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An Honors Thesis Proposal Human Papillomavirus and NF-B Expression in Human Epithelial Cells Author: Daniel Marker Advisor: Dr. Craig Woodworth March 7, 2005 Goal To determine the effect of the Human Papillomavirus on NF-B expression in human epithelial cells. Purpose NF-B is a protein that serves as a master switch for turning on certain immune and inflammatory responses within human epithelial cells. NF-B alters cell behavior in many ways; it inhibits apoptosis (programmed cell death), increases cell proliferation, and enhances inflammatory and immune response. Recent evidence suggests that activation of NF-B may contribute to the development of many human cancers. The focus of our research is to investigate whether the Human Papillomavirus (HPV), the major cause of cervical cancer, activates NF-B in human cervical cells. If NF-B is actually upregulated by the HPV virus, its inhibition may be useful as a method of either preventing carcinogenesis, or for increasing the effectiveness of chemotherapy. Background NF-B and Carcinogenesis Nuclear Factor-B is a family of closely related protein dimers which, when expressed, modify the expression of a number of genes involved in cell survival and proliferation. In the nucleus, these proteins bind to a specific DNA sequence known as the B box, and either act as promoters or silencers of the target genes. The active site on NF-B subunits is a central portion of the protein known as the reticuloendotherliosis (REL) domain. This domain is the site of DNA binding, Inhibition of B (IB) binding, and phosphorylation (1). Figure 1: Picture showing an example NF-B complex. The IB complex is shown in the center. The most common NF-B subunits are p50 and p65. The inactive complex formed by these subunits is shown in Figure 1. NF-B is activated by a number of different factors including cell stress, presence of pro inflammatory cytokines, and bacterial or viral infection (2). The infection pathway is the most relevant to our study, and it is shown below in Figure 2. When inactive, the NF-B subunits are found in the cytoplasm, bound to the IB complex. This prevents them from translocating to the nucleus. Some pathogens are known to label the IB complex for degradation, freeing the NFB subunits and allowing them to affect gene expression. This is a strong, fast acting, yet Figure 2: Diagram showing a common infection triggered activation scheme for NF-kB. The pathogen phosphorylates the IkB complex by inhibition of kB kinase (IKK), labeling it for ubiquitin dependent degradation. Once IkB is degraded, the subunits are free to translocate to the nucleus. Source: http://www.webbooks.com/MoBio/Free/images/Ch4H4.gif normally short lived response. The short duration of NF-B activation is due to the fact that NFB subunits also up regulate the production of IB, which binds and inactivates the active subunits in the nucleus (1). This is a very important feedback inhibition pathway for the regulation of NF-B. When it is activated, NF-B affects the transcription of a number of important genes involved in immune response, inflammation, and the cell life cycle. It produces a major inflammatory response by turning on genes which encode for the pro inflammatory cytokines IL- 1, IL-6, IL-8, and TNF (1). NF-B also activates a number of anti apoptotic genes, including c-IAP1 and c-IAP2 (3). Finally, it can increase cell proliferation by increasing the production of cyclin D1 (4). Recently, NF-B has been shown to play a role in a number of human cancers (5). Its anti apoptotic properties and ability to increase the rate of cell proliferation are both aspects exhibited strongly in carcinogenic cells. Also, the Rel homology domain of the NF-B proteins is present in a number of known potent oncogenes, the V-Rel oncogene being the most studied. Activation of the V-Rel oncogene was shown to induce the formation of lymphomas in chickens, and its similarity to NF-B was the starting point for most NF-B cancer research. Finally, a number of human cancers have already been shown to exhibit high levels of NF-B activity, including cancers of the blood, breast, prostrate, head and neck (5). It is for these reasons that NF-B has become a factor of interest in tumor formation, and why it was chosen for this study. Human Papillomavirus and Cervical Cancer Human papillomaviruses (HPV) is a small family of DNA viruses which normally cause benign growths on the hands and feet, commonly known as warts. Of the approximately one hundred strains of this viral family, a small subset is sexually transmitted and has the ability to form genital warts. A few of these sexually transmitted viruses have the ability to cause cervical carcinogenesis, and are therefore referred to as “high risk.” In fact, high risk HPV infection is the leading risk factor for cervical cancer. The most common high risk strain found in cervical cancer is HPV-16, which is the virus proposed to be used in this study. The viral genome is roughly 8kB long, and is broken down into two sections (shown in Figure 3). The section known as the late (L) region composes a large amount of the viral genome and encodes the viral capsid. It is not expressed until much later in the life cycle of the infected cell, in order to evade immune detection. Figure 3: Diagram showing the layout of the HPV-16 genome. LCR stands for long control region, which is responsible for the proper replication of viral DNA. The axis is labeled in the number of base pairs. The early (E) is responsible for hijacking the cell’s molecular machinery in order to reproduce the viral particles. The two genes in this region that are of greatest interest to this study are E6 and E7, because they are retained in most cervical cancers. The E6 protein is known to label p53, a protein vital for preventing DNA damage and inducing apoptosis, for ubiquitin dependent degradation. It has also been shown to induce the production of telomerase, an enzyme required for bypassing senescence (6). E7 is known to bind and inactivate the Retinoblastoma protein. This allows for uncontrolled cell division (7). Proposed Research My proposed research will aim to establish a link between HPV16 genes and NF-B expression in cervical cells. We propose to measure NF-B activity in normal cervical cells in the presence of HPV16 genes using a reporter gene assay. A reporter gene assay utilizes specialized cloned DNA plasmids. These plasmids contain the gene for an easily measurable protein, in this case firefly luciferase, the protein which under certain chemical conditions causes fireflies to glow. A specific genetic sequence which acts as the promoter for NF-B, called a B box, is cloned in front of the luciferase gene. This results in the production of luciferase in direct proportion to the amount of NF-B activation. Therefore, by measuring the concentration of NF-B luciferase, we can determine the relative amount of NF-B activation. In order to make the results even more specific, another reporter gene is introduced which produces a luminescent protein called Renilla. A promoter for a gene which is expressed equally in all cells in cloned into this plasmid. The concentration of this protein is used to normalize the NF-B signal. The renilla and luciferase protein concentrations will then be measured and compared using a luminometer, which is a specialized fluorescence spectrophotometer. In order to introduce the reporter and experimental DNA into the cells, we plan to use the lipofection transfection technique. The overview of this technique is shown in Figure 4. The transfection reagents will be purchased from Invitrogen. The lipofection transfection method involves coating the plasmid DNA with a positively charged lipid. This is necessary because the cervical cells will not take up the raw plasmid DNA. The lipofection technique covers the DNA with a positively charged lipid, which allows it to be taken up and expressed by the cells. Figure 4: Diagram showing the transfection technique. The DNA, shown in the bottom left hand tube, is mixed with the lipofectAMINE, shown in the top left. The positively charged lipofectAMINE coats the negatively charged DNA, forming a lipid complex. This complex is taken up by the cells. This methodology will allow us to measure NF-B under a number of conditions. The specific issues which are to be addressed using these techniques are as follows: 1. To examine the differences in baseline NF-B activity in normal cells originating from different areas of the cervix; 2. to examine the effect of the introduction of the HPV-16 genome on NF-B activity in these cells; 3. and to determine the effect of single HPV-16 genes, specifically E6 and E7, on NF-B expression in these cells. Preliminary Results Reporter Gene Assay Optimization In order to obtain the most accurate results possible, the lipofection and reporter gene assay techniques needed to be optimize. To do this, we performed an experiment which varied the amount of DNA and lipofectAMINE introduced into the cells during transfection. We then examined the NF-B signal produced under these different conditions. The most desirable conditions are those in which there is a strong luciferase and renilla signal, yet a low activation of NF-B. As NF-B is activated under conditions of cell stress, an effective optimization is difficult to obtain. The results from a representative optimization experiment are shown in Figures 5 and 6. These results show that as the amount of either DNA or lipofectAMINE is increased, the signal strength of both the signal proteins increases. However, the increase in DNA or lipofectAMINE also increases the stress placed on the cells, resulting in higher baseline NF-B activation. For these reasons, we choose to use a mid level amount of DNA (no more that 0.5mg total) and a mid level amount of lipofectAMINE (2ml). These values result in a strong protein signal, while keeping NF-B activation to a minimum. These guidelines were Relative Activation and Signal Strength used in all subsequent experiments. 16.000 14.000 12.000 10.000 8.000 6.000 4.000 2.000 0.000 0.1 0.25 1 Amount of DNA (in mg) NF-kB Activity NF-kB Luciferase Signal Renilla Signal Figure 5: Diagram showing the relation between amount of total DNA used and NF-B activation and signal activity. The amount of lipofectAMINE used was kept constant at 0.3ml. Relative Activation and Signal Strength 80.000 70.000 60.000 50.000 40.000 30.000 20.000 10.000 0.000 0.3 1 2 3 Amount of LipofectAMINE (in ml) NF-kB Activity NF-kB Luciferase Signal Renilla Signal Figure 6: Diagram showing the relation between amount of lipofectAMINE used and NF-B activation and signal activity. The amount of DNA used was kept constant at 0.25mg. NF-B Activity and Carcinogenic Progression After our experimental technique was optimized, we looked to verify results from an earlier study in our lab which showed that NF-B activity increased with carcinogenic progression. In order to do this, we examined basal NF-B activity in normal, immortal, and tumor cells. Normal cells were cultured directly from normal human foreskin or cervix, and showed no signs of carcinogenesis. Immortal cells stably express the HPV16 E6 and E7 genes. They will continue to divide for an indefinite amount of time, however, they will not form tumors in transplanted into mice. This places these cells at a sort of mid point between normal cells and cancer cells. Cancer cells were either cultured from human cervical tumors, or created in the laboratory environment. NF-B activity was measured in four normal strains, five immortal lines, and three cancer lines. The results are shown in Figure 6. Average Ratio of Activation 16.00 14.00 12.00 10.00 8.00 6.00 4.00 2.00 0.00 Normal Immortal Cancer Figure 7: Diagram comparing NF-kB activity in cells of different levels of carcinogenesis. These results support the earlier findings in our lab which showed that NF-B activity increased as cells transform from the normal stage to the immortal stage. They also show that cervical cancer cells have a high NF-B activity. These results serve as an effective lead in to my main project, which will examine the direct effect of high risk HPV16 genes on NF-B activity in normal cervical cells. Timeline My experiments can only be performed on newly cultured human cervical cells (pass one, two or three). After this many passes, the cells start to differentiate or senesce, making them unusable. Therefore, the timeline of my project will depend largely upon when our lab receives new cervical samples. I have also already performed a number of important experiments relating to my project this past summer, and am now looking to verify those results through repetition. Sources Cited 1. Ghosh, S., M. May, and E. Kopp. NF-B and Rel proteins: evolutionarily conserved mediators of immune responses. Annual Reviews of Immunology. 1998, 16: 225-260. 2. Pahl, H. Activators and target genes of Rel/NF-B transcription factors. Oncogene. 1999, 18: 6853-6866. 3. Karin, M., Y. Cao, F. Greten, and Z. Li. NF-B in cancer: from innocent bystander to major culprit. Nature Reviews Cancer. 2002, 2: 301-310. 4. Joyce, D., C. Albanese, J. Steer, M. Fu, B. Bouzahzah and R. Pestell. NF-B and cell regulation: the cyclin connection. Cytokine and Growth Factor Reviews. 2001, 12: 73-90. 5. Baldwin, A. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kB. Journal of Clinical Investigation. 2001, 7: 241-246. 6. Mantovani, F. and Banks, L. The Human Papillomavirus E6 protein and its contribution to malignant progression. Oncogene. 2001, 20: 7874-7887. 7. Munger, K., J. Basile, S. Duensing, A. Eichten, S. Gonzalez, M. Grace, and V. Zacny. Biological activities and molecular targets of the human papillomavirus E7 oncoprotein. Oncogene. 2001, 20: 7888-7898.