Katsuda Koh, Kataoka Masafumi, Uno Futoshi, Murakami Takayoshi, Kondo Tadashi, Roth Jack A, Tanaka Noriaki, Fujiwara Toshiyoshi
Division of Surgical Oncology, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan.
Oncogene. 2002 Mar 27;21(13):2108-13. doi: 10.1038/sj.onc.1205272.
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.
p16肿瘤抑制基因在人类癌症组织和细胞系中经常失活。我们之前报道过，腺病毒载体（Adv/p16）表达的野生型p16可在非小细胞肺癌（NSCLC）细胞系中诱导p53依赖性凋亡细胞死亡。在此我们展示了Adv/p16感染诱导凋亡的潜在机制。携带野生型p53基因的人类NSCLC细胞系A549用Adv/p16感染后，在感染第3天导致半胱天冬酶-3激活，同时伴随其底物聚（ADP-核糖）聚合酶（PARP）的裂解。视网膜母细胞瘤（Rb）细胞周期调节蛋白在半胱天冬酶-3激活后也发生裂解；当Rb水平显著降低时，凋亡开始。当A549细胞用半胱天冬酶抑制肽N-乙酰-天冬氨酸-谷氨酸-缬氨酸-天冬氨酸-醛（Ac-DEVD-CHO）预处理后，Adv/p16介导的凋亡和Rb裂解受到极大抑制。此外，p53表达的负调节因子MDM2在Adv/p16感染3天后上调，随后MDM2被半胱天冬酶-3裂解；Ac-DEVD-CHO处理可抑制MDM2裂解。这些数据表明，除了半胱天冬酶-3激活外（此处原英文有误，已修正为“除了半胱天冬酶-3激活外”），Rb裂解是Adv/p16诱导人类NSCLC细胞凋亡性细胞死亡的一种机制。我们的结果支持Adv/p16作为治疗表达野生型p53的p16缺失型人类NSCLC的临床相关性。
