AIM:There are conflicting data about p53 function on cellular sensitivity to the cytotoxic action of 5-fluorouracil (5-FU). Therefore the objective of this study was to determine the combined effects of adenovirus-med...AIM:There are conflicting data about p53 function on cellular sensitivity to the cytotoxic action of 5-fluorouracil (5-FU). Therefore the objective of this study was to determine the combined effects of adenovirus-mediated wild-type (wt) p53 gene transfer and 5-FU chemotherapy on pancreatic cancer cells with different p53 gene status. METHODS:Human pancreatic cancer cell lines Capan-1^(p53mut), Capan-2^(p53wt),FAMPAC^(p53mut),PANC1^(p53mut),and rat pancreatic cancer cell lines AS^(p53wt) and DSL6A^(p53null) were used for in vitro studies.Following infection with different ratios of Ad- p53-particles (MOI) in combination with 5-FU,proliferation of tumor cells and apoptosis were quantified by cell proliferation assay (WST-1) and FACS (PI-staining).In addition,DSL6A syngeneic pancreatic tumor cells were inoculated subcutaneously in to Lewis rats for in vivo studies. Tumor size,apoptosis (TUNEL) and survival were determined. RESULTS:Ad-p53 gene transfer combined with 5-FU significantly inhibited tumor cell proliferation and substantially enhanced apoptosis in all four cell lines with an alteration in the p53 gene compared to those two cell lines containing wt-p53.In vivo experiments showed the most effective tumor regression in animals treated with Ad-p53 plus 5-FU.Both in vitro and in vivo analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by 5-FU. CONCLUSION:Our results suggest that Ad-p53 may synergistically enhance 5-FU-chemosensitivity most strikingly in pancreatic cancer cells lacking p53 function.These findings illustrate that the anticancer efficacy of this combination treatment is dependent on the p53 gene status of the target tumor cells.展开更多
OBJECTIVE: To determine the efficacy of adenovirus mediated suicide gene transduction combined with prodrug 5-fluorocytosine (5FC) as a therapeutic protocol for pancreatic cancer. METHODS: Cytosine Deaminase(CD) gene...OBJECTIVE: To determine the efficacy of adenovirus mediated suicide gene transduction combined with prodrug 5-fluorocytosine (5FC) as a therapeutic protocol for pancreatic cancer. METHODS: Cytosine Deaminase(CD) gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD and pAdEasy-1 were recombined in bacteria. The newly recombined adenovirus (Ad)-CD containing green fluorescent protein (GFP) were packaged and propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic carcinoma cell line-Patu8988 was infected with this virus, then 5FC was added. XTT assay was used to estimate relative numbers of viable cells. In vivo model of pancreatic cancer was established by injecting 1.0 x 10(7) Patu8988 cells subcutaneously in Balb/c nude mice. When tumors were palpable, Ad-CD was injected into each tumor and 5FC was administered. RESULTS: Positive clones were selected using endonuclease to digest the recombinants and the concentration of viral liquids containing the CD gene was 2 x 10(11) pfu /ml. Significant cytotoxic activity as shown for 5FC in the CD gene transduced 8988 cell line, while little effect was found in the nontransduced pancreatic carcinoma cells. Antitumor effect was observed in Patu8988 xenograft nude mice with in situ CD gene transduction. CONCLUSIONS: CD gene mediated by adenovirus has high infectivity and may be useful for gene therapy in pancreatic carcinoma. These data demonstrate the use of an enzyme prodrug strategy in experimental pancreatic cancer.展开更多
文摘AIM:There are conflicting data about p53 function on cellular sensitivity to the cytotoxic action of 5-fluorouracil (5-FU). Therefore the objective of this study was to determine the combined effects of adenovirus-mediated wild-type (wt) p53 gene transfer and 5-FU chemotherapy on pancreatic cancer cells with different p53 gene status. METHODS:Human pancreatic cancer cell lines Capan-1^(p53mut), Capan-2^(p53wt),FAMPAC^(p53mut),PANC1^(p53mut),and rat pancreatic cancer cell lines AS^(p53wt) and DSL6A^(p53null) were used for in vitro studies.Following infection with different ratios of Ad- p53-particles (MOI) in combination with 5-FU,proliferation of tumor cells and apoptosis were quantified by cell proliferation assay (WST-1) and FACS (PI-staining).In addition,DSL6A syngeneic pancreatic tumor cells were inoculated subcutaneously in to Lewis rats for in vivo studies. Tumor size,apoptosis (TUNEL) and survival were determined. RESULTS:Ad-p53 gene transfer combined with 5-FU significantly inhibited tumor cell proliferation and substantially enhanced apoptosis in all four cell lines with an alteration in the p53 gene compared to those two cell lines containing wt-p53.In vivo experiments showed the most effective tumor regression in animals treated with Ad-p53 plus 5-FU.Both in vitro and in vivo analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by 5-FU. CONCLUSION:Our results suggest that Ad-p53 may synergistically enhance 5-FU-chemosensitivity most strikingly in pancreatic cancer cells lacking p53 function.These findings illustrate that the anticancer efficacy of this combination treatment is dependent on the p53 gene status of the target tumor cells.
基金ThisworkwassurpportedbyScientificCommitteeFoundationofShanghai (No 9941190 44 )
文摘OBJECTIVE: To determine the efficacy of adenovirus mediated suicide gene transduction combined with prodrug 5-fluorocytosine (5FC) as a therapeutic protocol for pancreatic cancer. METHODS: Cytosine Deaminase(CD) gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD and pAdEasy-1 were recombined in bacteria. The newly recombined adenovirus (Ad)-CD containing green fluorescent protein (GFP) were packaged and propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic carcinoma cell line-Patu8988 was infected with this virus, then 5FC was added. XTT assay was used to estimate relative numbers of viable cells. In vivo model of pancreatic cancer was established by injecting 1.0 x 10(7) Patu8988 cells subcutaneously in Balb/c nude mice. When tumors were palpable, Ad-CD was injected into each tumor and 5FC was administered. RESULTS: Positive clones were selected using endonuclease to digest the recombinants and the concentration of viral liquids containing the CD gene was 2 x 10(11) pfu /ml. Significant cytotoxic activity as shown for 5FC in the CD gene transduced 8988 cell line, while little effect was found in the nontransduced pancreatic carcinoma cells. Antitumor effect was observed in Patu8988 xenograft nude mice with in situ CD gene transduction. CONCLUSIONS: CD gene mediated by adenovirus has high infectivity and may be useful for gene therapy in pancreatic carcinoma. These data demonstrate the use of an enzyme prodrug strategy in experimental pancreatic cancer.
