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Down-regulation of bcl-2 is associated with p16INK4-mediated apoptosis in non-small cell lung cancer cells

Abstract

We introduced a functional p16 cDNA into non-small cell lung cancer (NSCLC) cell lines expressing different combinations of normal and mutated p16, p53, and Rb genes via a recombinant adenovirus to determine the effect of exogenous p16 expression on cell growth. Analysis of p16-deficient cells infected with Adv/p16 identified growth arrest of the cells in the G0–G1 phase early on. Apoptosis was identified to occur by the 5th day after infection which corresponded with increased p16 expression, reduced Rb expression, and increased Rb hypophosphorylation, but only occurred in cells expressing functional p53. Further analysis indicated that the expression of the anti-apoptotic protein bcl-2 was greatly reduced in the NSCLC cell lines H460 and A549 (both −p16, +p53, +Rb), again only by the 5th day after Adv/p16 infection, but no affect on Bax expression was observed. H1299 cells (−p16, −p53, +Rb) infected with Adv/p16 only exhibited apoptosis by an additional infection with Adv/p53 which also corresponded with a down-regulation of bcl-2. In addition, the infection of A549 cells with Adv/p16 followed by a subsequent infection with Adv/Rb lead to a significant decrease in apoptosis which correlated with an increase in bcl-2 expression. These studies suggest that p16 is capable of mediating apoptosis in NSCLC cell lines expressing wild-type p53, through a direct down-regulation of Rb and an indirect down-regulation of the anti-apoptotic protein bcl-2.

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Abbreviations

Adv:

adenovirus

CMV:

cytomegalovirus enhancer/promoter

FACS:

Fluorescence Automated Cell Sorting

MOI:

multiplicity of infection

NSCLC:

non-small lung cell cancer

Rb:

retinoblastoma susceptibility gene

TUNEL:

terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling

β-gal:

E-coli β-galactosidase gene

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Acknowledgements

We would like to thank Ms. Monica Spears for help in the preparation of this manuscript. We would also like to thank Ms. Karen Ramirez for her help in the FACS analysis of cells. This study was partially supported by grants from the National Cancer Institute Training (T32-09599-08) (F Spitz); by M.D. Anderson Clinical Fellows Research Award (F Spitz); by gifts to the Division of Surgery from Tenneco and Exxon for the Core Laboratory Facility; by the M.D. Anderson Cancer Center Support Core Grant (CA16672); Specialized Program of Research Excellence (SPORE) in Lung Cancer (P50-CA70907); and by a grant from the Mathers Foundation.

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Kataoka, M., Wiehle, S., Spitz, F. et al. Down-regulation of bcl-2 is associated with p16INK4-mediated apoptosis in non-small cell lung cancer cells. Oncogene 19, 1589–1595 (2000). https://doi.org/10.1038/sj.onc.1203466

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