AIM： To investigate the expression of gastrin-releasing peptide （GRP） and GRP-receptor mRNA in non-tumor tissues of the human esophagus, gastrointestinal tract, pancreas and gallbladder using molecular biology tech...AIM： To investigate the expression of gastrin-releasing peptide （GRP） and GRP-receptor mRNA in non-tumor tissues of the human esophagus, gastrointestinal tract, pancreas and gallbladder using molecular biology techniques. METHODS： Poly A^＋ mRNA was isolated from total RNA extracts using an automated nucleic acid extractor and, subsequently, converted into single-stranded cDNA （sscDNA）. PCR amplifications were carried out using genespecific GRP and GRP-receptor primers. The specificity of the PCR amplicons was further confirmed by Southern blot analyses using gene-specific GRP and GRP-receptor hybridization probes. RESULTS： Expression of GRP and GRP-receptor mRNA was detected at various levels in nearly all segments of the non-tumor specimens analysed, except the gallbladder. In most of the biopsy specimens, coexpression of both GRP and GRP-receptor mRNA appeared to take place. However, expression of GRP mRNA was more prominent than was GRP-receptor mRNA. CONCLUSION： GRP and GRP-receptor mRNAs are expressed throughout the gastrointestinal tract and provides information for the future mapping and determination of its physiological importance in normal and tumor cells.展开更多
AIM： To analyse αv integrin expression induced by gas- trin in pancreatic cancer models.METHODS： αv integrin mRNA expression in human pan- creatic cancer cells was analysed using a ＂cancer genes＂ array and confi...AIM： To analyse αv integrin expression induced by gas- trin in pancreatic cancer models.METHODS： αv integrin mRNA expression in human pan- creatic cancer cells was analysed using a ＂cancer genes＂ array and confirmed by real-time reverse transcription- polymerase chain reaction （PCR）. Western blotting and semi-quantitative immunohistochemistry were used to examine protein levels in human pancreatic cancer cell lines and pancreatic tissues, respectively, The role of αv integrin on gastrin-induced cell adhesion was examined using blocking anti-αv integrin monoclonal antibodies. Adherent cells were quantified by staining with crystal violet.RESULTS： Using a ＂cancer genes＂ array we identi- fied c^v integrin as a new gastrin target gene in human pancreatic cancer cells. A quantitative real-time PCR approach was used to confirm αv integrin gene expression. We also demonstrate that Src family kinases and the PI 3-kinase, two signalling pathways specifically activated by the CCK-2 receptor （CCK2R）, are involved in gastrin-mediated αv integrin expression. In contrast, inhibition of the ERK pathway was without any effect on αv integrin expression induced by gastrin. Our results also show that gastrin modulates cell adhesion via αv integrins. Indeed, in vitro adhesion assays performed on fibronectin show that gastrin significantly increases adhesion of pancreatic cancer cells. The use of blocking anti-αv integrin monoclonal antibodies completely reversed the increase in cell-substrate adhesion induced by gastrin. In addition, we showed in vivo that the targeted CCK2R expression in the pancreas of Elas-CCK2 mice, leads to the overexpression of αv integrin. This process may contribute to pancreatic turnout development observed in these transgenic animals.CONCLUSION： αv integrin is a new gastrin target in pancreatic cancer models and contributes to gastrin effects on cell adhesion.展开更多
AIM： To explore the effect of gastrin 17 （G17） on β-catenin/T cell factor-4 （Tcf-4） signaling in colonic cancer cell line Colo320WT. METHODS： The pCR3.1/GR plasmid, which expresses gastrin receptor, cholecystok...AIM： To explore the effect of gastrin 17 （G17） on β-catenin/T cell factor-4 （Tcf-4） signaling in colonic cancer cell line Colo320WT. METHODS： The pCR3.1/GR plasmid, which expresses gastrin receptor, cholecystokinin-2 receptor （CCK-2R）, was transfected into a colonic cancer cell line Colo320 by Lipofectamine ^TM 2000 and the stably expressing CCK-2R clones were screened by G418. The expression levels of gastrin receptor in the Colo320 and the transfected Colo320WT cell line were assayed by RTPCR. Colo320WT cells were treated with G17 in a time-dependent manner （0, 1, 6, 12, 24 and 48 h）, then with L365,260 （Gastrin17 receptor blocker） for 30 rain, and with G17 again for 12 h or L365,260 for 12 h. Expression levels of β-catenin in a TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells treated with G17 were detected by co-immuniprecipation and Western blot. Immunocytochemistry was used to examine the distribution of β-catenin in CoLoWT320 cells. Expression levels of c-myc and cyclin D1 in Colo320WT cells treated with G17 were assayed by Western blot. RESULTS： Expression levels of β-catenin in the TX-100 solution fraction decreased apparently in a time- dependent fashion and reached the highest level after G17 treatment for 12 h, while expression levels of β-catenin in the TX-100 insoluble fraction were just on the contrary. Immunocytochemistry showed that β-catenin was translocated from the cell membranes into the cytoplasm and nucleus under G17 treatment. Expression levels of c-myc and cyclin D1 in the G17- treated Colo320WT cells were markedly higher compared to the untreated Colo320WT cells. In addition, the aforementioned G17-stimulated responses were blocked by L365,260.CONCLUSION： Gastrin17 activates β-catenin/Tcf-4 signaling in Colo320WT cells, thereby leading to over- expression of c-myc and cyclin D1.展开更多
AIM： TO investigate in vivo, whether CCK2 receptors （CCK2R） regulate proteins known to play a crucial role in cell proliferation and cancer development and analyse in vitro the molecular mechanisms that lead to Src...AIM： TO investigate in vivo, whether CCK2 receptors （CCK2R） regulate proteins known to play a crucial role in cell proliferation and cancer development and analyse in vitro the molecular mechanisms that lead to Src activation; in particular, to identify the domains within the CCK2R sequence that are implicated in this activation. METHODS： The expression and activation of Src and ERK were studied in vivo using immunofluorescence and western-blot techniques. We used pancreatic tissues derived from wild type or Elas-CCK2 mice that expressed the CCK2R in pancreatic acini, displayed an increased pancreatic growth and developed preneoplastic lesions. The pancreatic tumor cell line AR4-2J expressing the endogenous CCK2R or COS-7 cells transiently transfected with wild type or mutant CCK2R were used as in vitro models to study the mechanism of Src activation. Src activation was measured by in vitro kinase assays, ERK activation by western blot using antiphospho-ERK antibodies and the involvement of Src in gastrin-induced cell proliferation by MTT test. RESULTS： We showed in vivo that the targeted CCK2R expression in the pancreas of Elas-CCK2 mice, led to the activation of Src and the ERK pathway. Src was activated upstream of the ERK pathway by the CCK2R in pancreatic tumoral cells and contributed to the proliferative effects mediated by this receptor. In vitro results demonstrated that activation of the Src/ERK pathway by the CCK2R required the NPXXY motif, located within the CCK2R sequence at the end of the 7^th transmembrane domain, and suggested the putative role of Gq in this mechanism. CONCLUSION： Deregulation of the Src/ERK pathway by the CCK2R might represent an early step that contributes to cell proliferation, formation of preneoplastic lesions and pancreatic tumor development.展开更多
Gastrin(gas)is a peptide hormone that stimulates gastric acid secretion by gastric parietal cells and stimulates gastric motility.The cholecystokinin B receptor(cckbr)can act as a receptor for gastrin,conveying regula...Gastrin(gas)is a peptide hormone that stimulates gastric acid secretion by gastric parietal cells and stimulates gastric motility.The cholecystokinin B receptor(cckbr)can act as a receptor for gastrin,conveying regulatory information on gastrin,but there are fewer studies on its function in fish.The Lateolabrax maculatus is one of the marine aquaculture species in China,it widely distribute in coastal areas.In the study,we cloned the genes of Lateolabrax maculatus gastrin(Lm-gas)and Lateolabrax maculatus cholecystokinin B receptor(Lm-cckbr).The results showed that the full-length gene of Lm-gas is 638bp and the carboxy-terminal conserved domain(DFGRR)is the core functional domain of gastrin protein.The Lm-cckbr gene has a total nucleotide sequence of 2066 bp,and the open reading frame encodes a total of 453 amino acids.The result of protein sequence alignment showed that the similarity between Lm-cckbr protein and other different species was 50.11%-89.67%.The PCR results showed that Lm-gas and Lm-cckbr were expressed in brain and stomach.Further localization by immunehistochemical staining showed that Lm-gas protein was located in the mucosal layer of the gastric wall,but the expression signal was weak in the brain.Hunger causeed a significant decrease in these two genes.The results provided basic research data for further study on the function of Lm-gas and its recepter Lm-cckbr in the in the central nervous system and digestive system of Lateolabrax maculatus.展开更多
基金Supported by the Molecular Biology Program (Grant No.21407)Laboratory Medicine Center-LMC, University Hospital Linkoping, Swedenthe Development Foundation of Region Skane, Sweden
文摘AIM： To investigate the expression of gastrin-releasing peptide （GRP） and GRP-receptor mRNA in non-tumor tissues of the human esophagus, gastrointestinal tract, pancreas and gallbladder using molecular biology techniques. METHODS： Poly A^＋ mRNA was isolated from total RNA extracts using an automated nucleic acid extractor and, subsequently, converted into single-stranded cDNA （sscDNA）. PCR amplifications were carried out using genespecific GRP and GRP-receptor primers. The specificity of the PCR amplicons was further confirmed by Southern blot analyses using gene-specific GRP and GRP-receptor hybridization probes. RESULTS： Expression of GRP and GRP-receptor mRNA was detected at various levels in nearly all segments of the non-tumor specimens analysed, except the gallbladder. In most of the biopsy specimens, coexpression of both GRP and GRP-receptor mRNA appeared to take place. However, expression of GRP mRNA was more prominent than was GRP-receptor mRNA. CONCLUSION： GRP and GRP-receptor mRNAs are expressed throughout the gastrointestinal tract and provides information for the future mapping and determination of its physiological importance in normal and tumor cells.
文摘AIM： To analyse αv integrin expression induced by gas- trin in pancreatic cancer models.METHODS： αv integrin mRNA expression in human pan- creatic cancer cells was analysed using a ＂cancer genes＂ array and confirmed by real-time reverse transcription- polymerase chain reaction （PCR）. Western blotting and semi-quantitative immunohistochemistry were used to examine protein levels in human pancreatic cancer cell lines and pancreatic tissues, respectively, The role of αv integrin on gastrin-induced cell adhesion was examined using blocking anti-αv integrin monoclonal antibodies. Adherent cells were quantified by staining with crystal violet.RESULTS： Using a ＂cancer genes＂ array we identi- fied c^v integrin as a new gastrin target gene in human pancreatic cancer cells. A quantitative real-time PCR approach was used to confirm αv integrin gene expression. We also demonstrate that Src family kinases and the PI 3-kinase, two signalling pathways specifically activated by the CCK-2 receptor （CCK2R）, are involved in gastrin-mediated αv integrin expression. In contrast, inhibition of the ERK pathway was without any effect on αv integrin expression induced by gastrin. Our results also show that gastrin modulates cell adhesion via αv integrins. Indeed, in vitro adhesion assays performed on fibronectin show that gastrin significantly increases adhesion of pancreatic cancer cells. The use of blocking anti-αv integrin monoclonal antibodies completely reversed the increase in cell-substrate adhesion induced by gastrin. In addition, we showed in vivo that the targeted CCK2R expression in the pancreas of Elas-CCK2 mice, leads to the overexpression of αv integrin. This process may contribute to pancreatic turnout development observed in these transgenic animals.CONCLUSION： αv integrin is a new gastrin target in pancreatic cancer models and contributes to gastrin effects on cell adhesion.
基金Supported by the National Natural Science Foundation of China, No. 30470782
文摘AIM： To explore the effect of gastrin 17 （G17） on β-catenin/T cell factor-4 （Tcf-4） signaling in colonic cancer cell line Colo320WT. METHODS： The pCR3.1/GR plasmid, which expresses gastrin receptor, cholecystokinin-2 receptor （CCK-2R）, was transfected into a colonic cancer cell line Colo320 by Lipofectamine ^TM 2000 and the stably expressing CCK-2R clones were screened by G418. The expression levels of gastrin receptor in the Colo320 and the transfected Colo320WT cell line were assayed by RTPCR. Colo320WT cells were treated with G17 in a time-dependent manner （0, 1, 6, 12, 24 and 48 h）, then with L365,260 （Gastrin17 receptor blocker） for 30 rain, and with G17 again for 12 h or L365,260 for 12 h. Expression levels of β-catenin in a TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells treated with G17 were detected by co-immuniprecipation and Western blot. Immunocytochemistry was used to examine the distribution of β-catenin in CoLoWT320 cells. Expression levels of c-myc and cyclin D1 in Colo320WT cells treated with G17 were assayed by Western blot. RESULTS： Expression levels of β-catenin in the TX-100 solution fraction decreased apparently in a time- dependent fashion and reached the highest level after G17 treatment for 12 h, while expression levels of β-catenin in the TX-100 insoluble fraction were just on the contrary. Immunocytochemistry showed that β-catenin was translocated from the cell membranes into the cytoplasm and nucleus under G17 treatment. Expression levels of c-myc and cyclin D1 in the G17- treated Colo320WT cells were markedly higher compared to the untreated Colo320WT cells. In addition, the aforementioned G17-stimulated responses were blocked by L365,260.CONCLUSION： Gastrin17 activates β-catenin/Tcf-4 signaling in Colo320WT cells, thereby leading to over- expression of c-myc and cyclin D1.
基金Supported by funds from INSERM, the "Association pour la Recherche contre le Cancer" Grants, No. 3664, the "Region Midi Pyréeés". Audrey Ferrand was supported by the Ligue Nationale contre le cancer
文摘AIM： TO investigate in vivo, whether CCK2 receptors （CCK2R） regulate proteins known to play a crucial role in cell proliferation and cancer development and analyse in vitro the molecular mechanisms that lead to Src activation; in particular, to identify the domains within the CCK2R sequence that are implicated in this activation. METHODS： The expression and activation of Src and ERK were studied in vivo using immunofluorescence and western-blot techniques. We used pancreatic tissues derived from wild type or Elas-CCK2 mice that expressed the CCK2R in pancreatic acini, displayed an increased pancreatic growth and developed preneoplastic lesions. The pancreatic tumor cell line AR4-2J expressing the endogenous CCK2R or COS-7 cells transiently transfected with wild type or mutant CCK2R were used as in vitro models to study the mechanism of Src activation. Src activation was measured by in vitro kinase assays, ERK activation by western blot using antiphospho-ERK antibodies and the involvement of Src in gastrin-induced cell proliferation by MTT test. RESULTS： We showed in vivo that the targeted CCK2R expression in the pancreas of Elas-CCK2 mice, led to the activation of Src and the ERK pathway. Src was activated upstream of the ERK pathway by the CCK2R in pancreatic tumoral cells and contributed to the proliferative effects mediated by this receptor. In vitro results demonstrated that activation of the Src/ERK pathway by the CCK2R required the NPXXY motif, located within the CCK2R sequence at the end of the 7^th transmembrane domain, and suggested the putative role of Gq in this mechanism. CONCLUSION： Deregulation of the Src/ERK pathway by the CCK2R might represent an early step that contributes to cell proliferation, formation of preneoplastic lesions and pancreatic tumor development.
基金This project was supported by the National Key Research and Development Program of China(2017YFC0506003)Blue Granary Science and Technology Innovation Project,and Shanghai Collaborative Innovation Center of Animal Genetics and Breeding(ZF1206).
文摘Gastrin(gas)is a peptide hormone that stimulates gastric acid secretion by gastric parietal cells and stimulates gastric motility.The cholecystokinin B receptor(cckbr)can act as a receptor for gastrin,conveying regulatory information on gastrin,but there are fewer studies on its function in fish.The Lateolabrax maculatus is one of the marine aquaculture species in China,it widely distribute in coastal areas.In the study,we cloned the genes of Lateolabrax maculatus gastrin(Lm-gas)and Lateolabrax maculatus cholecystokinin B receptor(Lm-cckbr).The results showed that the full-length gene of Lm-gas is 638bp and the carboxy-terminal conserved domain(DFGRR)is the core functional domain of gastrin protein.The Lm-cckbr gene has a total nucleotide sequence of 2066 bp,and the open reading frame encodes a total of 453 amino acids.The result of protein sequence alignment showed that the similarity between Lm-cckbr protein and other different species was 50.11%-89.67%.The PCR results showed that Lm-gas and Lm-cckbr were expressed in brain and stomach.Further localization by immunehistochemical staining showed that Lm-gas protein was located in the mucosal layer of the gastric wall,but the expression signal was weak in the brain.Hunger causeed a significant decrease in these two genes.The results provided basic research data for further study on the function of Lm-gas and its recepter Lm-cckbr in the in the central nervous system and digestive system of Lateolabrax maculatus.
