Effect of exogenous p16ink4a and hRb1 genes on cell cycle regulation of osteosarcoma cell

Abstract

To study the effect on regulation of cell cycle of osteosarcoma cell line MG63 tranceduced with exogenous p16ink4a and hRb1 genes, pIRES-p16ink4a-hRbl, PIRES-p16ink4a and pIRES-hRb1 plasmids were constructed by gene recombination technology. The recombinant plasmid was transferred into osteosarcoma cell line MG63 by metafectene, and the resistant clones were selected by G418 selective medium. mRNA and protein expression of osteosarcoma cell line were assayed by RT-PCR and Western-Blot respectively. Cell cycle and apoptosis were analyzed by subG1 flow cytometric. Cell proliferation was tested by MTT. In the genome of these transfected target cells, the expression of p16ink4a and hRb1 mRNA and protein were detected respectivelyin vitro. It was demonstrated with subG₁ flow cytometric analysis and MTT method that p16ink4a and hRb1 genes cooperation more significantly inhibited cell growth and induced a more marked G1 arrest and apoptosis than p16ink4a/hRb1 alone (P<0.01). Coexpression of exogenous p16ink4a with hRb1 broke the regulatory feedback loop of p16ink4a-cyclinD₁/CDK-hRb1 and played a more significant role in inhibiting cell growth as well as inducing cell apoptosis than p16ink4a or hRb1 did alonein vitro.

Keywords

p16ink4a / rRb1 / Cell cycle / Flow cytometric

References

[1] ChatterjeeS J, GeorgeB, GoebellP J, et al. . Hyperphosphorylation of pRb: a mechanism for RB tumour suppressor pathway inactivation in bladder cancer. J Pathol, 2004, 203: 762-762

[2] SharplessE, ChinL. The INK4a/ARF locus and melanoma. Oncogene, 2003, 22: 3092-3092

[3] NakamuraM, SakakiT, HashimotoH, et al. . Frequent alterations of the p14(ARF) and p16(INK4a) genes in primary central nervous system lymphomas. Cancer Res, 2001, 61: 6335-6335

[4] FrostS J, SimpsonD J, ClaytonR N, et al. . Transfection of an inducible p16/CDKN2A construct mediates reversible growth inhibition and G1 arrest in the AtT20 pituitary tumor cell line. Mol Endocrinol, 1999, 13: 1801-1801

[5] GongJ, TraganosF, DarzynkiewiczZ. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal Biochem, 1994, 218: 314-314

[6] ParkY B, ParkM J, KimuraK, et al. . Alterations in the INK4a/ARF locus and their effects on the growth of human osteosarcoma cell lines. Cancer Genet Cytogenet, 2002, 133: 105-105

[7] IidaK, NoboriT, MatsumineA, et al. . Effect of retinoblastoma tumor suppressor gene expression on chemosensitivity of human osteosarcoma cell lines. Oncol Rep, 2003, 10: 1961-1961

[8] SteinerM S, ZhangY A. Adenoviral vector containing wild type p16 suppresses prostate cancer growth and prolongs survival by inducing cell senescence. Cancer Gene Ther, 2000, 7: 360-360

[9] NaruseI, HeikeY, HamaS, et al. . High concentrations of recombinant adenovirus expression p16 gene induces apoptosis in lung cancer cell lines. Anticancer Res, 1998, 18: 4275-4275