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Drug Induced Apoptosis Investigation 622555

Introduction
Leukaemia is a broad term which covers a variety of diseases which cause cancer in the blood and bone marrow. There are two main types of Leukaemia which are acute Leukaemia and chronic Leukaemia, these two types are then subdivided into more types. According to X. Frank Zhao et al Environmental exposure to radiation, chemicals and microorganisms has the potential to cause leukaemia. Mutations of DNA, like other cancers, is what results in Leukaemia. Leukaemia is treatable by three methods, chemotherapy, medical radiation therapy, or hormone treatments. One such drug used to treat Leukaemia is Etoposide. Etoposide is a cytotoxic agent or anticancer drug that belongs to the topoisomerase inhibitor classification which are designed to interfere with the action of topoisomerase enzymes . Synthesised first in 1966 Etoposide is derived from podophyllotoxin, a toxin found in the American Mayapple.it functions as an anticancer drug by forming a ternary complex with topoisomerase II enzyme which aids in DNA unwinding and DNA itself. Topoisomerase II enzyme prevents DNA from being able to re-litigate which in turn causes DNA to break. This enzyme is relied upon by cancerous cells more than healthy cells as there division is more rapid. Errors in DNA synthesis are then caused by this and cell apoptosis is promoted. There are other drugs in the topoisomerase inhibitor class that specifically target topoisomerase II enzyme and work in a similar way, these include, teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid,[5] and HU-331, a quinolone synthesized from cannabidiol. Apoptosis, as mentioned before, is a course of action of programmed cell death (PCD) which is caused by biochemical episodes, effecting cell morphology and resulting in cell death, this occurs in multicellular organisms. Unlike necrosis which is cell death caused through trauma, PCD is an automated function controlled by the organism. This automated function can be manipulated however, by administering certain drugs. Mitotic index is the count of the reproduction state within a cell population. It is extremely useful in investigating cancer treatment as it shows how many cells are in the process of mitosis and therefore operating in a normal fashion. By using it you can predict the overall response and effectiveness of chemotherapy. By utilising the mitotic index and also using electrophoresis this experiment will ascertain how well Jurkat cells respond to etoposide treatment.

Method
Firstly the DNA will be extracted from Jurkat cells using a DNA extraction kit (from Norgen). 200l of digestion buffer was added to a frozen pellet of Jurkat cells that had either been treated or not been treated with etoposide.12l of Proteinase K and 1 l of RNase A were also added. The Proteinase K and RNase A break down the protein the DNA is coiled around and the RNA respectively. The mixture was then incubated at 55C for 1 hour.200l of binding solution was then added lysate followed by 200l of 100% ethanol. A spin column was then assembled along with a collection tube and the mixture was then added using a 1ml pipette. The column was then centrifuged at 8,000RPM for 3 minutes. The flow through was then discarded and the spin column was reassembled with the collection tube. After this 500l of wash solution was added to the column and centrifuged for 1 minute at 13,000RPM, the flow through was then discarded. The last step was then repeated again

only centriguging for 2 minutes.the column was then spun at 13,000RPM for a further minute to completely dry the column. The next step of the process is to extract the DNA from the resin. This is done by adding 200l of elution buffer to the centre of the resin bed, this extracts the DNA from the resin bed. The column was the centrifuged at 6,000RPM for 1 minute passing the elutin buffer through the resin hydrating the DNA. The column was then centrifuged for a further 2 minutes at 13,000RPM to collect all of the elution volume, this contains the purified DNA genome.20l of the DNA sample was then added to a new centrifuge tube along with 4l of loading buffer IV. 5l of the DNA sample was then added to lanes 1 and 9 of a prepared agarose gel electrophoresis apparatus with added midori green to make it fluoresce. 20 of the DNA sample was then added to the wells in the gel. The loading buffer containing sucrose and bromophenol blue applied earlier helps to give the sample density so it sinks into the wells better and also gives it colouration so its progress can be tracked through the gel. Alternatives to this buffer include loading buffer II containing ficoll and loading buffer III containing glycerol. The gel was then observed under UV light. Onto a sample of CytospinTM Jurkat cells on microscope slide provided a Giemsa stain was applied. After this the slides were observed under a light microscope.

Results
Figure 1: Analysis of DNA from Jurkat cells by agarose gel electrophoresis.

Figure 1: Analysis of DNA from Jurkat cells by agarose gel electrophoresis. analysis of DNA from Jurkat cells. Cells were treated with 100 M etoposide for 24 hours (lanes 912), or left untreated (lanes 2-5). Hyperladder I (H, lanes 1 and 8) was used as a DNA marker. Figure 1 shows that the etoposide has successfully treated the jurkat cells. This is because after viewing the gel under UV light the DNA from the treated cells has travelled further through the agarose gel than the untreated DNA. This occurs because the pH of 8.3 used for the electrophoresis ionizes the phosphate groups within the DNA giving it a strong negative charge. This in turn facilitates active migration towards the positive anode.

Figure 2: Giemsa-stained Jurkat cells.

Figure 2: Giemsa-stained Jurkat cells. Cytospin preparations of Jurkat cells that had been exposed to 100 M etoposide for 24 hours (B) or left untreated (A) were stained with Geimsa stain for 20 minutes. Images were captured at 1000X magnification using a light microscope equipped with a camera. Figure 2 also shows that etoposide treatment was succsessful as the treated cells show no whole cells indicating that apoptosis has occurred. The untreated cells however, show cells that have fully formed membranes and also show cells in mitotic states. This indicates that cells were living at the point of slide preparation. Figure 3: table to show percentage of Jurkat cells treated or untreated with etoposide in mitotic states after Giemsa staining. Jurkat cells Treated with etoposide Untreated with etoposide Percentage of cells in active mitosis 0 0 0 0 0 0 0 0 0 0 0

12

15

10

12

11

11

14

Mean = 10.1% untreated cells in mitotic states and 0% treated cells in mitotic states.

Standard deviation = 101.6

Discussion
The objective of this lab report was to ascertain whether etoposide can be used to successfully treat Leukaemia in Jurkat cells. The findings of this experiment would indicate that the objective of this report has been achieved and the etoposide does indeed successfully treat Jurkat cells. The agarose gel electrophoresis proves this correct as the etoposide has fragmented the Jurkat cell DNA , allowing them to move through the agarose gel freely as the fragments of DNA are small enough. The same cannot be said for the untreated Jurkat cell DNA which is too large to travel through the gel. It is also noticed that when observed under a light microscope the Geimsa stained slides of untreated Jurkat cells showed whole cells and also cells in states of mitosis. The Geimsa stained slides of treated Jurkat cells when viewed under a light microscope showed fragmented cells and no cells in mitotic states indicating apoptosis had occurred and the DNA had been successfully unwound. This data is shown in Fig 3. Acridine orange/ethidium bromide (AO/EB) double staining test is an alternative to traditional Giemsa staining, as shown by C. CINIGLIA et al when investigating apoptosis and necrosis in plant cells. Acridine orange ethidium bromide (AO/EB) double staining test is quick, reliable and can also be used to examine cancer cells. Another alternative to the Giemsa stain is the Romanowsky Giemsa stain, the application of this however can be troublesome. There are alternatives to Giemsa staining of cells and agarose gel methods being used to study the ability of drugs to induce apoptosis. These methods include the MiCK assay as used by Kravtsov et al and the SCGE assay used by P Lebailly et al. If the concentration of the DNA was required it could be ascertained by measuring the Absorbance of the sample in spectrophotometer and then using the following equation. 50g/ml xg/ml 1 = A

In conclusion this lab report and experiment have successfully proven that etoposide does indeed treat cancer cells effectively.

References
P Lebaillya, b, C Vigreuxa, T Godarda, F Sichela, E Bara, J.Y LeTalara, M Henry-Amara, b, P Gauduchona. (1997). Assessment of DNA damage induced in vitro by etoposide and two fungicides (carbendazim and chlorothalonil) in human lymphocytes with the comet assay. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 375 (2), p205-215. Kravtsov VD, Daniel TO, Koury MJ.. (1999). Comparative analysis of different methodological approaches to the in vitro study of drug-induced apoptosis.. Am J Pathol. . 155(4), p1327-39. Horobin, R. W. How Romanowsky stains work and why they remain valuable - including a proposed universal Romanowsky staining mechanism and a rational troubleshooting scheme. Biotechnic & Histochemistry. Feb2011, Vol. 86 Issue 1, p36-51. Behera, B.; Mathur, P.; Gupta, B. Blood culture gram stain, acridine orange stain and direct sensitivity-based antimicrobial therapy of bloodstream infection in patients with trauma. Indian Journal of Medical Microbiology. Apr2010, Vol. 28 Issue 2, p138-142

By: Ciniglia, C.; Pinto, G.; Sansone, C.; Pollio, A. Acridine orange/Ethidium bromide double staining test: A simple In-vitro assay to detect apoptosis induced by phenolic compounds in plant cells. Allelopathy Journal. 2010, Vol. 26 Issue 2, p301-308

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