Synergistic antitumor effect by coexpression of chemokine CCL21/SLC and costimulatory molecule LIGHT.

To establish a more efficient treatment for immunotherapy against solid tumors, we have evaluated the antitumor effect by coexpression of a chemokine CCL21/secondary lymphoid tissue chemokine and a costimulatory molecule LIGHT in colon carcinoma C26. C26 cells expressing either CCL21 or LIGHT exhibited a significantly reduced tumor growth in vivo, and mice inoculated with these cells showed a prolonged survival, but eventually all these mice died. In contrast, C26 cells expressing both CCL21 and LIGHT exhibited a minimal tumor growth in vivo, and all these mice survived healthily with a tumor remission and consequently acquired a strong protective immunity. A markedly increased infiltration of mature dendritic cells (DCs), and CD8(+) T cells was observed in the tumor mass, and their spleen cells showed a greatly enhanced cytotoxic T lymphocyte (CTL) activity against C26 tumor and interferon (IFN)-gamma production. Neutralization of IFN-gamma or depletion of CD8(+) or CD4(+) T cells significantly reduced the antitumor activity. These results suggest that the combined treatment with CCL21 and LIGHT is able to induce a synergistic antitumor effect to eradicate tumor completely by greatly enhancing tumor-infiltration of lymphocytes including mature DCs and CD8(+) T cells, resulting in markedly augmented CTL activity and IFN-gamma production.

Positive modulation of IL-12 signaling by sphingosine kinase 2 associating with the IL-12 receptor beta 1 cytoplasmic region.

IL-12 is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses. IL-12 stimulation results in the activation of Janus kinase 2 and tyrosine kinase 2 and, subsequently, STAT4 and STAT3. In addition, mitogen-activated protein kinase kinase 6/p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways have been recently demonstrated to be activated by IL-12 and play an important role in IL-12 signaling. To further elucidate the molecular mechanism underlying IL-12 signaling, we have performed a yeast two-hybrid screening and identified mouse sphingosine kinase 2 (SPHK2) as a molecule associating with the mouse IL-12Rbeta1 cytoplasmic region. Analyses of various mutants of each molecule revealed that the region including the proline-rich domain in SPHK2 is probably responsible for the binding to IL-12Rbeta1, while the regions including the carboxyl terminus and Box II in the IL-12Rbeta1 cytoplasmic region appear to be involved in the binding to SPHK2. Transient expression of wild-type SPHK2 in T cell hybridoma augmented IL-12-induced STAT4-mediated transcriptional activation. Ectopic expression of dominant-negative SPHK2 in Th1 cell clone significantly reduced IL-12-induced IFN-gamma production, while that of wild-type SPHK2 enhanced it. In contrast, the expression minimally affected IL-12-induced proliferation. A similar decrease in IL-12-induced IFN-gamma production was observed when dominant-negative SPHK2 was expressed in activated primary T cells using a retroviral expression system. These results suggest that SPHK2 associates with the IL-12Rbeta1 cytoplasmic region and probably plays a role in modulating IL-12 signaling.

Efficient gene transfer to hepatoblastoma cells through asialoglycoprotein receptor and expression under the control of the cyclin A promoter.

Specific gene delivery into hepatoma cells by liposomes and specific gene expression under the control of the cyclin A promoter were examined in HepG2 cells, a hepatoblastoma cell line that overexpresses cyclin A. A plasmid carrying the luciferase gene under the cyclin A promoter sequence was condensed with poly-L-lysine and encapsulated into anionic asialofetuin-labeled liposomes (AF-liposomes), which were preferentially taken up by hepatocytes through the action of the asialoglycoprotein receptor (AgpR). AF-liposomes delivered plasmids to the hepatoma cells by receptor-mediated endocytosis through the AgpR, and transgene expression could be achieved under the control of the cyclin A promoter. Furthermore, a fusogenic lipid, DOPE, as a liposomal component was required for the enhancement of transfection efficiency of AF-liposomes.

Turnover of acinar and islet cells in the pancreas of monosodium glutamate-treated obese mice.

OBJECTIVE: Subcutaneous administrations of monosodium glutamate (MSG) to neonatal animals result in obesity and induce the toxicity on the central nervous system, and furthermore, have an effect on entero-pancreatic hormone. The effect of MSG on the cell turnover of organs, especially the pancreas, has received little attention until now. This study was designed to examine the effect of MSG on pancreatic cell turnover by immunohistochemistry and [(3)H]thymidine autoradiography. RESEARCH METHODS AND PROCEDURES: Male JcI-ICR strain mice were SC injected with MSG (2 mg/g body weight daily) for 5 days after birth, received 112 repeated injections of [(3)H]thymidine at 6-hour intervals for 28 days after birth, and then were killed immediately thereafter, or 30, 60, or 120 days after the last injection. Autoradiography was performed on sections immunostained for glucagon, insulin, and somatostatin. RESULTS: After continuous labeling, most pancreatic cells were labeled, and thereafter, labeling of cells decreased in control and MSG-treated mice. The mean grain counts of acinar cells in MSG-treated mice decreased more slowly than those in control mice. On the other hand, those of islet cells, including glucagon, insulin, and somatostatin cells, decreased more rapidly in MSG-treated mice than those in control mice. DISCUSSION: Cell turnover of acinar cells was decelerated and that of islet cells including glucagon, insulin, and somatostatin cells was accelerated in MSG-treated mice pancreas. MSG-induced hypothalamic lesions exert the contrary influences on the cell turnover of acinar and islet cells.

Suppression of tumor growth through introduction of an antisense plasmid of macrophage migration inhibitory factor.

The potential role in cell growth of Macrophage migration inhibitory factor (MIF) has been studied, however, the mechanism of its anti-tumor effect is poorly understood. Antisense-MIF plasmids were directly injected into colon 26 tumors embedded in the back of mice. Furthermore, the role of MIF in the cell cycle was assessed with regard to retinoblastoma (Rb) protein and transcription factor E2F. Plasmids containing sense- and antisense-MIF genes were transfected into human colon cancer KM12SM cells in vitro. To examine the Rb protein-E2F pathway, plasmids containing each specific cis-acting enhancer for Rb protein and E2F with luciferase reporter genes, pRB-luc and pE2F-luc, respectively, were used. Antisense MIF treatment significantly reduced the tumor size. In vitro cell proliferation was significantly suppressed by the antisense treatment as examined by BrdU uptake. Transcriptions of Rb protein were 8.4x10(3) (RLU), 9.5x10(3) and 24.3x10(3) in the antisense MIF, PBK, and the sense MIF, respectively. As for E2F, transcription activities were 3.8x10(3), 3.6x10(3) and 7.7x10(3), respectively. These results indicate the possibility that MIF may promote tumor growth, in which the activation-inactivation mechanism of the Rb protein-E2F pathway could be profoundly involved.

Cell proliferation and renewal of normal hepatocytes and bile duct cells in adult mouse liver.

BACKGROUND/AIMS: The source of new cells in the normal adult liver has been controversial. Some investigators have hypothesized the streaming liver model. On the other hand, others reject this hypothesis. We examined hepatic cell kinetics by a special labeling method with [3H]thymidine. METHODS: ICR mice received 112 repeated injections of [3H]thymidine at 6 h intervals for 28 days after birth and were killed immediately thereafter, or 100, 200 or 300 days after the last injection. Immediately after killing the animals, samples of the liver were taken and autoradiography was performed. RESULTS: After continuous labeling, more than 90% of the cells in the liver were labeled. Mean grain counts of hepatocytes decreased to half over approximately 100 days. Those of bile duct cells decreased at a slower rate (50%) than hepatocytes. Mean grain counts of hepatocytes decreased over the regions, although those in perivenular region decreased more rapidly in comparison to those in periportal region. CONCLUSIONS: The present study indicated that most cells in the liver arise postnatally. The changes in labeling of cells show that there is no special zone for proliferation of hepatocytes and they renew in all regions of the hepatic lobule, suggesting (i) that hepatocytes are supplied by postnatal replication and (ii) streaming of hepatocytes from periportal to pericentral regions does not occur in the adult mouse liver. The bile duct cells renewed more rapidly than hepatocytes.

Cell kinetics of slow renewing cell populations in mice stomach.

BACKGROUND: The renewal rates of parietal and chief cells in the gastric mucosa and smooth muscle cells of muscularis propria have not been examined as precisely as superficial epithelial cells. To examine cell renewal of these cells, continuous labeling with tritiated ([3H])-thymidine was performed. METHODS: Mice received 112 repeated injections of [3H]-thymidine at 6-hour intervals for 28 days after birth and were killed immediately thereafter, or 60, 120, 200 or 300 days after the last injection. RESULTS: After continuous labeling, most cells in the stomach were labeled. At 60 days, unlabeled parietal cells in the neck area of the gland and unlabeled chief cells in the middle part of the gland appeared. Thereafter, the area of unlabeled cells expanded downwards to the bottom of the gland. Times required for labeling of total cell populations of parietal and chief cells to half were less than 60 days and more than 200 days, respectively. At 300 days, most parietal cells and about half of the chief cells remained labeled in the bottom of the gland. The labeling index of smooth muscle cells was about 100% for 300 days. CONCLUSIONS: The time required for the newly formed parietal and chief cells to reach the lower end of the gland was more than 300 days. As a total cell population, the renewal rate of parietal cells was more rapid than that of chief cells. However, in terms of the downward migrating cell population, the renewal rate of parietal cells was a little slower than that of chief cells. Smooth muscle cells showed almost no renewal.

Heterogeneous cell renewal of pancreas in mice: [(3)H]-thymidine autoradiographic investigation.

INTRODUCTION: Although cell kinetics of the gastrointestinal mucosa has been extensively examined, that of the pancreas has not been fully analyzed. AIM: To determine the renewal rate of pancreatic cells directly. METHODOLOGY: Postnatal proliferative activity and cellular renewal of the parenchymal cells in ICR mouse pancreas were studied by immunohistochemistry and [(3)H]-thymidine autoradiography. RESULTS: In the single labeling experiments, the proliferative activity of the parenchymal cells in pancreas showed peaks at a few days after birth, decreased thereafter, and reached a low level at 2 months after birth. Continuous labeling experiments revealed that, in the adult pancreas, the half lives of acinar cells, islet cells, and duct epithelial cells were approximately 70 days, 47 days, and 40 days, respectively. Moreover, in the exocrine pancreas, acinar cells of the peri-insular region proliferated more actively than those of the tele-insular region. The renewal rate of glucagon cells was more rapid than that of insulin cells or somatostatin cells. Large ducts showed a high rate of cell renewal in comparison with small ducts. CONCLUSION: The results of this study indicate that cell renewal rates of the pancreas are not homogeneous, but heterogeneous.