Abstract
BackgroundThe tumor suppressor gene CDKN2A generates at least three different transcriptional variants, each of which is thought to encode a tumor suppressor. However, the inhibitory activities of these variants have not yet been compared in the same cells. Protein therapy is known to have several advantages over gene therapy. Thus, investigation of the exogenous protein molecule of the most effective suppressor may yield meaningful information regarding protein-based cancer therapy.MethodsThe inhibitory effects of p16INK4a, p14ARF and p12 were studied in the human lung cancer cell line A549 which lacks the CDKN2A locus. The eukaryotic expression plasmids of the three transcriptional variants were constructed and stably transfected into the cells. RNA and protein expression by the plasmids was confirmed using RT-PCR and fluorescence immunocytochemistry, respectively. Cell growth inhibition and cell-cycle redistribution after transfection were investigated based on growth curve and flow cytometry analyses. An exogenous His-tag fusion p16INK4a protein was obtained and purified by affinity chromatography. Cell growth inhibition and cell cycle arrest induced by the expression of p16INK4a protein were measured in A549 cells transduced with the exogenous protein.ResultsWhile all three variants suppressed cell growth, p16INK4a had the strongest effect. Marked G1-phase accumulation and S-phase inhibition were induced by p16INK4a and p14ARF but not by p12. Exogenous p16INK4a protein was successfully expressed and purified and transduction of the fusion protein into A549 cells inhibited cell growth by G1→S arrest.ConclusionsAmong the three transcript variants, p16INK4a has a greater inhibitory effect than p14ARF and p12; exogenous p16INK4a protein should be further investigated for use in cancer therapy as a protein agent.
Highlights
The tumor suppressor gene CDKN2A generates at least three different transcriptional variants, each of which is thought to encode a tumor suppressor
The protein products of these transcripts function via different pathways. p16INK4a binds to the cyclin-dependent kinases CDK4/6, thereby inhibiting the phosphorylation of the retinoblastoma protein and causing cell-cycle arrest at the G1 phase [5]. p14ARF interacts with MDM2, which targets p53 for degradation, thereby inducing p53-dependent cell-cycle arrest in both G1 and G2 phases [6,7]. p53 participates in a wide range of activities including growth arrest, DNA repair and apoptosis and nearly 50% of human tumors have defects in p53 [8]
Plasmids construction and stable transfection Full-length fragments of complementary DNA corresponding to p16INK4a, p14ARF and p12 were obtained by reverse transcription polymerase chain reaction (RT-PCR) from AGZY and H446 cells and normal pancreas tissue, respectively, which were positive for the respective transcript
Summary
The tumor suppressor gene CDKN2A generates at least three different transcriptional variants, each of which is thought to encode a tumor suppressor. The tumor suppressor gene CDKN2A, located at 9p21, generates at least three structurally and functionally unrelated transcriptional variants: p16INK4a, p14ARF and p12 [3]. P16INK4a binds to the cyclin-dependent kinases CDK4/6, thereby inhibiting the phosphorylation of the retinoblastoma protein (pRB) and causing cell-cycle arrest at the G1 phase [5]. P14ARF interacts with MDM2, which targets p53 for degradation, thereby inducing p53-dependent cell-cycle arrest in both G1 and G2 phases [6,7]. Less is known about p12; pRBindependent growth suppression by p12 was reported in pancreatic cells, but the tumor suppressive and cell-cycle effects of this protein are as yet unclear [4]
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