Early Phases of P53-Enhanced Apoptosis Are Reversible - Essay Example

Early Phases of P53-Enhanced Apoptosis Are Reversible Hypothesis The hypothesis in this study is that it is postulated that the early stages of the process of apoptosis is indeed reversible. According to the initial results from our laboratory, it has been revealed that DNA repair is normally activated very early in the p53- Enhanced apoptosis, and this early stage may actually be reversible. The current work is based on these early works, and through more evidence it has confirmed that indeed the early phases of a p53-Enhanced apoptosis are reversible. Definition of terms Kerr et al are accredited with the definition of process of apoptosis, and define it as a process in which cells because of a natural process known as morphological changes. These changes encompass the condensation of chromatin, nuclear blebbing, cytoplasmic, and the eventual demise of cellular without the loss of the membrane. Abbreviations P53ts_ temperature-sensitive p53 p53mut_constitutivelymutant p53 PS_ phosphatidylserine Aph_ aphidicolin FITC_uoresceine isothyocyanate FACS_uorescent activated cell sorting HU_hydroxyurea Background Over time, the field of apoptosis has received much attention, and has become a major field of research. As the understanding of the way the process of apoptosis takes place, it has become easier to effectively define the manner in which the late and earlier phases this process take place. The materialization of the phosphatidylserine (PS) starts from the inner to the outer parts of the cell membrane is one of the earliest features of the process of apoptosis. Those cells having PS on the outer surfaces may be inked so as to enhance the easy of identification as well as possible removal of phagocytes and cells. Another early feature of an apoptotic cell is the actual split of the genomic DNA into very large fragments of about 50-300 kb. There are so many cell types that show this kind of process, and they include U937 leukemia cells, thymocytes, and the MOLT-4 human T lymphoblastoid cells. Generally, it is assumed robust DNA fragments are as a result of a split of the looped domains at the attached points of the nuclear matrix, and they can occur independently. The tumor suppressor, p53 is the best example of a molecule that can result into an induction the apoptosis process. This molecule is initiated by a myriad of changes to the cellular material, such as changed DNA, hypoxia, heat shock, as well as nucleotide depletion. Until now, the mechanism upon which the Enhanced P53 apoptosis takes place is still not known well, but it is clear that the transcriptional activation of a variety of apoptosis-enhancing genes is involved. These kinds of genes include bax, fats, KILLER/DR5, and more others. On top of the role it plays in the induction of apoptosis , P53 is also responsible to enhancing the process of DNA repair. Currently, it is believed that the apoptosis induction process is irreversible. According to the preliminary results from the laboratory, the process of DNA repair is normally enhanced very early in the p53- Enhanced apoptosis, and this early stage may actually be reversible. The current work is based on these early works, and through more evidence it has confirmed that indeed the early phases of a p53-Enhanced apoptosis can be reversed. Figure 1. Results P53 Activation Externalizes PS According to the preliminary studies in the laboratory, it has been noted that early phases of a p53-Enhanced apoptosis may actually be reversible, and the DNA repair could be playing a big role in this process of reversibility. To further strengthen our hypothesis, we have gone further to analyze the p53-Enhanced apoptosis as well as its reversibility. We have incorporated the temperature-sensitive p53 cell line (p53ts) upon which p53 normally activates at 30 degrees. In addition, we sought to examine the early stages of apoptosis such as the externalization of phosphatidylserine (PS). The FITC-labeled Annexin-V antibody was used to analyse the percentage of the apoptotic cells by flow cytometry. As observed in figure 1a and b, the incubation of the p53ts at 30 degrees for six hours is responsible for the increased levels of the annexin-positive apoptotic, as well the levels of FITC-labeled cells. Through quantitation, it has been revealed that at the no-permissive temperature of 37 degrees, the p53ts contains 10% annexin-V positive cells. Nevertheless, after the eruption of six hours at the permissive temperature of 30 degrees, there is an increase in the number of annexin-V positive cells to about 31% (Figure 1c). in a similar manner, the analysis of mutant 37 degrees and 30 degrees show that there is only 6% annexin-V positive cells at 37 degrees, and with no changes with the incubation at 30 degrees. As per these results, the activation of p53 leads the flipping and apoptosis of PS within 6 hours in the p53ts cells. Figure 2. If the apoptotic stimulus is removed PS-positive cells can survive Next, we the reversibility of each PS-positive apoptotic cells by fluorescent-activated cell sorting analysis. Also, it was observed that the p53ts cells were incubated either at 30 degrees or 37 degrees after a period of 6 hours, and were incubated with FITC-labeled annexin-V antibody. By use of MoFlow cell sorter, 1500 PS-positive apoptotic cells were removed from the population cells and then sorted into 24-well culture dishes. In addition, the FITC-negative control cells from the dishes that were incubated at 37 degrees were sorted to indicate the number of surviving, non-apoptotic cells that usually replete and grow during this process. Figure 2c shows that annexin-V positive, apoptotic cells can survive and grow if the apoptotic stimulus is removed. This is clearly a sign that the early phases of p53-Enhanced apoptosis are indeed reversible. Reversible Apoptotic Cells Are Not Resistant To Further Apoptosis Induction Hypothetically, it is assumed that the reversibility of apoptotic cells is possible because those selected cells have already undergone genetic alterations which have resulted into apoptosis resistance. So as to effectively address this concern, we incubated p53ts cells at 30 degrees and we then allowed them to grow. We then followed up this with the re-exposure at 30 degrees for a total of 4 cycles (see figure3). As indicated in figure 3a, these cells are still able to undergo apoptosis. Therefore, it is not easy reversible apoptotic cells to be selected because of their resistance to the classical unscheduled DNA synthesis assay that utilizes autoradiography to examine the H-thymidine incorporation into nuclear DNA in the complete absence of DNA replication. Figure 4a shows that without aphidicolin or HU, there is H-thy incorporation into the nuclei at 30 degrees. The inclusion of HU into the cells leads to an almost complete inhibition of H-thy incorporation (figure 4b). On the other hand, figure 4c indicates that the addition of HU and aphidicolin altogether leads to 100% inhibition. Figure 3. Figure 4. Aphidicolin Accelerates P53-Enhanced Apoptosis If we found out that DNA repair would modulate p53-Enhanced apoptosis, we concluded that inhibiting DNA repair should catalyze the death process. To examine this hypothesis, we utilized the classic DNA repair inhibitor aphidicolin. We used two methods to analyze the process of cell death, and one of them was the colony formulation assay. Figure 6 indicates that colony formation stays at control levels up to six hours at 30 degrees. Material and Methods Cell culture Originally, MOD cells were obtained from a mouse mammary carcinoma and contained truncated p53 mRNA as well as nonfunctional p53 protein. These cells we stably transfected using either a constitutively mutant p53 or a temperature-sensitive p53. The p53 plasmid leads in a valine substituted for alanine at amino acid #135 of the mouse protein. On the other hand, the p53mut construct consists of a cysteine to a phenylalanine substitution at amino acid #132. Annexin-V Assay All the cells were incubated AT 30 or 37 degrees. From here, these cells were then removed from the respective dishes and were washed using PBS and the cells were then spun again. After washing the cells using PBS, the cells were suspended in a modified 1x binding buffer. The reaction was performed for a period of 15 minutes at room temperature. After the end of the reaction, 400 µl of modified 1x binding buffer was added into the solution. From here, an analysis was performed. In order to obtain the percentage of FITC-V-positive cells, the flow cytometry was conducted and the number of FITC-V-positive cells was obtained. UDS Assay At 30 or 37 degrees, cells were incubated at DMEM for 6 hours with or without hdroxyurea or aphidicolin. In the last hour of each incubator, H-thymidine was added to each well. After the end of the 6 hours, all the incubators a rinse in the DMEM were continued at either 30 or 37 degrees, with or without aphidicolin or hydoxyurea. Cells were considered to be positive if there was five grains appearing over the nucleus. The background was on the other hand determined by doing an analysis of the number of grains in the region with no cells corresponding to the actual size of one cell. Colony Formation Assay 2000 cells were all plated on 30mm-diameter dishes and were allowed to attach overnight at 37 degrees. These dishes were either left at 37 degrees for 72 hours or were put at 30 degrees for 2,4,6, or 12 hours with or without the inclusion of aphidicolin, and were then returned to 37 degrees for the remainder of the 72 hours. Those colonies that were considered positive contained five or more cells. That number of cells that were obtained in the 72 hours at 37 degrees dishes was set at 100% and then was compared with the 30 degree-treated dishes. DNA Fragmentation Analysis The cells were either incubated at 37 or 30 degrees for indicated periods, with or without the addition of aphidicolin. Then, media was removed and digestion buffer was added. Using a rubber, the cells were scraped off the plates and incubated at 50 degrees overnight. After this, DNA was treated using RNase, followed by another phenol/chloroform extraction and finally treated by ethanol precipitation. The DNA concentration was determined using measurement. Western Blots The cells were either incubated at 37 or 30 degrees for indicated periods, then media was removed. Next, RIPA buffer was added. The extracts were then scraped off the plates, and then were boiled for five minutes; and then they were spun at 10,000 r.p.m for 30 minutes. According to the manufacturer’s instructions, ECL/ECL-Plus method detects the protein antibodies complexes. Statistical Analysis All the statistical data was performed by the analysis of variance (ANOVA), and the Stats Plus Program (Human Systems Dynamics) was used to perform the task. Conclusion Apoptosis is referred as an orderly event that takes place in a reproducible manner in a variety of cell types that are Enhanced by many different agents as well as physiological situations. Through the continued gaining of more knowledge on this topic, it is very possible that this knowledge will be useful in the diagnosis and treatment of various diseases. In our current study, we are focusing on p53, a molecule that takes place in the process of apoptosis as well as the maintenance of the genomic integrity. According to figure in our studies, there is a possibility that the early stages of p53-Enhanced apoptosis are indeed reversible if the apoptosis stimulus is removed. Further, we conclude that the process of DNA repair is activated very early in the process of p53-Enhanced apoptosis. On the same note, the study indicates that the inhibition of DNA repair results into the accelerated apoptosis. This strongly suggests that DNA has a role in the reversibility of the early apoptotic phenotypes. How the Article Incorporates Visual This article is presented in clear and elaborative manner, with the incorporation of well taken pictures. These figures are an extra incentive to the overall understanding of the concept being addressed. With every concept or idea being addressed, there is a direct reference to the corresponding figure for better understanding. The concept being addressed seems to be clear when you refer it in the figure that is corresponding to it. Nevertheless, some of the figures used seem not to be clear because of the color or the quality of the picture. I will suggest that in future articles, there is need to use more quality pictures. With poor quality pictures, it becomes very difficult to view some of the results as indicated in the main document. Why I Am Interested In This Topic/Article? My interest in the topic stems from the willingness to understand how cells die and the existing ways through which they die. As observed by Manfredi et al. (153), there is a tendency of cells to continuously die in the living organisms. This tendency in which cells continuously die in the bodies of living organisms has been a hot topic that in the recent times has attracted lots of scientific studies. Since there has been no agreement on how this process takes place, I will also like to study further with the motif of adding further insight into the topic. Works Cited Manfredi, A.A., Iannacone, M., D’Auria,F., & Rovere-Querini,P. The Disposal of Dying Cells In Living Tissues. 2002. Viewed 9th May 2015. < http://www.iannaconelab.com/wp- content/uploads/2011/03/Apoptosis-2002-Manfredi.pdf>. Read More