A cytotoxic monoclonal autoantibody from the BB rat which binds an islet cell surface protein

The BB rat provides an excellent animal model for type 1 (insulin-dependent) diabetes mellitus. Cytotoxic autoantibodies against pancreatic beta cells have been found in the sera of both patients with type 1 (insulin-dependent) diabetes and BB rats. These antibodies have been implicated in the pathogenesis of the disease. In this study, a monoclonal autoantibody, designated KT1, has been developed by the fusion of spleen cells from a BB rat and a mouse myeloma cell line. KT1 was found to be of the immunoglobulin M isotype and reacted specifically with islet cells. In microcytotoxicity assays KT1 was shown to mediate complement-dependent lysis of approximately 30% of a rat insulinoma cell line and 25% of rat pancreatic islet cells in culture. It did not cause lysis of the other cell lines tested. KT1 has been demonstrated, by indirect immunofluorescence, to bind specifically to a cell surface antigen on live and acetone-fixed islet cell cultures from Wistar rat neonates and to rat insulinoma cells. Western blotting experiments revealed reaction to a 68-kDa protein from rat insulinoma cell extracts. This monoclonal antibody may have clinical relevance as it exhibits properties similar to the islet cell surface antibodies present in the sera of BB rats.     

Development of cytotoxic islet cell antibodies in rats following damage of the pancreas by complete Freund's adjuvant combined with a nondiabetogenic dose of streptozotocin

The possible relationship between destruction of pancreatic beta cells and islet cell surface antibodies (ICSA) was examined in a rat model using complete Freund's adjuvant (CFA), a lymphocyte activator, in combination with the beta cell toxin, streptozotocin (STZ). In addition to this treatment, the rat insulinoma cell line, RIN5AH, as a readily accessible source of insulin-producing cells, was utilized to potentiate the production of ICSA. Intraperitoneal injections of CFA to male Lewis rats, followed 24 h later by a single nondiabetogenic dose of STZ, produced a 47% (p less than 0.01) reduction in pancreatic insulin content associated with degranulation and necrosis of insulin-immunoreactive cells. Eight weeks after treatment, ICSA were detectable that mediated the complement-dependent lysis of neonatal rat islet cells. Injections of RIN5AH cells, following treatment with CFA/STZ, did neither increase the severity of histopathological changes in the exocrine pancreas nor the extent of beta cell necrosis, but gave rise to higher levels of cytotoxic ICSA. Immunization with RIN cells alone, although increasing ICSA levels above those of the other experimental groups, produced no major histopathological changes. These results indicate that ICSA are the consequence of beta cell damage, and they are not capable of promoting or initiating beta cell necrosis in this model.     

Development of cytotoxic islet cell antibodies in rats following damage of the pancreas by complete freund’s adjuvant combined with a nondiabetogenic dose of streptozotocin

The possible relationship between destruction of pancreatic beta cells and islet cell surface antibodies (ICSA) was examined in a rat model using complete Freund’s adjuvant (CFA), a lymphocyte activator, in combination with the beta cell toxin, streptozotocin (STZ). In addition to this treatment, the rat insulinoma cell line, RIN5AH, as a readily accessible source of insulin-producing cells, was utilized to potentiate the production of ICSA. Intraperitoneal injections of CFA to male Lewis rats, followed 24 h later by a single nondiabetogenic dose of STZ, produced a 47% (p<0.01) reduction in pancreatic insulin content associated with degranulation and necrosis of insulin-immunoreactive cells. Eight weeks after treatment, ICSA were detectable that mediated the complement-dependent lysis of neonatal rat islet cells. Injections of RIN5AH cells, following treatment with CFA/ STZ, did neither increase the severity of histopathological changes in the exocrine pancreas nor the extent of beta cell necrosis, but gave rise to higher levels of cytotoxic ICSA. Immunization with RIN cells alone, although increasing ICSA levels above those of the other experimental groups, produced no major histopathological changes. These results indicate that ICSA are the consequence of beta cell damage, and they are not capable of promoting or initiating beta cell necrosis in this model.     

DNA fragmentation is an early event in cytokine-induced islet beta-cell destruction

Summary The cytokines, interleukin 1, tumour necrosis factor, and interferon gamma are cytotoxic to islet beta cells, however, their mechanisms of beta-cell killing are not fully characterized. Since DNA damage is a mechanism of cytokine-induced cell death in some cell types, we sought evidence for cytotoxic effects of cytokines at a nuclear level in islet beta cells by measuring DNA fragmentation in rat islets and islet beta-cell lines. The individual cytokines, interleukin 1 (10 U/ml), tumour recrosis factor (10³ U/ml) and interferon gamma (10³ U/ml) inhibited insulin release from rat islets, but did not cause DNA fragmentation or destroy islet cells; by contrast, combination of the three cytokines induced DNA fragmentation and islet-cell death. Cytokine-induced DNA fragmentation preceded cell lysis in islet beta-cell lines (RINm5F, rat insulinoma cells; and NIT-1, NOD/Lt mouse transgenic beta cells), whereas in non-islet cell lines (GH-3, rat pituitary; and PC-12, rat adrenal) the cytokines induced cell lysis and no or late DNA fragmentation. Nicotinamide prevented both DNA fragmentation and destruction of RINm5F islet cells by the cytokines. These findings identify DNA as an early target of cytokine action in islet beta cells, and implicate DNA fragmentation as a mechanism of cytokine-induced beta-cell destruction.Abbreviations IDDM Insulin-dependent diabetes mellitus - IL-1 interleukin-1 - TNF tumour necrosis factor - IFN interferon gamma - NO nitric oxide - l-NMMA l-NG-monomethyl arginine     

Immunohistochemical localization of betacellulin, a new member of the EGF family, in normal human pancreas and islet tumor cells

Betacellulin (BTC) purified from mouse beta cell tumor (betaTC-3) is a new member of the epidermal growth factor (EGF) family which can bind receptor tyrosine kinase, EGF receptor (erbB1) and erbB4. It has been demonstrated that proBTC mRNA was abundantly expressed in human pancreas tissue, and that BTC converted amylase-secreting rat acinar cell line (AR42J) into insulin-secreting cells, suggesting that BTC might be important for the growth and/or differentiation of islet cells. However, the cell type producing BTC in the pancreas has not been clarified. In this study, we examined the localization of BTC in human pancreas and islet cell tumors. Immunohistochemistry using specific antibodies to human BTC revealed that this protein was produced in alpha cells and duct cells, and probably in beta cells in normal adult pancreas. Furthermore, strong immunoreactivity to BTC was detected in primitive duct cells of the fetal pancreas, and both insulinoma and glucagonoma cells also showed positive immunoreactivity to BTC. EGF receptor (erbB1) and erbB4 were expressed mainly in islet and duct cells, and duct cells, respectively. These results demonstrate the localization of BTC and its receptors, and suggest that BTC may be one of the factors that have physiologically important roles such as growth and differentiation of islet cells in the human pancreas.     

Proteasome inhibitors synergize with tumor necrosis factor-related apoptosis-induced ligand to induce anaplastic thyroid carcinoma cell death

CONTEXT: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive types of cancer characterized by complete refractoriness to multimodal treatment approaches. Therapeutic strategies based on the simultaneous use of proteasome inhibitors and death receptor ligands have been shown to induce apoptosis in several tumor types but have not yet been explored in ATC. OBJECTIVE AND METHODS: The aim of this study was to investigate the ability of the proteasome inhibitor Bortezomib to induce apoptosis in ATC cell lines. Bortezomib was used as a single agent or in combination with TNF-related apoptosis-induced ligand (TRAIL), a member of the TNF family that selectively induces tumor cell apoptosis. The molecular effects of Bortezomib were investigated by analyzing the expression of key regulators of cell cycle and apoptosis and the activation of different apoptotic pathways. RESULTS: Bortezomib induced apoptosis in ATC cells at doses achieved in the clinical setting, differently from conventional chemotherapeutic agents. Simultaneous treatment with low doses of Bortezomib and TRAIL had a synergistic effect in inducing massive ATC cell apoptosis. Bortezomib increased the expression of cytotoxic TRAIL receptors, p21 (WAF/CIP1) and proapoptotic second mitochondria-derived activator of caspases/direct inhibitor of apoptosis binding protein with low pI, and reduced the expression of antiapoptotic mediators such as cellular Fas-associated death domain-like IL-1beta converting enzyme inhibitory protein, Bcl-2, Bcl-X(L), and inhibitor of apoptosis-1, thus resulting in cell death induction through the mitochondrial apoptotic pathway. CONCLUSIONS: The combination of proteasome inhibitors and TRAIL synergizes to induce the destruction of chemoresistant neoplastic thyrocytes and could represent a promising therapeutic strategy for the treatment of anaplastic thyroid carcinoma.     

CIK对地西他滨增敏乳腺癌MDA-MB-231细胞的杀伤机制探讨

目的 观察地西他滨（DAC）增敏细胞因子介导的杀伤细胞（CIK）对人乳腺癌MDA-MB-231细胞株的细胞毒作用,并探讨其增敏机制.方法 取对数生长期的乳腺癌MDA-MB-231及正常乳腺MCF-10A细胞,MMT法检测DAC及TRAIL对乳腺癌细胞的增殖抑制率,LDH法检测DAC单用或联合肿瘤坏死因子相关凋亡诱导配体（TRAIL）及CIK对乳腺癌细胞的杀伤率,流式细胞仪检测细胞凋亡率.结果 DAC对乳腺癌细胞MDA-MB-231及正常乳腺上皮细胞MCF-10A无明显杀伤作用.TRAIL对MDA-MB-231有明显杀伤作用,作用24h的IC50约为100 ng/mL,然而对MCF-10A无明显促凋亡影响.5∶1、10∶1、20∶1效靶比的CIK对MDA-MB-231的杀伤率分别为10.7％±1.2％、17.8％±2.1％、37.2％±3.4％,对MCF-10A无杀伤作用.经50 μmol/L的DAC预处理乳腺癌细胞MDA-MB-231 24 h后,5∶1、10∶1、20∶1效靶比的CIK对MDA-MB-231的杀伤率分别为18.6％±2.6％、26.3％±4.5％、51.6％±6.6％,与相应单独效靶比的CIK杀伤作用比较,P均＜0.01.结论 DAC增敏CIK对乳腺癌MDA-MB-231细胞的杀伤敏感性,对正常的乳腺上皮细胞无影响,DAC联合CIK可能成为治疗乳腺癌患者的新途径.     

Multiple low-dose streptozotocin-induced diabetes in the mouse: further evidence for involvement of an anti-B cell cytotoxic cellular auto-immune response

Summary Anti-B cell auto-immunity may play a role in the pathogenesis of diabetes in mice resulting from multiple subdiabetogenic doses of the pancreatic B cell toxin, streptozotocin. In the present study we have investigated the cytotoxic anti-B cell response in these mice. A major role for B lymphocytes, macrophages, or their products in the cytotoxic response originally detected in vitro was eliminated by passing splenocytes from the mice treated with multiple subdiabetogenic doses of streptozotocin over a nylon wool column. The removal of the adherent cells enhanced the cytotoxicity against a rat insulinoma cell line in vitro by that expected due to enrichment of T-lymphocytes by approximately twofold. The induction of diabetes after multiple subdiabetogenic doses of streptozotocin is strain dependent. Mice of five strains were immunized with rat insulinoma cells, but only splenocytes from the two strains susceptible to multiple subdiabetogenic doses of streptozotocin demonstrated a significant cytotoxic response against the rat insulinoma cells in vitro. Mice pre-immunized with either the rat insulinoma cells or with syngeneic islets labelled in vitro with the hapten trinitrophenol developed hyperglycaemia more rapidly than control mice after multiple subdiabetogenic doses of streptozotocin. In the latter experiment the control mice immunized with complete Freund's adjuvant alone also became hyperglycaemic after a modified multiple subdiabetogenic dose of streptozotocin that did not cause diabetes in non-immunized mice. In mice pre-treated with either adjuvant or cyclophosphamide and then given a modified multiple subdiabetogenic dose of streptozotocin (35 mg/kg × 5 rather than 40 mg/ kg) the degree of hyperglycaemia was reduced and there was no protective effect of cyclophosphamide. However, the mice pre-treated with adjuvant again developed hyperglycaemia more rapidly and to a much higher level than did the mice given multiple subdiabetogenic doses of streptozotocin only. These additional data further support the hypothesis that B-cell destruction after multiple subdiabetogenic doses of streptozotocin results from triggering of an immune response against these insulin-producing cells.     

Role of tumor necrosis factor-alpha and interferon-gamma as growth factors to the human fetal beta-cell

The effects of the cytokines tumor necrosis factor-alpha and interferon-gamma on the adult beta-cell have been well described: a reduction of insulin secretion and content and death of the cell. For this reason and because these cytokines may be released from activated lymphocytes and macrophages that infiltrate islets in insulin-dependent diabetes, they have been implicated in the pathophysiology of this form of diabetes. As to whether the human fetal beta-cell, which differs from the adult beta-cell in not releasing insulin in response to the nutrient glucose and not being adversely affected by the toxin streptozotocin, is similarly affected is unknown. To examine this question we cultured monolayers of a single cell suspension of human fetal pancreas in the presence or absence of 1000 U/mL of these cytokines for 7 days. Chronic insulin release was enhanced for the first 2 days of culture, but unchanged thereafter. Acute insulin release in response to the secretagogue theophylline (10 mM) was enhanced on day 7, but not earlier. There was an increase in the insulin content of the cells by the fourth day, probably due to an increase in the number of beta-cells present (45 +/- 5% vs. 22 +/- 3%). Microscopically, non-beta-cells also seemed to increase in number; there was an increase in both DNA and cell number by the seventh day. In contrast to these beneficial effects on the human fetal beta-cell, treatment of adult rat insulinoma cells, represented by RIN-m5F cells, resulted in inhibition of insulin secretion during the first day of culture and subsequent death of 86% of the cells by the sixth day of culture. It is hypothesized that the functional immaturity and lack of normal (adult) metabolic activity of the human fetal beta-cell somehow confers protection on these cells from the cytotoxic effects of tumor necrosis factor-alpha and interferon-gamma. Indeed, our findings suggest that these cytokines may be trophic for the developing beta-cell.     

Increased vulnerability of newly forming beta cells to cytokine-induced cell death

Aims/hypothesis Beta cell destruction in type 1 diabetes is apparently mediated by the release of cytokines. We questioned whether cytokine-induced apoptosis preferentially kills replicating beta cells. Materials and methods In the first experiment, rat insulinoma (RIN) cells were studied for 36 h by time-lapse video microscopy. Cells were exposed to three doses of a cytokine mixture (maximal concentration: IL-1β 50 U/ml; TNF-α 1,000 U/ml; IFN-γ 1,000 U/ml) or vehicle and analysed for the total cell number (2-h intervals) and timing of each cell death and division. In the second experiment, isolated human islets were incubated with the same cytokine mixture for 24 h and examined for replication and paired (postmitotic) apoptosis. Results In the first experiment, after application of cytokines, apoptosis occurred most frequently immediately after the next or subsequent cell mitosis (p<0.05). In the second experiment, cytokines caused increased apoptosis in human islets, with an increase in the proportion of postmitotic apoptotic pairs (p<0.001). Conclusions/interpretation Cytokine-induced beta cell death preferentially affects newly forming beta cells, which implies that replicating beta cells might be more vulnerable to cytokine destruction. Efforts to expand beta cell mass in type 1 diabetes by fostering beta cell replication are likely to fail unless cytokine-induced apoptosis is concurrently suppressed.     

Pancreatic adenocarcinoma cell lines show variable susceptibility to TRAIL-mediated cell death.

BACKGROUND AND AIMS: Programmed cell death via the Fas receptor/Fas Ligand and DR4, DR5/TRAIL plays a major role in tumor escape and elimination mechanisms. It also promises to be an effective therapy alternative for aggressive tumors, as has been recently shown for colon, breast, and lung cancer cells. We attempted to clarify the role of these molecules in aggressivity of pancreatic carcinomas and to identify possible pathways as targets for therapy. METHODS: Five pancreatic cell lines were investigated for the expression of FasL/Fas, DcR3, DR4, DR5/TRAIL, DcR1, DcR2, and other death pathways related molecules such as Bax, bcl-xL, bcl-2, FADD, and caspase-3 by flow cytometry, immunoblotting, and RT/PCR, both semiquantitative and real time (TaqMan). The susceptibility of these cell lines to apoptosis mediated by recombinant TRAIL was investigated. The effect of therapeutic agents (gemcitabine) on their susceptibility to TRAIL induced apoptosis was studied as well. RESULTS: Pancreatic adenocarcinomas expressed high levels of apoptosis-inducing receptors and ligands. They showed differential susceptibility to cell death induced by TRAIL, despite expressing intact receptors and signaling machineries. Treatment with commonly used therapeutic agents did not augment their susceptibility to apoptosis. This could be explained by the fact that they expressed differentially high levels of decoy receptors, as well as molecules known as inhibitors of apoptosis. CONCLUSIONS: The data suggest that pancreatic carcinoma cells have developed different mechanisms to evade the immune system. One is the expression of nonfunctional receptors, decoy receptors, and molecules that block cell death, such as bcl2 and bcl-xL. The second is the expression of apoptosis-inducing ligands, such as TRAIL, that could induce cell death of immune cells. The success in treating malignant tumors by recombinant TRAIL might apply to some but not all pancreatic tumors because of their differential resistance to TRAIL-induced cell death.     

Killing effect of TNF-related apoptosis inducing ligand regulated by tetracycline on gastric cancer cell line NCI-N87

AIM: To clone the cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) into a tetracycline-regulated gene expression system, the RevTet-On system, transduce expression vectors into a gastric carcinoma cell line-NCI-N87 and examine the effects of controlled expression of TRAIL in vitro on the gastric carcinoma cells. METHODS: The full-length cDNA of TRAIL was inserted into a vector under the control of the tetracycline-responsive element (TRE) to obtain the plasmid pRevTRE-TRAIL, which was transfected into a packaging cell line PT67. In addition, vector pRev-Tet On and pRevTRE were also transfected into PT67 separately. After hygromycin and G418 selection, the viral titer was determined. The medium containing retroviral vectors was collected and used to transduce a gastric carcinoma cell line NCI-N87. The resulting cell line NCI-N87-Tet On TRE-TRAIL and a control cell line, NCI-N87 Tet On-TRE, were established. TRAIL expression in the cell line was induced by incubating cells with doxycycline (Dox), which is a tetracycline analogue. The killing effect on gastric carcinoma cells was analyzed after induction. RESULTS: The recombinant plasmid pRev-TRE-TRAIL was constructed. After hygromycin or G418 selection, the producer cell lines PT67-TRE, PT67-TRE-TRAIL and PT67-Tet On were obtained,with titers of about 10(8)CFU.L(-1). By transducing NCI-N87 cells with retroviral vectors from these cell lines, stable cell lines NCI-N87-Tet-On TRE-TRAIL (NN3T) and control cell line NCI-N87-Tet-On-TRE (NN2T) were established. The growth curves of the selected cell lines were the same with the wild type NCI-N87. When Dox was added, cell death was obvious in the test groups (29%-77%), whereas no difference was observed in control and wild type cell lines. With the addition of a medium from the test group, human leukemia cell line Jurkat was activated till death (83%), indicating the secretion of active TRAIL proteins from the test cells to the medium. CONCLUSION: With the use of the RevTet-On system, a regulated expression system for TRAIL was constructed. Using this system, the selected killing effect of TRAIL on gastric carcinoma cell line NCI-N87 could be observed.     

Unresponsiveness of insulinoma cells to secretin: significance of the secretin test in patients with insulinoma

It is well known that B cells in the pancreas release insulin when stimulated by secretin, but there have been few reports on the response of insulinoma cells to secretin. In five patients with insulinoma, changes in serum immunoreactive insulin (IRI) concentration were measured after the intravenous injection of secretin into the peripheral vein before and after extirpation of the insulinoma. The extirpated insulinomas were cultured and tested for their response to secretin. The rise in serum IRI in response to secretin in patients with insulinoma was significantly slower and smaller than in normal volunteers. After removal of the insulinoma, the response to secretin became prompt and increased with time. Cultured insulinoma cells did not release insulin when stimulated by secretin. Therefore, it is concluded that the response of insulinoma cells to secretin is quite different from that of normal beta cells, and that the function of beta cells in the insulinoma-bearing pancreas is suppressed by the autonomous hypersecretion of insulin by the insulinoma. The extent of the decrease in function of the beta cells in patients with insulinoma can be estimated by the intravenous secretin test. Thus, the secretin test is sometimes useful in the differentiation of hypoglycemia due to insulinoma from that due to beta cell hyperplasia or alimentary hyperinsulinemia.     

Modestly increased beta cell apoptosis but no increased beta cell replication in recent-onset type 1 diabetic patients who died of diabetic ketoacidosis

Aims/hypothesis Type 1 diabetes is characterised by a deficit in beta cell mass thought to be due to immune-mediated increased beta cell apoptosis. Beta cell turnover has not been examined in the context of new-onset type 1 diabetes with diabetic ketoacidosis. Methods Samples of pancreas were obtained at autopsy from nine patients, aged 12 to 38 years (mean 24.3±3.4 years), who had had type 1 diabetes for less than 3 years before death due to diabetic ketoacidosis. Samples of pancreas obtained at autopsy from nine non-diabetic cases aged 11.5 to 38 years (mean 24.2±3.4 years) were used as control. Fractional beta cell area (insulin staining), beta cell replication (insulin and Ki67 staining) and beta cell apoptosis (insulin and TUNEL staining) were measured. Results In pancreas obtained at autopsy from recent-onset type 1 diabetes patients who had died of diabetic ketoacidosis, the beta cell deficit varied from 70 to 99% (mean 90%). The pattern of beta cell loss was lobular, with almost all beta cells absent in most pancreatic lobules; islets in lobules not devoid of beta cells had reduced or a near-normal complement of beta cells. Beta cell apoptosis was increased in recent-onset type 1 diabetes, but to a surprisingly modest degree given the marked hyperglycaemia (30 mmol/l), acidosis and presumably high NEFA. Beta cell replication, scattered pancreatic beta cells and beta cells in exocrine ducts were not increased in recent-onset type 1 diabetes. Conclusions/interpretation These findings do not support the notion of active beta cell regeneration by replication in new-onset type 1 diabetes under conditions of diabetic ketoacidosis. The gluco-lipotoxicity reported in isolated human islets may be less evident in vivo.     

Monoclonal antibody-mediated cytotoxicity against rat Beta cells detected in vitro does not cause Beta-cell destruction in vivo

Summary Two monoclonal Beta-cell surface antibodies M10H6 und K14D10 were obtained by fusion of spleen cells of Balb/c mice with the myeloma cell line P₃0. The monoclonal antibody M10H6 was induced by immunization with rat insulinoma cells finally boostered with disintegrated rat islets, whereas the K14D10 was generated after immunization with porcine proinsulin. Both monoclonals belong to the IgG_2A isotype and were screened with insulin-producing rat insulinoma cells by an indirect immunofluorescence test as well as by a cellular enzyme linked immunosorbent assay. In addition to the cell surface binding on living Beta cells the monoclonals react with islets on cryostat sections of rat pancreas. The anti-islet cytotoxic potential of these monoclonals was measured by ⁵¹Chromium-release in the presence of complement or Fc-receptor bearing leucocytes using ⁵¹Chromium-labelled rat islet cells as target. Both antibody secreting hybridomas were propagated in syngeneic mice resulting in high levels of islet cell surface antibodies in ascites and sera from the recipient. High anti-islet cytotoxicity was mediated by ascites fluid, but no mouse developed hyperglycaemia. Furthermore, the repeated injections of the monoclonals into rats did not exert a diabetogenic action and failed to reduce the pancreatic insulin content although the attraction of the K14D10 to the pancreatic islets in vivo could be demonstrated. We conclude that islet cell surface antibody-mediated Beta-cell lysis in vitro may not be relevant to Beta-cell destruction in vivo.     

Osteoprotegerin and RANKL differentially regulate angiogenesis and endothelial cell function

Osteoprotegerin (OPG) a soluble tumor necrosis factor receptor family molecule protects endothelial cells from apoptosis in vitro and promotes neovascularization in vivo. In this study, we assessed the role of OPG and its ligands, receptor activator of nuclear factor-κB ligand (RANKL) and tumor necrosis factor-related apoptosis inducing ligand (TRAIL), in microvessel formation using the rat aortic ring model of angiogenesis. OPG was found to promote a twofold increase in angiogenic sprouting in the aortic ring model, and this effect was inhibited by pre-incubation with a fivefold molar excess of either RANKL or TRAIL. While TRAIL had no effect upon angiogenesis on its own, RANKL was found to potently inhibit basal and vascular endothelial growth factor-induced angiogenesis. OPG increased the rate of endothelial cell proliferation in sprouting microvessels; in contrast, RANKL inhibited proliferation. RANKL was found to induce endothelial apoptosis at days 6, 7, and 10 in the aortic ring model and after incubation with human umbilical vein endothelial cells (HUVECs). Signaling studies showed that OPG induced ERK1/2 and Akt phosphorylation in HUVECs while RANKL had no effect. Our results indicate that OPG is a positive regulator of microvessel formation, while RANKL is an angiogenic inhibitor due to effects on regulation of endothelial cell proliferation, apoptosis, and signaling.     

Gastrin in the perinatal rat pancreas and gastric antrum: immunofluorescence localization of pancreatic gastrin cells and gastrin secretion in monolayer cell cultures.

The presence and development of immunoreactive gastrin (IRGa) in the fetal and neonatal pancreas and pyloric antrum of the rat were studied. IRGa appeared in both organs at least as early as the 16th day of fetal life. Antral IRGa increased rapidly and continuously in the neonatal period, while pancreatic IRGa concentration increased and was maintained at a relatively constant level from days 5 to 35. Monolayer cell cultures of the neonatal rat pancreas were used to evaluate the role of cyclic AMP mediated release of gastrin. The addition of N6,O2'-dibutyryl cyclic AMP (4 mM) or theophylline (4 mM) to the culture medium induced significant release of gastrin. The stimulation of adenylate cyclase with cholera toxin (10 ng/ml) also resulted in significant gastrin release. Long-term cultures (18-24 days) were shown to release gastrin continuously at a relatively constant rate. The cellular localization of pancreatic gastrin in 7-day-old cultures was performed by immunological techniques, using fluorescein-labeled antibodies to gastrin. The gastrin-containing cells were located at the periphery of most of the endocrine cell clusters. Immunofluorescence techniques for insulin and glucagon also showed that the alpha cells had a similar peripheral distribution, although they were more frequent in number. In contrast, insulin-containing cells were numerous and were present in all areas of the endocrine cell clusters. The studies support the following conclusions: a) Gastrin is present in the rat pancreas, even as early as late fetal life; b) Gastrin-producing cells are present and functionally competent in monolayer cell cultures of the neonatal rat pancreas for prolonged periods of time (24 days); c) Gastrin is released from these cells when intracellular levels of cyclic AMP are increased; d) By immunofluorescence methods, the gastrin-producing cells in pancreatic cell cultures are found to be located at the periphery of the endocrine cell clusters.     

Interferon-gamma: pleiotropic effects on a rat pancreatic beta cell line

We have recently shown that interferon-gamma (IFN-gamma) markedly upregulates the expression of the class I major histocompatibility proteins on pancreatic beta cells and have therefore postulated that interferon-gamma may enhance cytotoxic lymphocyte-mediated beta cell damage in insulin-dependent diabetes mellitus. To further explore the interaction between interferon-gamma and the pancreatic beta cell we have used the RIN-m5F insulinoma line to define the effects of interferon-gamma on major histocompatibility protein expression, (pro)insulin and protein synthesis and cell growth. Interferon-gamma induced a dose-dependent increase in the expression of the class I major histocompatibility proteins on the RIN-m5F cells, the maximal increase (10-fold) being seen at an interferon-gamma concentration of 1 U/ml. The induction of class I proteins by interferon-gamma was nearly completely abolished by cycloheximide. Expression of class II (Ia) proteins was not detected either in the presence or absence of interferon-gamma. (Pro)insulin and protein synthesis were decreased by 60% and 40%, respectively, in RIN-m5F cells cultured with interferon-gamma (10 U/ml). Furthermore, the growth of RIN-m5F cells was significantly inhibited, and corresponding changes in cell morphology were evident, after 3 days of exposure to interferon-gamma (10 U/ml). These findings indicate that, in addition to its potential role in amplifying cytotoxic T cell activity against the pancreatic beta cell, IFN-gamma may also directly inhibit beta cell function and growth. Several mechanisms could therefore account for an ability of IFN-gamma to compromise beta cell function and contribute to the pathogenesis of insulin-dependent diabetes.     

Modulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by chemotherapy in thyroid cancer cell lines.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many human cancer cells but not in normal cells. Thyroid cancer cells, however, appear to be relatively resistant to TRAIL-induced apoptosis. We therefore investigated the effect of chemotherapy on TRAIL-induced apoptosis in thyroid cancer cells. We used six thyroid cancer cell lines: TPC-1, FTC-133, FTC-236, FTC-238, XTC-1, and ARO82-1. We used flow cytometry to measure apoptosis, dimethyl-thiazol-diphenyltetrazolium bromide (MTT) assay to measure antiproliferation effects and Western blot to determine the expression of Bcl family proteins. Troglitazone, paclitaxel, geldanamycin, and cycloheximide were used for pretreatment. We used the Student's t test and analysis of variance (ANOVA) for statistical analysis. All thyroid cancer cell lines, except the TPC-1 cell line, were resistant to TRAIL, and growth inhibition was less than 20% at concentration of 800 ng/mL of TRAIL. In both TPC-1 (TRAIL-sensitive) and FTC-133 (TRAIL-resistant) thyroid cancer cell lines, pretreatment with troglitazone, cycloheximide, and paclitaxel enhanced TRAIL-induced cell death significantly but pretreatment with geldanamycin did not. There were no significant changes in Bcl-2, Bcl-xl, and Bax protein expression after troglitazone treatment. In conclusion, TRAIL in combination with troglitazone, paclitaxel, and cycloheximide induces apoptosis in thyroid cancer cells at suboptimal concentrations that cannot be achieved using TRAIL alone.