[Effects of wild-type INK4a/ARF gene on biological behavior of lung adenocarcinoma cell line A549].

BACKGROUND

p16INK4a and p14ARF, encoded by gene INK4a/ARF located at chromosome 9p21, are cyclin dependent kinase (CDK) inhibitors. Both p16INK4a and p14ARF are cell cycle regulatory proteins and play an important role in Rb and p53 passways respectively. In this study, wild-type INK4a/ARF gene was transfected into human lung adenocarcinoma cell line A549, in which this gene site was lost, and the effects on the cell's biological behavior were investigated.

METHODS

The recombinant eukaryotic expression plasmids pcDNA3-p16INK4a and pcDNA3-p14ARF were transfected into A549 by cationic liposome method. By RT-PCR, immunocytochemistry and Western blot after G418 selection, A549 cells that could stably express p16INK4a and p14ARF were obtained. As a control, the parental cell and negative control cell with plasmid pcDNA3-LacZ were used. Inhibition of proliferation was measured by MTT assay. The cell growth curve was drawn according to cell counts. Cell cycle distribution was measured by flow cytometry (FCM), the apoptosis indexes were observed at the same time. The colony formation rate was counted by staining the cells with Coomassie brilliant blue.

RESULTS

The introduction of exogenous INK4a and ARF caused significantly growth inhibition of A549. By FCM, more percentage of A549-p16INK4a-p14ARF cells couldn't pass through the checkpoint G1. The percentage of A549-p16INK4a-p14ARF cells inhibited at G0/G1 was 59.9%, 50.3% for A549-vector and 51.2% for A549. The statistical differences were significant between A549-p16INK4a-p14ARF cell and A549-vector cell (P=0.025) and between A549-p16INK4a-p14ARF cell and A549 cell (P=0.043). The apoptosis index of A549-p16INK4a-p14ARF cell was 8.0% and 2.7% for both A549-vector and A549 cell (P < 0.01). The colony formation ability of A549-p16INK4a-p14ARF was weaker than that of A549-vector and A549, they were 63%, 87% and 85% respectively.

CONCLUSIONS

The wild-type INK4a/ARF gene can be co-introduced effectively into A549 cell by cationic liposome method. The reexpression of p16INK4a and p14ARF in A549 can inhibit the growth and enhance the apoptosis. This trial will be helpful in using gene therapy of lung cancer in the future.