The Activation of P53 in Both Mutant Strain XY1054 and Wild Type Caenorhabditis Elegans Using UV Radiation at 254 nm

Ankur Gupta and Ajay RajanIntroduction

Cancer refers to over 100 different diseases characterized by the uncontrollable growth of abnormal cells, which spread throughout a person’s body through the bloodstream and destroy normal body tissue. An effective cure for cancer is yet to be found, making it the 2nd leading cause of death in the United States 11. However, there have been advances in search for the cure for cancer. Most noticeable of these advances is chemotherapy. In chemotherapy, patients are administered one or more cytotoxic antineoplastic drugs and is usually given in conjugation with other forms of cancer treatments such as radiation therapy and surgery. This treatment targets cells that divide with exceptional speed.

While cancerous cells are almost identical to normal cells, key differences in certain substructures and molecules within cancerous cells give them their unique set of characteristics. Normal cells have regulated periods of growth, division, and death, but a mutation in cell DNA can cause some cells, cancerous cells, to divide rapidly without control or regulation and live for longer periods of time, crowding out normal cells. Their abnormal growth and division cause them to rapidly group together in one location forming a tumor, a cancerous growth. Some of these cells then enter the bloodstream and are relocated throughout the body, disrupting surrounding tissues both physically and metabolically.

On the 17th chromosome of human DNA lies the TP53 gene, which codes for tumor protein 53 (abbreviated as p53). p53 is a tumor suppressor protein, meaning that it prevents cancer by regulating the cell cycle. p53 has three main functions in a cell: it activates DNA repair proteins when DNA has sustained damage, it arrests growth by holding the cell cycle at the G1/S regulation point on DNA damage recognition giving DNA repair proteins sufficient time to repair the DNA, and if the damage to the DNA is irreparable, it initiates apoptosis, programmed cell death. Arnold Levine first discovered the protein in 1979. Later research has shown that mutation to the TP53 gene is very common in cancerous cells. In fact, it is the most common mutation to cell DNA found in cancerous cells and is found in over fifty percent of cancers. The TP53 gene is prone to such mutations from carcinogens in the environment, like tobacco smoke. These mutations to TP53 deactivate the gene and prevent the cell from producing the protein p53, allowing cancerous cells to divide uncontrollably. Some people may also be born without an actively functioning TP53 gene. Those that inherit only one copy of the TP53 gene (Li-Fraumeni syndrome) are predisposed to developing cancer later in life 6.

Some researchers are now looking to the TP53 gene in their search for the cure for cancer. In theory, re-activating the TP53 gene would kill all cancerous cells in the body, but researchers have not yet found a safe and effective way to activate the gene in cancerous cells. It is now a known fact that this gene is activated by diverse cellular stresses such as DNA damage, abnormal oncogenic events, and hypoxia (Li, Kon, Jiang, Tan, Ludwig, Zhao, Baer, and Gu 2012) 1. Thus, placing stress on cancerous cells, through stressors like radiation, seems like the most promising route to activating the TP53 gene. Some research shows that ionizing radiation may be able to place enough stress on cancerous cells to activate the TP53 gene, allowing p53 proteins to be produced and initiate apoptosis to their respective cells. But ionizing radiation is not a feasible option because there is a high risk that patients will develop acute radiation syndrome, which results from exposure to ionizing radiation. A safer way to activate the gene might be to re-activate the gene through non-ionizing radiation, which poses a much smaller

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threat towards the health of the patients. Thus in this experiment, UV radiation at 254 nm was tested as the activating agent, because 254 nm is the closest wavelength non-ionizing UV radiation can be without actually becoming ionizing.

Caenorhabditis elegans are a free-living, transparent, 1mm long nematode. They are one of the most commonly used model organisms in research today, along with the Drosophila melanogaster (fruit fly), Escherichia coli (E. coli bacteria), and Arabidopsis thaliana (mustard plant). C. elegans are simple, cheap to breed, easy to grow in lab, grow in bulks, convenient for genetic analysis, can be frozen, and are multicellular eukaryotic organisms. Embryogenesis in C. elegans occurs every 12 hours, making age synchronization of these nematodes quick and simple. RNA interference, which disrupts the function of specific genes, is a straightforward process in C. elegans because it can be accomplished simply by feeding the worms transgenic bacteria expressing double-stranded RNA complementary to the gene of interest. Their relative simplicity makes gene loss-of-function experiments in C. elegans the easiest of all animal models, and, thus, scientists have been able to knock out 86% of the roughly 20,000 genes in the worm. Thus the TP53 gene (the C. elegan ortholog is Cep-1) could easily be de-activated in a C. elegan, allowing researchers to test different ways of re-activating the gene. For these reasons, C. elegans were chosen as the model organism in this experiment.

The research question of this project is whether UV radiation at 254 nm will activate the TP53 gene in a strain of Caenorhabditis elegans in which a large part of the reading frame of the TP53 gene is removed? The hypothesis is that UV radiation at 254 nm will activate the P53 gene in a strain of Caenorhabditis elegans in which a large part of the reading frame of the P53 gene is removed.

Materials and MethodsCaenorhabditis elegans strains and cultureTwo strains of Caenorhabditis elegans were separated into two different groups: the control group consisting of wild type Caenorhabditis elegans and the experimental group consisting of the mutant strain XY1054. In this mutant strain of Caenorhabditis elegans, a large part of the reading frame of the C. elegan’s TP53 gene, known as Cep-1 in C. elegans, is removed, preventing its cells from manufacturing p53 proteins. The mutant strain XY1054 was purchased from the Caenorhabditis Genetics Center (University of Minnesota), and the wild type C. elegans were purchased from Schmahl Science Workshop. Six 60 mm Nematode Growth Media (NGM) plates, spread with the Escherichia coli strain OP50 growth media (the food source for the C. elegans), were prepared as described by Stiernagle (2006) 7. Age SynchronizationThirty-six gravid mutant C. elegans were transferred to the three of the six NGM plates, and thirty-six gravid wild type C. elegans were transferred to the other three NGM plates. Each NGM plate was labeled with the type of C. elegan (mutant or wild type) and the selected exposure time to UV radiation at 254 nm. The C. elegans were then given a 2 hour egg lay period during which they were incubated at 20oC. After the egg lay period, the adult C. elegans were removed from the NGM plates, and the progeny were incubated at 20oC for 3 days. UV RadiationThe NGM plate containing mutant C. elegans set for an exposure time of five minutes was placed under a UV transilluminator at 254 nm for five minutes. Then the NGM plate containing wild type C. elegans set for an exposure time of five minutes was placed under the UV transilluminator at

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254 nm for five minutes. Next, the plate of mutant C. elegans set for an exposure time of 10 minutes was placed the UV transilluminator for ten minutes, and so was the plate of wild type C. elegans set for a ten minute exposure period. The last plate of mutant C. elegans and wild type C. elegans, each with an exposure time of fifteen minutes, were exposed to UV radiation at 254 nm for 15 minutes each.Freeze-CrackAll six NGM plates were placed in a box of dry ice, separated from the ice by a thin sheet of metal. The C. elegans were washed from the NGM plates with M9 buffer and collected in 1.5 mL tubes (1 plate per tube). They were centrifuged at 2000 rpm for 2 minutes, and the supernatant was removed from the tubes with micropipettes. This process was repeated until all of the bacteria were gone and the solution was clear. The C. elegans were then rinsed with distilled water and spun until they settled at the bottom of the tube. Four hundred and fifty microliters of the water was removed from each tube, leaving the bottom 50 µl of water heavily concentrated with C. elegans. Each lab-made poly-lysine slides was assigned a number and a tube, and with the use a glass pipette, 10 µl of the solution in the tubes was placed on the corresponding slide. Using a permanent marker, a circle was drawn around the drop of solution, signaling the area concentrated with C. elegans. An unlabeled poly-lysine cover slide was then placed down on the slide. The slides were put on a flat piece of dry ice for 24 hours (Duerr 2006) 3.Methanol and Acetone FixationA coplin jar was filled with 100% acetone and placed on dry ice. Another coplin jar was filled with 100% methanol and also placed on dry ice. Both jars were allowed to chill for 15 minutes. The cover slides were then removed from the poly-lysine slides, and all of the slides were immersed in the ice-cold methanol for 5 minutes and then in ice-cold acetone for 5 minutes. The slides were then dried with napkins. The slides were washed with 1x PBS and once again dried with napkins (Duerr 2006) 3.Immunohistochemistry Twenty microliters of 0.5% hydrogen peroxide, which was diluted with PBS buffer, was placed on the marked area of the slide concentrated with C. elegans. The slides were incubated for 2 minutes, and then were washed in 1x PBS for five minutes twice. The slides were incubated at 20oC for one hour in 1.5% blocking serum in PBS. The slides were then incubated with the primary antibody overnight at 4oC. The slides were washed in 1x PBS for 5 minutes three times and then dried. Two hundred microliters of the biotinylated secondary antibody solution, which was made by adding 75 µl of blocking serum and 5 mL of 1x PBS to 25 µl of biotinylated secondary antibody, was added to each slide. The slides were incubated for one hour and were then washed in 1x PBS for five minutes three times. One drop of Avidin biotinylated horseradish peroxidase enzyme (Avidin HRP), which was prepared by adding 2.5 mL of 1x PBS and 50 µl of Avidin enzyme to 50 µl of biotinylated horseradish peroxidase, was added to each slide, and the slides were incubated for 30 minutes. The slides were then washed in 1x PBS for five minutes three times. The supervising mentor prepared the peroxidase substrate, due to the fact that it is carcinogenic, by adding 5 drops of 10x substrate buffer, 1 drop of 50x peroxidase substrate, and 1 drop of 50x DAB chromogen to 1.6 mL of distilled water, and added 1 drop of the peroxidase substrate to each slide. The slides were then washed in distilled water and dried with napkins. Three drops of mounting medium were added to each slide, and a cover slide was placed on every slide. The slides were observed under a light microscope, and a scoring procedure was conducted based on the intensity of the resulting brown color (Santa Cruz Biotechnology, Inc.) 9.

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Immunohistochemistry is a method of detecting the presence of specific proteins in cells or tissues (Abnova) 2. This process achieves such detection through a complex process involving antibodies and antigens. Antigens are the proteins in a cell that the researcher is trying to locate, p53 proteins in this case. Antibodies have a natural affinity for these antigens and will bind to them at specific sites called epitopes. The first step of immunohistochemistry is to freeze the organism or tissue through fixation while retaining antigenicity and maintaining tissue structure. Epitopes may also be cross-linked and covered, and pretreatment with antigen retrieval agents may be necessary to re-open the cross-linked epitopes and allow the antibodies to bind. This step was not of use, however, in this experiment. Several endogenous substances may also interfere with immunohistochemistry results, such as endogenous peroxidase, endogenous fluorescence, endogenous antibody binding capability and endogenous biotin, so blocking the endogenous material with the blocking serum prior to staining is crucial to avoid acquiring false positive staining. Once the fixation process is completed, the primary antibody, specifically Cep-1, is added to the organisms or tissues. The primary antibodies will then bind to the antigens, the p53 proteins, at their epitopes. The biotinylated secondary antibody solution is then added to the organisms or tissues, and this will bind to the primary antibody. Avidin HRP serves as a marker, and when added to the organisms or models, it will bind to the secondary antibodies. The Avidin HRP then catalyzes a color-producing reaction, in this case a brown color, which becomes visible in the cells. This resulting color confirms the presence of the targeted antigens, the p53 proteins (Innova Biosciences) 4.

ResultsScores and FindingsThe findings of this experiment show the effect on different times of exposure to UV radiation at 254 nm has on both wild type C. elegans and on the mutant strain XY1054. The scoring system used in this experiment is explained in Figure 5 in the Illustrations. The mutant C. elegans that were exposed to UV radiation for five minutes had a strong presence of p53 proteins, as it received a score of 3. This means that fifty to seventy-five percent of the TP53 genes in the cells of the C. elegans were successfully activated. The wild type C. elegans that were exposed to UV radiation for five minutes had a lower presence of p53 proteins, as it received a score of 2. The mutant C. elegans that were exposed to ten minutes of UV radiation received a score of 2, while the wild type C. elegans that were exposed to ten minutes received a score of 3. This finding implies that UV radiation at longer times actually decreases its effect. This finding is further supported by the results of the C. elegans exposed to fifteen minutes, as the mutant C. elegans received a score of 1 and the wild type C. elegans a score of 3. The intensity of the color was also recorded because it shows where the p53 proteins were more clustered (high intensity) and where they were more spread out (low intensity). The intensity is the qualitative data in this experiment, and the intensity of each set of C. elegans can be found in Figure 1 in the Illustrations. The scoring is the quantitative data.Analysis of ResultsThe goal of this experiment was to test whether UV radiation at 254 nm could activate the TP53 gene in C. elegans with inactive TP53 genes, and the hypothesis was supported, as UV radiation

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at 254 nm did activate the TP53 gene in the mutant C. elegans. Different times of exposure were selected in an attempt to determine the ideal exposure time, which is five minutes, and to test if the C. elegans would not be able to survive the stress of UV radiation. As previously stated, exposure time negatively correlates with the activation of the TP53 gene in mutant C. elegans. However in wild type C. elegans, the score increased from 2 to 3 when the exposure time increased from 10 to 15. Thus these results show that UV radiation had less of an effect on the C. elegans as the time of exposure increased. Experimental ErrorIn the experiment, many of the slides were thrown out because the numbers on the slides smeared when dipped in the methanol, acetone, and PBS buffer. Originally, thirty-six slides were present, each filled with roughly twenty to thirty worms, but only seven slides were deemed usable after the incident and were thus worked with after. Those slides were thought to be usable because their numbers remained intact and because with those seven slides, there would be data on the effect of the three different exposure times to both of the types of C. elegans, mutant and wild type. Through the use of a T test, it has been determined that these results are statistically significant. UV RadiationThere are two main forms of UV radiation, ionizing and non-ionizing. Non-ionizing radiation has enough energy to move atoms in a molecule around or cause them to vibrate, but not enough to remove electrons. Ionizing radiation, on the other hand, has enough energy to remove tightly bound electrons from atoms, thus creating ions (United States Environmental Protection Agency) 5. Examples of ionizing radiation include ultraviolet light, gamma rays, and x-rays, and examples of non-ionizing radiation include radio waves, infrared waves, and microwaves. Ionizing radiation is extremely harmful to humans, as it can result in radiation sickness and cancer due to its potential to its ability to ionize atoms and mutate DNA. Thus, non-ionizing radiation is the more practical option when activating the TP53 gene due to the fact it is not nearly as harmful to humans as ionizing radiation.Strain XY105 MutationThe TP53 gene in the mutant strain of C. elegans was deactivated by removing a large chunk of the mutant strain’s cell’s open reading frame. The open reading frame identifies protein coding regions in the DNA and allows the translation of genes through deletion and transcription. Thus, it reads the TP53 gene, which codes for the p53 protein. The open reading frame specific to the TP53 gene starts with the nucleotides ATG and ends with the nucleotides TAA. In the mutant C. elegans, the start codon ATG is removed along with a large chunk of the open reading frame. This prevents the TP53 gene from being translated and does not allow p53 proteins to be manufactured. SummaryThe results of this experiment thus support the hypothesis. The TP53 gene was activated in the mutant C. elegans, and the results show that the longer the radiation time, the less effective it is on the cells. The activation of the TP53 gene was evident in the resulting brown color, as Avidin HRP attached to the biotinylated secondary enzyme, which was attached to the primary antibody. The primary antibody was attached to the epitopes of the antigen, the p53 proteins produced by the activated TP53 gene. The findings were statistically significant, according to a T test. It is important that the UV radiation used to activate the TP53 gene was non-ionizing, because non-ionizing radiation does not put humans at risk of diseases like radiation sickness. A large chunk of the open reading frame, including the start codon, was removed from the mutant C. elegans,

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not allowing it to read the TP53 gene and thus manufacture p53 proteins. Immunohistochemistry is the method used to detect the presence of p53 proteins in the C. elegans, which signifies that the TP53 gene has been activated by UV radiation at 254 nm. The results of this experiment prove that UV radiation at 254 nm for short periods of time, around 5 minutes, can activate fifty to seventy-five percent of TP53 genes in cells with inactive TP53 genes.

Illustrations

Figure 1

Slide # Type Exposure Time (min) Score Intensity

1 Mutant 15 1 Low

2 Wild Type 15 3 High

3 Mutant 5 3 High

6 Mutant 5 2 Low

9 Mutant 10 2 High

10 Wild Type 10 2 Low

11 Wild Type 5 2 Low

Figure 1: Results of experiment. Note: While being washed in the PBS buffer, many worms were washed off, rendering certain slides ineffectual. These slides are slide # 4,5,7,8,12.

Figure 2

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Figure 2: Mutant Caenorhabditis elegan (exposed to UV radiation for 10 minutes) with brown granules, proving that the TP53 gene has been activated in many of its cells

Figure 3

Figure 3: Wild type Caenorhabditis elegans (exposed to UV radiation for 5 minutes) with some

brown granules, proving that the TP53 gene has been activated in some of its cells

Figure 4

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Figure 4: NGM plate of mutant Caenorhabditis elegans being exposed to UV radiation at 254 nm for 10 minutes

Figure 5Score 0 1 2 3 4

Positive Cells

<10%

10-25%

25-50%

50-75%

>75%

Figure 4: The scoring system used in this experiment. The number of positive cells was roughly estimated and given a score according to these values.

DiscussionDNA Damage and the Effects of Ionizing Radiation

According to these results, every cancerous cell in a C. elegan with cancer would, in theory, be destroyed if that C. elegan were exposed to UV radiation at 254 nm, because p53 proteins would be produced and would initiate apoptosis in each cancerous cell.

Non-ionizing UV radiation at 254 nm was the tested method because it will not harm patients, unlike ionizing radiation. This harm can refer to a wide range of diseases, such as parathyroid adenoma and posterior subscapular cataract. However, radiation therapy is used in conjunction with chemotherapy as a form of cancer treatment. Researchers are still trying to determine how radiation is aiding in the destruction of cancerous cells. Ionizing radiation causes many mutations in the gene sequence by deleting or transcribing the codes. Damage to DNA can tear apart the DNA’s double helix structure, and as the two strands attempt to reconnect, abnormalities to the DNA occur causing certain defects and diseases like cancer.

Chronic side effects of DNA damage, such as memory loss and fibrosis, can result in long periods of treatment, during which the chronic conditions worsen as the patient constantly struggles with the acute side effects (“National Cancer Institute”) 8. These acute side effects include skin irritation and fatigue. All of these symptoms can result from radiation exposure to the skin. Researchers are trying to improve radiation therapy in order to reduce the side effects.

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Researchers are now looking into certain chemicals that have the potential to prevent cells from reacting to radiation. Ionizing radiation, compared to non-ionizing radiation, puts the health of patients at higher risk, thus making non-ionizing radiation preferable when dealing with humans and even C. elegans. Uniqueness of Research ProjectUtilizing non-ionizing radiation is what makes the research unique. The majority of research today is looking to ionizing radiation to activate the TP53 gene. Despite the stronger effect of ionizing radiation on the cell, ionizing UV radiation, as previously stated, will be extremely harmful to the patients exposed to it. It causes damage to living tissue which can result in mutation, radiation sickness, cancer, and cell death. Non ionizing radiation is a far better and safer option. Possible Cure for CancerNon-ionizing radiation has the potential to cure cancer. As the results of this experiment shows, non-ionizing radiation can activate the TP53 gene in cells with inactive TP53 genes. Thus, the cells will then be able to produce p53 proteins and initiate apoptosis for all of the cancerous cells in the body. Unlike popular belief, ionizing radiation is not the only viable option for the cure for cancer. Just because ionizing radiation can alter DNA and non-ionizing radiation cannot, ionizing radiation is not the best path to take. Ionizing radiation is too harmful to humans, and until researchers can find a way to negate such harmful effects, ionizing radiation can never be a feasible option. Non-ionizing radiation is safe and risk-free compared, because it cannot mutate DNA, to ionizing radiation, and it can also activate the TP53 gene. The exposure time would also be quite small, as these results show that non-ionizing radiation actually activates more TP53 genes when administered for smaller lengths of time. It is not yet known the ideal exposure time for ionizing radiation in order for it to have the maximum effect it can on cancerous cells. Thus, non-ionizing radiation is currently the better way to go when searching for the cure for cancer because it is safer than ionizing radiation and can also activate the TP53 gene in cells with an inactive TP53 gene. The exposure time to non-ionizing radiation would also have to be quite small for maximum effect, making it an even safer and more effective method for treating cancer.

Conclusion and Future WorkThe research question of this project was whether UV radiation at 254 nm could activate

the TP53 gene in C. elegans with inactive TP53 gene, and the hypothesis was that UV radiation would be able to activate the TP53 gene in this mutant strain. The hypothesis was supported.

The results are statistically significant, as verified by a T test. The results prove that non-ionizing radiation does activate the TP53 gene in the XY2054 strain, but the exposure time negatively correlates with the number of p53 proteins. Thus, the longer the exposure time, the less effective it is at activating TP53 genes. This result is somewhat in accord with many other findings, that UV radiation does activate the TP53 gene, but in this experiment, non-ionizing radiation was used, not ionizing radiation (which a majority of researchers today are experimenting with). Non-ionizing radiation does have the potential to cure cancer because it is very safe in comparison to ionizing radiation and can activate the TP53 gene.

An experimental error did occur during the experiment in which twenty-nine of the thirty-six slides had to be discarded. However this did not affect the data, as the slides remaining allowed data to be collected for all three exposure times tested against both types of C. elegans.

Several questions remain unanswered, but the biggest question is why shorter exposure times activated more TP53 genes than longer exposure times.

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     Appendices

1. Origin of p53 gene: Arnold Levine hypothesized the p53 gene to be the target of a SV40 virus, which induces tumors. But after several tests, Levine and his colleagues realized that this gene was a tumor killer.  2. p53 gene structure: Composed of 39 amino acids

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