This Research was completed with the University of Toledo
Anthony Wayne High School
2801 W Bancroft St, Toledo, OH 43606
Nineteen million people are projected to have cancer by 2024 so understanding tumorigenesis is of foremost importance. Of solid tumors, 90 percent are aneuploid, signifying an abnormal number of chromosomes usually caused by malfunctioning mitotic checkpoints. TRIP13 AAA-ATPase is a crucial silencing protein in this checkpoint. Therefore, it was hypothesized that TRIP13 overexpression is already present in cancerous cells and is a novel instigator of cancerous aneuploidy. Testing this required artificial overexpression of the gene because prior experimentation found while overexpression increased chromosome counts in regular cells, cells which already possessed the overexpression lacked a change in ploidy. This means cancerous cells with unchanging chromosome counts, following artificial overexpression, have an irregular overexpressed TRIP13 gene inherently present. Therefore, MCF-7 (cancerous breast) cells and MCF-10a (normal human breast) cells were cultivated and overexpressed with TRIP13. However, the failure of MCF-10a cells to reach appropriate confluence led to its abandoning as a control and MCF-7 cells were cultivated alone, with and without overexpression. Through microscopic chromosome spread analysis and the identification of a novel new Giemsa stain (instead of the standard DAPI), an average (standard deviation) of 45 (5) chromosomes was observed for the cells with overexpression and 50 (2) for those without, and a chi-squared statistical tests indicated no significant difference between these counts, supporting the original hypothesis. The next step is to analyze the chromosomes after under expression and compare it against the results from normal somatic cells to distinctly identify an inducer of tumorigenesis and apply it to treat and prevent cancer.
I would like to thank Dr. Liu for letting me use his lab equipment and taking me into his ongoing research. I would also like to thank Dr. Moenk for helping with making the chromosome spreads and using lab equipment. Lastly, I would like to thank my parents for encouraging my interest in the field of science and for supporting my research endeavors.
Chromosome instability (CIN) is widely believed to be the cause of cellular aneuploidy and with aneuploidy present in over 90% of solid tumors, CIN is being considered a direct inducer of cancer (American Cancer Society, 2016). This theory is strongly supported by the overexpression of key spindle assembly checkpoint (SAC) regulatory proteins, namely mitotic arrest deficient 2 (MAD2). The SAC ensures the kinetochores of chromosomes are securely attached to spindle fibers prior to beginning anaphase. If the checkpoint finds the kinetochores failed to adhere to a spindle fiber then a barrier is put up to stop the progression of the cell cycle (Giam & Rancati, 2015). What this means is that if the checkpoint is inactivated, chromosome division will initiate immediately after metaphase and cause asymmetrical division of the chromosomes. This will allow sister chromatids to still be pulled apart, but only if kinetochores had sufficient time to bond to their spindle fibers. This results in chromosomes being spread unevenly between the two sides of the cleavage furrow and, following telophase, a positive and negative aneuploidy will persist in each respective cell. The cell with negative aneuploidy (too few chromosomes) will become hemizygous for many genes and lack genes necessary for protein production. This will then lead to cell apoptosis. Meanwhile, the cells with excess genes will either die or continue to grow as a super cell with a higher than average ploidy (Manchado, Guillamot, & Malumbres, 2016). Ultimately it will divide over time to become a tumor.
The significance of this is illustrated through MAD2 (SAC protein) which when overexpressed in mice, resulted in the formation of tumors after only twelve months (Giam & Rancati, 2015). This is essential data because it reasserts that the SAC is what leads to cancerous aneuploidy, while in the past controversy persisted over whether the SAC or the CENP-E were responsible for cancerous aneuploidy. The CENP-E is a motor protein which is used to align chromosomes to accept spindle fibers and can therefore result in similar cellular aneuploidy as illustrated above. When CENP-E proteins were modified in mice, tumors did still form, but at a much lower rate. In fact, in the liver, the CENP-E reduced cases of tumors in tumor prone mice (Giam & Rancati, 2015). This indicates that CENP-E is little more than a motor protein whose overactivity increases the chance of an error, while the SAC proteins consistently increases tumorigenesis by promoting anaphase prematurely.
The MAD2 protein, while a strong aneuploid inducer, does not render consistent results of tumor formation, suggesting that this protein is not the only one involved in tumor formation. However, its relation to the SAC does provide a novel connection to another protein, TRIP13 AAA-ATPase, which may also be related to tumor formation. This connection has to do with the p31 (comet), which is the component of the kinetochore where the the MAD2 protein binds in order to function (Hagan et al., 2011). The function of this component can be arrested through nocodazole induction and will result in subsequent mitotic arrest, demonstrating the importance of the p31 to the mitotic process (Henegariu et al., 2001). TRIP13 also binds to p31 and is one of the most expressed genes in the centromere-kinetochore component, indicating its importance to this process (Tipton et al., 2012).
Until recent breakthrough research made the connection between TRIP13 and mitosis, TRIP13 was largely considered an essential protein for meiosis. In fact, TRIP13 was found to aid in chromosome crossing and consequential genetic diversification (Roig et al., 2010). The role of TRIP13 in meiosis was solidified to an extent that when comparing the number of chiasmata before and after TRIP13 was removed, significant discrepancy between them persisted. However, this research also found that TRIP13 only performed these functions when combined with the Pch2 gene, meaning that there was a possibility that TRIP13 had another function (Roig et al., 2010). This secondary function was finally determined through its correlation with p31 and positioned TRIP13 AAA-ATPase as a mitotic checkpoint silencing protein.
Currently all that is known about the TRIP13 AAA-ATPase role in mitosis is that it silences the SAC to allow normal cell division (Wang et al., 2014). The SAC works by checking for E3 ubiquitin ligase anaphase-promoting complexes/cyclosomes of the microtubule spindles and recent research has found that although the p31 site deals with proteins from both the E3 site and kinetochores themselves, TRIP13 is localized exclusively on the kinetochores. Furthermore, it was determined that TRIP13 is the protein which makes contact with the E3 site in order to reduce the delay between metaphase and anaphase. Therefore, TRIP13 is the final protein to turn the SAC on or off and is a hallmark of chromosomal instability (Hagan et al., 2011).
Documenting the presence of TRIP13 in cancer cells will not only provide insight into genetically induced tumorigenesis, but will also strengthen the theory that cancer generation is a result of the workings of the SAC. Overall then, the purpose of this study is to asses if chromosome counts change following artificial overexpression of TRIP13 in order to validate/invalidate to the null hypothesis that TRIP13 overexpression in cancerous cell will lead to no change.
MATERIALS AND METHODS
Both human breast cancer (MCF-7) and mammary (breast) epithelial (MCF-10a) cell lines were used for this procedure. The MCF-10a line was used to provide a control of cells that lacked the TRIP13 overexpression both naturally and artificially, but they had a relatively slow mitotic rate with high growth medium requirements, meaning they could not survive at confluency levels above 50%. This resulted in low cell concentrations for the chromosome spread and overall unusable results. However, a control still persisted in the form of MCF-7 cells lacking the artificial TRIP13 overexpression which were to be compared against the chromosome spreads from the artificially overexpressed MCF-7 cells (cell with artificial overexpression premade by other members of lab team with standard viral transfection. The success of the transfection was checked with DNA and RNA electrophoresis).
Three cell cultures were prepared for each treatment: giemsa staining on MCF-7 cells with overexpression, giemsa staining on unaltered MCF-7 cells, and DAPI staining on unaltered MCF-7 cells. These cells were grown in ten centimeter plates with Dulbecco’s modified Eagle’s Medium in 37ºC humidified incubators until confluent. Three hours prior to harvest, cells were arrested in prometaphase with a nocodazole treatment. Some plates were also treated with doxycycline to select for the cells containing the TRIP13 artificial expression. The cells were then detached from the plates through aspiration of the original medium and a ten minute trypsin incubation at 37ºC.
The cells were transferred to a centrifuge tube to be pelleted (100 g for five minutes at room temperature). The medium was then aspirate (100 times the cell pellet volume of PBS was added to the tube) and the cells were resuspended. This was followed by another run in the centrifuge, aspiration, and resuspension of new PBS. This suspension was pipetted into a 1.5 mL microtube with Geneticin (G418)/hydramacin-1 (antibiotics to reaffirm the selection for TRIP13 inducible cells already made by doxycycline).
Initial Hypotonic Treatment
The PBS was aspirated from the tube, leaving a residual amount. Cells were resuspended via slow vortexing while adding 0.075M KCl (stored at 37ºC) dropwise until 3/4 of the tube was full.
Final Hypotonic Treatment
Due to cells not bursting to reveal their chromosomes during microscopic observation, the original hypotonic solution (KCl) was replaced with 1.2 mL of a new solution made from 40% growth medium and 60% Toledo City tap water.
The cells were incubated at room temperature for five minutes and later spun down at 100 g for two minutes. The medium was then aspirated, leaving a residual amount for resuspension through slow vortexing while adding 1.0 mL of fresh ice cold fixative (3:1 methanol:acetic acid) dropwise. This spinning, aspiration, and addition/vortexing of fixative was repeated. Following this the cells were placed on ice for 5 minutes and the spinning, aspiration, and addition/vortexing of fixative was repeated once more.
30 µL of the resulting cell suspension was dispensed on each slide (usually two can be derived from each tube) and the slides were put in an incubator for five minutes to allow the fixative to evaporate.
Initial Chromosome Spread Protocol
Following the drying of the slides, they were held face down above the steam of a hot water bath for three seconds and placed on a hot metal plate face up to make the cells competent for staining, which consisted of two drops of DAPI fluorescence stain evenly added on top of the cells. After a five minute incubation at room temperature the slides were rinsed with drops of deionized water to get rid of excess stain. A mounting medium was then added and was sealed through the usage of nail polish around a coverslip. After the nail polish dried, the slides were stored in the freezer.
Final Chromosome Spread Protocol
Due to the original protocol inflating chromosome counts with extraneous staining (see Figure 2), an altered protocol was developed that featured a new 5% Giemsa (Merck) stain instead of the original DAPI. This meant that following the drying of the fixative, 40µL of the Giemsa was added per slide and the slide was then dried in an incubator. Just as before, excess stain was removed with drops of deionized water, but instead of mounting medium, immersion oil was used prior to adding the coverslip. Lastly, nail polish was added and following drying of the polish, the slides were stored at -20°C.
Slides were viewed on a fluorescence microscope with computer imaging capabilities at 20x and 40x to ensure cells were present and they had swollen to cause cytolysis. If this test was passed cells were viewed at 60x which required immersion oil to be dropped on the slides. Fluorescence filters were turned on for all objectives when dealing with slides treated with the DAPI fluorescence stain, but the Giemsa operated on purely bright light.
Chromosome patches were chosen to be photographed digitally through slide book software if they seemed to be isolated patches from one cell with distinct centromeres. Counting of the chromosomes was accomplished using slide book’s computer aided tools (See Figure 1). The number of chromosomes were then compared between (those from cells with and without artificial overexpression of TRIP13) using a chi-squared statistical tests (?=0.05).
The Chromosome spread protocol utilized was based on the protocol designed by Henegariu et al., 2001, but was heavily modified to create the best possible spread for cancerous cells. The method designed by Henegariu et al. was a generic template for noncancerous cells.
When using the DAPI stain, chromosome counts were more variable than those derived with the Giemsa stain (Standard Deviation of 10 compared to SD of 1.4) and featured higher counts of chromosomes (see Figure 2). This discrepancy was caused due to the DAPI coloring other cell fragments outside of the chromosomes, creating blurry images, and leading to the usage of only the Giemsa stain (see Figure 1).
The combined mean (standard deviation) of trials 1 – 3 (see figures 3 – 5) with only the use of the Giemsa stain was 50 (2) chromosomes per unaltered cell which differed from the mean of 59 (1) derived from the initial trial comparing the two stains (see figure 2). Considering the same protocols were used in both trials, it could be deduced that chromosome numbers vary based on the amount of time they were allowed to grow in cultures (time to reach confluence).
Meanwhile, cells with the artificial TRIP13 overexpression averaged a lower number of chromosomes (45 at SD of 6 to 50 at SD of 2.3), but all trials maintain a lower chi-squared value (6.36) than than the critical value (7.82, ?=0.05), showing no difference between the control and TRIP13 overexpression slides (See Figures 2-5). All slides observed were the MCF-7 slides because MCF-10a slides render almost no chromosomes and the few that are visible are not in clusters for counting. Instead they are present as isolated chromatids which do not accurately represent the count of chromosomes present within a single cell.
The experiment did support the hypothesis, but also identified a new protocol for chromosome spread of cancerous cells. Notably, it was determined that the DAPI stain was not ideal for chromosomal analysis as it stained extra fragments of lysed cells in such a fashion that chromosome counts were neither precise nor accurate. Furthermore, discrepancies between control results showed that the time taken to reach confluency plays a role in the aneuploidy of the cells; the original control cells averaged 59 chromosomes and took one extra day to reach confluency than the cells isolated in the later trials which averaged 50 chromosomes. This indicates that positive cancerous aneuploidy increases over time and could be a focus of later research involving MCF-7 cells.
Results of our experiment supported the hypothesis as there was no significant ploidy difference between the chromosomes from the cells with and without artificial TRIP13 overexpression. This indicates that cancerous cells most likely have an irregular TRIP13 gene and are thus not affected by the artificial treatment. Interestingly, the standard deviation was higher for the TRIP13 overexpressed cells when compared to the control cells, indicating TRIP13 overexpression may have an effect on chromosomal stability in addition to ploidy counts which is promising as it shows TRIP13 is an integral protein for many aspects of tumorigenesis.
Another outcome of the results is that the cellular workings of the SAC in relation to TRIP13 can further be hypothesized. Since TRIP13 silences the SAC when only a few mitotic spindles are secured to kinetochores, both a positive and negative aneuploidy will be formed in the daughter cells. Theoretically, the cells with negative aneuploidy will die and some of the cells with the positive aneuploidy will grow into cancerous cells. From here the data seems to show that the cancerous cells with an inherently high number of chromosomes form daughter cells with enough chromosomes to negate the detrimental effects of negative aneuploidy on cell survival. This phenomenon seems to be the reason chromosome counts balance although artificial overexpression has been applied on top of the inherently present overexpression.
Overall, TRIP13 seems to have proven itself as an integral part in chromosomal instability as the model of TRIP13 AAA-ATPase being responsible for silencing the SAC remains intact. The next step in the research of TRIP13 induced tumorigenesis is a combination of Fluorescence In Situ Hybridization (FISH), MCF-10a cell analysis, and an analysis of cancerous cells with TRIP13 under expression. If the results of this research continue to hold true, the FISH testing will simply reiterate the information derived, the MCF-10a testing will study the ability of TRIP13 to induce cancer within non-cancerous breast tissues, and the under expression will hopefully offer insight for future treatments of tumors. Implications of this research within the near future could include under expressing this gene via gene therapy for the purpose of a possible treatment or even preventative measure for cancer, all to be controlled by the newly discovered mitotic gene, TRIP13.
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Wang et al. (2014). Thyroid hormone receptor interacting protein 13 (TRIP13) AAA-ATPase is a novel mitotic checkpoint-silencing protein. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/25012665