Adenovirus-mediated E2F-1 gene transfer induces an apoptotic response in human gastric carcinoma cells that is enhanced by cyclin dependent kinase inhibitors.
E2F -1 is a transcription factor that regulates cell cycle progression into S-phase. Deregulation of E2F-1 activity has been associated with cellular commitment to apoptosis. Also critical in the regulation of S-phase are the actions of the cyclin dependent kinases, Cdk2 and cdc2. Inhibition of these cyclin dependent kinases has been similarly associated with disrupting orderly S-phase progression and causing subsequent apoptosis in certain cancer cells. In this study, we examine the ability of adenovirus-mediated E2F-1 overexpression to induce apoptosis in human gastric carcinoma cells. Furthermore, we investigate the effect of the cyclin dependent kinase inhibitors, olomoucine and roscovitine, on E2F-1-mediated apoptosis in human gastric carcinoma cells. AGS and SNU-1 gastric adenocarcinoma cells were infected with adenoviral vectors expressing E2F-1 (Ad5CMVE2F-1) or control viruses expressing beta-galactosidase (Ad5CMVLacZ) or lacking a transgene (Ad5). Gastric adenocarcinoma cells were then independently treated with roscovitine or olomoucine. Finally, gastric adenocarcinoma cells were infected with the various adenoviral vectors in combination with roscovitine or olomoucine. E2F-1 overexpression resulted in an 85% reduction in cell viability at 72 h compared to controls. Combining E2F-1 overexpression with roscovitine resulted in >99% reduction in cell viability by 72 h. Overexpression of E2F-1 resulted in premature S-phase entry and G2/M arrest at 24 h, followed by apoptosis by 72 h. Combining E2F-1 overexpression with roscovitine resulted in an earlier G2/M arrest, followed by a more complete, widespread apoptotic response by 24 h. Caspase 3/CPP32 activation and PARP cleavage in response to E2F-1 overexpression, alone and in combination with roscovitine, implicate the caspase cascade in E2F-1-mediated apoptosis of gastric cancer cells. Bax levels also increased in response to E2F-1 gene transfer, alone and in combination with roscovitine. E2F-1 overexpression induces widespread apoptosis in human gastric carcinoma cells. Combining E2F-1 overexpression with cyclin-dependent kinase inhibitors results in an enhanced apoptotic response, causing nearly complete gastric tumor cell death within 72 h. E2F-1 gene therapy in combination with cyclin dependent kinase inhibitors is a potentially active chemogene therapy strategy for the treatment of human gastric cancer.
Atienza
C
C
Department of Surgery, Division of Surgical Oncology, University of Louisville School of Medicine, J. Graham Brown Cancer Center, Louisville, Kentucky 40202, USA.
Elliott
M J
MJ
Dong
Y B
YB
Yang
H L
HL
Stilwell
A
A
Liu
T J
TJ
McMasters
K M
KM
D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D018797:Cell Cycle Proteins; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D018014:Gene Transfer Techniques; D006801:Humans; D007701:Kinetin; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D011687:Purines / Q000494:pharmacology; D056465:Retinoblastoma-Binding Protein 1; D000077546:Roscovitine; D013274:Stomach Neoplasms; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011687:Purines; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D051028:bcl-2-Associated X Protein; D000077546:Roscovitine; C090046:olomoucine; D011065:Poly(ADP-ribose) Polymerases; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D020169:Caspases; D007701:Kinetin