title: Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549.
abstract: BACKGROUND
  The 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.
  
  
  METHODS
  The 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.
  
  
  RESULTS
  While 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.
  
  
  CONCLUSIONS
  Among 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.
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin 150081, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Jing
authors.initials: J
authors.lastname: Zhu
authors.forename: Jing
authors.initials: J
authors.lastname: Bai
authors.forename: Hui
authors.initials: H
authors.lastname: Jiang
authors.forename: Fangli
authors.initials: F
authors.lastname: Liu
authors.forename: An
authors.initials: A
authors.lastname: Liu
authors.forename: Peng
authors.initials: P
authors.lastname: Liu
authors.forename: Guohua
authors.initials: G
authors.lastname: Ji
authors.forename: Rongwei
authors.initials: R
authors.lastname: Guan
authors.forename: Donglin
authors.initials: D
authors.lastname: Sun
authors.forename: Wei
authors.initials: W
authors.lastname: Ji
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.forename: Yan
authors.initials: Y
authors.lastname: Jin
authors.forename: Xiangning
authors.initials: X
authors.lastname: Meng
authors.forename: Songbin
authors.initials: S
authors.lastname: Fu
mesh_terms: D017398:Alternative Splicing; D015153:Blotting, Western; D002453:Cell Cycle; D049109:Cell Proliferation; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D014644:Genetic Variation / Q000235:genetics*; D006801:Humans; D007124:Immunoenzyme Techniques; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology / Q000517:prevention & control*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D012333:RNA, Messenger