Positive tetracycline control of expression of p15INK4B from an Epstein–Barr autonomous plasmid in a human melanoma cell line

• Published in: Gene 2000-01, Vol.242 (1), p.249-256
• Main Authors: Pacheco, Theresa R, Maxwell, Françoise, Vasiloudes, Panos E, Robinson, William A, Norris, David A, Maxwell, Ian H
• Format: Article
• Language: English
• Subjects: Autonomous plasmid; Carrier Proteins - genetics; Cell cycle; Cell Cycle - drug effects; Cell Cycle - genetics; Cell Cycle Proteins; cyclin-dependent kinase inhibitor; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; doxycycline; Doxycycline - pharmacology; Epstein-Barr virus; Flow Cytometry; Gene Expression Regulation - drug effects; Herpesvirus 4, Human - genetics; Humans; INK4A gene; INK4A protein; INK4B gene; Luciferases - genetics; Melanoma; Mutation; p15 protein; p15INK4B; p15INK4B gene; p16 protein; Plasmids - genetics; Positive tetracycline regulation; Recombinant Fusion Proteins - drug effects; Recombinant Fusion Proteins - genetics; Retinoblastoma Protein - genetics; Simplexvirus - enzymology; tetracycline; Tetracycline - pharmacology; Thymidine Kinase - genetics; Trans-Activators - genetics; Transfection; Tumor Cells, Cultured - drug effects; Tumor Cells, Cultured - metabolism; Tumor Suppressor Proteins
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(99)00514-4

Homozygous deletions in the region of chromosome 9p21 are frequent in human melanoma. Mutations in the p16INK4A cyclin-dependent kinase inhibitor (CDI) gene at this locus have implicated the product of this gene as a tumor suppressor. Less attention has been focused on the homologous, closely linked p15INK4B gene. To facilitate study of the phenotypic effects of restoring expression of the latter in aggressive melanoma cells lacking INK4 expression, we inserted the cDNA encoding p15INK4B into an autonomously maintained plasmid under positive tetracycline control (‘TET ON’ system). Similarly regulated luciferase and herpes thymidine kinase sequences were used as controls. We demonstrate that this system enabled efficient, and reasonably uniform, induction of p15INK4B expression in a human melanoma cell line exposed to the tetracycline derivative, doxycycline. Flow cytometry showed that this induction resulted in substantial accumulation of cells in the G0/G1 phase of the cell cycle. This system will facilitate detailed analysis of the cell cycle inhibitory mechanisms of this CDI in human melanoma cells.