Activation of NF-B in Epithelial Cells Expressing
HPV Oncogenes
Daniel Marker1 and Craig Woodworth2
Department of Biology, Clarkson University
NF-B is a transcription factor that serves as a master switch for turning on certain immune and
inflammatory responses. NF-B alters cell behavior in many ways; it inhibits apoptosis (programmed cell
death), increases cell proliferation, and increases inflammatory and immune response. Recent evidence
suggests that activation of NF-kB contributes to the development of several types of human cancer,
including cancers of the blood and certain breast cancers. The focus of this research project is to
investigate whether NF-B is activated in normal human cervical and keratinocyte cells expressing high
risk HPV (namely HPV16) oncogenes, specifically E6 and E7.
In order to examine this question, a number of experiments were performed which involved
transfecting cultured cells with E6, E7, combination E6/E7, or the full HPV16 genome, resulting in
transient expression of these genes, and their resulting proteins. After a period of time, resulting NF-B
activity was measured using a reporter gene assay, which utilizes a reporter plasmid that contains the NFB promoter cloned in front of the firefly luciferase gene. The protein encoded by the luciferase gene
fluoresces in the presence of certain chemicals, making it easy to determine the level of its expression
based on the level of luminescence.
The first goal of this study was to optimize the transfection protocol in such a way that high
transfection rates were achieved, along with reduced cytotoxicity. After this was accomplished, earlier
results showing that NFkB activity increased with increased carcinogenic progression were verified by
repeating the experiment with a number of normal, immortal and tumor lines. Next, the basal activity and
inducibility of NFkB was examined in epithelial cells of different origins: human foreskin (HFK), endo
cervix, transformation zone cervix, and ecto cervix. The effect of the HPV16 virus and known inducer of
NFkB TNFα on NFkB expression in these cells was also examined. Results show that the HFK cells
have a much higher basal NFkB activity than cells from the cervical regions, and that differences in basal
activity of the cervical cells were negligible. However, activity in HFK cells was not affected by the
presence of the viral genome, while activity and inducibility in the cervical cells was greatly increased.
Finally, the effect of the separate E6 and E7 genes was examined. The preliminary results are
inconclusive as to whether it is the E6 or E7 gene which causes the largest increase in NFkB activity;
however, these genes both separately and combined cause at least some significant increase in NFkB
activity when they are expressed within epithelial cells of varying origin. The results of this study could
be used to propose a new mechanism through which high risk HPV infection can lead from benign to
malignant tumors.
1
Daniel Marker, Bio molecular Science, class of 2006
2
Craig Woodworth, Associate Professor, Department of Biology
3
Cancer Research (1990) 50:3709-3715.
Talk