Interleukin 1beta-converting enzyme (caspase-1) is overexpressed in adenocarcinoma of the pancreas.

We investigated the expression of interleukin 1beta-converting enzyme (ICE; caspase-1) in human adenocarcinomas of the pancreas. Immunohistochemistry and Western blot analyses revealed an overexpression of ICE in 71 and 80% of tumor cells, respectively. Also, on a mRNA level, ICE mRNA was overexpressed in 45% of the cases, as compared to normal pancreatic tissue. Interestingly, the overexpression of ICE in tumor cells correlated significantly with the overexpression of cyclin D1, epidermal growth factor, and epidermal growth factor receptor (P < 0.0005, P < 0.05, and P < 0.002, respectively), which are involved in cell cycle progression and proliferation in human pancreatic carcinoma. This is the first report concerning ICE expression in human carcinomas; however, the exact mechanism underlying these close correlations warrant further research.

Inhibition of epidermal growth factor-induced interleukin-1beta-converting enzyme expression reduces proliferation in the pancreatic carcinoma cell line AsPC-1.

It is suggested that interleukin-1beta-converting enzyme (ICE) and ICE-related proteases play an important role in programmed cell death (apoptosis). We investigated ICE expression in the human pancreatic carcinoma cell line AsPC-1 after stimulation with epidermal growth factor and found a time-dependent expression of active ICE induced by epidermal growth factor. Interestingly, ICE expression does not lead to apoptosis. Cell cycle analyses revealed that acetyl-Tyr-Val-Ala-Asp-chloromethylketone-specific and acetyl-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-T yr-Val-Ala-Asp-aldehyde-specific cell-permeable inhibitors of ICE significantly reduced the proliferation of AsPC-1 cells, which suggested a positive influence of ICE on the proliferation in human pancreatic carcinoma cells.

Differential expression of CD44 splice variants in human pancreatic adenocarcinoma and in normal pancreas.

Immunohistochemical screening of pancreatic adenocarcinomas from 24 different patients and 9 pancreatic carcinoma cell lines revealed variant CD44 expression in all specimens tested. In contrast to normal pancreatic tissue, carcinomas were strongly positive for epitopes encoded by variant exons v5, whereas v6 was expressed on carcinoma cells as well as normal ductal pancreatic cells. Analysis of RNA expression revealed clear differences between normal pancreatic tissue and tumor specimens. In normal pancreas, v6 and v3 solely and one major chain consisting of v6-v10 were expressed, whereas in pancreatic carcinoma, multiple splice variants were detected. In about 80% of all carcinoma cases and all cell lines tested, the exon v5 appeared in the chain containing at least v4-v10. These data thus far suggest that not the presence alone but the chain composition of the CD44 variant chains could be important for their altered function because one of the major differences between normal and cancer tissue is the linkage of CD44v5 to the CD44v6-containing chain.

Overexpression of cyclin D1 in human pancreatic carcinoma is associated with poor prognosis.

We have investigated the expression of cyclin D1 in adenocarcinoma of the pancreas and the relevance of cyclin D1 expression to clinical outcome. In comparison to normal pancreas, Southern blot analyses revealed amplification of the cyclin D1 coding gene in 25% of the cases, whereas with reverse transcription-PCR, overexpression of mRNA was observed in 82% of the examined tissues. Immunohistochemically, we could demonstrate nuclear overexpression in tumor cells in 68.4%, and this protein accumulation correlated significantly with poor prognosis [median survival, 18.1 versus 10.5 months; P < 0.01 (chi2 test)].

Increased angiogenin expression in pancreatic cancer is related to cancer aggressiveness.

We have investigated the expression of angiogenin (ANG) in pancreatic cancer and the relevance of ANG expression to the progression of pancreatic cancer. In comparison to normal pancreas, increased ANG mRNA expression was observed in 80.0% of the cases of pancreatic cancer by in situ hybridization, and increased ANG protein expression was observed in 86.7% of the cases of pancreatic cancer using Western blot analysis. The mean serum ANG concentration of pancreatic cancer patients (566.6 +/- 191.9 ng/ml) was significantly higher (P < 2.0 x 10(-8)) than that of healthy volunteers (359.0 +/-t 59.9 ng/ml). Increased ANG mRNA expression as well as elevated serum ANG concentration correlated with poor prognosis. These findings suggest that ANG expression is up-regulated in pancreatic cancer patients and that ANG contributes to the aggressiveness of pancreatic cancer.

Molecular oncology in pancreatic cancer.

Cancer of the pancreas still has a very poor prognosis despite improved diagnostic methods and therapeutic regimens. The reasons for the aggressiveness of this cancer are not known, and the molecular mechanisms that govern the growth of pancreatic cancer cells are still not clearly defined. During the past two decades the development of new molecular biological techniques has offered new perspectives for a better understanding of pancreatic cancer. Tumor markers such as CA19-9 and CEA are used for diagnosis and for following the postoperative course of cancer patients. Characterization of pancreatic cancer cells using several molecular biological techniques has revealed overexpression or altered expression of growth factors and adhesion molecules, implying altered cell-cell and growth-regulatory interactions. In pancreatic cancer mutations in oncogenes and tumor suppressor genes are frequently detected in p53 and K-ras. This article reviews the possible molecular approaches for diagnosis, prognosis, or even therapy of pancreatic cancer.

CD44v6 cell surface expression is a common feature of macrophages and macrophage-like cells - implication for a natural macrophage extravasation mechanism mimicked by tumor cells.

Soluble CD44standard (sCD44s) and CD44v6 (sCD44v6) cannot only be detected in sera of patients with pancreatic carcinoma but also of healthy blood donors. To investigate whether sCD44s and sCD44v6 are derived from white blood cells, we stimulated whole blood with phytohemagglutinin and interleukin-2, which induced expression of CD44v6 only on monocytes. For further investigations, we used the promyelocytic leukemia cell line Hl-60. Only Hl-60 cells differentiating along the macrophage pathway showed increased expression of CD44s and CD44v6. Furthermore, only macrophages showed increased secretion of sCD44s and sCD44v6. Our data suggest that CD44s and CD44v6 are common adhesion molecules on macrophages and macrophage-like cells.

Inhibition of caspase-1 induces cell death in pancreatic carcinoma cells and potentially modulates expression levels of bcl-2 family proteins.

Caspase-1 (interleukin-1beta-converting enzyme) is reported to play an important role in the regulation of apoptosis. We investigated the inhibition of caspase-1 by the cell permeable caspase-1 inhibitor Ac-AAVALLPAVLLALLAP-YVAD.CHO in pancreatic carcinoma cells. Inhibition of caspase-1 induced a non-apoptotic/"necrotic-like" cell death in AsPC-1, BxPC-3, MiaPaCa-2 and Panc-1 cells. Expression levels of bcl-2 and bax were up-regulated in caspase-1 inhibitor-treated cells while that of bcl-x(L) remained unaltered. Our observations support our previous findings that caspase-1 is potentially involved in anti-apoptotic processes in pancreatic carcinoma.

The induction of apoptosis in proliferating human fibroblasts by oxygen radicals is associated with a p53- and p21WAF1CIP1 induction.

The role of reactive oxygen species (ROS) generated by hypoxanthine/xanthine oxidase (HX/XO) in the induction of apoptosis was studied in the human fibroblast cell line WI38. Apoptosis but not necrosis was observed in proliferating fibroblasts after 48 h incubation with 1 mM HX and 0.05 U/ml XO. Induction of apoptosis was hindered by catalase. Cell-cycle analysis revealed a reduction of cells in the S/G2 phase 24 and 48 h after stimulation, suggesting that ROS induce a G1 arrest in proliferating fibroblasts. This was supported by an accumulation of p53 and the cdk inhibitor p21WAF1/CIP1. Since apoptosis was not inducible in senescent fibroblasts our data indicate that ROS mainly induces apoptosis in proliferating cells.

Increased oxidative stress in the RAW 264.7 macrophage cell line is partially mediated via the S-nitrosothiol-induced inhibition of glutathione reductase.

We investigated whether endogenously or exogenously produced nitric oxide (NO) can inhibit cellular glutathione reductase (GR) via the formation of S-nitrosothiols to decrease cellular glutathione (GSH) and increase oxidative stress in RAW 264.7 cells. The specificity of this inhibition was demonstrated by addition of a NO-synthase inhibitor, and met- or oxyhemoglobin. Using isolated GR we found that only certain NO donors inhibit this enzyme via S-nitrosothiol. Furthermore, we found that cellular GSH decrease is paralleled by an increase of superoxide anion production. Our results show that the GR enzyme is a potential target of S-nitrosothiols to decrease cellular GSH levels and to induce oxidative stress in macrophages.

Exogenous, but not endogenous, nitric oxide increases proliferation rates in senescent human fibroblasts.

We investigated the effects of endogenously produced and exogenously applied nitric oxide (NO) on cell proliferation rates and cell cycle regulation in senescent human fibroblasts (WI38). Induction of inducible nitric oxide synthase by tumor necrosis factor-alpha, interferon-gamma and interleukin-1beta inhibited cell proliferation and led to a G1 arrest. These effects were partially reversible by N(G)-monomethyl-arginine (NMA). Addition of the NO donors sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP) increased cell proliferation rates as well as the S/G2 fraction. This points to a functional role of NO in cell cycle regulation and cell proliferation in human fibroblasts which depends on the mode of NO generation as well as the culture conditions used.

The role of polymorphonuclear leukocytes and oxygen-derived free radicals in experimental acute pancreatitis: mediators of local destruction and activators of inflammation.

Using a retrograde infusion sodium taurocholate pancreatitis model in the rat treatment with oxygen radical scavengers or monoclonal anti-ICAM-1 antibody decreased tissue damage and polymorphonuclear leukocytes (PMN) infiltration. Scavengers or anti-ICAM-1 treatment attenuated the activating capacity of blood PMNs following zymosan stimulation. The local production of oxygen free radicals in the pancreas by systemic infusion of hypoxanthine and regional infusion of xanthine oxidase did not induce acute pancreatitis, although an increase of infiltrating PMNs was observed. Our data suggest that oxygen free radicals and infiltrating PMNs aggravate acute pancreatitis and that both are important mediators of local destruction and systemic activation of PMNs.

Prognostic significance of soluble interleukin-2 receptor-alpha in adenocarcinoma of the pancreas.

Soluble interleukin-2-receptor-alpha (sIL-2Ralpha) serum concentrations were examined in chronic pancreatitis patients, patients with cystadenocarcinoma of the pancreas, patients with adenocarcinoma of the pancreas and healthy blood donors. sIL-2Ralpha serum concentrations in pancreatic cancer patients were significantly higher than those of normal control subjects or chronic pancreatitis patients. In patients with adenocarcinoma of the pancreas no significant differences were found between sIL-2Ralpha and tumor size, grading, resectability and lymph node involvement. In Kaplan-Meier regression analysis patients with adenocarcinoma of the pancreas with low sIL-2Ralpha levels (<500 U/ml) lived significantly shorter than patients with sIL-2Ralpha concentrations above 500 U/ml (P < 0.01), suggesting that determination of sIL-2Ralpha serum concentrations could provide additional important information about prognosis.

p53 genetic alterations, protein expression and autoantibodies in human colorectal carcinoma: A comparative study.

This study investigated a total number of 120 colorectal malignant tumor tissues by applying a new quantitative luminometric assay (LIA)-mat, immunohistochemistry (IHC) (n=100), PCR/SSCP (n=42), and sequencing (n=7). Sera were collected from 235 patients suffering from colorectal carcinoma in addition to 195 healthy individuals as a control group. Manual ELISA kit was developed to detect p53 autoantibodies in the sera of those patients. Our data demonstrated that the LIA-mat yields reliable estimates of p53 expression in soluble cell extracts as compared with results obtained by immunohistochemistry which showed positive immunostaining in 63% of the studied cases. Using a cut-off value of 1.8 ng/mg protein, 65 tumors out of 120 (54%) were classified to be positive by LIA-mat, manifesting protein overexpression, while 22 out of 42 (52%) tumor samples showed p53 gene alteration when applying single strand conformation polymorphism (SSCP) analysis on polymerase chain reaction products. In tumor samples without a p53 gene alteration, the median soluble p53 protein level was 4.3 ng/mg protein, whereas the median p53 protein level for tumor samples with p53 gene alteration was 7.5 times higher. Despite a significant correlation between the outcome of LIA and SSCP, a disagreement was found in 30% of cases. We found no significant correlation between p53 protein overexpression and clinicopathological findings except for distant metastasis (p=0.33), indicating p53 immunoreactivity to be an independent prognostic factor. Our data showed that 18% of patients suffering from colorectal cancer developed autoantibodies against p53 in their sera which might be an early indicator for tumor development and distant metastasis.

p53 in relation to therapeutic outcome of locoregional chemotherapy in pancreatic cancer.

Since celiac artery infusion (CAI) led to an increase in survival in palliative chemotherapy in pancreatic cancer, we treated 26 patients with adjuvant CAI following resection for advanced pancreatic cancer. Catheters were placed angiographically into the celiac artery and remained there for five consecutive days. One cycle of chemotherapy consisted of mitoxantrone, 5-fluorouracil (5-FU), folinic acid, and cis-platinum. This treatment was repeated five times in monthly intervals. Median survival times in patients who received CAI are 21 months for all patients, whereas in patients who did not receive adjuvant treatment median survival is 10.5 months. In all patients p53 expression of the carcinomas was determined by immunohistochemistry. In 11/26 patients a p53 overexpression was observed. Although p53 overexpression turned out to be associated with poor prognosis in the patients who underwent adjuvant regional cancer treatment, p53 is not a sufficient prognostic parameter in pancreatic carcinoma, since p53 overexpression was more frequent in undifferentiated tumors and in palliative resected tumors.

Low serum levels of CD44, CD44v6, and neopterin indicate immune dysfunction in chronic pancreatitis.

INTRODUCTION: In autoimmune diseases, malignancies, and inflammatory conditions, a correlation of serum levels of CD44, interleukin-2 receptor (IL-2r), and neopterin with disease activity could be shown. AIMS: To assess the immune parameters in chronic pancreatitis in correlation to clinical data to evaluate the potential role of immune dysfunction as a risk factor. METHODOLOGY: Levels of IL-2r, sCD44, sCD44v6, and neopterin were measured using the enzyme-linked immunosorbent assay in 63 patients with chronic pancreatitis who underwent surgery between 1992 and 1995 in our institution. Clinical data were evaluated prospectively before surgery, and a follow-up investigation was conducted in 1997. RESULTS: Mean serum levels of CD44, CD44v6, and neopterin were significantly lower in patients with chronic pancreatitis compared with the control group. The mean level of IL-2r was also lower in chronic pancreatitis, but this difference was not significant. However, no influence of immunosuppressive factors such as alcohol consumption, cigarette smoking, or diabetes could be detected on the levels of IL-2r, CD44, CD44v6, and neopterin. CONCLUSION: In accordance with other diseases of reduced immunoreactivity, depressed serum levels of biomarkers in chronic pancreatitis are caused by reduced T-lymphocyte and macrophage activation. By ruling out a significant influence of concomitant immunosuppressive factors, we conclude that the inflammatory process itself is the source of the depressed immune function, which might be restored by surgical resection.

Overexpression of caspase-1 (interleukin-1beta converting enzyme) in chronic pancreatitis and its participation in apoptosis and proliferation.

Caspase-1, formerly designated interleukin-1beta converting enzyme, was the first described member of a group of cysteine proteases called caspases. It is suggested that caspases play an important role in apoptosis, but recent observations could show that caspase-1 might also be involved in cellular proliferation. We investigated the expression of caspase-1 in 38 chronic pancreatitis tissues, six pancreatitis tissues from patients with pancreatic carcinoma and nine normal pancreatic tissues by immunohistochemistry. Western blot analysis was used to confirm the immunohistochemical findings. We found a clear expression of caspase-1 in chronic pancreatitis, but not in normal pancreatic tissues. Interestingly, we found expression of caspase-1 in three distinct morphologic compartments: (i) in atrophic acinar cells (31 of 35; 89%), (ii) proliferating cells of ductal origin (33 of 38; 87%), and (iii) in acinar cells redifferentiating to form tubular structures (26 of 31; 83%). These immunohistochemical findings were confirmed by Western blot analysis, which showed an expression of caspase-1 in 85% of the tissues. No correlation was found between any of the examined clinicopathologic features and the caspase-1 expression in chronic pancreatitis. In conclusion, the expression of caspase-1 is a frequent event in chronic pancreatitis and its distribution pattern may reflect two functions of this protease: on one hand its participation in the apoptotic pathway in atrophic acinar cells and, on the other hand, its role in proliferation and differentiation in proliferating duct cells.

Increased angiogenin expression in obstructive chronic pancreatitis surrounding pancreatic cancer but not in pure chronic pancreatitis.

We previously demonstrated the increased expression of angiogenin (ANG) in pancreatic cancer and its relation to cancer aggressiveness; however, the expression patterns and the roles of angiogenin in chronic pancreatitis are still unknown. We investigated the expression of ANG both in the tissues and in the sera of chronic pancreatitis patients (pure chronic pancreatitis) by using in situ hybridization, Western blot analysis, and enzyme-linked immunosorbent assay. In situ hybridization revealed no detectable ANG messenger RNA (mRNA) signals in all tissues of pure chronic pancreatitis and normal pancreas. Only a small amount of protein band expression was obtained in all of the protein lysates of pure chronic pancreatitis and normal pancreas. Accordingly, there was no significant difference between the mean serum ANG concentration of chronic pancreatitis patients (352.1+/-72.5 ng/ml) and that of healthy volunteers (357.6+/-45.2 ng/ml). By contrast, acinar cells and interstitial fibroblasts in the tissues surrounding pancreatic cancer showed increased ANG mRNA expression. Strong protein band expression was obtained in the protein lysates of pancreatic cancer surrounding tissue, and mean serum ANG concentration was increased in pancreatic cancer patients. These findings suggest that ANG expression is increased in pancreatic cancer surrounding tissue but is not increased in pure chronic pancreatitis, and that ANG is potentially involved in the pancreatic cancer microenvironment rather than the establishment of pure chronic pancreatitis.

Overexpression of intercellular adhesion molecule-1 (ICAM-1) in pancreatic adenocarcinoma in comparison with normal pancreas.

To elucidate the role of intercellular adhesion molecules (ICAMs), which has not been well understood in pancreas, we investigated the localization and expression of ICAM-1 by immunohistochemistry and in situ hybridization (ISH) in pancreatic adenocarcinoma and in normal pancreas. The localizations of ICAM-2 and ICAM-3 were also investigated by immunohistochemistry. In normal pancreas, acinar cells, duct epithelial cells, and Langerhans islet cells failed to stain with anti-ICAM-1, anti-ICAM-2, and anti-ICAM-3 antibodies. These cells showed no expression of ICAM-1 mRNA. On the other hand, various percentages of carcinoma cells were stained with anti-ICAM-1 antibody, while no carcinoma cells were stained with anti-ICAM-2 and anti-ICAM-3 antibodies. ICAM-1 mRNA expression was also observed in carcinoma cells, and ICAM-1 mRNA expression was associated with localization of the ICAM-1 protein. These results suggest that ICAM-1 expression is up-regulated in pancreatic adenocarcinoma cells and that ICAM-1 is involved in malignant processes in pancreas.

Epidermal growth factor induces cyclin D1 in human pancreatic carcinoma: evidence for a cyclin D1-dependent cell cycle progression.

INTRODUCTION: We recently showed that cyclin D1 is overexpressed in human pancreatic carcinoma cells, and that this overexpression correlates significantly with a poor prognosis. AIMS: To assess the interrelations of epidermal growth factor (EGF), EGF receptor (EGFR), and cyclin D1 in human pancreatic carcinoma. METHODOLOGY AND RESULTS: In pancreatic carcinoma cell lines (BxPC-3, AsPC-1), cell cycle analysis revealed an increase in cells in the S/G1 phase between 18 and 30 hours after stimulation with 50 ng/mL EGF. Cyclin D1 mRNA increased after 2 hours, corresponding to an increase in cyclin D1 protein, with the maximum level between 7.5 and 10 hours after stimulation, as demonstrated by Western blot analysis. We performed immunohistochemical analysis on 61 adenocarcinoma tissues for the expression of EGF, EGFR, and cyclin D1 and demonstrated an overexpression in the tumor cells in 51%, 54%, and 62.3%, respectively, whereas normal human pancreas stained negative for all of the three factors. Interestingly, EGF and EGFR expression correlated significantly with the cyclin D1 expression in human pancreatic tumor cells (p < 0.001 and p < 0.01, respectively). CONCLUSION: These results demonstrate that cyclin D1 overexpression in the tumor cells of pancreatic carcinoma tissue is at least partly dependent on the mitogenic effects of EGF signaling through the EGFR.