Effects Of Cell Growth And Chemosensitivity After INK4a And ARF Genes Co-transfection In Human Lung Adenocarcinoma Cell Line A549

OBJECTIVE Two functionally and structurally different proteins, p16^INK4a and p14^ARF, encoded by INK4a/ARF gene located at chromsome 9p21 are cyclin dependent kinase(cdk) inhibitors. Both p16^INK4a and p14^ARF are cell cycle regulatory proteins and simultaneously play important roles through two most important cell cycle regulation pathways so far identified, p16^INK4a-CDK4/6-cyclin D-Rb-E2F and p14^ARF-MDM2-p53. More and more evidences have been accumulating to show that the exogenous p16^INK4a or p14^ARF can inhibit the cell growth and/or induce the apoptosis. But it is still unclear what effects they can play if combine the gene therapy with the conventional chemotherapy in cancer treatment. Here we show that cationic liposome-mediated gene co-transfection of INK4a and ARF into human lung adenocarcinoma cell line A549 can suppress the growth of A549 and induce apoptosis. Besides that, we investigate the effects on the chemosensitivity after the transfection. Methods The established human lung adenocarcinoma cell line A549 was used in our experiment. A549 cell has a deletion at the INK4A/ARF locus but retains intact wild-type p53, RB, CDK4/6, cyclin D, E2F and MDM2. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and sodium bicarbonate at 37℃in an incubator with 95% air and 5% CO₂. The eukaryocyte expression plasmids pcDNA3-p16^INK4a,pcDNA3-p14^ARF and the blank control plasmid pcDNA3-LacZ were used for transfection into A549.Transfection was performed according to the liposomal formulation DOTAP transfection reagent in 6-well plates and selected by G418.The cells containing both plasmids pcDNA3-p16^INK4a and pcDNA3-p14^ARF analyzed by RT-PCR, ICC and western blotting and the cells were used for the following experiments and named as A549-p16^INK4a-p14^ARF.The negative control cells transfected with pcDNA3-LacZ was named A549-vector. After the selection, the biological behavior of the A549-p16INK4a-p14ARF, A549-vector and A549 cells were measured. The growth curve was made according to the counting results. Flow cytometry(FCM) was performed to determine the cell cycle distribution and apoptosis index. Every experiment was repeated for three times. Colony formation rate was got by staining the cells with Coomassie brilliant blue and the surviving colony with more than 50 cells was available. To further investigate whether transfection of INK4a and ARF could confer increased cell's sensitivity to chemotherapeutic drugs. After the above job was finished, 2.0×103cells of A549-p16INK4a-p14ARF, A549-vector or A549 cells were added into the 96-well plates respectively and incubated for further 24hs.Then the medium was moved and replaced with the fresh medium containing the five commonly-used cytotoxic drugs (ADM, CDDP, NVB, TPT, Taxol) under different concentrations. The concentration of each drug varied from 0.001 to 10 times of the peak plasma concentration (PPC). After incubation for another 72h,the MTT assay was performed to measure the absorption values. RESULTS 1. The cells containing the products of both INK4a and ARF genes detected by RT-PCR and ICC. The results were consistent with the expectation. Western blot analysis was in agreement with the RT-PCR and ICC analysis results. 2. After the transfection, the introduction of exogenous INK4a and ARF caused significantly growth inhibition in comparison with the A549-vector and A549 cells. The colony formation ability of A549-p16INK4a-p14ARF is significantly weaker than that of A549-vector and A549.They were 63%, 87% and 85% respectively. There was no statistical difference between A549-vector and A549. By FCM, comparing with A549 and A549-vector, more percentage of A549-p16INK4a-p14ARF cells were unable to pass through the checkpoint G1 and were arrested at G0/G1 phase. The proportion of S phase and G2-M phase decreased at the same time. The percentage of A549-p16INK4a-p14ARF cells inhibited at G1 was 59.9%, 50.3% for A549-vector and 51.2% for A549. Besides that, the apoptosis index got a remarkable increase after the transfection. 3. After the transfected cells were treated with five different anticancer drugs, the dose-dependent inhibition of cell proliferation was obvious. The apoptosis indexes of A549 treated by ADM and CDDP were higher than treated by other three drugs. The IC50 valuesof ADM and CDDP remarkably decreased from 1.04±0.03μg/ml to 0.43±0.02μg/ml and from 1.03±0.03μg/ml to 0.54±0.02 μg/ml respectively. The IC50 values of NVB,TPT and Taxol were similar with that of untreated cells. CONCLUSIONS 1. It is efficient to co-transfect INK4a and ARF into human lung adenocarcinoma cell line A549 by the method of cationic liposome. 2. The exogenous INK4a and ARF expression can induce the arrest of A549 at G1 checkpoint and reduce the colony formation. At the same time, the transfection lead to a remarkable increase of apoptosis. 3. The transfection of INK4a and ARF into A549 can increases the chemosensitivity for ADM and CDDP but cann't change it for TPT, Taxol or NVB. This result might be the reason that different drugs have different mechanisms of anticancer.