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C-terminal deletion mutant p21WAF1/CIP1 enhances E2F-1–mediated apoptosis in colon adenocarcinoma cells

Abstract

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 p21WAF1/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. IC25 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 p21WAF1/CIP1 (Ad-p21WT). 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 p21WT. 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.

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Acknowledgements

We are grateful to Dr Brent French for providing the Ad5CMV-LacZ, Dr TJ Liu for providing the Ad5CMV-E2F-1, and Dr Wafik S El-Diery for providing both p21 adenoviral vectors AdCMV-WAF1 and AdCMV-WAF1-341. We thank Sherri Matthews for expert assistance with manuscript preparation. This study was supported by Grant 96-55 from the American Cancer Society, Grant 96-46 from the Alliant Community Trust Fund, the Mary and Mason Rudd Foundation Award, and the Center for Advanced Surgical Technologies (CAST) of Norton Hospital.

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Correspondence to Kelly M McMasters.

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Elliott, M., Stilwell, A., Dong, Y. et al. C-terminal deletion mutant p21WAF1/CIP1 enhances E2F-1–mediated apoptosis in colon adenocarcinoma cells. Cancer Gene Ther 9, 453–463 (2002). https://doi.org/10.1038/sj.cgt.7700458

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