"The Effect of NF-kB on Immortalization of Cervical Cells"
The Effect of NF-kB on Immortalization of Human Cervical Cells by Papillomaviruses Bethanny Smith-Packard1 and Craig D. Woodworth2 Department of Biology, Clarkson University Nuclear factor-B (NF-B) is a transcription factor that regulates cell proliferation, inflammatory and immune responses, and apoptosis (programmed cell death). Increased activation of NF-B is found in many human cancers, including cervical cancer, which is the second most common cancer in women worldwide. Human papillomavirus (HPV) is the major risk factor for cervical cancer. The HPV-16 E6 and E7 proteins are selectively retained and expressed in most cervical cancers, and they cooperate to induce normal human cells to become immortal. Previous studies in our lab have shown that the HPV E6 and E7 proteins activate NF- B. The hypothesis that this research project is focusing on is whether increased activation of NF-B enhances immortalization by HPV-16 E6 and E7. To examine this question, cervical epithelial cells were isolated from human biopsies and grown in cell culture. Immortalization assays are being performed by stably cotransfecting cervical cells with the HPV-16 E6 and E7 genes, an antibiotic resistance gene (puromycin or hygromycin), and either pIkB or p65. pIkB is an expression vector containing a dominant negative mutant that prevents normal activation of NF-kB, and p65 is a vector that over expresses the p65 subunit of NF-kB causing overactivation. Stable transfections are being performed using lipofection. In this process, lipids surround the plasmid DNA to facilitate penetration through the cell membrane and integratation into the host cell DNA. Immortalization assays are long-term experiments and they are ongoing. To date, we have prepared and characterized all of the recombinant plasmid DNAs. We have also determined the optimal concentration of puromycin and hygromycin to use for selection. Experiments are currently underway to 1) optimize the protocol for stable transfection of normal cervical cells, and 2) determine whether the NF-kB mutants inhibit or enhance immortalization by HPV-16 E6 and E7 genes. 1 Class of 2007, Bio Molecular Science, Clarkson University Honors Program 2 Associate Professor of Biology, Clarkson University 117