C-terminal deletion mutant p21(WAF1/CIP1) enhances E2F-1-mediated apoptosis in colon adenocarcinoma cells.
The present study was designed to investigate the efficacy of combination gene therapy using adenoviral vectors expressing gene products shown to possess apoptotic activity: E2F-1 (Ad-E2F-1) and a C-terminal deletion mutant of p21(WAF1/cIP1) (Ad-p21(-PCNA)), on growth inhibition and apoptosis of human colon cancer cells in vitro and in vivo. Marked E2F-1 and p21(-PCNA) overexpression in response to adenovirus infection was evident by Western blot analysis. IC(25) concentrations of each virus were used for each treatment in vitro to detect cooperative effects on cell death. Coexpression of E2F-1 and p21(-PCNA) resulted in an additive effect on cell death compared to infection with either virus alone. Cell cycle analysis, poly(ADP-ribose) polymerase (PARP) cleavage and analysis of cell morphology also revealed that coinfection with both Ad-E2F-1 and Ad-p21(-PCNA) enhanced cellular apoptosis compared to either virus alone. Interestingly, E2F-1 protein expression was markedly enhanced in the E2F-1/p21(-PCNA) adenovirus combination compared to Ad-E2F-1 infection alone. However, these same effects were not evident in cells coinfected with Ad-E2F-1 and an adenovirus expressing wild-type human p21(WAF1/CIP1) (Ad-p21(WT)). The increase in E2F-1 expression with coexpression of E2F-1 and p21(-PCNA) was not a result of increased E2F-1 protein stability, but was related to increased transcriptional activity from the CMV promoter. Cell cycle analysis revealed G1 arrest 72 hours following single-gene therapy with either the wild-type or mutant p21, whereas increased accumulation of cells in G2/M phase was demonstrated in the E2F-1-overexpressing cells. In the combined therapies, E2F-1/p21(-PCNA) treatment still resulted in G1 arrest, but E2F-1 was able to counteract the G1 arrest when coinfected with p21(WT). These results provide further evidence of the importance of the p21:PCNA-binding domain in mediating the complex cell cycle interaction between E2F-1 and p21. Simultaneous intratumoral injection of Ad-E2F-1 and Ad-p21(-PCNA) dramatically reduced tumor burden of SW620 xenografts compared to either treatment alone in our in vivo model but not in HT-29 colon cancer xenografts. When combined with Ad-p21(-PCNA), E2F-1 adenovirus therapy resulted in approximately 95% decrease in tumor volume of SW620 tumor xenografts compared with controls (P<.05). In conclusion, although simultaneous delivery of E2F-1 and p21(-PCNA) transgenes results in increased E2F-1 expression and enhanced apoptosis of both SW620 and HT-29 colon cancer cells in vitro, this combination was only effective in the treatment of SW620 metastatic colon cancer in vivo. This may represent a potentially useful combination gene therapy strategy for metastatic colon cancer.
Elliott
Mary Jane
MJ
Department of Surgery, University of Louisville, James Graham Brown Cancer Center, Louisville, KY 40202, USA.
Stilwell
Ariel
A
Dong
Yan Bin
YB
Yang
Hai Liang
HL
Wong
Sandra L
SL
Wrightson
William R
WR
Martin
Robert C G
RC
McMasters
Kelly M
KM
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D016923:Cell Death; D002469:Cell Separation; D002470:Cell Survival; D003110:Colonic Neoplasms / Q000235:genetics* / Q000473:pathology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics*; D003587:Cytomegalovirus / Q000235:genetics; D053938:DNA Fragmentation; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D005434:Flow Cytometry; D016195:G2 Phase; D017353:Gene Deletion; D015316:Genetic Therapy; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008938:Mitosis; D009154:Mutation; D009368:Neoplasm Transplantation; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics*; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000378:metabolism
C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C493754:E2f1 protein, mouse; D012333:RNA, Messenger; D014157:Transcription Factors; D001616:beta-Galactosidase