Human Pancreatic Tumor Cell Lines Research Articles

Cannabinoids Induce Apoptosis of Pancreatic Tumor Cells via Endoplasmic Reticulum Stress–Related Genes

Pancreatic adenocarcinomas are among the most malignant forms of cancer and, therefore, it is of especial interest to set new strategies aimed at improving the prognostic of this deadly disease. The present study was undertaken to investigate the action of cannabinoids, a new family of potential antitumoral agents, in pancreatic cancer. We show that cannabinoid receptors are expressed in human pancreatic tumor cell lines and tumor biopsies at much higher levels than in normal pancreatic tissue. Studies conducted with MiaPaCa2 and Panc1 cell lines showed that cannabinoid administration (a) induced apoptosis, (b) increased ceramide levels, and (c) up-regulated mRNA levels of the stress protein p8. These effects were prevented by blockade of the CB(2) cannabinoid receptor or by pharmacologic inhibition of ceramide synthesis de novo. Knockdown experiments using selective small interfering RNAs showed the involvement of p8 via its downstream endoplasmic reticulum stress-related targets activating transcription factor 4 (ATF-4) and TRB3 in Delta(9)-tetrahydrocannabinol-induced apoptosis. Cannabinoids also reduced the growth of tumor cells in two animal models of pancreatic cancer. In addition, cannabinoid treatment inhibited the spreading of pancreatic tumor cells. Moreover, cannabinoid administration selectively increased apoptosis and TRB3 expression in pancreatic tumor cells but not in normal tissue. In conclusion, results presented here show that cannabinoids lead to apoptosis of pancreatic tumor cells via a CB(2) receptor and de novo synthesized ceramide-dependent up-regulation of p8 and the endoplasmic reticulum stress-related genes ATF-4 and TRB3. These findings may contribute to set the basis for a new therapeutic approach for the treatment of pancreatic cancer.

Gemcitabine plus celecoxib (GECO) in advanced pancreatic cancer: a phase II trial

Single agent gemcitabine (GEM) is the standard treatment of pancreatic adenocarcinoma. Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor. Recent studies in human pancreatic tumor cell lines suggest an involvement of COX-2 in tumor-dependent angiogenesis and provide the rational for inhibition of the COX pathway as an effective therapeutic approach. The aim of this study is to evaluate the toxicity and activity of gemcitabine plus celecoxib. Forty-two consecutive patients with histologically or cytologically confirmed pancreatic adenocarcinoma entered the trial. Twenty-six patients (pts) were metastatic, 16 pts had locally advanced disease. The schedule consisted of GEM 1,000 mg/m2 (as a 30 min iv infusion) on days 1, 8 every 3 weeks and celecoxib 400 mg bid. Four pts (9%) achieved a partial response and 26 (62%) had stable disease, gaining a total disease control in 30 pts (71% [95% CI, 58-84%]). Overall clinical benefit response was experienced by 23 pts (54.7% [95%CI, 38.6-70.1%]). Neither grade 4 neutropenia nor grade 3-4 thrombocytopenia was observed. Grade 3 neutropenia was detected in 19% of pts. Grade 3 non-hematological toxicity was as follows: hepatic toxicity 7%, nausea 2.3%. Three pts (7%) and 5 pts (12%) had respectively a minimum creatinine increase and edema. Median survival was 9.1 months (95% CI, 7.5-10.6 months). GEM in combination with celecoxib showed low toxicity, good clinical benefit rate and good disease control. Further clinical investigation is warranted.

Fluvastatin synergistically enhances the antiproliferative effect of gemcitabine in human pancreatic cancer MIAPaCa-2 cells

The new combination between the nucleoside analogue gemcitabine and the cholesterol-lowering drug fluvastatin was investigated in vitro and in vivo on the human pancreatic tumour cell line MIAPaCa-2. The present study demonstrates that fluvastatin inhibits proliferation, induces apoptosis in pancreatic cancer cells harbouring a p21ras mutation at codon 12 and synergistically potentiates the cytotoxic effect of gemcitabine. The pharmacologic activities of fluvastatin are prevented by administration of mevalonic acid, suggesting that the shown inhibition of geranyl-geranylation and farnesylation of cellular proteins, including p21rhoA and p21ras, plays a major role in its anticancer effect. Fluvastatin treatment also indirectly inhibits the phosphorylation of p42ERK2/mitogen-activated protein kinase, the cellular effector of ras and other signal transduction peptides. Moreover, fluvastatin administration significantly increases the expression of the deoxycytidine kinase, the enzyme required for the activation of gemcitabine, and simultaneously reduces the 5′-nucleotidase, responsible for deactivation of gemcitabine, suggesting a possible additional role of these enzymes in the enhanced cytotoxic activity of gemcitabine. Finally, a significant in vivo antitumour effect on MIAPaCa-2 xenografts was observed with the simultaneous combination of fluvastatin and gemcitabine, resulting in an almost complete suppression and a marked delay in relapse of tumour growth. In conclusion, the combination of fluvastatin and gemcitabine is an effective cytotoxic, proapoptotic treatment in vitro and in vivo against MIAPaCa-2 cells by a mechanism of action mediated, at least in part, by the inhibition of p21ras and rhoA prenylation. The obtained experimental findings might constitute the basis for a novel translational research in humans.

Parthenolide and sulindac cooperate to mediate growth suppression and inhibit the nuclear factor-κB pathway in pancreatic carcinoma cells

Activation of the transcription factor nuclear factor-kappa B (NF-kappa B) has been implicated in pancreatic tumorigenesis. We evaluated the effect of a novel NF-kappa B inhibitor, parthenolide, a sesquiterpene lactone isolated from the herb feverfew, in three human pancreatic tumor cell lines (BxPC-3, PANC-1, and MIA PaCa-2). Parthenolide inhibited pancreatic cancer cell growth in a dose-dependent manner with substantial growth inhibition observed between 5 and 10 micromol/L parthenolide in all three cell lines. Parthenolide treatment also dose-dependently increased the amount of the NF-kappa B inhibitory protein, I kappa B-alpha, and decreased NF-kappa B DNA binding activity. We have previously shown that nonsteroidal anti-inflammatory drugs (NSAID) suppress the growth of pancreatic cancer cells. To determine whether inhibition of the NF-kappa B pathway by parthenolide could sensitize pancreatic cancer cells to NSAID inhibition, BxPC-3, PANC-1, and MIA PaCa-2 cells were treated with parthenolide and the NSAID sulindac, either alone or in combination. Treatment with the combination of parthenolide and sulindac inhibited cell growth synergistically in MIA PaCa-2 and BxPC-3 cells and additively in PANC-1 cells. In addition, treatment with the parthenolide/sulindac combination lowered the threshold for apoptosis. Increased levels of I kappa B-alpha protein were detected, especially in MIA PaCa-2 cells, after treatment with parthenolide and sulindac compared with each agent alone. Similarly, decreased NF-kappa B DNA binding and transcriptional activities were detected in cells treated with the combination compared with the single agents, demonstrating cooperative targeting of the NF-kappa B pathway. These data provide preclinical support for a combined chemotherapeutic approach with NF-kappa B inhibitors and NSAIDs for the treatment of pancreatic adenocarcinoma.

Gemcitabine (Gem) plus celecoxib in advanced pancreatic carcinoma: a phase II study

4103 Background: GEM is the most active single agent in the treatment of pancreatic carcinoma. Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor. Recent studies in human pancreatic tumor cell lines suggest an involvement of COX-2 in the control of tumor-dependent angiogenesis and provide the rational for inhibition of the COX pathway as an effective therapeutic approach for pancreatic cancer. The aim of this study is to evaluate the toxicity, safety and efficacy of this combination regimen. Methods: From January 2001 to July 2002, 32 consecutive patients (18 male/14 female) entered the trial. Median age was 63 years (range 48–76), median PS 1 (range 0–2); 22 patients (pts) were metastatic, 10 pts had locally advanced disease. Median basal CA19.9 value was 747 U/mL (range 1–50000). A total number of 210 cycles (median 6,5 per patient; range 2–12) were administered. The schedule consisted of GEM 1000 mg/m2 (as a 30 minutes iv infusion) on days 1,8 every 3 weeks and Celecoxib 400 mg bid. Results: Grade 3 NCI-CTC haematological toxicity was neutropenia in 9% of pts. Neither grade 4 neutropenia nor grade 3–4 thrombocytopenia were observed. Grade 3 non-haematological toxicity was as follows: hepatic toxicity 9%, nausea 3%. Although the high dose of celecoxib, neither gastric toxicity nor renal failure were observed. Only 3 pts (9%) had a minimum creatinine increase. Clinical benefit was obtained in 46% of pts, 38% had no basal pain and 16% didn't achieve any symptom control. Median survival was 9,1 months; 1-year survival rate was 36%. Conclusions: The goals we achieved with celecoxib-GEM combination are very low toxicity, good quality of life and improved overall survival. Accrual is ongoing. No significant financial relationships to disclose.

Gemcitabine (Gem) plus celecoxib in advanced pancreatic carcinoma: a phase II study

4103 Background: GEM is the most active single agent in the treatment of pancreatic carcinoma. Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor. Recent studies in human pancreatic tumor cell lines suggest an involvement of COX-2 in the control of tumor-dependent angiogenesis and provide the rational for inhibition of the COX pathway as an effective therapeutic approach for pancreatic cancer. The aim of this study is to evaluate the toxicity, safety and efficacy of this combination regimen. Methods: From January 2001 to July 2002, 32 consecutive patients (18 male/14 female) entered the trial. Median age was 63 years (range 48–76), median PS 1 (range 0–2); 22 patients (pts) were metastatic, 10 pts had locally advanced disease. Median basal CA19.9 value was 747 U/mL (range 1–50000). A total number of 210 cycles (median 6,5 per patient; range 2–12) were administered. The schedule consisted of GEM 1000 mg/m2 (as a 30 minutes iv infusion) on days 1,8 every 3 weeks and Celecoxib 400 mg bid. Results: Grade 3 NCI-CTC haematological toxicity was neutropenia in 9% of pts. Neither grade 4 neutropenia nor grade 3–4 thrombocytopenia were observed. Grade 3 non-haematological toxicity was as follows: hepatic toxicity 9%, nausea 3%. Although the high dose of celecoxib, neither gastric toxicity nor renal failure were observed. Only 3 pts (9%) had a minimum creatinine increase. Clinical benefit was obtained in 46% of pts, 38% had no basal pain and 16% didn't achieve any symptom control. Median survival was 9,1 months; 1-year survival rate was 36%. Conclusions: The goals we achieved with celecoxib-GEM combination are very low toxicity, good quality of life and improved overall survival. Accrual is ongoing. No significant financial relationships to disclose.

Transfection of the Multiple Endocrine Neoplasia Type 1 Gene to a Human Endocrine Pancreatic Tumor Cell Line Inhibits Cell Growth and Affects Expression of JunD, δ-Like Protein 1/Preadipocyte Factor-1, Proliferating Cell Nuclear Antigen, and QM/Jif-1

In the absence of metastases or overgrowth to adjacent organs, the lack of reliable markers for malignancy is a well-recognized problem for clinicians managing patients with endocrine tumors. Apart from inactivation of the multiple endocrine neoplasia type 1 (MEN1) gene, the molecular mechanisms involved in tumorigenesis of the endocrine organs and MEN1-associated nonendocrine lesions are vastly unknown. To try to learn more about down-stream effects on MEN1 gene inactivation, we used the BON1 cells, showing low levels of endogenous menin, and transfected them with a MEN1 gene construct. On restoring the menin expression, we recorded inhibition of cell growth. We also performed macroarray and present data on differentially expressed genes in the transfected cells, after corroboration by Northern blots and quantitative PCR. JunD was up-regulated in menin-expressing clones, whereas delta-like protein 1/preadipocyte factor-1 (involved in differentiation and growth of the pancreatic endocrine cells), proliferating cell nuclear antigen, and QM/Jif-1 (a negative regulator of c-Jun) became down-regulated. These findings might contribute to the understanding of the tissue-specific features of MEN1. We also show that homozygous inactivation of the MEN1 gene statistically correlates to higher expression of delta-like protein 1/preadipocyte factor-1, proliferating cell nuclear antigen, and QM/Jif-1, as well as lower MEN1 expression, in a limited sample of malignant endocrine pancreatic tumors.

Adenovirus-mediated retinoblastoma 94 gene transfer induces human pancreatic tumor regression in a mouse xenograft model.

Gene transfer of a truncated variant of the retinoblastoma (RB) gene encoding a M(r) 94000 protein that lacks the NH(2)-terminal 112 amino acid residues, termed RB94, has been shown to inhibit proliferation of several human tumor cell types. We have assessed its therapeutic effectiveness on pancreatic cancer, one of the most aggressive and therapy-resistant types of cancer. For this purpose, preclinical studies aimed to evaluate the therapeutic potential of RB94 gene transfer in pancreatic cancer were carried out. We have compared the antiproliferative effects of adenovirus-mediated gene transfer of RBwt and RB94 at the in vitro and in vivo levels in three RB-positive human pancreatic tumor cell lines: (a). NP-9; (b). NP-18; and (c). NP-31. We have also examined their effects on cell cycle and their capacity to induce apoptosis. In vitro results indicate that RB94 gene transfer has stronger antiproliferative effects compared with RBwt. RB94 transduction correlated with accumulation at the S-G(2) phase of the cell cycle in the three cell lines tested and induction of apoptosis in two of them. In vivo studies show significant decreases in the growth rate of tumors treated with Ad-RB94 when compared with those treated with Ad-RBwt. Moreover, terminal deoxynucleotidyl transferase-mediated nick end labeling analyses of Ad-RB94-treated tumor sections revealed that only RB94 is able to significantly induce apoptosis. RB94 gene expression has antiproliferative effects also in human pancreatic tumor cells, being more effective than wild-type RB in preventing tumor growth.

Purification and characterization of recombinant sTRAIL expressed in Escherichia coli.

As a potential anti-tumor protein, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has drawn considerable attention. This report presented the purification and characterization of soluble TRAIL, expressed as inclusion bodies in E. coli. sTRAIL inclusion bodies were solubilized and refolded at a high concentration up to 0.9 g/L by a simple dilution method. Refolded protein was purified to electrophoretic homogeneity by a single-step immobilized metal affinity chromatography. The purified sTRAIL had a strong cytotoxic activity against human pancreatic tumor cell line 1990, with ED50 about 1.5 mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had a structure similar to that of native protein with beta-sheet secondary structure. This efficient procedure of sTRAIL renaturation may be useful for the mass production of this therapeutically important protein.

MUC4 mucin expression in human pancreatic tumours is affected by organ environment: the possible role of TGFbeta2.

MUC4 is highly expressed in human pancreatic tumours and pancreatic tumour cell lines, but is minimally or not expressed in normal pancreas or chronic pancreatitis. Here, we investigated the aberrant regulation of MUC4 expression in vivo using clonal human pancreatic tumour cells (CD18/HPAF) grown either orthotopically in the pancreas (OT) or ectopically in subcutaneous tissue (SC) in the nude mice. Histological examination of the OT and SC tumours showed moderately differentiated and anaplastic morphology, respectively. The OT tumour cells showed metastases to distant lymph nodes and faster tumour growth (P<0.01) compared to the SC tumours. The MUC4 transcripts in OT tumours were very high compared to the undetectable levels in SC tumours. The SC tumour cells regained their ability to express MUC4 transcripts after in vitro culture. Immunohistochemical analysis using MUC4-specific polyclonal antiserum confirmed the results obtained by Northern blot analysis. Interestingly, the OT tumours showed expression of TGFβ2 compared to no expression in SC, suggesting a possible link between MUC4 and TGFβ2. The MUC4 expression, morphology, and metastasis of human pancreatic tumour cells are regulated by a local host microenvironment. TGFβ2 may serve as an interim regulator of this function.

Restoration of SMAD4 by gene therapy reverses the invasive phenotype in pancreatic adenocarcinoma cells.

SMAD4 is a critical cofactor in signal transduction pathways activated in response to transforming growth factor-beta (TGF-beta)-related ligands, regulating cell growth and differentiation. The roles played by SMAD4 inactivation in tumours highlighted it as a tumour-suppressor gene. However, restoration of the TGF-beta antiproliferative pathway following SMAD4 gene transfer in null-tumour cell lines is controversial. Herein, we report the inhibitory effects of SMAD4 on pancreatic tumour invasion and angiogenesis. Adenoviral transfer of this gene in a panel of SMAD4 homozygous-deleted human pancreatic tumour cell lines restored SMAD4 protein expression and function. Although it did not affect proliferation significantly in vitro, SMAD4 inhibited in vivo tumour growth in immunodeficient mice. In this xenograft setting, differential suppression of tumour growth in vivo was mediated, at least in part, through downregulation of vascular endothelial growth factor and expression of gelatinases. We documented the reduced invasion and angiogenesis histologically and by intravital microscopy, and gained mechanistic insight at the messenger and protein level. Finally, we found a negative reciprocal regulation between SMAD4 and ETS-1. ETS-1 is considered a marker for tumour invasion. Upon SMAD4 deletion, we detected high expression levels of ETS-1 in pancreatic tumour cells, suggesting the shift of the pancreatic tumour toward an invasive phenotype.

Two New Epimeric Pairs of Acetogenins Bearing a Carbonyl Group from Annona cherimolia Seeds

Further studies on the seeds of Annona cherimolia have led to the isolation of two pairs of Annonaceous acetogenins, a mixture of epimers of annomolon A (1) and 34-epi-annomolon A (1'), and a mixture of epimers of annomolon B (2) and 34-epi-annomolon B (2'), containing a rare gamma-hydroxymethyl-gamma-lactone moiety and a carbonyl group. Their structures were elucidated on the basis of spectroscopic and chemical methods, and their absolute stereochemistry was solved by preparing their respective per-Mosher ester derivatives. These acetogenins showed substantial cytotoxicity, comparable to that of adriamycin, against a human pancreatic tumor cell line (MIA PaCa-2).

Polymers for induction of revascularization in the rat fascial flap: application of vascular endothelial growth factor and pancreatic islet cells.

One of the major obstacles in transplanting avascular tissue or metabolically active cells for ischemic diseases is the loss of transplanted cells due to lack of oxygen and nutrients in the early posttransplantation period. Biodegradable polymeric tissue engineering scaffolds and hydrogels have a potential to incorporate cells or cellular organoids such as islets of Langerhans and growth factors. In this study, we tested the efficiency of two types of polymeric materials to carry recombinant human vascular endothelial growth factor (rhVEGF) or pancreatic tumor cell lines, namely Ins-1 and AR42J, for the induction of new vessels. Chitosan hydrogel fibers with micropores were prepared and molded into a cylinder construct (5 mm phi; 8 mm height). Macroporous PLGA scaffolds with a pore size of 250-400 microm were prepared and cut into cylinders (6 mm phi; 3 mm height). Both chitosan and PLGA constructs were loaded with rhVEGF (3 microg) or seeded with the cell lines (5 x 10(5) cells and 3 x 10(5) cells/construct, respectively, for AR42J and INS-1 cells), and transplanted into the fascial flaps of Wistar rats. At distinct time points up to 4 weeks postimplantation, polymers were explanted, fixed, and vessel density was counted on sections stained with anti-Factor-VIII antibody. Additionally, the kinetics of rhVEGF release from PLGA microspheres (phi of 50-80 microm) was determined using VEGF Elisa. Endogenous VEGF release from pancreatic rat cell lines was also determined. Light microscopy study was performed on H&E-stained paraffin sections of the islet-polymer samples. The vascular density of rhVEGF-loaded chitosan constructs was increased fourfold 2 weeks after subcutaneous transplantation compared with rhVEGF-unloaded controls (465 +/- 144 vs. 104 +/- 80 vessels per mm2, p < 0.05). Protein leakage occurred, but was not observed after 2 weeks. Higher insulin content was detected in rat islet grafts transplanted following VEGF application. More than 50% of total rhVEGF was released on the first day of in vitro culture of PLGA microspheres. rhVEGF secretion had another, but smaller, peak on the third day followed by a constant release. By comparison, endogeneous VEGF secretion of pancreatic tumor cells was measured within a 3-day culture period. Biodegradable polymer scaffolds and hydrogels may have potential use as solid supports to induce angiogenesis for pancreatic cell transplantation.

Overexpression of pRB in human pancreatic carcinoma cells: function in chemotherapy-induced apoptosis.

Human pancreatic adenocarcinomas are highly resistant to chemotherapy. The p16 tumor-suppressor protein is inactivated in more than 90% of human pancreatic cancers. The p16 protein transcriptionally inhibits expression of retinoblastoma tumor-suppressor gene pRB. The pRB protein transcriptionally inhibits expression of the p16 gene. Because pRB normally prevents apoptosis, we investigated whether pRB is involved in resistance to chemotherapy-induced apoptosis in pancreatic cancer cells. pRB expression was examined by immunohistochemistry in 106 human pancreatic tissue specimens. The human pancreatic tumor cell line Capan-1 (pRB+/p16-) was stably transfected with p16 to functionally inactivate pRB. pRB gene expression was examined by western and northern blot analyses, and pRB function was assessed by electrophoretic mobility shift assays and promoter transactivation studies for the transcription factor E2F. Changes in cell sensitivity to chemotherapy were measured by assays for cytotoxicity and apoptosis. pRB was overexpressed in pancreatic ductal adenocarcinomas but was hardly detectable in other pancreatic malignancies, chronic pancreatitis, or nontransformed human pancreatic tissue. Expression of p16 in Capan-1 cells resulted in the loss of pRB gene and protein expression concomitant with increased activity of the transcription factor E2F, which was not detected in wild-type or control-transfected Capan-1 cells. Wild-type and control-transfected Capan-1 cells were resistant to chemotherapy-induced apoptosis, but pRB-depleted (i.e., p16-transfected) Capan-1 cells were highly sensitive. The effect was specific to pRB depletion because two other human pancreatic cancer cell lines that retained high pRB expression after p16 transfection were resistant to chemotherapy-induced apoptosis. Overexpression of pRB is associated with human pancreatic duct-cell cancer and may allow pancreatic cancer cells to evade chemotherapy-induced apoptosis.

Hsp 27 is better associated with the expression of inducible thermotolerance in human pancreatic tumor cell lines than hsp 70, p53 or p21/waf1/cip1

We evaluated the levels of p53, p21/waf1/cip1, hsp 27 and hsp 70 proteins in confluent cultures of human pancreatic tumor cell lines during the expression of inducible thermotolerance (IT). Two of these cell lines have wild type, whereas the other two have mutant, p53 status. Three cell lines expressed a significant amount of IT ( p<0.05 and better, one-tailed Student's t-test); the fourth manifested little IT. Hsp 70 was upregulated in all cell lines. In contrast, hsp 27 was upregulated only in the cell lines that expressed IT. There was no correlation between either p53, or p21, protein levels and propensity to express IT. We conclude that hsp 27 is better associated with the expression of IT than hsp 70 in these cell lines.

Adenovirus-mediated wt-p16 reintroduction induces cell cycle arrest or apoptosis in pancreatic cancer.

Pancreatic cancer has long carried poor prognosis. The development of new therapeutic approaches is particularly urgent. Inactivation of the tumor-suppressor gene p16(INK4a/CDKN2), a specific inhibitor of the cyclin-dependent kinases CDK4 and CDK6, is the most common genetic alteration in human pancreatic cancer, making it an ideal target for gene replacement. Here we transfected tumor cells using a recombinant adenovirus containing the wt-p16 cDNA (Ad5RSV-p16). The overexpression of p16 decreased cell proliferation in all four human pancreatic tumor cell lines (NP-9, NP-18, NP-29, and NP-31). However, G1 arrest and senescence were observed in only three. In contrast, the fourth (NP-18) showed a significant increase in apoptosis. This differential behavior may be related to the differences found in the expression level of E2F-1. Experiments on subcutaneous pancreatic xenografts demonstrated the effectiveness of p16 in the inhibition of pancreatic tumor growth in vivo. Taken together, our results indicate that approaches involving p16 replacement are promising in pancreatic cancer treatment.

Differentially Expressed cDNAs in PLCβ3-Induced Tumor Suppression in a Human Endocrine Pancreatic Tumor Cell Line: Activation of the Human Mismatch Repair Protein 3 Gene

Phospholipase Cβ3 (PLCB3) is located to chromosome 11q13 in the vicinity of the multiple endocrine neoplasia type1 (MEN1) gene and shows loss of expression in some neuroendocrine tumors. Transfection of PLCB3 to neuroendocrine cell lines induces growth suppression and phenotypic alterations, but the mechanisms remain unclear. To investigate the underlying events behind this tumor suppression, we performed an RT-Differential cDNA Display of total RNA from BON-1 (human endocrine pancreatic tumor cell line) transfected with PLCB3 and compared to wild type and BON-1 transfected with vector without insert. PLCB3 transfection resulted in increased expression of 4 genes and decreased of 2. The two inhibited were homologous to S100A3 and Chromogranin A. One of the four activated cDNAs could be identified as human mismatch repair protein 3 mRNA (hMSH3), and another was homologous to TIS/MA-3 mRNA (mouse topoisomerase suppressor inhibited gene/mouse apoptosis gene-3). Differential expression of these genes may contribute to the PLCB3-induced tumor suppression of neuroendocrine tumor cell lines.

Inhibition of farnesyltransferase increases TGFbeta type II receptor expression and enhances the responsiveness of human cancer cells to TGFbeta.

Several small GTPases of the Ras superfamily have been shown to antagonize TGFbeta signaling in human tumor cell lines. Some of these GTPases are post-translationally modified by farnesylation, a lipid modification catalyzed by farnesyltransferase and required for the proteins to attach to membranes and to function. In this study, we investigated the effect of the farnesyltransferase inhibitor FTI-277 on TGFbeta-regulated cell growth and transcription. Treatment of the human pancreatic tumor cell line, Panc-1, with FTI-277 enhanced the ability of TGFbeta to inhibit both anchorage-dependent and -independent tumor cell growth. FTI-277 also enhanced the ability of TGFbeta to induce transcription, as measured by p3TP-lux reporter activity and collagen synthesis. The enhancement of TGFbeta responses by FTI-277 correlated with the stimulation of transcription and protein expression of type II TGFbeta receptor (TbetaRII). Consequently, FTI-277-treated cells exhibited a higher level of TGFbeta binding to its receptor. Thus, inhibition of protein farnesylation stimulates TbetaRII expression, which leads to increased TGFbeta receptor binding and signaling as well as inhibition of tumor cell growth and transformation.

De novo expression of vascular endothelial growth factor in human pancreatic cancer: evidence for an autocrine mitogenic loop.

The role of vascular endothelial growth factor (VEGF) and its receptors in tumor angiogenesis has been well established. We analyzed the expression pattern and biologic significance of VEGF and its receptors in human pancreatic cancer. VEGF, KDR/flk-1, and flt-1 expression were examined by immunohistochemistry, in situ hybridization, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and receptor phosphorylation. VEGF-stimulated mitogenesis was investigated by mitogen-activated protein kinase (MAPK) phosphorylation, transactivation of a c-fos promoter reporter construct, DNA synthesis assays, and stable transfection of a dominant-negative flk-1 complementary DNA (cDNA) construct. Compared with normal pancreas and chronic pancreatitis, VEGF and its receptors were overexpressed in pancreatic cancer. KDR and flt-1 were detected not only in endothelial cells but also in tumor cells. VEGF expression was observed in all human pancreatic tumor cell lines examined, and the KDR/flk-1 and flt-1 receptor was detected in 2 cell lines. VEGF treatment results in phosphorylation of MAPKs, transactivation of a c-fos promoter construct, and growth stimulation in KDR/flk-1-expressing cell lines, which could be blocked by VEGF antagonists. Furthermore, stable transfection of a dominant-negative flk-1 cDNA significantly inhibited tumor cell growth. These results not only support the important role of the VEGF/VEGF receptor system in pancreatic tumor biology but also suggest the existence of an autocrine/paracrine mitogenic loop for pancreatic cancer cells.

Interferon regulatory factor‐2 point mutations in human pancreatic tumors

Interferon regulatory factor (IRF)-2, a member of the IRF family, is a transcription factor involved in the regulation of various interferon and virus-stimulated genes and other genes. For example, IRF-2 is an activator of the interferon (IFN)-γ-inducible MHC class II transactivator (CIITA) type IV promoter. It cooperates with IRF-1 in the activation of the CIITA type IV promoter and can co-occupy the IRF-E of the promoter with IRF-1. In a previous study, we identified an inactivating point mutation in the DNA binding domain of IRF-2 expressed in a human pancreatic tumor cell line that does not express CIITA or MHC class II in response to IFN-γ. To further assess the potential impact of IRF-2 mutations in tumorigenesis, we screened fresh pancreatic tumor explants and identified 2 IRF-2 point mutations in the 2 alleles of IRF-2 from a single tumor specimen. Both mutations occurred in the DNA binding domain of IRF-2. DNA binding assays demonstrated that the IRF-2 point mutations impaired IRF-2 DNA binding. The transactivation function of the mutant IRF-2s was similarly impaired. This is the first report of IRF-2 mutations in human tumor explants. Int. J. Cancer 87:803–808, 2000. © 2000 Wiley-Liss, Inc.