Drug Screening Lab Report
Drug Screening Lab Report
Cell Biology Laboratory PCB 4023 Laboratory 6: Screening drugs to treat diseases Mayoral, J. INTRODUCTION: CHOLESTEROL IN HUMANS Humans need certain amount of cholesterol for the regular maintenance of metabolic processes like the synthesis of cell membranes, hormones and some vitamins. Cholesterol enters the body through the food we eat or it is synthesized in our bodies, mainly in the liver. A proper equilibrium of cholesterol levels in the plasma is controlled by the low-density lipoprotein receptors (or, LDL receptors) located on the outside of different cells and that facilitate the endocystosis of LDL-cholesterol. Once inside, the cholesterol is either stored, used by the cells or expelled by the body. Synthesis of the receptors in the cell is regulated by the level of free intracellular cholesterol; if it is in excess for the needs of the cell, then the transcription of the receptor gene will be inhibited leading to fewer receptors expressed and less cholesterol uptake. An excess intake of certain foods or a miss regulation in any of the metabolic processes controlling cholesterol levels may lead to undesirable levels in blood or the cytoplasm of cells, inducing foam cell formation in Source: https://slideplayer.com/slide/5242016/ atherosclerosis. CASE STUDY: CHOLESTEROL ACCUMULATION AND FOAM CELL FORMATION In humans, LDL is directly involved in the development of atherosclerosis, which is the process responsible for the majority of cardiovascular diseases; in this condition, the inside of the arteries narrows down after a buildup of LDL-cholesterol (fat and cholesterol). The development of foam cell formation is one of the early steps in the pathologic process of atherosclerotic plaque development. The continued development of foam cells and the formation of plaques result in deposits that will cause blood clots and ultimately this may cause a heart attack or a stroke. In this laboratory, we will work on a case study, a rare condition in which cells from patients take up abnormally high amounts of cholesterol. This accumulation has been reported to happen in different cell types including fibroblasts, macrophages and others. For example, in macrophages, this accumulation of cholesterol inside of the cells transforms the Cell Biology Laboratory PCB 4023 macrophages into the so called foam cells; these fatty droplet deposits are responsible for the foamy appearance of the macrophage, and thus their name. Unfortunately, the mechanism by which this abnormal accumulation of cholesterol occurs is not fully understood. Although it is not clear to what extent cell uptake of cholesterol contributes to vessel wall cholesterol accumulation, cell cholesterol accumulation has pathologic consequences. In the specific case of macrophages, accumulation of cholesterol promotes macrophage release of metalloproteinases and expression of tissue factor, processes that can promote plaque rupture and subsequent plaque thrombosis. Additionally, macrophage uptake of extracellular unbound LDL may directly contribute to vessel wall cholesterol accumulation by preventing the LDL from exiting the vessel wall through transport back either into the blood or into the lymphatic’s vessels. One hypothesis is that excess cholesterol is uptaken bound to LDL via LDL receptors. However, several studies have shown that patients and animals lacking the LDL receptors nerveless show foam cells in their atherosclerotic plaques; furthermore, LDL receptors in this individuals show to be down regulated as a response to high level of intracellular cholesterol. Thus, a different mechanism may be responsible for this pathology. In the 1970’s, in a study with normal subjects and patients with hypercholesterolemia two researchers reported for first time that modified LDL maybe the cause for the abnormal uptake of cholesterol. Interestingly, they also suggested a non-receptor mediated, fluid uptake of LDL in patients with mutant fibroblasts that lacked the LDL receptors. In this laboratory, we will evaluate this last hypothesis. Students will assess if any of the fluid uptake mechanisms is responsible for this disease. For the purpose of this study, we isolated a fibroblast cell line from a patient with this pathology. SCREENING OF COMPOUNDS In order to identify the mechanism by which cells accumulate intracellular cholesterol, students will use the following drugs: Compound A (Amiloride) is a selective Na+/H+ antiport inhibitor of macropinocytosis but not any of the micropinocytic processes. Compound B (Nystatin) is an inhibitor of lipid raft-caveolae micropinocytosis in mammalian cells Compound C (Chlorpromazine) is an inhibitor of the clathrin-coated pit formation in micropinocytosis by a reverse translocation of clathrin from the plasma membrane to the intracellular vesicles. EXPERIMENTAL PROTOCOL: In this lab session, students will work with a fibroblast cell line isolated from a sick patient with cholesterol plaque accumulation. To test the possible intake of nonspecific material through the cell membrane, students will assess the incorporation of cholesterol-LDL conjugated to horseradish peroxidase (HRP) in the cell. The class will be divided in several groups in order to assess the 3 types of pinocytic processes as possible candidates for this pathology. We will screen several compound to find a cure for this rare pathology. Cell Biology Laboratory PCB 4023 EQUIPMENT: provided by the lab coordinators – Culture plates with patient fibroblasts 37°C incubator Cuvettes Spectrophotometer REAGENTS: prepared in advance by lab coordinator – 0.2 % Triton-X PBS PBS+0.2% BSA (ice-cold) HRP stock 50 mg/mL Cell scrapers DMSO Amiloride Nystatin Chlorpromazine Eppendorf tubes (3 per group) 0.1N H2SO4 PROCEDURE: 1. Take out a plate with cells from the incubator. 2. Remove 750 µl of media using your pipette. 3. Pre-incubate cells with 50 µl of the treatment assigned to your group for 30 min, 37°C. 4. After the pre-incubation period, add 20 µl of LDL-cholesterol-HRP; Place the lid and mix gently by swirling the plate consistently for 10 seconds. Start your timer for 30 minutes exactly when you add the HRP solution. 5. Incubate your cells at 37°C for 30 min. 6. After the 30 minutes, remove media immediately and wash the cell monolayer 4 times with 2 mL of ice-cold PBS containing 0.2% BSA. Gently swirl the plate for each washing step and finally remove the volume of the last wash (vacuum trap is ok). 7. Add 750 µl PBS containing 0.2 % Triton-X. Incubate 10 min at 4°C. 8. Detach the cells thoroughly from the bottom of the plate using a cell scraper (follow your instructor’s instructions). 9. Transfer-pipette the cells into a 1.5 ml Eppendorf tube. 10. Sonicate your cells for 7 seconds using a power of 17.5 (follow your instructor’s instructions). Cell Biology Laboratory PCB 4023 11. Place your cells on ice for 2 minutes. 12. Centrifuge your Eppendorf with your cells at 11,000 rpm for 3 min. Bring them back to ice. 13. Take 500 µl for the top of the solution (be careful to do not disturb the pellet) and bring it into a clean Eppendorf. 14. Label the tube “Extract” 15. Prepare a 1:5 dilution of your cell extract in a new Eppendorf tube. Prepare a total volume of 250 µl. 16. Label the new tube “Extract 1:5”. Keep it at 4°C until you are ready to use it for the enzymatic assay. HRP Assay 17. Add 700 µl of the substrate solution (o-phenylenediamine and H202) into a clean cuvette. 18. Add 10 µl of your lysate (diluted 1:5) from step 15. Start the timer as soon as you add the lysate to the reaction! 19. Mix by inverting the cuvette several times using Parafilm. Incubate the reaction at room temperature for exactly 8 minutes in the dark, and observe the appearance of orange/yellow color. 20. Stop the reaction by adding 400 µl of 0.1 N H2S04 to your cuvette. Mix by inverting the cuvette several times using Parafilm. 21. Quantify the results using the spectrophotometer at λ = 450 nm. Record your absorbance and share it with the class on the board. Keep your cuvette until the instructor authorizes you to dispose the content in the right waste container. 22. Discuss the results with your class. QUESTIONS: Testing and applying your understanding. 1.) According to the results obtained in class, what is the mechanism that fibroblasts with the pathology studied use to uptake abnormal levels of cholesterol? Is this a receptor mediated mechanism or unspecific? 2.) How is HRP important in this experiment? 3.) Do you have a negative control? Why? What reagents should your negative control contain for this experiment? 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