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
2
Class of 2007, Bio Molecular Science, Clarkson University Honors Program
Associate Professor of Biology, Clarkson University
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