Backgrownd and aim: Gene therapy using diphteria toxin (DT) is a promising tool for cancer cell killing. It causes a rapid cell death catalysing ADP-ribosylation of elongating factor 2 and kills cells by an apoptosis-mediated pathway. Our aim was to test the ability of the DT catalytic subunit (fragment A comprising 1-193 aa residues) (DTA) to kill 5 different pancreatic cancer cell lines, four obtained from primary tumors (BxPC3, PANC1, PSN1 and MIAPaCa2) and one from hepatic metastasis (CAPAN1). Methods: DTA was subcloned in an eukariotic expression vector (pRc) under the control of a constitutive promoter (RSV). Lipofectamine 2000 was used to transfer the DNA vector into the cells. The efficiency of transfection was evaluated by FACS analysis, using a FITC-oligonucleotide as tracer, 24 hours after transfection. The efficiency of transcription was evaluated by FACS analysis and western blotting after 48 hours from transfection with a reporter gene (GFP) cloned in pRc. Study design was: 1. transfection of 250.000 cells for 6 h with 4 ug DNA; 2. after 24 h cells were seeded in quadruplicate in a 96-well plate; 3. cell growth was evaluated daily for 3 days by the cell viability XTT test. Results: More than 50% of cells were efficiently transfected. GFP transcription was recorded in four out of the five cell lines studied: CAPAN- 1 did not translate the inserted gene. In agreement this cell line was resistant to DTA gene transfer. A complete growth inhibition was achieved after DTA gene transfer in BxPC3, PANC1, PSN1 and MIAPaCa2 (figure). Conclusions: DTA expression is lethal for pancreatic cancer cells and this supports the potential use of this toxin for pancreatic cancer gene therapy. The lack of gene translation found in CAPAN-1 cells, a metastatic cell line, might be consequent to the absence of specific transcription factors recognizing the RSV promoter.
Engineering diphteria toxin in pancreatic cancer cells: A promising tool for therapy
FOGAR, PAOLA;GRECO, ELIANA;ZAMBON, CARLO-FEDERICO;BASSO, DANIELA;PEDRAZZOLI, SERGIO;PLEBANI, MARIO
2006
Abstract
Backgrownd and aim: Gene therapy using diphteria toxin (DT) is a promising tool for cancer cell killing. It causes a rapid cell death catalysing ADP-ribosylation of elongating factor 2 and kills cells by an apoptosis-mediated pathway. Our aim was to test the ability of the DT catalytic subunit (fragment A comprising 1-193 aa residues) (DTA) to kill 5 different pancreatic cancer cell lines, four obtained from primary tumors (BxPC3, PANC1, PSN1 and MIAPaCa2) and one from hepatic metastasis (CAPAN1). Methods: DTA was subcloned in an eukariotic expression vector (pRc) under the control of a constitutive promoter (RSV). Lipofectamine 2000 was used to transfer the DNA vector into the cells. The efficiency of transfection was evaluated by FACS analysis, using a FITC-oligonucleotide as tracer, 24 hours after transfection. The efficiency of transcription was evaluated by FACS analysis and western blotting after 48 hours from transfection with a reporter gene (GFP) cloned in pRc. Study design was: 1. transfection of 250.000 cells for 6 h with 4 ug DNA; 2. after 24 h cells were seeded in quadruplicate in a 96-well plate; 3. cell growth was evaluated daily for 3 days by the cell viability XTT test. Results: More than 50% of cells were efficiently transfected. GFP transcription was recorded in four out of the five cell lines studied: CAPAN- 1 did not translate the inserted gene. In agreement this cell line was resistant to DTA gene transfer. A complete growth inhibition was achieved after DTA gene transfer in BxPC3, PANC1, PSN1 and MIAPaCa2 (figure). Conclusions: DTA expression is lethal for pancreatic cancer cells and this supports the potential use of this toxin for pancreatic cancer gene therapy. The lack of gene translation found in CAPAN-1 cells, a metastatic cell line, might be consequent to the absence of specific transcription factors recognizing the RSV promoter.Pubblicazioni consigliate
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