Cancer Biology Experimental Design Report-Free-Samples for Students

Question: Write an Cancer Biology Experimental Design Report. Answer: Introduction: The appearance of characteristic morphological features, and the occurrence of specific energy dependant biochemical mechanisms which indicate towards the process of programmed cell death, are referred to as apoptosis. It is responsible for a number of biological functions such as normal cell turnover, appropriate development of the immune system, development of embryo, hormonal atrophy, and chemically induced cell death (Elmore 2007). This process also helps in maintaining tissue homeostasis, and forms normal part of growth and development events. Also, appropriate apoptosis involves differential and appropriate response of the different types of cells to physiological and pathological stimuli. However, any disturbance in the homeostatic equilibrium could either result in unwarranted cellular proliferation or atrophy characterized with faster cellular death (Hejmadi 2009). Therefore, the dysfunction of the homeostatic pathways, often leads to terminal proliferation and differentiation of cells, resulting in cancer. The development and progression of cancers have often been attributed to the suppressed apoptotic mechanisms during carcinogenesis. The tumour cells either acquire resistance to apoptotic pathways by the means of anti-apoptotic proteins, or evade the immune surveillance (Fulda 2009). For example, the uncontrolled expression of Bcl-2 gene results in failure of cell death, contributing to cancer. However, apoptosis also presents therapeutic opportunities for cancer. The comprehension and analysis of the cell cycle signalling pathways could help identify the possible mechanism of cell cycle which could be exploited for arresting or controlling the cell cycle and apoptotic mechanisms (Gerl Vaux 2005). The table below shows the features of cells undergoing apoptosis. Morphological features Biochemical features Physiological significance Blebbing of membranes Chromatin aggregation in nuclear membrane Initial stages involve cytoplasmic shrinkage and nuclear condensation Later stages involve cellular fragmentation Pore formation in mitochondria resulting in leakage Tightly regulated stepwise activation of enzymatic processes Energy dependent processes involving uptake of ATP Mono and oligonucleosomal DNA fragmentation in a non-random fashion Release of multiple factors such as cytochrome c, AIF into mitochondrial cytoplasm Activation of caspase cascade Altering membrane symmetry Absent inflammatory responses Phagocytosis by either macrophages or adjacent cells Affect individual cells Induced by physiological stimuli Table 1: Characteristics of cells undergoing apoptosis (Source: UCCS, 2017) Experiments: Identification of transformed cells: MTT assay Rationale: For the purpose of screening of the transformed cells, over expressing the BKLY protein, the cell based MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay is proposed. MTT is positively charged and has the ability to penetrate the eukaryotic cells. The viable cells exhibiting active metabolic activity possess the capability to convert MTT into purple colored formazan compound, having maximum absorbance near 570nm. On the other hand when the cells no longer remain viable they lose the capability to convert MTT into formazan. MTT is regarded to have the capability to measure mitochondrial activity as MTT cleavage occurs in the living cells only by the mitochondrial enzyme succinate dehydrogenase (Hughes Mehmet 2003). Control: To examine whether BKLY has any effect on apoptosis of the cells we would need to compare the data from positive, negative and blank controls with the experimental cells. The negative control will be consist of untreated cells, positive control will consist of the cells treated with cytotoxic chemical, and blank will contain no cells. The control and experimental cell lines will be maintained under similar conditions of temperature and incubation time periods. Conclusion: If the BKLY protein is involved in the apoptotic process then differences in metabolic activity of the apoptotic cells which have taken up the plasmids will help in identification of the transfected cells. The change in colour from yellow water soluble tetrazolium MTT to purpled colored insoluble formazan will help in identifying the non-viable cells, indicating towards transfection with BKLY gene. Identification of cells showing protein expression: Annexin V staining assay Rationale: In order to detect the cells expressing the protein, the immunohistochemical technique using Annexin V is suggested. Annexin V is a calcium dependent, phospholipid binding protein, which has the capability to serve as an excellent tool for detecting the ongoing process of apoptosis in cells. The process of apoptosis involves translocation of phosphatidylserinefrom inner leaflet to outer leaflet of the plasma membrane. The integrity of the phosphatidylserine exposed membrane is thus breached. The calcium dependant binding of Annexin V with the breached membrane is further tested using vital dyes such as Propidium Iodide. These dyes can penetrate the membrane only when the membrane integrity is breached. However, it must be noted that Annexin V shows preferable binding tendencies with apoptotic cells even in condition of excess necrosis. Hence, it is crucial to use combination of Annexin V assay with Propidium Idodie, a DNA marker. This further allows monitoring the progress ion of different stages of apoptosis. The staining procedures follow fluorescence microscopy (Petrovsky et al. 2003). Controls: To examine whether BKLY has any effect on apoptosis of the cells we would need to compare the data from positive, negative and blank controls with the experimental cells. The negative control will be consist of untreated cells, positive control will consist of the cells treated with Annexin V, and blank will contain no cells. The control and experimental cell lines will be maintained under similar conditions of temperature and incubation time periods. Conclusion: The untreated and treated cell lines when observed under fluorescence microscope help identify the cells expressing the protein. The treated cell lines will show increase in population of cells undergoing apoptosis, identified by increasing membrane permeability. Identification of nature of apoptotic properties: Flow Cytometry Rationale: For the purpose of identifying the apoptotic properties of the protein, the method of flow cytometry is proposed. Flow cytometry helps in successful quantification of apoptosis, besides distinguishing the apoptotic cells from non-apoptotic ones. The microscopic particles suspended in the fluid stream are counted, examined, and sorted, by means of DNA staining. The apoptotic cells which have been stained using a fluorescent dye, when passes through the single wavelength light beam, scatter light up to some extent. The forward scatter versus side scattering of the light helps distinguish between the apoptotic and non-apoptotic cells. The identification of the immunophenotype of the cell is facilitated by the altered morphology of the plasma membrane of apoptotic cells. The phosphatidylserine is present on the outer membrane in the apoptotic cells, which would be detected by Annexin V. Propidium Iodide would act as the DNA specific fluorochrome, helping differentiate between necrotic and apoptotic cells. However, the technique could prove to be time consuming (Archana et al. 2013). Controls: The proposed experiment would involve three controls. Control 1 will consist of the unstained cells, Control 2 will consist of those stained with Annexin V only, and Control 3 will be stained using PI only. Thereby allowing the differentiation between healthy (unstained), apoptotic (positive for Annexin), and necrotic cells (positive for both Annexin and PI) respectively. This will allow the researcher to draw accurate conclusions by forming basis of comparison and analysis of the experimental cells. Conclusions: The apoptotic cells would appear as having hypo-diploid structure of DNA contents, and will be represented in sub-G1 peaks on the histograms. The decreased staining ability of fluorochrome with respect to the apoptotic DNA is attributed to the endonuclease activity of apoptotic cells resulting in extraction of low molecular weight DNA. References: Archana, M., Yogesh, T.L. Kumaraswamy, K.L., 2013. Various methods available for detection of apoptotic cells- A review. Indian journal of cancer, 50(3), p.274. Elmore, S., 2007. Apoptosis: A review of programmed cell death. Toxicol Pathol., 35(4), pp.495516. Available at: https://www.ncbi.nlm.nih.gov/pubmed/19003982. Fulda, S., 2009. Tumor resistance to apoptosis. Int J Cancer., 124(3), pp.511515. Gerl, R. Vaux, D.L., 2005. Apoptosis in the development and treatment of cancer. Carcinogenesis, 26(2), pp.263270. Hejmadi, M., 2009. Introduction to cancer biology, Bookboon. Hughes, D. Mehmet, H., 2003. Cell Proliferation and apoptosis, Garland science. Petrovsky, A. et al., 2003. Near-infrared fluorescent imaging of tumor apoptosis. Cancer Res, 63, pp.193642. UCCS, 2017. Differences between necrosis and apoptosis. Apoptosis, Cell Death, and Cell Proliferation Manual, p.4. Available at: https://www.uccs.edu/Documents/rmelamed/apoptosis_003_004.pdf [Accessed February 11, 2018].