Activation of caspase-3 and cleavage of Rb are associated with p16-mediated apoptosis in human non-small cell lung cancer cells.
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.
Katsuda
Koh
K
Division of Surgical Oncology, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan.
Kataoka
Masafumi
M
Uno
Futoshi
F
Murakami
Takayoshi
T
Kondo
Tadashi
T
Roth
Jack A
JA
Tanaka
Noriaki
N
Fujiwara
Toshiyoshi
T
D000256:Adenoviridae; D017209:Apoptosis; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D004789:Enzyme Activation; D005822:Genetic Vectors; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*; D009687:Nuclear Proteins; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D016160:Retinoblastoma Protein / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured
D061945:Caspase Inhibitors; D019941:Cyclin-Dependent Kinase Inhibitor p16; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases