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Activation of caspase-3 and cleavage of Rb are associated with p16-mediated apoptosis in human non-small cell lung cancer cells

Abstract

The p16 tumor suppressor gene is frequently inactivated in human cancer tissues and cell lines. We previously reported that wild-type p16 expression from an adenovirus vector (Adv/p16) induced p53-dependent apoptotic cell death in non-small cell lung cancer (NSCLC) cell lines. Here we show the potential mechanism of apoptosis induced by Adv/p16 infection. Infection of human NSCLC cell line A549, which carries the wild-type p53 gene, with Adv/p16 resulted in activation of caspase-3, accompanied by the cleavage of its substrate poly (ADP-ribose) polymerase (PARP), on day 3 of infection. The retinoblastoma (Rb) cell cycle regulator protein was also cleaved after activation of caspase-3; when the levels of Rb significantly diminished, apoptosis began. When A549 cells were pretreated with the caspase-inhibitory peptide N-acetyl-asp-Glu-Val-Asp-CHO (aldehyde) (Ac-DEVD-CHO), Adv/p16-mediated apoptosis and Rb cleavage were greatly inhibited. Furthermore, MDM2, a negative regulator of p53 expression was upregulated 3 days after Adv/p16 infection, and MDM2 was subsequently cleaved by caspase-3; MDM2 cleavage was inhibited by Ac-DEVD-CHO treatment. These data implied that cleavage of Rb, in addition to activation of caspase-3, represented a mechanism by which Adv/p16 induced apoptotic cell death in human NSCLC cells. Our results support the clinical relevance of Adv/p16 as a treatment for p16-null human NSCLC that express wild-type p53.

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Acknowledgements

We thank Dr Ta-Jen Liu for the gift of Adv/p16. We also thank Tohru Tanida, Yoshiko Takata and Kyoko Nasu for their technical support. This work was supported in part by grants from the Ministry of Education, Science, and Culture, Japan; by grants from the Ministry of Health and Welfare, Japan (Health Sciences Research Grants [Research on Human Genome and Gene Therapy]); by grants from the National Cancer Institute and the National Institutes of Health SPORE (2P50-CA70970-04) [JA Roth]; (P01 CA78778-01A1) [JA Roth]; by gifts to the Division of Surgery, from Tenneco and Exxon for the Core Laboratory Facility; by the UT M.D. Anderson Cancer Center Support Core Grant (CA 16672); by a grant from the Tobacco Settlement Funds as appropriate by the Texas State Legislature (Project 8), and The W. M. Keck Center for Cancer Gene Therapy [JA Roth].

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Correspondence to Toshiyoshi Fujiwara.

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Katsuda, K., Kataoka, M., Uno, F. et al. Activation of caspase-3 and cleavage of Rb are associated with p16-mediated apoptosis in human non-small cell lung cancer cells. Oncogene 21, 2108–2113 (2002). https://doi.org/10.1038/sj.onc.1205272

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