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Cancer is a big topic on the minds of many scientists today, because of its broad-reaching effects on the human race. Because of this, there are many drugs out there that are being researched to combat the effects of cancer. One drug in particular, which is the drug of choice in this study, is noscapine. This drug, which is a derivative of an opiate, has been used in the past for antitussive (anti-cough) purposes in over the counter drugs (3). Recently, however, this drug has been shown to have anti-cancer properties in larger doses than given in over the counter drugs (2). It binds to the protein tubulin and changes its conformation, which affects the cell production of microtubules (3). During mitosis, the cells form microtubules when the cell starts to divide and after the cell has divided, it proceeds to break down the microtubules. Noscapine and other anti-microtubule drugs disrupt the breaking down process of the microtubules and the cells become overcrowded with microtubules and are unable to complete the division process (2). Cancer cells, which uncontrollably divide until a tumor develops, can be hindered by this process.
This inhibition of microtubules in particularly of interest to the study of inhibiting virus replication as well, because of their vast reliance on cell machinery to replicate. A recent study on Tobacco Mosaic Virus showed that when there is reduced microtubule dynamics in a plant cell, it is less susceptible to TMV. This is interesting because there has been a debate on whether microtubules are even involved at all in the virus replication cycle. Their conclusions included that although the reduced microtubule dynamics did not totally inhibit viral infection, it did in fact reduce the cell to cell movement of the Tobacco Mosaic Virus (1). This suggests that there might be a mechanism in cell to cell movement that involves cell microtubule machinery.
The virus used in this experiment is called Vesicular Stomatitis Virus, and is part of the Rhabdoviridae family. These viruses are found in many places in the world, and infect plants and animals. They are nonsegmented RNA viruses of the negative sense strand. Typically the VSV infections in humans have been in cases where humans have been in contact with infected animals in the laboratory. They developed flu-like symptoms and occasionally developed lesions in the mouth (4).
Methods and Results
Preparation of Media. This experiment began with the preparation of the culture medium, DMEM, which contains five percent FBS, or fetal bovine serum, and ten micrograms per liter gentamycin, which is an antibiotic. This was calculated to be 500 mL total media, including 0.5 mL of gentamycin and 25 mL of FBS. The bottle began with 500 mL, so about 25 mL of media was discarded and the gentamycin and FBS were added to solution. The other media prepared was the infection media: PBS with 1 percent FBS. This was made by adding 0.4 mL of FBS to 40 mL of PBS.
Maintenance of Cells. There were two types of cells used in this experiment. The BHK cells were used for the mass production of the virus. These cells needed to be split every 3 to 4 days. The other type of cells used was Vero cells. These cells were used in the plaque assay portion of the experiment to determine the amount of VSV virus in solution. These cells needed to be split only once a week. Changes were eventually made to this procedure to accommodate the slower than expected growth of the BHK cells.
Splitting Cells. Check cells under microscope for confluence. They should generally be around 100 percent confluent before splitting. The media that was in the flask was drawn out by a suction pipette. Then about 10 mL of PBS was added to the flask and rocked back and forth to wash the cells. The PBS was then drawn out in the same manner as the original media. 2 mL of STV was added to the plate to remove the cell adhesions to the bottom of the flask. The cells were incubated for about 5 minutes with the STV at 37 degrees Celsius. After about five minutes, the cells were taken out and the flask was shaken a bit to dislodge the cells from the bottom of the flask. Around 8 mL of media was added to the flask to deactivate the STV. After all cells were suspended in the media, 1 mL was drawn out of the flask and pipetted into a new flask. To this new flask, around 6 mL of media was added, or just enough to cover the bottom of the flask. Changes to this procedure included changing from a flask to culture cells to a Petri dish.
Infection. First, a formula was used to determine the amount of virus needed for a multiplicity of infection (MOI) of 0.1 (See Appendix). This means for every ten cells, there is a virus. It was determined that the amount of virus needed was 45 micro liters. The virus was then diluted in about 7 mL of the PBS/FBS infection solution prepared earlier, or enough to cover the bottom of the flask, similar to the media. The media present in the BHK flask was then drawn out and discarded and the media containing the virus was added. The flask was then incubated for one hour at 35 degrees Celsius and during this time, the flask was rocked back and forth a few times every 20 minutes. Once the one hour incubation period had ended, the virus media was removed and washed with about 10 mL of the DMEM culture media. After, about 6 mL of media was added to the flask for normal culturing. This flask was incubated at 35 degrees Celsius for 3 days.
Harvest Virus. After the virus had ample time to incubate with the cells, the cloudy media containing dead cells and virus was collected into several small glass vials and stored in a freezer at -80 degrees Celsius.
Plaque Assay. Vero cells were prepared in a 6-well culture dish and allowed to grow to confluence. Then, ten-fold dilutions were made of the virus that was grown up in the BHK flasks. Each of the six tubes that were made contained 1.8 mL of FBS/PBS mixture. To the first tube, 0.2 mL of virus stock was added and the tube was vortexed well. This was named the 10-1 dilution. After this, 0.2 mL of the 10-1 dilution was added to the next tube and this became the 10-2 dilution. This was continued until the 10-6 dilution was obtained. In performing this dilution, care was taken that each tube was vortexed well after adding solutions to the tubes, and that the pipette tip was changed after each dilution. Once the dilutions were made, the media in the 6-well dish was removed and 0.8 mL of a virus dilution was added to each well. The 10-4, 10-5, and 10-6 dilutions were used on the dish twice each so that there were two wells containing each dilution. This dish was then incubated for one hour at 35 degrees Celsius and the dish was rocked back and forth every 20 minutes. Meanwhile, the soft agar was prepared while the virus was incubating with the cells. To prepare the soft agar, Solution A and Solution B were made (See Appendix). After each was prepared, both solutions were brought to 45-50 degrees Celsius in a water bath. Once the virus incubation time was almost over, the two solutions were mixed together and cooled down to at least body temperature or below. Then, 4 mL of the solution A and B mix was added to each well. Then, the agar was allowed to cool to room temperature until the agar hardens. The plates were then incubated overnight and checked daily until CPE was visible. Changes made to this procedure included using Petri dishes instead of the 6-well dish and also removing the virus containing media before adding the soft agar. Once the plates were ready and CPE was visible, the plate was held upside down and the agar was carefully scraped off the plate until all the agar is gone from the dish. Once the agar was gone, the dish was flooded with crystal violet/formalin solution (See Appendix). After about 45 minutes, the crystal violet solution was removed from the plate and the plaques visible in each well were counted.
Drug/Virus Infection. The cell count was determined by first removing the media from a confluent plate of BHK cells. The cells were washed with PBS. After, trypsin was added and the cells were incubated for 10 min at 37 degrees Celsius similar to the cell splitting technique. After this, about 3 mL of media was added to make a total of 4 mL solution. The cells were added to the hemacytometer to count. To use the hemacytometer, draw out about ten micro liters from the cell solution using a micro pipette. Then slowly pipette the solution onto the groove in the side of the hemacytometer. Stop adding when liquid is pulled onto the center of the device under the microscope slide. Place under microscope and look for the four squares with a crisscross in the center. Once found, count the cells in each of the four squares and average the total amount. Once the cells were counted, a formula was used to find out how many cells were present in solution (See Appendix). Once the amount of cells was determined, the same formula used in the infection protocol earlier was used to calculate the amount of virus needed to have an MOI of 0.1. A dilution was made of the virus in PBS/FBS, placing 180 micro liters of virus into 2 mL of the PBS/FBS solution. Then, the media was removed from the BHK cells and the cells were washed with PBS. To each plate, 1 mL of the virus solution was added and incubated for one hour at 35 degrees Celsius, rocking every 20 minutes. Once the infection was complete, the innoculum was removed from the plates and the cells were again washed with PBS and covered in DMEM media and placed back in the incubator. Then, the drug and control were prepared by first adding 4 micro liters of 100 mM noscapine to 40 mL of media. 4 hours after the virus was added, the cells were taken out of the incubator and washed 4 times with PBS. Then the drug and control were added to the plates. The cells were placed into the incubator at 35 degrees overnight. After exactly 24 hours from when the virus was first added to the cells, the media was pulled off and stored in a vial at -80 degrees Celsius.
Results. When splitting the cells, the BHK cells were not growing at the rate expected, so the procedure was changed to split the BHK cells less often than the guidelines spelled out. After the first round of splitting cells, some of the cells were contaminated. The media, PBS, and STV were tested to see if this was the culprit, but the results were inconclusive. All materials were then thrown away and we started from scratch. Some of the cells were okay, and we went on to infect them. After the infection, the media appeared cloudy and when viewed under the microscope the cells were floating around. Once we tried the plaque assay for this, this is when the cells for sure showed signs of contamination, so we had to throw those out. The next few times we tried to start up BHK cells, they were contaminated. Once we finally got the cells grown without contaminating them, we performed the plaque assay for the virus only culture, but the results were inconclusive because all of the cells died. The lab instructor performed a plaque assay and obtained a count of 2.5 x 10-6 PFU per mL. The plaque assay with drug culture resulted in all cells in the flask dying and had inconclusive results as well.
The entire experiment was riddled with contamination. This is most likely because of inconsistent handling and sterilization techniques. Many changes were made to try and improve on these techniques. The materials were divided into separate aliquots for each person to cut down on use of the main bottles and to minimize the chance of contamination. Pipette tips were changed more often. The six well dishes were changed to the Petri dishes to ease the infection process and allow us to focus on one plate at a time.
The results for the drug plaque assay experiment were inconclusive because all of the cells died. It is easy to quickly assume that it was something wrong in the procedure since the same thing happened with the plaque assay for the pure virus without drug. But in actuality, the first plaque assay was repeated and yielded results, so it is safe to assume that the agar was most likely too hot when poured into the plate and probably killed the cells. The plaque assay with the drug was repeated three times. It is more likely that the drug had some sort of cytotoxic effect on the cells since the cells died every time the experiment was repeated. If this experiment is repeated in the future, I would suggest that the drugs be diluted further to reduce the possible cytotoxic effects on the cells. Since the drug targets microtubules, it affects all cells, not just cancer cells. Although it seems that this would defeat the purpose of being able to be used on human cells, it is possible that the concentration is just too high for these specific types of cells and that the drug would still be effective and less toxic in a much less concentrated dose.
Maurice O. Ouko et al. Tobacco mutants with reduced microtubule dynamics are less susceptible to TMV. The Plant Journal 2010: 1-10. http://ezproxy.gsu.edu:2211/cgi-bin/fulltext/123306582/PDFSTART
MedInsight. Noscapine: A Safe Cough Suppressant with Newly Discovered Effects in Treating Cancer and Stroke. PC-REF 2007: 1-12. http://www.pcref.org/MedInsight%20-%20PCREF%20Noscapine%20Review.pdf
NCI Drug Dictionary. Noscapine. National Cancer Institute. http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=469778
Rose J.K., Whitt M.A. Rhabdoviridae: The Viruses and Their Replication. 4th edn. Lippincott Williams and Wilkins: New York. 665-684.