Marko Kornmann

Concomitant over-expression of vascular endothelial growth factor and its receptors in pancreatic cancer.

Vascular endothelial growth factor (VEGF) is a potent angiogenic polypeptide that activates 2 distinct high‐affinity tyrosine kinase receptors, flk‐1/KDR and flt‐1. In the present study, we characterized the expression of VEGF and its receptors flk‐1/KDR and flt‐1 in the normal human pancreas and in human pancreatic cancer tissues and cell lines. VEGF, flk‐1/KDR and flt‐1 mRNA levels were elevated in cancer tissues compared with normal pancreas. By immuno‐histochemistry, VEGF, flk‐1/KDR and flt‐1 immunoreactivity co‐localized in many of the cancer cells within the tumor mass. Three (AsPC‐1, Capan‐1 and MIAPaCa‐2) of 6 pancreatic cancer cell lines expressed flk‐1/KDR mRNA and protein, and 4 cell lines (AsPC‐1, Capan‐1, T3M4 and PANC‐1) expressed flt‐1 mRNA transcripts. Binding studies with 125I‐labeled VEGF165 indicated that only Capan‐1 cells exhibited high levels of specific binding. Furthermore, VEGF enhanced the growth of Capan‐1 cells but was without effect in the other cell lines. VEGF also enhanced mitogen‐activated protein kinase (MAPK) phosphorylation and c‐fos induction in Capan‐1 cells, whereas the MAPK kinase inhibitor PD98059 abolished the growth‐stimulatory effect of VEGF. These data indicate that human pancreatic cancers have the capacity to over‐express VEGF and its receptors and suggest that in some instances VEGF may directly promote pancreatic cancer growth via the MAPK pathway. Int. J. Cancer 85:27–34, 2000.

Inhibition of basal and mitogen-stimulated pancreatic cancer cell growth by cyclin D1 antisense is associated with loss of tumorigenicity and potentiation of cytotoxicity to cisplatinum.

Cyclin D1 belongs to a family of protein kinases that have been implicated in cell cycle regulation. Recent studies have demonstrated that elevated cyclin D1 levels correlate with decreased survival in human pancreatic cancer. In this study we expressed in a stable manner a cyclin D1 antisense cDNA construct in PANC-1 human pancreatic cancer cells. Expression of the antisense construct caused a decrease in cyclin D1 mRNA and protein levels and in cyclin D1-associated kinase activity. Antisense expressing clones displayed significantly increased doubling times, decreased anchorage-dependent and -independent basal growth, and complete loss of tumorigenicity in nude mice. EGF, FGF-2, and IGF-I enhanced mitogen-activated protein kinase activity in antisense expressing clones, but failed to stimulate their proliferation. In contrast, all three growth factors were mitogenic in parental cells. Furthermore, the inhibitory effect of cisplatinum on cell proliferation was enhanced markedly in the antisense expressing clones. These findings indicate that cyclin D1 overexpression contributes to abnormal growth and tumorigenicity in human pancreatic cancer and to the resistance of pancreatic cancer to chemotherapeutic agents.

Role of fibroblast growth factors and their receptors in pancreatic cancer and chronic pancreatitis.

The fibroblast growth factor (FGF) family is a group of homologous heparin-binding polypeptides that has been implicated in a variety of human neoplasms and presently includes 14 members. FGF signaling is mediated by a dual-receptor system, consisting of four high-affinity tyrosine kinase receptors, termed fibroblast growth factor receptors (FGFRs), and of low-affinity heparan sulfate proteoglycan receptors that enhance ligand presentation to the FGFRs. Several FGFs, including FGF‐1, ‐2, ‐3, ‐4, ‐5, ‐6, and ‐7, and several FGFR variants, among them the 2 immunoglobulin-like form and the IIIc splice variant of FGFR‐1 and the keratinocyte growth factor receptor, a splice variant of FGFR‐2, are expressed in human pancreatic cancer cell lines and are overexpressed in human pancreatic cancers or in the pancreas of chronic pancreatitis and, therefore, may play important roles in the pathobiology of these pancreatic diseases. This review summarizes the current information on the involvement of the FGF family and their receptors in human pancreatic cancer and chronic pancreatitis.

Actinomycin D induces apoptosis and inhibits growth of pancreatic cancer cells

Pancreatic cancer cells are usually resistant to apoptosis induced by cytotoxic drugs, by activation of surface receptors such as Fas and TNF receptor or by serum or growth factor withdrawal. Actinomycin D (actD) is an inhibitor of RNA synthesis and acts as a potent inducer of apoptosis in several cell lines. In the present study, we investigated the effects of actD on PANC‐1 pancreatic cancer cells. ActD caused apoptosis in PANC‐1 cells in a dose‐dependent manner, as determined by cell growth assays, DNA laddering and TUNEL assays. Induction of apoptosis correlated with activation of the JNK/SAPK pathway and increased expression of Bax but not Bad or p53. PANC‐1 cells were completely resistant to Fas antibody and TNF‐α. In contrast, TRAIL decreased the growth of PANC‐1 cells by 22%. Low concentrations of actD (10 ng/ml) enhanced the cytotoxic effects of all 3 cytokines. EGF, FGF‐2 and IGF‐I did not protect PANC‐1 cells from actD‐mediated apoptosis. ActD (10 ng/ml) also inhibited the growth of CAPAN‐1 and T3M4 pancreatic cancer cells but not MiaPaCa‐2 cells. Our observations suggest that actD may act via JNK/SAPK and Bax to promote apoptosis in PANC‐1 cells and that it may inhibit the growth of other pancreatic cancer cell lines. Int. J. Cancer 86:399–407, 2000.

Fibroblast growth factor-5 stimulates mitogenic signaling and is overexpressed in human pancreatic cancer: evidence for autocrine and paracrine actions.

Fibroblast growth factor (FGF)-1 and -2 are overexpressed in human pancreatic cancer. In this study the role of FGF-5 in human pancreatic cancer was investigated, as FGF-5 has a classical signal sequence for secretion not found in FGF-1 or -2. Northern blot analysis with a 306 bp FGF-5 cDNA revealed the presence of 4.0 kb and 1.6 kb FGF-5 mRNA transcripts in both normal and cancerous pancreatic tissues. Densitometric analysis indicated that 4.0 kb and 1.6 kb FGF-5 mRNA transcripts levels were increased 2.4- and 2.7-fold in the cancers by comparison with normal tissues, respectively (P<0.002, P<0.0001). Immunohistochemistry and in situ hybridization demonstrated that FGF-5 localized in the cancer cells, stromal fibroblast and inflitrating macrophages. FGF-5 mRNA was also detected in COLO-357 human pancreatic cancer cells. Furthermore, secreted FGF-5 protein was present in conditioned medium of COLO-357 cells. Exogeneous FGF-5 (0.37 nM) increased the growth of COLO-357 cells by 48% (P<0.0001) and increased mitogen-activated protein kinase activity. COLO-357 cells expressed the IIIc isoform of the type I FGF receptor, the preferred FGF receptor for FGF-5. These observations suggest that FGF-5 may participate in autocrine and paracrine pathways promoting pancreatic cancer cell growth in vivo.

Increased Cyclin D1 in Human Pancreatic Cancer Is Associated with Decreased Postoperative Survival

Cyclin D1 belongs to a family of protein kinases that have been implicated in cell cycle regulation. In the present study we characterized cyclin D1 expression in 6 cultured human pancreatic cancer cell lines and in normal and cancerous human pancreatic tissues. A 4.4-kb cyclin D1 mRNA transcript was present in all cell lines and in all pancreatic tissues. Cyclin D1 mRNA levels were 2.1-fold higher in the pancreatic cancers than in normal pancreatic tissues (p < 0.0002). Cancer patients with lower cyclin D1 levels (n = 16) had a median survival of 15.5 months whereas patients with higher levels (n = 16) had a median survival of 6.5 months (p < 0.007). These data indicate that cyclin D1 expression may serve as a predictor of postoperative survival in pancreatic cancer patients, and raise the possibility that treatment modalities blocking cyclin D1 activity may have a future role in the therapy of these patients.

Altered expression of insulin-like growth factor II receptor in human pancreatic cancer

The insulin-like growth factor-II (IGF-II) receptor (IGF-IIR) is a single-chain transmembrane protein identical to the mannose-6-phosphate receptor. In the present study we examined IGF-IIR expression in normal and cancerous human pancreatic tissues. In the normal pancreas, moderately strong IGF-IIR immunoreactivity was present in the cytoplasm of islet cells, and mild cytoplasmic immunoreactivity was evident occasionally in ductal and acinar cells. Some ductal cells also exhibited nuclear IGF-IIR immunoreactivity. In the pancreatic cancers, regions of strong IGF-IIR immunoreactivity were present in the duct-like cancer cells within the tumor mass, often exhibiting nuclear localization. Expression of IGF-IIR mRNA in the cancer cells was confirmed by in situ hybridization. By comparison with normal pancreatic tissues, 7 of 12 pancreatic cancers exhibited a 5.6-fold increase in IGF-IIR mRNA levels, whereas in 3 cancers the IGF-IIR transcript was below the level of detection. Furthermore, all six tested cultured human pancreatic cancer cell lines expressed the IGF-IIR mRNA transcript. Our data indicate that IGF-IIR is over expressed in a significant number of human pancreatic cancers, where it has a tendency to localize in the nucleus, and raise the possibility that IGF-IIR may contribute to the pathobiology of pancreatic cancer.

Enhanced fibroblast growth factor 5 expression in stromal and exocrine elements of the pancreas in chronic pancreatitis

Background—Fibroblast growth factor 5 (FGF-5) belongs to a group of mitogenic and angiogenic heparin binding growth factors but its potential role in chronic inflammatory conditions is not known. Aims—To compare FGF-5 expression in the normal pancreas and in the pancreas of patients with chronic pancreatitis (CP) and to characterise FGF-5 expression and secretion in TAKA-1 cells, an immortalised Syrian hamster pancreatic duct cell line. Methods and results—Northern blotting revealed the presence of a 4.0 kb FGF-5 mRNA transcript in both normal and CP tissue samples. Densitometric analysis indicated that the transcript levels were increased by a factor of 1.44 in CP tissue samples compared with normal tissue samples (p=0.039). By immunohistochemisty and in situ hybridisation, FGF-5 was faintly expressed in ductal and islet cells in the normal pancreas. In contrast, in CP tissue samples, there was abundant expression of FGF-5 in ductal, acinar, and islet cells, as well as in periductal fibroblasts. FGF-5 was also expressed in TAKA-1 cells as determined by Northern blotting. By immunoblotting of heparin-sepharose precipitates, TAKA-1 cells were shown to secrete FGF-5 into the medium. Conclusion—Exocrine and stromal derived FGF-5 has the potential to participate in autocrine and paracrine pathways that may contribute to the pathobiology of chronic pancreatitis.

TGF‐β‐1 up‐regulates cyclin D1 expression in colo‐357 cells, whereas suppression of cyclin d1 levels is associated with down‐regulation of the type I TGF‐β receptor

Transforming growth factor‐β1 (TGF‐β1) inhibits cell growth in susceptible cells by interacting with a family of protein kinases that control cell cycle progression. In the present study, we investigated the effects of TGF‐β1 on cyclin D1 expression and activity in COLO‐357 human pancreatic cancer cells. TGF‐β1 increased cyclin D1 mRNA and protein levels. Nuclear runoff transcription and protein synthesis inhibition by cycloheximide revealed that this increase was, in part, due to increased cyclin D1 mRNA synthesis. Despite its stimulatory effects on cyclin D1 levels, TGF‐β1 inhibited cyclin D1‐associated kinase activity and the growth of COLO‐357 cells. Furthermore, suppression of cyclin D1 expression with a cyclin D1 antisense cDNA resulted in loss of TGF‐β1‐mediated growth inhibition in association with reduced induction of cyclin D1, p21Cip1 and plasminogen activator inhibitor‐1 (PAI‐1). Concomitantly, there was a marked decrease in the levels of the type I TGF‐β receptor (TβRI). Our findings suggest that in some cell types cyclin D1 expression may be important for TGF‐β1‐mediated signaling and that cyclin D1 may be involved in the transcriptional regulation of TβRI. Int. J. Cancer 83:247–254, 1999.

Coexpression of FAS and FAS-ligand in chronic pancreatitis: Correlation with apoptosis

Activation of the Fas receptor by Fas-ligand (FasL) results in apoptosis, and dysregulation of this pathway may contribute to abnormal cell proliferation and cell death. The aim of this study was to compare the expression of Fas and FasL in the normal pancreas and chronic pancreatitis (CP). By Northern blotting, Fas messenger RNA (mRNA) levels were increased in CP in comparison to the normal pancreas. Immunostaining revealed that faint Fas and FasL immunoreactivity was present in ductal and islet cells of the normal pancreas. In CP, there was faint Fas and strong FasL immunoreactivity in the proliferating ductal cells. Additionally, many of these ductal cells in the CP samples exhibited an apoptotic signal, as determined by DNA 3´-OH end labeling. These findings suggest that activation of apoptosis through the Fas receptor may contribute to the pathobiology of CP.