. Several cell lines expressed HB-EGF mRNA transcripts, and the transcript level was enhanced by HB-EGF, as well as by 12-O-tetradecanoylphorbol-13-acetate and transforming growth factor-alpha (TGF-alpha). By comparison with the normal pancreas, HB-EGF mRNA levels were increased in human pancreatic cancer tissues. These findings suggest that HB-EGF may participate in aberrant autocrine and paracrine activation of the EGF receptor, thereby contributing to pancreatic cancer cell growth.

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Time-resolved fluoroimmunoassays were used for the detection of pancreatic group I and synovial-type group II phospholipases A2 in sera of patients suffering from chronic renal failure before and after haemodialysis. The concentration of group I phospholipase A2 was ten-fold higher in sera of uraemic patients than in healthy controls. There was no significant difference in the concentrations of group I phospholipase A2 in serum before and after haemodialysis. The concentration of group II phospholipase A2 was only marginally increased in sera of uraemic patients, compared with healthy controls. There was no significant difference in the concentrations of group II phospholipase A2 before and after haemodialysis. The results indicate that the metabolism of group I phospholipase A2 differs from that of group II phospholipase A2 in chronic renal failure.

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Collagen and non-collagen protein metabolisms in rat acute pancreatitis induced by ethionine were investigated with an incorporation study of 3H-proline to those proteins. Enhanced collagen metabolism, which may provide a basis for parenchymal cells regeneration, was observed in the initial phase of acute pancreatitis. This phenomenon was followed by the increase of non-collagen protein metabolism, and histologically mitosis of pancreatic acinar cell was detected concomitantly. Thereafter the enhancement of collagen metabolism continued to the convalescent phase of acute pancreatitis and this change was thought to be related to the resolution and the elimination of excess of collagenous materials and simultaneous increase of non-collagen protein synthesis, which may result in the maturation of acinar cell, was observed. It was thus assumed that the restoration from acute pancreatitis was initiated by the enhanced collagen metabolism with following parenchymal cell regeneration, and was finished by the maturation of acinar cells.

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Serum and ascitic phospholipase A2 (PLA2) activities in rat acute pancreatitis induced by sodium taurocholate (TCA) were investigated with special reference to their heat stabilities and were compared to those in serum of carrageenan induced granuloma in rats. PLA2 activity was measured by previously reported radiochemical method and was separated into two forms, heat stable and labile PLA2, based on the stability to preincubation at 55 degrees C for 5 minutes. While a heat stable PLA2 predominantly increased in ascitic fluid, the elevations of both heat stable and labile PLA2 activities in serum were observed in TCA induced pancreatitis. On the other hand, serum PLA2, which was mainly composed of heat labile form, elevated with the increase of leukocyte count in carrageenan induced granuloma rats. These facts suggested that serum PLA2 activity might increase in acute inflammatory changes other than in pancreatic diseases. And it is assumed that PLA2 derived from extrapancreatic origins as well as pancreatic secretory PLA2 may also contribute to high PLA2 level in acute pancreatitis, because pancreatic secretory PLA2 was generally accepted to be heat stable.

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A 55-year-old man with addiction of alcohol was admitted to our hospital with hematoemesis. After admission, the rupture of esophageal varices was observed and it was treated with endoscopic injection sclerotherapy. On the 3rd hospital day, the patient showed alcohol withdrawal syndrome and therefore haloperidol was administered intramuscularly and intravenously. After a half day of this treatment, high fever, diaphoresis, hypotension, tachycardia, muscular rigidity and tremor developed. With the laboratory data including high serum levels of CK, LDH, GOT and GPT, neuroleptic malignant syndrome (NMS) was suspected. Regardless of intensive care, hepatic failure, DIC and acute renal failure promptly developed, and he died on the 11th hospital day. Neuroleptics may cause serious side effects, such as NMS, when the physical status of patients was deteriorated. Especially in exhausted patient such as our case, even the small dose of neuroleptics caused NMS within short term. Thus, it seemed to be important for clinicians to pay attention to choice of neuroleptics.

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Urinary excretions of hydroxyproline and fibronectin fragment (FN fragment) were serially investigated in the patients with acute pancreatitis or acute exacerbation of chronic pancreatitis. While urinary excretion of FN fragment showed the maximal level on the first day of admission, high levels of urinary hydroxyproline were observed on the second to fifth day. As to the changes in the individuals, peak level of urinary FN fragment always preceded that of hydroxyproline. And it was assumed that the elevation of FN fragment excretion on the early phase of pancreatitis reflected tissue damages of pancreas itself and complicated organs, and following elevation of hydroxyproline showed enhanced collagen metabolism induced by acute inflammation and tissue damage. According to the severity of pancreatitis, urinary excretion of FN fragment on the first day increased, and it was therefore suggested that urinary FN fragment would be one of the parameters for the assessment of the severity of acute pancreatitis.

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Under low choline diet, chronic pancreatic injury was induced by the intraperitoneal administration of ethionine of 10 mg/100 g body weight, 3 times a week for sequential 8 weeks in rats and histological changes, pancreatic hydroxyproline and collagenase activity were studied. Histologically, acute inflammatory changes were observed mainly in the early days, and infiltration of fibroblastoid cells was increased on the 28th day. Then, fat droplets deposition became remarkable. Though hydroxyproline concentration increased gradually after the injection of ethionine and showed maximal level on the 28th day when the slight fibrosis was noticed, it was decreased on the 56th day when collagenous tissue was replaced by fat droplets. On the other hand, collagenase activity showed two peaks, in the early phase and on the 56th day, and it showed bottom level on the 28th day. Collagen synthesis relatively dominated over degradation on the 28th day. It is suggested that the unbalance of collagen metabolism between synthesis and degradation may result in fibrosis.

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Heat stability of human serum phospholipase A2 (PLA2) was investigated comparing to those of porcine pancreatic PLA2 (p-PLA2) and PLA2 in human duodenal aspirate. PLA2 activity was measured by radiochemical method using phosphatidylcholine as a substrate. While the activities of PLA2 in duodenal aspirate and p-PLA2 solution were not influenced by the heat treatment of 55 degrees C, PLA2 activity in normal human serum was remarkably decreased by the preincubation at 55 degrees C for only 5 minutes. From the result of preincubation temperature, serum PLA2 activity began to decrease at 50 degrees C and the decrease of PLA2 activity was remarkable above 55 degrees C of preincubation temperature. As a conclusion, PLA2 in human serum was consisted of heat labile and stable form, and latter was thought to be derived from pancreas. Thus serum PLA2 level should be assessed with consideration of incubation temperature of PLA2 assay and whether pretreatment of heating was done or not.

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To investigate the mechanisms of serum amylase abnormalities in liver disease, we determined serum amylase levels, S-type isozyme proportion, clinical symptoms, and laboratory data in 38 cases of histologically confirmed liver cirrhosis and 19 controls. Of the 12 patients who were hyperamylasemic (12/38, 32%), 5 showed S-type isozyme dominance (5/12, 42%), whereas in the 26 normoamylasemic cirrhosis patients, only 5 were S-type isozyme dominant (5/26, 19%). Isozyme percentages were significantly higher (P less than 0.01) in the dominant-S-type cases than in the controls, and S-type dominance was found more frequently in the hyperamylasemic than in the normoamylasemic cirrhosis cases. Only ascites and esophageal varices were observed more frequently as clinical symptoms in the dominant-S-type cases. Our results suggest that amylase is not produced in the human liver, but that the decreased clearance rate of amylase, especially the S-type isozyme, may be a cause of hyperamylasemia and S-type isozyme dominance in cirrhosis.

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