Columbia Undergraduate Science Journal Open-Access Publication | http://cusj.columbia.com 42 cusjVolume 5Spring 2011 Columbia Undergraduate Sci J http://cusj.columbia.edu V IR O LO G Y cusjcolumbia undergraduate science journalResearch Articles 43cusj Volume 5Spring 2011 Columbia Undergraduate Sci J http://cusj.columbia.edu V IR O LO G Y overall survival rates. Preventing glioma invasion has the potential to convert this highly malignant tumor into a focal disease, which could then be e!ectively treated with focal therapies, such as radiation and surgery (Madsen, 2003; Gillespie, 1999). Invasion and Migration of Malignant Gliomas Tissue invasion by malignant gliomas is a multi-step process. Central to this is the ability of transformed cells to crawl through the extracellular matrix. "e initial step requires receptor-mediated adhesion of tumor cells to matrix proteins, followed by a second phase of deg- radation of the matrix by tumor-secreted proteases. "e ability of invasive glioma cells to navigate these diverse anatomic structures and molecular substrates raises the question of whether speci#c mechanisms and phenotypes of invasive cells are involved in dispersion following spe- ci#c pathways (Gillespie, 1999). Gliomas typically invade the brain by migrating long distances through white matter tracts and by in#ltrating cortex and subcortical gray matter structures. When mi- grating through brain white matter, gliomas move in a manner remarkably similar to that previously described for neural progenitor cells. Given the submicrometer size of the extracellular spaces that are present in the tight- ly packed neuropil of the brain, most other motile cells would likely #nd it impossible to migrate because there would be no room to allow a lamellipodium to form. "e unique form of motility shared by gliomas and neural pro- genitors re$ects adaptations that these cells have to make to a uniquely challenging environment with small e!ec- tive pore sizes (Beadle, 2008). One of these adaptations is in how glioma cells use myosin II, the major source of cytoplasmic contractile force (Gillespie, 1999). Myosin II is absolutely required for migration in the brain, where its major role is to push the bulky nucleus and cell body through the small pores found within the brain matrix. "is feature distinguishes glial cells from other cancers because other cancers do not require myosin II for the collapse of their nucleus. "e progenitor-like motility of glioma cells represents a method of migration that is spe- ci#cally adapted for traversing the densely packed neuro- pil of the mature brain (Beadle, 2008). Poliovirus Poliovirus, among other human pathogenic viruses, is being studied increasingly along with the development of novel therapeutic agents against malignant glioma. Po- liovirus, the causative agent of poliomyelitis, is classi#ed within the enterovirus genus of the family Picornaviri- dae. Poliovirus is composed of a RNA genome and a pro- tein capsid. "e genome is single-stranded positive-sense RNA genome that is about 7500 nucleotides long, while the viral particle is about 30 nanometers in diameter with icosahedral symmetry. Because of its short genome and its simple composition, poliovirus is widely regarded as the simplest signi#cant virus. First isolated in 1909 by Karl Landsteiner and Erwin Popper, poliovirus has be- come one of the most well-characterized viruses, and has become a useful model system for understanding the bi- ology of RNA viruses. "ere are three serotypes of poliovirus, PV1, PV2, and PV3, each with a slightly di!erent capsid protein. "e capsid proteins de#ne cellular receptor speci#city and vi- rus antigenicity. PV1 is the most common form encoun- tered in nature, however all three forms are extremely infectious. Wild polioviruses can be found in approxi- mately 10 countries. PV1 is highly localized to regions in India, Pakistan, Afghanistan, and Egypt, but follow- ing outbreaks of poliomyelitis in 2003–2004 it remains widespread in West and Central Africa. Wild poliovirus type 2 has probably been eradicated; it was last detected in October 1999 in Uttar Pradesh, India. Wild PV3 is found in parts of only #ve countries (Nigeria, Ni- ger, Pakistan, India, and Sudan) (Merrill, 2004). Poliovirus, like other picornaviruses, is character- ized by a single positive strand genomic RNA. In- fection occurs via the fecal-oral route; meaning that one ingests the virus, which then replicates within the alimentary tract. In 95% of cases only a primary, transient presence of the virus in the bloodstream occurs (called a viremia) and the poliovirus infec- tion is asymptomatic. In about 5% of cases, the virus spreads, and replicates in other sites such as brown fat, the reticuloendothelial tissues, and mus- cle. "is sustained replication causes a secondary viremia, and leads to the development of minor symptoms such as fever, headache and sore throat. Paralytic poliomyelitis occurs in less than 1% of poliovirus infections. Paralytic disease occurs when the virus enters the central nervous system (CNS) and replicates in motor neurons within the spinal cord, brain stem, or motor cortex, resulting in the selective destruction of motor neurons; leading to either temporary or permanent paralysis and, in rare cases, to respiratory arrest and death (Pliaka, 2007). "ere are two key mechanism by which poliovirus evades the immune system. First, it is capable of surviving the highly acidic conditions of the gastrointestinal tract, allowing the virus to infect the host and spread throughout the body via the lymphatic system. Second, because it can replicate very quickly, the virus overwhelms the host organs before an immune response can be mounted. In addition, there are more than two such mechanisms; additional ones include inhibition of RNA and protein synthesis, and inhibition of protein secretion, both of which interfere with the IFN respsonse. Fully assembled poliovirus leaves the con#nes Poliovirus Receptor CD155 Over-Expression E!ect on Migration in C6 Glioma Cells Zahra Bhaiwala1,*, Christine Marshall2 1Columbia College, Columbia University, New York, NY 10027, 2Phillips Academy, Andover, MA, 01810 Abstract Cell migration plays a key role in brain cancer invasion, an early step in metastasis, and proteins that regulate migration are often up-regulated in tumor cells. The poliovirus receptor CD155 has recently been shown to affect migration levels of select malignant glioma strains, fueling the exploration of treating brain cancer with oncolytic virus recombinants. In the beginning phase of experimentation, we analyzed the migratory behavior of control C6 gliomas through two rounds of [YHUZÄS[LY�HZZH`Z��0U�VYKLY�[V�L_WSVYL�[OL�M\UJ[PVU�VM�*+����PU�NSPVTH�TPNYH[PVU��^L�[OLU�JVUK\J[LK�[^V�V]LY�L_WYLZZPVU� experiments with the use of a full-length cDNA expression vector and compared the resulting migration rates to control KH[H��>L�MV\UK�[OH[�HU�H]LYHNL�VM��������JVU[YVS�JLSSZ�JYVZZ�V\Y�[YHUZÄS[LYZ�HM[LY�����OV\YZ��HUK�HU�H]LYHNL�VM�������JLSSZ� [YHUZMLJ[LK�^P[O�[OL�7=9�L_WYLZZPVU�]LJ[VY��6\Y�YLZ\S[Z�YLÅLJ[LK�H�������PUJYLHZL�PU�JLSS�TPNYH[PVU�K\L�[V�HU�PUJYLHZLK� WYLZLUJL�VM�[OL�WYV[LPU�*+�����0U�VYKLY�[V�JVUÄYT�[OH[�*+����^HZ�L_WYLZZLK�PU�[OL�YH[�*�Z��^L�Z\IZLX\LU[S`�JVUK\J[LK� two Western Blots: one comparing control C6s and transfected C6s, and the other running control C6s next to a human U87 glioma control cell line. The blots establish that CD155 is indeed present in both of these human and rat strains of glioma, and that our over-expression was successful. Key words: Glioblastoma multiforme; tumor migration; poliovirus receptor CD155; protein over-expression. Introduction After the human brain completes its develop- ment soon after birth, the vast majority of its cells enter the G0 phase, in which they never divide again. One exception to this rule is when a brain tumor develops as a result of abnormal, unregu- lated growth of cells. "e atypical brain cells re-enter the cell cycle because of alterations in any of a large number of genes that control cell division and growth. Astrocy- tomas and oligodendrogliomas are types of gliomas that are the most common primary tumors of the adult brain. Primary brain tumors arise from cells of the brain itself rather than traveling, or metastasizing, to the brain from another location in the body like other known forms of cancer. Tumors are generally classi#ed in four grades, with grade 1 being the most benign and grade 4 being the most malignant. Signs that the tumor is growing rap- idly include cells undergoing division (mitosis), the pres- ence of newly-formed blood vessels (angiogenesis), and evidence that the tumor is outgrowing its blood supply (necrosis). Typically, malignant gliomas show an area of central necrosis surrounded by a highly cellular rim of viable tumor. Malignant gliomas have remained resistant to therapy and are capable of spreading long distances within the brain (Madsen, 2006). In glioblastoma multiforme (GBM), the most aggres- sive form of brain cancer, tumor cells disperse so exten- sively that common treatment approaches such as surgical resection or radiation therapy are not e!ective in check- ing progression. Unfortunately, signi#cant in#ltration of normal brain matter is not limited to the most malignant of tumors. "e invasive behavior expressed by all levels of malignant gliomas has limited the e!ectiveness of local therapies and contributes to the high mortality rate seen in these tumors. "e position of the tumor is also cru- cial. Brain stem gliomas are particularly di%cult to treat, regardless of their grade. "e brain stem is such a com- plicated and delicate part of the brain that completely re- moving the tumor is highly unlikely. Unfortunately, high doses of radiation are not recommended for treatment, as this may cause too much damage to the normal brain stem (Giese, 2003). Having an agent that blocks migration is key to man- aging glioblastoma multiforme. Interventions to control the spreading of glioblastoma multiforme have the poten- tial to slow the clinical course of the disease and improve Copyright: © 2011 The Trustees of Columbia University, Co- lumbia University Libraries, some rights reserved, Porter, et al. Received Jan. 2, 2011. Accepted Jan. 26, 2011. Published April 1, 2011. *To whom correspondence should be addressed: Columbia College, Columbia University, New York, NY 10027. zsb2104@columbia.edu Columbia Undergraduate Science Journal Open-Access Publication | http://cusj.columbia.com 44 cusjVolume 5Spring 2011 Columbia Undergraduate Sci J http://cusj.columbia.edu V IR O LO G Y cusjcolumbia undergraduate science journalResearch Articles 45cusj Volume 5Spring 2011 Columbia Undergraduate Sci J http://cusj.columbia.edu V IR O LO G Y Materials and Methods somal entry site (IRES) within the 5’ untranslated region of the viral genome, which is active in cells of neuronal origin and allows translation of the viral genome without a 5’ cap. Non-transductional targeting involves altering the genome of the virus so it can only replicate in cancer cells. !is can be done by either transcription targeting, where genes essential for viral replication are placed un- der the control of a tumor-speci"c promoter, or by at- tenuation, which involves introducing deletions into the viral genome that eliminate functions that are dispens- able in cancer cells, but not in normal cells. Gromeier et al. replaced the normal poliovirus IRES with a rhinovirus IRES, altering tissue speci"city (Gromeier, 2000). !e re- sulting PV1(RIPO) virus was able to selectively destroy malignant glioma cells, while leaving normal neuronal cells untouched. Although the poliovirus itself has been demonstrated in previous studies to have an oncolytic e#ect in certain types of gliomas, it is still somewhat unclear what the actual role of the receptor CD155 might have, if any, in this relatively new discovery of cancer treatment through viral infection. In glioblastoma, the most aggressive form of brain cancer, tumor cells disperse so extensively that current treatment approaches such as surgical resection or radiation therapy have little e#ect in checking pro- gression. !e survival of patients with malignant gliomas, and a median survival time of patients with malignant gliomas is still anticipated as a bleak 12 to 14 months. Invasive cells remaining after the surgical resection sig- ni"cantly contribute to the demise of the patient. Any successful treatment will have to treat the invasive por- tion of the tumor and the core lesion. Speci"cally target- ing invasive glioma cells remains an interesting concept because invasiveness must be a common characteristic of most tumor cells remaining after surgical resection. Be- cause it has been increasingly shown that glioma invasion is regulated by distinct trigger mechanisms, downstream e#ector molecules of the invasion process represent the best treatment targets. It has been previously found that CD155/PVR was highly expressed in both U87 human glioma cells and primary glioblastoma tumor tissue, and that inactivation of CD155/PVR reduced cell migration in vitro (Sloan, 2005). !ese "ndings suggest a novel role for CD155/PVR in regulating motility and has prompted me to explore this function further in the C6 rat glioma cells we have here in lab by conducting an over-expres- sion experiment to mirror the relationship that has been found in the knockdown experiments. Cell Culture C6 rat glioma cell lines (Canoll Lab, Columbia Univer- sity) were cultured in Dulbecco’s Modi"cation of Eagle’s Medium (DMEM) with 4.5 g/L glucose, L-glutamine, and sodium pyruvate supplemented with 10% Fetal Bo- vine Serum and 1X Penn Strep (Mediatech). Cells were incubated at 37°C in a NAPCO Series 8000 WJ CO2 in- cubator under a humidi"ed 5% CO2 atmosphere. Cells were passaged every seven days onto fresh poly-L-lysine- coated T75 $asks, and medium was changed every three days. Transfection of cDNA expression vector We obtained glycerol stocks of E. coli transformed with a 4.395 kb pCMV-SPORT6 expression vector (Image ID: 3902226, American Type Culture Collection), which used a 50 µg/µl ampicillin marker and was stored inde"- nitely at -80°C. DNA was isolated according to standard plasmid preparation procedures and absorbance was mea- sured at 260 nm via spectrophotometer (BioMate). One day prior to transfection, the cells were trypsin- ized from the T75 $ask and added to 10 mL of growth medium. !e mixture was then spun in a tabletop centri- fuge (2000rpm for 5 minutes) and resuspended in 5 mL of antibiotic-free growth medium. Cells were tritu- rated and then counted by inserting 10 µl into a he- mocytometer and approximately 5x105 cells were plated into 60 mM tissue culture dishes containing 3 ml of growth medium. In two separate sterile eppendorf tubes, the ap- propriate amount of DNA (based on absorbance value) and 20 µl of Lipofectamine 2000 reagent were diluted in 50 µl of serum-free medium. Both tubes were mixed gently and incubated for 5 min- utes at room temperature. !e tubes were com- bined, incubated for another 20 minutes and the entire mixture was added to the transfection dishes. 6 hours after transfection, the media was changed to regu- lar growth media (DMEM + 10% FBS + P/S). Cells were allowed to grow for 48 hours prior to trans"lter assays. Transfilter Migration Assay In preparation for the migration assay, the C6 cells were trypsinized from the tissue culture dishes, added to 5 mL growth medium, and spun at 1600 rpm for 5 minutes. Cells were resuspended in 1-2 mL of serum-free medium and kept on ice. 3µm pore Fluoroblok trans"lters (BD Biosciences) were coated with 10% type 1 rat tail colla- gen (BD Biosciences) in sterile de-ionized water at 37°C for 1 hour. 500 µl of serum-containing growth medium was added to the bottom of each well beneath the "lter to stimulate migration in a single direction (top to bottom). Cells were counted and the number was adjusted so that 5x105 cells were added to the top of each trans"lter in a 200 µl mixture. Cells were left in the incubator to run on the "lters for 5.5 hours. Post-migration, cells were "xed in 4% paraformalde- of its host cell 4 to 6 hours following initiation of infec- tion in cultured mammalian cells. !e mechanism of vi- ral release from the cell is unclear, but each dying cell can release up to 10,000 polio virions (Merrill, 2004). CD155/PVR !e cellular life cycle of poliovirus is initiated by bind- ing to the cell surface receptor CD155. !e virion is taken up via endocytosis, and the viral RNA is released. Translation of the viral RNA occurs by an IRES-mediat- ed mechanism. !e polyprotein is then cleaved, yielding mature viral proteins. !e positive-sense RNA serves as template for complementary negative-strand synthesis, producing double-stranded replicative form (RF) RNA. Many positive strand RNA copies are produced from the single negative strand. !e newly synthesized positive- sense RNA molecules can serve as templates for transla- tion of more viral proteins, or can be enclosed in a capsid to form progeny virions. Lysis of the infected cell results in release of infectious progeny virions. !e presence of CD155 is thought to de"ne the ani- mals that can be infected by poliovirus. CD155 has only been thought to be on the cells of humans, higher primates, and Old World monkeys. Polio- virus is however strictly a human pathogen, and does not naturally infect any other species. CD155 is a Type I transmembrane glycoprotein in the im- munoglobulin superfamily. Commonly known as Poliovirus Receptor (PVR) due to its involve- ment in the cellular poliovirus infection in pri- mates, CD155’s normal cellular function is in the establishment of intercellular adherens junctions between epithelial cells. Like many other receptor molecules used by picornaviruses, CD155 is a long, high- ly glycosylated, single-span surface molecule. While its predominant function is related to cellular adhesion and activation, CD155 also activates natural killer cells and has been reported to play a role in cell motility and tumor cell invasion. It consists of 3 successive Ig-like domains (D1, D2, and D3), a transmembrane domain and a C- terminal cytoplasmic domain. !e human CD155 gene is expressed in 4 splice variants (Ơ, ơ, Ƣ, and Dž) of which 2 variants (ơ and Ƣ) are lacking the transmembrane domain and are released from the cell subsequent to their expres- sion. !e CD155Ơ and CD155Dž isotypes di#er only in their cytoplasmic domains and can both function as PV receptors Zhang, 2008). CD155 protein binds speci"cally to the extracellular matrix component vitronectin. Con"rming the activity pattern of the CD155 promoter in the developing spinal cord, morphogenic factors active in the $oor plate and no- tochord—the transcription factors sonic hedgehog (shh) and its downstream gli e#ectors—strongly activate the CD155 promoter and induce CD155 expression. Both shh and gli transcription factors have been implicated in the oncogenesis of neuroectodermal tumors. !us, the role of shh and gli transcription factors in CD155 gene regulation suggested that CD155 expression may occur ectopically in neuroectodermal malignancies. Evidence for CD155 expression in neuroectodermal tumors stems mainly from studies of neuroectodermal tumor cell lines that are susceptible to oncolytic poliovirus-based agents (Solecki, 2002). Oncolytic Applications Viral oncolysis has been recently recognized as a new development in the treatment of malignant glioma. On- colytic viruses must speci"cally target tumor cells, a prop- erty related to the fact that tumor cells often have aberrant innate immune reponses. !e "rst reported incidences of viral oncolysis, over a century ago, were due to nonin- tended exposure to naturally occurring viruses or after administration of live attenuated vaccine strains. In the last twenty years, new prospects for genetically manipu- lating viruses have opened possibilities for increasing the tumor speci"city and lowering the toxicity of oncolytic viral agents. !ese e#orts have given rise to oncolytic ad- enoviruses, herpesviruses, reoviruses, vesicular stomatitis virus, and most recently, polioviruses. !e antineoplas- tic e#ects of oncolytic viruses are subject to multifaceted interactions with the host cell. !e primary prerequisite for viral oncolysis is expression of cellular receptors me- diating viral entry in malignant cell types. In preparation for clinical applications using oncolytic viruses, analysis of viral receptor expression in the target tissue is highly desirable in order to select tumor types and patients most likely to respond favorably to therapeutic intervention. Cell adhesion molecules of the immunoglobulin su- perfamily are aberrantly expressed in malignant glioma. Amongst these, the human poliovirus receptor CD155 provides a molecular target for therapeutic interven- tion with oncolytic poliovirus recombinants. Analysis of 6 cases has indicated that CD155 over-expression is commonly associated with high-grade malignant glioma (HGL). CD155 expression levels in tumor tissues corre- sponded to those in primary tissue cultures derived from the tumors. Furthermore, CD155 expression in primary glioma explant cultures was equivalent to that found in established glioma cell lines used in preclinical evalua- tions of oncolytic poliovirus recombinants. Poliovirus has been genetically modi"ed through insertion of regu- latory sequences derived from human rhinovirus type 2 to selectively replicate within and destroy cancerous cells. Successful oncolysis with the use of poliovirus infection depends directly on the presence of CD155 in targeted tumors (Sloan, 2005). Poliovirus has a plus-strand RNA genome, the transla- tion of which depends on a tissue-speci"c internal ribo- Columbia Undergraduate Science Journal Open-Access Publication | http://cusj.columbia.com 46 cusjVolume 5Spring 2011 Columbia Undergraduate Sci J http://cusj.columbia.edu V IR O LO G Y cusjcolumbia undergraduate science journalResearch Articles 47cusj Volume 5Spring 2011 Columbia Undergraduate Sci J http://cusj.columbia.edu V IR O LO G Y hyde at 4°C for 20 minutes. After 3 washes with phos- phate-bu!ered saline (PBS) at room temperature, the cells were stained with the nuclei stain hoechst (Calbio- chem/EMD) at a 1000X dilution of 10µg/10µl for 30 minutes at 4°C. "e cells were washed again 3 times with PBS and then plated onto glass cover- slips for analysis. Data Analysis "e number of nuclei on the bottom of each of both control #lters was counted in two low power #elds (25X) of a Zeiss Axioplan microscope and av- eraged. "e nuclei on the top of the #lters was also counted in two low power #elds and averaged in order to further assess the rate of migration in normal C6 cell lines. Lysates Lysis Bu!er (RIPA bu!er) was prepared (1 M ph 8 Tris, 5 M NaCl, 20% NP40, 10% SDS, distilled H20 up to 50 mL) and stored at 4°C. 5X Running Bu!er (7.5 g Tris, 47 g Glycine, 25 mL 10% SDS, distilled H20 up to 500 mL), 5X Transfer Bu!er (9.5 g Tris, 47 g Glycine, distilled H20 up to 500 mL), and 10X TBS (6 g Tris, 44 g NaCl, dH20 up to 500 mL, pH 7.4) were prepared and kept at room temperature. 0.5 mL of EDTA was added to 4.5 mL of lysis buf- fer to make a 10X stock of protease inhibitor cocktail. "e cocktail was then diluted 1:10 in lysis bu!er (50µl to 450µl). Cells were washed twice with PBS and 250µl of the lysis bu!er mixture was added directly to the tis- sue culture dish. Cells were scraped and pipeted to an epindorph tube, vortexed for 10 seconds and returned to ice, and then vortexed for 30 seconds. Cells were then spun for ten minutes at 1600 rpm and the supernatant was pulled o! into a new epindorph. "e lysates were snap-frozen with liquid nitrogen and stored at -80°C un- til quanitation. Western Blot Analysis CD155 expression levels were determined by us- ing a Western blot procedure. 24 µl protein lysate was mixed with 8 µl 4X sample bu!er (20 mL 40% Glyc- erol, 4 g SDS, .01 g .02% Bromophenol-blue, 5 mL beta-mercaptoethanol (BME), 45mL dH20) and boiled for ten minutes. 10% stacking and separating acrylamide gels were prepared according to PAGE Recipe Calculator (Chang Bioscience) and placed into the chamber (VWR), followed by the addition of 1X running bu!er up to the brim of the apparatus. "e protein mixture, along with Prestained Standard (BioRad) was then loaded into the wells and run at 110V for approximately two hours. After the gel was run, it was placed directly into 1X transfer bu!er. "e nitrocellulose was prewet in sterile water to activate and the gels were subjected to overnight electro- phoretic transfer (.03amp) following standard procedures. Membranes were rinsed in sterile water and then blocked with 50 mL of Tris-bu!ered saline Tween-20 with 0.25 g BSA (TBSTB) + 5% milk for one hour on the shaker at room temperature. After soaking, the blots were treated with Anti-Necl-5 rat monoclonal antibody Clone 1A8- 8 (Gift of Dr. Yoshimi Takai, stored at -80°C) diluted 1:1000 in TBSTB and incubated overnight at 4°C. After the removal of the primary antibody and 3 consecutive ten-minute washes of TBST, the last with TBSTB + 5% milk, the membranes were treated with secondary Anti- Mouse IgG-Alkaline phosphatase antibody produced in goat (SIGMA, stored at 4°C) diluted 1:10,000 in TBSTB and set on the shaker for 1 hour. After three thorough rinses of the blots with TBST, they were treated with BM Purple (Roche) until the appearance of protein bands— after which they were stored at 4°C in TBST. With two successful Western Blots (See Figures 5 and 6), we were able to formulate a comprehensive picture of C6 glioma migration in$uenced by transfection of PVR. To determine if increased synthesis of CD155 af- fects the migration of C6 glioma cells, we captured two images of each control and transfection well, as well as one #eld each of the cells remaining on the top of the #lter (See Figures 1, 2, 3). It was not immediately preva- lent through qualitatively assessing cell numbers that a substantial increase in cell migration occurred between the control and transfected cells, but a detailed count re- vealed an increase. "e nuclei counts for the control #elds were 315, 355, 260, and 287, respectively. "e numbers of cells transfected with the PVR expression vector that transversed the matrix were 327, 367, 332, and 392. "e average of migrating control cells was 304.25, in comparison with the transfection aver- age of 354.5. Our data re$ect an average percent increase of 16.5%. By conventional standards ac- cording to a statistical T-test (two-tailed P-value of 0.0957), this di!erence is not considered to be remarkably signi#cant. It is important to note, however, that the second round of trans#lter assays yielded a 32.4% increase in glioma migration as a result of the transfection (See Figure 4). In the #rst Western Blot, which compared CD155 protein levels in C6 control cells to C6 cells transfected with the PVR over-expression vector, we could con#rm from the location of the bands (around 40 kDA, just like the mass of CD155) that the antibody did recognize the Figure 1 C6 Control cells remaining on top of the ÄS[LY�HM[LY�H�����OV\Y�TPNYH[PVU�WLYPVK� C6 Control cells at the bottom of ÄS[LY�HM[LY�����OV\Y�TPNYH[PVU�WLYPVK�� Figure 2 PVR-Transfected cells at the bottom of ÄS[LY�HM[LY�����OV\Y�TPNYH[PVU�WLYPVK� Figure 3 Figure 4 Migrating C6 cells: Control vs Transfection with PVR Expression Vector Avg. of Assay 1 Control: 335 Tfect: 347 Avg Percent Increase: 3.5% Avg of Assay 2 Control: 273.5 Tfect: 362 Avg Percent Increase: 32.4% Avg of All Control: 304.25 Tfect: 354.5 Avg Percent Increase: 16.5% Two-tailed P value = 0.0957 Image Content DNA Absorbance (µg/ul) Nuclei Count _______________________________________________________________________ _ Control 1A 315 Control 1B 355 Control 2A 260 Control 2B 287 Tfect 1A .3 327 Tfect 1B .3 367 Tfect 2A .4 332 Tfect 2B .4 392 Results Columbia Undergraduate Science Journal Open-Access Publication | http://cusj.columbia.com 48 cusjVolume 5Spring 2011 Columbia Undergraduate Sci J http://cusj.columbia.edu V IR O LO G Y cusjcolumbia undergraduate science journalResearch Articles 49cusj Volume 5Spring 2011 Columbia Undergraduate Sci J http://cusj.columbia.edu V IR O LO G Y human protein and that it is in fact present in the cells. Because the band on the right, the transfected band, is so much darker, we can also assume that our over-expression last term was successful and that any impact on migra- tion we saw was directly correlated to the up-regulation of CD155 in the C6 cells (Figure 5). In the second West- ern Blot, which compared C6 and U87 control glioma cells, we can observe two distinct, dark bands (again lo- cated around 40 kDA) (Figure 6). !is is a strong indica- tion that both C6 and U87 glioma cells naturally contain comparable and substantial levels of CD155 protein. We were attempting to mirror the results of a repu- table knockdown study conducted by the Department of Physiology at Tufts University School of Medicine, (Bos- ton, Massachusetts) and the Department of Neurobiol- ogy at Yale University School of Medicine (New Haven, Connecticut). !is study showed that a knockdown of CD155 by FALI in U87MG cells resulted in a sig- ni"cant (16 to 22%) decrease in trans-well migra- tion (Sloan, 2005). Because the siRNA expression vector was not available to conduct a knockdown experiment, our study would be used to perhaps mirror the relationship that has been found previ- ously in the knockdown of CD155. We expected that over-expression of CD155 would signi"cantly increase migration based on the expression of protein levels indicated by Western Blot analysis. If the over-expression were successful, we would expect that the knockdown that we were unable to conduct out of lack of a RNAi expression vec- tor would also decrease migration. Despite our predictions earlier in the year, there did not appear to be a tangible visible di#erence between the control pictures and the transfected pictures for this ex- periment as a whole. One may notice that in general the transfected cell "elds were clearer, and that the control "elds contained a lot of processes (especially in Trans"lter Assay 1) (Figure 3). !is could be possibly interpreted as the presence of CD155 speeding up the growth of cells. If a knockdown in U87 glioma decreased migration of cells, we could infer that CD155 possibly enhances the tumor and may serve as a catalyst for tumor growth. CD155 does not appear to a#ect the shape or size of the cells. Without the Western Blots, which were conducted to- wards the end of our study, we were not able to detect whether or not the transfection substantially increased the presence of CD155 or by how much. !is led us to question whether or not CD155 was actually present in C6 cells. We supposed that pursuing this inquiry would allow us to make further distinction between C6 cells and human strain glioma. At the very least, we would "nd our results to con"rm an expected di#erence between rat and human models. We predicted that increased CD155 expression in gli- oma cells was important for migration of the tumor cells in vivo. While our results indicate promise, only two suc- cessful experimental replicates were conducted and the variation observed was large—precluding a sound con- clusion on the role of CD155 in migration. An additional control, a transfection with a plasmid encoding another protein, would be needed to add to future experiments in order to determine if increased synthesis of any other protein would have the same e#ect on migration. We would also hope to obtain the siRNA in order to knock down PVR, which was not available at the time. !e challenge we faced during this experiment was the fact that CD155 is thought to be a uniquely human pro- tein, because poliovirus only naturally infects humans. While mice have been genetically engineered to produce References PVR, we had no predictions as to whether the C6 rat glioma cells would express CD155 at all. CD155 analysis has never been carried out before in C6 gliomas. Fur- thermore, the primary rat monoclonal antibody we used for our Western Blots was expected to react with Necl- 5, the mouse origin of the protein—and not with the human origin CD155. We were essentially testing to see whether our rat gliomas do naturally express the human protein, and if the mouse antibody would recognize the human homolog. Since the blots con"rm that CD155 is indeed present in C6 cells, in an amount comparable to that present in U87 human cells, we can con"rm that the mouse and human homologs of the protein are similar. Indeed, CD155 and Necl-5 are referred to in combina- tion within scienti"c dialogue and have virtually the same structure. Yet if the antibody treats CD155 and Necl-5 as the same protein, why is the poliovirus itself speci"c to humans? Not only do our "ndings shed light on the strict nature of speci"city characteristic to viruses, but these results suggest that CD155 has more than one function besides being the human receptor for poliovirus, and in concurrence with our assay counts, does e#ect migration on some level. Although some of our counts were not statistically signi"cant, what we do see is that the second assay with a higher concentration of DNA had an over- all much larger percentage di#erence than the control. If we know that the C6 cells contain the human protein and their migration is in$uenced by it, we can now po- tentially use rats as a model for CD155/PVR oncolytic treatment in humans. A study done by the Department of Molecular Genet- ics and Microbiology at the State University of New York (Stony Brook, NY) demonstrated for the "rst time that highly attenuated poliovirus recombinants can infect and propagate in cell lines derived from malignant gliomas and, most interestingly, halted tumor progression and eliminated tumors in athymic mice. !ey proposed that susceptibility of these malignant cells to poliovirus may be mediated by expression of the CD155 gene in glial neoplasms (Gromeier, 2000). As our understanding of the biology of brain cancer progresses, new knowledge about tumorigenesis and tumor biology can be used to diagnose, treat, and prevent this type of cancer. Our ex- periment may help to support the concept that oncolytic poliovirus recombinants may be the next possible treat- ment for malignant glioma. 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