The Raf/MEK/ERK pathway is one of the MAPK (Mitogen Activated Protein Kinase) pathways that control the cell cycle resulting in the meditation of cell growth, proliferation and differentiation. Paradoxically, the Raf/MEK/ERK pathway can also induce cell cycle arrest. How can the same pathway mediate these paradoxical cellular responses? One clue may be obtained by studying the cyclin-dependent kinase (cdk) inhibitor p21WAF/CIP1. When the Raf/MEK/ERK pathway induces cell proliferation, p21WAF/CIP1 is down-regulated by proteosomic degradation. However, when the pathway induces cell cycle arrest, p21WAF/CIP1 level is up-regulated via different mechanisms. The goal of my project was to determine whether expression of p21WAF/CIP1 is directly involved in mediating the pathway-induced cell cycle arrest. For that, it was necessary to establish a p21WAF/CIP1 over-expression system, which was the specific aim of my project this summer. I used a lentiviral expression system in order to effectively deliver a p21WAF/CIP1 gene into different cell lines. I successfully cloned the p21WAF/CIP1 gene into the pHAGE lentiviral vector through a series of cloning steps, including enzyme digestion, ligation, transformation, cracking, DNA purification and DNA sequencing. In the future, I will use this plasmid in different cell lines, TT, LNCaP and DMS53, to test whether expression of p21WAF/CIP1 alone can induce cell cycle arrest.
Yongik Lee, ’11
Kyungkido, South Korea
Major: Biochemistry and Molecular Biology
The Medical College of Wisconsin
Sponsor: Craig Tepper