Jean Lainé

MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-XL, and Mcl-1 and promotes survival of human pancreatic cancer cells

Background and aims: Growth factors are well known for their participation in the regulation of cell proliferation and survival. However, the intracellular signaling pathways by which growth factors promote survival are still poorly understood. In the present study, using the MIA PaCa‐2 cell line, a well‐established model of pancreatic cancer cells, we analyzed the roles of ERK1/2 activities in the regulation of cell survival and investigated some of the mechanisms involved. Methods: The ability of the MEK inhibitor PD98059 to modulate survival of the MIA PaCa‐2 cells was evaluated, and the responses were correlated with expression of Bcl‐2 homologs and caspases 1, 3, 6, 8, and 9 activities. Results. Herein, we showed that inhibition of ERK1/2 activities caused (1) a G1 arrest; (2) a down‐regulation of the expression levels of the anti‐apoptotic homologs Bcl‐2, Mcl‐1, and Bcl‐XL without affecting the pro‐apoptotic levels of Bax and Bak; (3) a promotion of caspases 3, 6, 8, and 9 activities; (4) a stimulation of PARP cleavage; and (5) a programmed cell death by apoptosis. Conclusion: Our data suggest that activation of the ERK pathway functions to protect pancreatic tumor cells from apoptosis as well as to regulate their progression in the cell cycle. J. Cell. Biochem. 79:355–369, 2000.

Localization of Cholecystokinin Receptor Subtypes in the Endocine Pancreas

This study was undertaken to clarify the controversy in the literature about pancreatic localization of the cholecystokinin (CCK) CCKA and CCKB receptors. With antibodies used by other investigators, we first established their specificity by Western blotting, indirect immunofluorescence, and confocal microscopy with each antibodys peptide antigen. Co-localization assays between the CCK receptors and the pancreatic hormones insulin, glucagon, and somatostatin revealed that the CCKA RAbs 1122 and R1-2 recognized insulin and glucagon cells in rat, pig, and human pancreas but not in the somatostatin cells. Conversely, the three CCKB RAbs tested, 9262, 9491, and GR4, identified the somatostatin cells. Abs 9491 and GR4 occasionally co-localized with glucagon, a feature that never occurred with Ab 9262. Finally, the specificity of Ab 9262 for the pancreatic CCKB R was confirmed in six different species. It co-localized with somatostatin but never with glucagon in these species. Our data suggest the use of Abs 1122 and 9262 to specifically identify and localize pancreatic CCKA and CCKB receptors, respectively. Confusion in the literature may result from the lack of specificity of most antibodies used, as established in this study.

Simultaneous kinetic determinations of lipase, chymotrypsin, trypsin, elastase, and amylase on the same microtiter plate.

Micromethods are described to determine in 10 min the activity of the five most common pancreatic zymogens: amylase, lipase, trypsin, chymotrypsin, and elastase. Progress of the reactions is monitored at 405 nm, allowing the kinetic determination of the five enzymes on a single 96-well microtiter plate. Amylase activity is measured by the release of p-nitrophenol from a chemically defined substrate. Linearity of the assay is from 10 to 360 U/L of amylase, and activities as low as 0.4 U/L can be easily measured by extending the period of incubation up to 24 h. Chymotrypsin, trypsin, and elastase activities are monitored by the release of p-nitroanilide from specific substrates, and activities are from 25 to 6,500, 15 to 260, and 20 to 600 U/L, respectively. Finally, lipase is determined by the clearing of a commercially available stabilized emulsion of triolein. The lipase determination can be performed from 90 to 3,600 U/L. When microplate methods were compared with conventional procedures, a perfect correspondence was found between the two types of procedure. Factors necessary to convert microplate results to those of conventional assays are provided. These microassays make possible the rapid and simultaneous determination of the three main types of pancreatic hydrolases (a glycohydrolase, three proteases, and a lipase) with < 5 microliters of pancreatic juice by kinetic analysis. They could be easily adopted as routine assays in most research laboratories.

Subcellular distribution and characterization of rat pancreatic phospholipase D isoforms.

This study was undertaken to characterize the biochemical properties of rat pancreatic phospholipase D (PLD). Based on Western blot analysis of pancreas subcellular fractions, PLD1 was detected as a protein of 120 kDa associated with the microsomal fraction, whereas PLD2 appeared as a 105-kDa protein enriched in the microvesicular fraction. In these fractions, a low level of PLD activity was measured with an exogenous substrate containing phosphatidylinositol-4,5-bisphosphate (PIP2), unresponsive to guanosine triphosphate (GTP)&ggr;S and adenosine diphosphate (ADP)-ribosylation factor (ARF). Addition of unsaturated but not saturated fatty acids stimulated an oleate-dependent PLD activity that colocalized with the PLD1 enzyme in the crude plasma membrane and microsomal fractions. The transphosphatidylation reaction was maximal with either 200–400 m M (1.2–2.3%) ethanol or 25 m M (0.23%) 1-butanol, with an optimal pH between 6.5 and 7.2. Lipids extracted from the pancreatic membranes were potent inhibitors of the HL60 cell PLD activity when compared with those isolated from HL60 cells. Oleate-dependent PLD activity was less susceptible to these inhibitions. A phospholipase A1 (PLA1) activity hydrolyzing phosphatidylethanol also was found in the pancreatic membrane fractions and was nearly absent in the HL60 cells. This activity was completely inhibited by 400 n M tetrahydrolipstatin (THL), a lipase inhibitor. Pancreatic PLD1 and PLD2 activities could be measured after a chromatographic separation from microsomal membranes and high-speed supernatants, respectively. Activities of both enzymes were inhibited by oleate and required the presence of PIP2 in the substrate vesicles. ARF1 strongly activated PLD1 in a dose-dependent manner, and PLD2 was slightly responsive. Indirect immunofluorescence revealed that PLD2 is distributed throughout the pancreas, with a more intense staining in the islets. This study presents for the first time biochemical characteristics of the pancreatic PLD activities and shows the presence of oleate-dependent PLD1 and PLD2 activities, as well as PLD1 and PLD2 proteins in this gland.

Efficient binding of regulated secretory protein aggregates to membrane phospholipids at acidic pH.

Some regulated secretory proteins are thought to be targeted to secretory granules through an acidic-dependent aggregation in the trans-Golgi network. In this report we use pancreatic zymogens, a paradigm of regulated proteins, to test this hypothesis, because they qualitatively aggregate upon acidification in vitro. Pig zymogens were found to start to aggregate significantly at pH approximately 6.0, a pH slightly lower than that at which rat zymogens aggregate, but still compatible with the pH of the cell-sorting compartments. When pig zymogen granule membranes were mixed with the zymogens in the aggregation assay, membranes that normally floated on 1 M sucrose were observed to be pelleted by the aggregating zymogens. Rat membranes were pelleted by pig zymogens and vice versa. Igs, typical constitutively secreted proteins, which needed chemical cross-linking to serve as an aggregated protein control, pelleted membranes almost independently of pH. Corresponding cross-linked zymogen-binding ability and pH dependence was unaffected by the chemical modification. Membranes treated with sodium carbonate, pH 11, or with protease K, were still pelleted by zymogens, suggesting that the aggregated zymogens bound to membrane lipids. This hypothesis was confirmed by the efficient pelleting of unilamellar vesicles composed of granule membrane lipids. Vesicles composed of single classes of phospholipids were also pelleted, but with various efficacies. We conclude that pancreatic zymogen aggregates, formed under the acidic conditions of the secretory pathway sorting compartments, have the capacity to bind firmly to membranes through their phospholipid constituents.

New method for determining total calcium content in tissue applied to skeletal muscle with and without calsequestrin

The concentration of total calcium in a skeletal muscle appears to be correlated with the muscle’s likely force requirements given by the ratio of body weight to muscle weight.

GI Hormones and Endocrine Pancreas: Growth

Information on the control of endocrine pancreas growth is not readily available since it has long been assumed that differentiated endocrine cells do not proliferate or regenerate. Studies on fetal pancreas development and pancreas regeneration emphasized the involvement and roles of transcription factors, growth factors, and hormones, including some gastrointestinal hormones.

Regulation of rat pancreatic CCKB receptor and somatostatin expressions by insulin

The cholecystokinin B receptor (CCK(B)R) is localized on pancreatic endocrine somatostatin delta-cells. Pancreatic somatostatin content was increased in diabetic rats. The mechanisms involved in this phenomenon are unknown, and we believe insulin is involved. In this study, four groups of rats were used: controls, streptozotocin-induced diabetic, streptozotocin-induced diabetic with insulin, and streptozotocin-induced diabetic with insulin and its cessation. Rats were killed after 7-28 days of treatment for diabetes, and somatostatin mRNA expression and pancreatic somatostatin content, CCK(B)R mRNA and protein expression evaluation in total pancreas and purified islets, and the cellular localization of somatostatin and CCK(B)R in islets was measured. Data indicate that diabetes is established after 7 days, is controlled by insulin, and reappears after treatment cessation. Pancreatic somatostatin mRNA expression and somatostatin content were increased during diabetes, normalized during insulin treatment, and reaugmented after treatment cessation. Gland and islet CCK(B)R mRNA and protein almost disappeared during diabetes; CCK(B) mRNA reappeared in response to insulin, but the protein did not. Confocal microscopy confirmed data obtained on somatostatin and CCK(B)R as established biochemically in the course of the treatments. In conclusion, these data strongly suggest that insulin can negatively control pancreatic somatostatin mRNA and hormone content and positively control CCK(B)R mRNA; the CCK(B)R protein appears to be delayed.