Introduction the ability to end cell proliferation,

Introduction The cell cycle is crucial to many mechanisms that exist in living things today such as the repairing of damaged cells, the growth of living things, and many others mechanisms. Within the cell cycle, there are sub-mechanisms that allow this process to perform its duty. There are two major growth phases, also called the G? and G? phases. When cells reach these two phases, they experience growth until they are large enough to continue to the next phase. After the first major growth phase comes the synthesis phase, in which is where a cell enters and its DNA is replicated. Towards the end of the cell cycle are three cell-dividing mechanisms called meiosis, mitosis, and cytokinesis. These three mechanisms allow for the cell to evenly divide into two cells with the same amount of DNA and with the same organisms within it. Caffeine, for centuries, has been commonly used to increase one’s energy in order to perform tasks with more efficiency and strength. Many medications within pharmacies and stores also contain caffeine. (Gabrielli et al., 2006) Furthermore, since caffeine has been known to have many uses, researchers all over the world have been studying caffeine in relation to medications, food, the cell cycle, etc. Some researchers claim that caffeine has the ability to end cell proliferation, to prevent chemical-induced delays in the cell cycle, and also to improve anti-cancer agents. Many other studies also suggest that caffeine can be used to repudiate any DNA damage that occurs in the second major growth phase of the cell cycle. (Gabrielli et al., 2006) Furthermore, caffeine is known to have some influence on the cell cycle. Many researchers have performed studies that relate caffeine to cell death, in which refers to the preventing of cells from continuing through the cell cycle properly. (Pardee & Schlegel, 1986) Caffeine also causes cell arrest, or the capturing of a cell within a single phase in the cell cycle and causing it to die there. Besides having effects on the cell cycle, researchers also suggest that caffeine has the ability to enhance anticancer drugs. (Wang et al., 2015) In plant cells, mitosis is most commonly known to occur in the root tip, or meristematic tissue, of the plant. The meristematic tissue, or meristem, of a plant is located within the tip of the root of a plant. It is the site for further mitosis during plant growth and is where researchers most often obtain their samples in order to create microscope slides. (Robinson, 2001) Specifically for a radish, the root apical meristem is located within the tip of the plant’s roots. The overall structure of a radish is divided into two systems: the shoot system and the root system. The shoot system is where the tap root and leaves are located. The root system, as implied in its name, is where the roots are located. (Page, 2017) In the root system, there exists the cortex, the root hairs, and the root cap. The cortex is known to be the ground tissue, or the outer tissue of the root, that surrounds the vascular tissue, in which is the center tissue of the root. Root hairs assist the root in absorbing water and minerals form the soil. The root cap is where hundreds of thousands of the radish’s cells cover up the tip of the root for protection. (Melton, 2016) The area above the root cap, in which is the meristem tissue, is where researchers would obtain cells for observing the the process of the cell cycle. Many plants, other than radishes, have been used to create microscope slides for observing the phases of mitosis. Onions are a great example since they are commonly used to create microscope slides. Onion cells require 12 hours, or 720 minutes, in order to complete one cell cycle. This information is then used to calculate the amount of time one phase of mitosis, prophase, metaphase, anaphase, or telophase, to be completed during the process. Many researchers have used this data in order to experiment the different factors that may affect or may be affected by the cell cycle and the duration of the time needed by the cell cycle. Through this specific research, the effects of caffeine upon the duration of the cell cycle will be researched. The data that will be obtained through this experimentation will be interpreted in order to make suggestions on caffeine’s effect on the cell cycle, specifically mitosis.Research  An experiment performed by Pardee and Schlegel in the year of 1986 related caffeine with the cell cycle within Syrian hamster fibroblast cells, or BHK cells. The two researchers also related their research to a similar research by a scientist named Nishimoto. According to Nishimoto, he suggests that a mutant, called the ts BN-2 mutant, can experience premature chromosome condensation and many other different early mitotic events under specific conditions. (Schlegel & Pardee, 1986) In Schlegel and Pardee’s experiment, they created figures that helped to illustrate the “grinded” appearance of their caffeine-induced process. They also explain that this “grinded” appearance was also observed in the premature chromosome condensation, or PCC, of synthesis phase cells, in which were claimed to have been combined with other cells in mitosis. (Schlegel & Pardee, 1986) Furthermore, their research suggests that a process, called indirect immunofluorescence, was able to produce phosphoproteins. DNA replication was also said to have stopped in the synthesis phase cells before caffeine had the ability to start premature chromosome condensation. Pardee and Schlegel further explained how caffeine has the ability to reverse the restricting of DNA synthesis, in which results from the research with two drugs called hydroxyurea and aphidicolin. They used these two drugs to relate to the essentialness of DNA synthesis inhibition to premature chromosome condensation. (Schlegel & Pardee, 1986) In Schlegel and Pardee’s experiment, premature chromosome condensation, or PCC, was observed to allow cells to be able to complete the synthesis phase of the cell cycle and enter the phases of mitosis and cytokinesis. Furthermore, their experiment and observations also suggest that caffeine can cause the translation of protein products of a certain RNA and it will also have the ability to free the link between the synthesis phase and the mitotic events. (Schlegel & Pardee, 1986) Aneuploidy is the presence of an abnormal number of chromosomes within a cell and is an important process leading to something called neoplasias. (Katsuki et al., 2008) In this experiment, the researchers suggest that caffeine-induced aneuploidy has the ability to experience a process called asymmetrical cell division, in which refers to the cell division where there is an uneven distribution of chromosomes among the two daughter cells. Caffeine was also observed to have caused exit delays within HeLa cells, U2OS cells, and primary fibroblast cells. (Katsuki et al., 2008) Many other researchers’ experiments and researchers suggest that caffeine-treated mitotic cells will demonstrate manya abnormal situations, such as the misalignment of chromosomes and cell arrests at different phases. However, in this experiment, MAD2, in which is the abbreviation for mitotic arrest deficient 2 depletion, is one factor that will rescue the delaying of the exits of mitosis. This result further indicates that, with the activation of the MAD2 spindle checkpoint, caffeine will cause an extension in the amount of time in which mitosis needs to be completed. When relating to aneuploidy, the cell cycle, in the presence of caffeine, resulted in aneuploid cell production and resulted in asymmetrical conditions. (Katsuki et al., 2008) A research performed in the year of 2013 turns its focus to the cell cycle, mitosis, and how caffeine is able to modulate a process called cisplatinum efficacy. Cisplatinum is a drug that is used to treat ovarian carcinoma and other cancers. Furthermore, cisplatinum efficacy is the process that is known to have the ability to improve the fight against ovarian cancer. In this experiment, present-day fluorescent-protein imaging was used in order to visualize caffeine-induced cisplatinum efficacy. In present day fluorescent-protein imaging, two colors are observed: green fluorescent proteins and red fluorescent proteins. The different between these two proteins is that green proteins are commonly expressed within the nucleus of a cell and the red proteins are referred to as those within the cytoplasm of the cell. Cells called 143B human osteosarcoma dual-color cells were used in order to observe mitotic and apoptotic changes. (Miwa et al., 2013) On the other hand, time-lapsing, in which was used to image HeLa cells, was used to express an indicator called the fluorescent ubiquitination-based cell cycle indicator, also known as FUCCI. This indicator was found to be located within the nucleus of the cells and is used to determine the growth phase, synthesis phase, and mitosis phases of the cell cycle. (Miwa et al., 2013) The researchers’ experiment suggested that caffeine has the ability to increase the prevention of cisplatinum on cell proliferation, in which is the process that yields more cells. Furthermore, the experiment also demonstrated that cisplatinum, under the influence of caffeine, can decrease the process of mitosis and caffeine-induced apoptosis in cells. With these results, the researchers suggest that when cisplatinum is in the presence of caffeine, it can intensify mitosis and can also increase apoptosis in cells. The final interpretation involved the ability of caffeine to modulate the cell cycle in cancer cells and its ability to improve the efficacy of anticancer drugs, such as cisplatinum. (Miwa et al., 2013)Researchers used caffeine in order to identify any indicators of signals that trigger apoptosis in mitotic checkpoint-arrested cells, in which refers to cells that can not complete a phase in the cell cycle and proceed to the next phase. Caffeine and Gö6976 both assist in the overcoming of a cell arrest checkpoint with equal efficiency. However, caffeine demonstrates its ability to cause rapid apoptosis compared to the role of another inhibitor, in which is called Gö6976. The cells treated by the inhibitor also were delayed in the phases of mitosis for a longer period of time than the caffeine-treated cells. (Gabrielli et al., 2006) It is suggested that caffeine, while assisting in the escape of cell arrest, was mostly likely participating in another activity as well. On the other hand, the inhibitor would have most likely assisted in the escaping from apoptosis rather than caffeine. Furthermore, the researchers of this experiment discussed that caffeine, according to their data, has two main effects on cell arrest in mitosis. Caffeine-treated cells have the ability to initiate apoptosis and can signal a process called proteasome-mediated destruction of cyclin B1 , in which refers to the breaking down of proteins into their amino acids. (Gabrielli et al., 2006) Further into this research, caffeine’s ability to induce apoptosis, specifically when cell arrests occur, suggests that the spindle checkpoint, or any phase of the cell cycle, can regulate some strong apoptotic responses. It also suggest that cells that are in cell arrest have a strong signal that initiates apoptotic to ensure that, if mitotic failure occurs, then cells will most likely enter a state of destruction. (Gabrielli et al., 2006) According to this research, cisplatin is known as an important DNA-damaging anticancer drug and has been used to treat many cancers, such as ovarian cancer. However, the effectiveness of the drug treatment is slowly decreasing due to the immunity, towards the drug, that has been developed by the cancer cells. In this research, the researchers refer to caffeine as an inhibitor of many biological activities such as ATM, in which is a kinase with an early name of  ataxia-telangiectasia mutated, and ATR, another kinase called the ataxia telangiectasia and Rad3-related protein. (Wang et al., 2015) These two proteins are important protein kinases that are involved in the processes of cell arrest and apoptosis. Two processes that help to determine caffeine’s effect on cisplatin-induced apoptosis and growth of lung cancer cells are caspase-3 activation and cell growth inhibition assays. Caspase-3 activation assay and cell growth inhibition assay are both indicators of activities of apoptosis. (Wang et al., 2015) The caspase-3 activation studies that were demonstrated in this research suggest that the presence of caffeine assisted in the increase in the activity of cisplatin-induced apoptosis in the cancer cells of the lungs. On the other hand, the cell growth inhibition studies indicated that caffeine’s presence caused a further increase for the cisplatin-induced cell growth inhibition. Furthermore, the results obtained suggested that caffeine allowed the increasing in the expression of a protein called the PUMA pro-apoptotic protein, in which regulates apoptosis. Caffeine was also observed and discussed as having the ability to enhance the first major growth phase’s cell population and to decrease the amount of cell arrests that occur in the cell cycle. So, caffeine is considered as increasing the elimination of cisplatin-induced lung cancer cells with the inhibition of ATR and ATM protein activations. (Wang et al., 2015) The effects of caffeine on the cell cycle appears in many different ways and occurs in many different caffeine-induced mechanisms such as those introduced by Gabrielli, Hiroshima, Imoto, Pardee, Patel, and Wang. Overall, the explanations of experiments, in which were performed with caffeine and cells, performed by other researchers. Each of the sources explain the effects of caffeine or caffeine induced processes on mitosis in cells. Generally, caffeine or caffeine induced processes have caused an arrest in the synthesis or growth phase. Most of the researchers had discussed about the process of caffeine induced premature chromosome condensation that had occurred during their experiment. According to Schlegel and Pardee’s experiment, caffeine has the ability release the link between the mitotic events from the DNA replication. Similar experiments from Gabrielli and Hiroshima also express the effects of caffeine on the entrance of cells into different phases of the cell cycle. Gabrielli and the other researchers of the experiment specifically demonstrated the results of caffeine-induced apoptosis in the spindle-checkpoint of mitosis. Their results and discussion suggested that caffeine has the ability to strong apoptotic responses, thus causing a rapid destruction of targeted cells. (Gabrielli et al., 2006) On the other hand, Hiroshima’s collaboration with a few other researchers also suggested, through the results of the experiment, that caffeine has the ability to regulate anticancer drugs such as cisplatinum. When comparing both Hiroshima and Gabrielli’s researches, there is an elucidated difference between the results of each experiment. (Hiroshima et al., 2013) Caffeine-induced premature chromosome condensation is also said to serve as a basis for the cause of programmed cell death. (Pardee & Schlegel, 1986) However, the effects of caffeine, as explained through the experiments that focus on caffeine-induced processes, are quite indirect. Caffeine-induced mechanisms have been demonstrated as having the ability to affect the way that the cells grow and how the cells travel through the cell cycle. The results of the multiple experiments and researchers that have been conducted strongly suggests that caffeine has the ability to affect the cell cycle, and future researchers will have the ability to use these results as reference to study more about the effects of caffeine on the cell cycle and to establish new theories to improve these effects.MethodsRadish growthThe site of the planting of the cherry-belle radish seeds was located in the basement of my home in Quincy, Massachusetts. During the season of fall, the temperature of Quincy approximates 50°F – 60°F during the day and 30°F – 50°F during the night. As a result, the cherry-belle radishes had to be planted indoors under a ultraviolet growing light. Pots were labeled and separated into four groups: the control group, the ? cup caffeine group, the ? cup caffeine group, and the ½ cup caffeine group. The amount of radish seeds were then evenly distributed among the pots. Approximately four inches of soil was place at the bottom of each pot in order to allow the roots of the radishes to have more room to grow. Then, radish seeds would be place on top of the four inches of soil and would be watered over slightly. About ¼ – ½ inches of soil was used to cover up the seeds and this soil was watered over. Each separated group of pots were water with the corresponding amount of caffeine that was labeled on the side of each pot. Germination of the cherry-belle radish seeds began four to seven days after planting. The radish took a period of approximately 25 to 30 days in order to complete their growth. The harvested radishes were separated into their corresponding groups and placed in the refrigerator, covered with a damp towel, in order to keep them fresh. Microscope slide preparationThe site of microscope slide preparation was North Quincy High School in Quincy, Massachusetts. Each group was brought into the school and placed into a refrigerator when not in use. When cutting the cherry-belle radishes for samples with a razor blade, the cutting occurred at the tip of the root of the radish, where the meristem tissue is located. The tip of the root was cut and carefully sliced into small slivers. Once this action was completed, the slivers were carefully set aside and a glass slide and glass cover were obtained. A 25-milliliter beaker was filled 10 milliliters of distilled water and a pipette was obtained. The pipette was used to drop four to five drops of distilled water in the center of the glass slide. Tweezers were used in order to carefully transfer the slivers from the laboratory table onto the drops of water. A glass slide was then gently placed on top of the samples in the water and a stain, called methylene blue, was used to stain the slivers. Finally, the prepared slide was placed aside for approximately 10 seconds and was placed under a microscope for further examination.Slide examinationOnce the slide was placed on the platform of a light microscope, under low power (4x), the sample was located on the microscope slide. Then, using medium power (10x), one area of the sample was set into focus with the corresponding knobs on the microscope. Finally, the microscope was turned to high power (40x) in order to examine the cells of the radishes. Some microscope slides were rejected due to the large amount of stain that was absorbed by the cells. Many slides were also rejected due to the failure to find any signs of mitosis, in which was a result of the cutting of the wrong area on the root tip. When looking at visible cells, the phases of mitosis were seen occurring in the center of each plant cell. Each phase of mitosis, prophase, metaphase, anaphase, and telophase, were counted and tallied inside the science fair notebook data table. Due to the large amount of cells seen under the microscope, each sample was divided into around 3 areas of cells. When necessary, a second-time counting was performed to assure accuracy. For each group in each run, approximately ten microscope slides were made. When an area of a sample is divided into two to three areas, each area would represent one set of data. As a result, one hundred trials was obtained for all three runs in this experiment.

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