Progression of pancreatic cancer

Although apoptosis pancreatic [beta]-cells is a critical step in the development of type 1 diabetes (1,2), it has not yet been clearly elucidated which molecules are the real effectors of pancreatic [beta]-cell death. We have recently published in vitro and in vivo data suggesting that [gamma]-interferon (IFN-[gamma]) and tumor necrosis factor (TNF)-[alpha] synergism is responsible for apoptosis of pancreatic [beta]-cells (3). IFN-[gamma] seems to sensitize otherwise resistant pancreatic islet cells to TNF-[alpha]-mediated apoptosis, and TNF-[alpha] is thought to exert the final apoptosis on pancreatic islet cells. The role of TNF-[alpha] as the final death effector molecule is consistent with other studies that use genetic ablation models (4,5). However, other data showing the opposite effect of TNF-[alpha] in autoimmune diabetes have been published, reflecting the complexity of the pathogenesis and probably different role of cytokines in the different stages of the disease progression (6,7).

Although TNF-[alpha] is one of the most important death effector molecules, most primary or immortalized cells are not susceptible to apoptosis by TNF-[alpha] alone because of the concomitant activation of the antiapoptotic process by TNF-[alpha] (8-10). Many studies have implicated nuclear factor (NF)-[kappa]B as an important player in the protection of target cells against TNF-[alpha]-induced apoptosis (11-13). However, other studies have reported increased cell death by NF-[kappa]B activation using neuronal cells, pancreatic islet cells/insulinoma cells, or others (14-20), reflecting a complex interplay of cytokines and transcriptional factors that could be different according to the cell types or modes of cell death.

This investigation was carried out to determine the role of NF-[kappa]B activation in cytokine-induced pancreatic [beta]-cell death. We found evidence supporting the role of NF-[kappa]B in the protection of pancreatic [beta]-cells against TNF-[alpha]-induced apoptosis.

RESEARCH DESIGN AND METHODS

Cell line and reagents. MIN6N8 cells, SV40 T-transformed insulinoma cells derived from nonobese diabetic (NOD) mice (provided by Prof. Jun-ichi Miyagaki, Osaka University, Osaka, Japan) (21), were grown in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum, 2 mmol/l glutamine, and penicillin-streptomycin (Life Technologies, Gaithersburg, MD). Recombinant rat IFN-[gamma] that is active on murine cells was provided by Dr. van der Meide (TNO Primate Center, the Netherlands). For some experiments, routine IFN-[gamma] (R&D Systems, Minneapolis, MN) was used to confirm the results obtained using rat IFN-[gamma] Recombinant mouse TNF-[alpha] and interleukin (IL)-1[beta] were also purchased from R&D Systems. Caspase inhibitors (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone [z-VAD-fmk]) were purchased from Enzyme Systems Products (Livermore, CA). Carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132) and Hoechst 33342 dye were from Calbiochem (La Jolla, CA). All other chemicals were obtained from Sigma (St. Louis, Mo) unless stated otherwise.

Isolation of mouse pancreatic islets. Islets were isolated from overnight-fasted ICR mice by the collagenase digestion technique as previously described (3). In brief, after 2.5 ml collagenase P (0.8 mg/ml) was injected into the bile duct of anesthetized mice, swollen pancreas was gently pulled out and other attached tissues were removed. The pancreas was then digested in collagenase P solution at 37[degrees]C for 15 min with gentle shaking. After stopping the digestion with cold Hanks' balanced salt solution, tissue was passed through a 400-[micro]m screen and centrifuged on 25, 23, 21.5, and 11.5% Ficoll gradients. Islets collected from the interface were washed with M199 media, and individual islets were handpicked using micropipettes. The islets were treated with trypsin-EDTA for 5 min to yield single islet cells before treatment with TNF-[alpha]. All animal experiments in this work were done in accordance with the institutional guidelines of Samsung Medical Center.

Western blot analysis. Cells were lysed in triple-detergent lysis buffer (50 mmol/l Tris HCl, pH 8.0, 150 mmol/l NaCl, 0.02% sodium azide, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholoate, and 1 mmol/l phenylmethylsulfonyl fluoride) as described (22). Protein concentration in cell lysate was determined using a protein assay kit (Bio-Rad, Hercules, CA). An equal amount of protein for each sample was separated by 10 or 12% SDS-PAGE and transferred to a Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham, Piscataway, NJ). After blocking with 5% skim milk, the membranes were sequentially incubated with anti-I[kappa]B[alpha] antibody (Ab) (Santa Cruz Biotechnology, Santa Cruz, CA) and horseradish peroxides-conjugated anti-rabbit IgG (Vector, Burlingame, CA), followed by enhanced chemiluminescence detection (Amersham). In experiments exploring the cleavage of caspases, CM1 Ab (provided by Idun Pharmaceuticals, La Jolla, CA) was used as the primary Ab.

Immunofluorescence staining. MIN6N8 cells or primary islet cells seeded on chamber slides (Lab Tek; Nalge Nunc International, Naperville, IL) were fixed in 4% paraformaldehyde at room temperature for 30 min said than in cold methanol at -20[degrees]C for 10 min. After three washes with PBS, nonspecific binding was blocked with 2% horse serum/0.5% Triton X-100/0.02% Na[N.sub.3] in PBS for 20 min. Fixed cells were then sequentially incubated with anti-p65 Ab (Santa Cruz), biotinylated anti-mouse IgG, and streptavidin fluorescein isothiocyanate (Vector). Stained cells were examined on a fluorescent microscope, in some experiments, anti-FLAG (Stratagene, La Jolla, CA) or anti-insulin Ab (Dako, Glostrup, Denmark) was used as the primary Ab.

Electrophoretic mobility shift assay. Nuclear extracts were prepared from MIN6N8 cells treated with cytokines as previously described (23). Synthetic double-stranded oligonucleotides of the consensus NF-[kappa]B binding sequence GAT CCC AAC GGC AGG GGA (Promega, Madison, WI) were end-labeled with [[gamma]-[sup.32]P]ATP using T4 polynucleotide kinase. Nuclear extract was incubated with the labeled probe ill the presence of poly(dI-dC) in a binding buffer containing 20 mmol/l HEPES at room temperature for 30 min. For supershift assays, a total of 0.2 [micro]g Ab against the p65 or p50 subunit of NF-[kappa]B (Santa Cruz) was included in the reaction. DNA-protein complexes were resolved by electrophoresis in a 5% nondenaturing polyacrylamide gel and visualized by antoradiography.

MTT assay, Cells were seeded on 96 well microtiter plates (3 X [10.sup.4]/well for MIN6N8 cells, 2 X [10.sup.4]/well for single islet cells) and treated with cytokines for indicated Lime periods. In some experiments, cells were pretreated with caspase inhibitors or MG132 for 15-30 min before cytokine treatment. After cytokine treatment, medium was removed and 12.2 mmol/l 3-[4,5-dimethylthiazol-2-1]-2,5-diphenyltetrazolium bromide (MTT) was added, followed by incubation in a C[O.sub.2] incubator at 37[degrees]C for 2 h. After a brief centrifugation, supernatant was carefully removed, and DMSO was added. After insoluble crystals were completely dissolved, absorbance at 540 nm was read using a Thermomax microplate reader (Molecular Devices, Sunnyvale, CA). In some experiments, a trypan blue exclusion test or Hoechst staining was used instead of MTT assays as a measure of cell death. A trypan blue exclusion test was performed by incubating cells in 0.02% trypan blue solution (Life Technologies) for 5 min before observation on a light microscope. Morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with 4.1 [micro]mol/l DNA-binding bisbenzimide Hoechst 33342 fluorochrome, followed by an examination oil a fluorescence microscope. N-monomethyl L-arginine (NMMA) was added to the culture in experiments to study the role of nitric oxide (NO) in insulinoma cell death. NO production was measured using the Griess reaction, in brief, 50 [micro]l of sample aliquots were mixed with 50 [micro]l Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride, and 2% phosphoric acid) in 96-well plates and incubated at 25[degrees]C for l0 min. The absorbance at 550 nm/was measured on a microplate reader. NaN[O.sub.2] was used as the standard for calculation.