Identification of Apoptotic Proteins

1. Identification of Apoptotic Proteins Emily Young Carr

2. Purpose • To determine if cells under tumor-like stress die by apoptosis. • To test if Bcl-XL and Bax are involved when cells die under tumor-like stress.

3. Background Information • Apoptosis is a naturally occurring process in which a no longer functional cell dies. (Campbell & Reece, 2008). • The cell’s decision of whether to live or die occurs in the mitochondrion (Campbell & Reece, 2008). The release of cytochrome c from the mitochondrion triggers apoptosis.(Weinberg, 2006). • The protein Bcl-XL inhibits apoptosis by stopping the passage of cytochrome c out of the mitochondrion (Adams & Cory, 1998).

4. Background Information cont. • The protein Bax promotes apoptosis by heightening the mitochondrial membrane’s permeability so cytochrome ccan be released(Marzo, 1998). • To identify a specific protein, size markers are used. • Bax is typically seen at about 21 kDa (Cell Signaling, 2011) however Bax is known to cleave during apoptosis to a molecular weight of 18 kDa (Cao, 2003). • Bcl-XL is located at about 28 kilodaltons (Cell Signaling, 2011). • Recent research has shown that cells under tumor-like stress die due to lipid deprivation (Young, 2012). • Lipids rescue viability in cells under tumor-like conditions. Therefore the apoptotic process is halted by the presence of lipids in cells (Young, 2012).

5. The diagram illustrates the release of cytochrome c triggering the apoptotic cycle. The role of anti- and pro- apoptotic proteins in this occurrence is also shown.

6. Hypothesis • Cells under lipid deprivation die through the mechanism of apoptosis. • If both proteins are involved, then higher levels of the pro-survival protein Bcl-XL will be found in both the replete and limited serum and oxygen with fatty acid supplementation conditions while higher levels of the pro-apoptotic protein Bax will be found in the limited serum and oxygen condition.

7. Procedure •  1. The plates were set up in a Bio-Rad apparatus. • 2. 20 mL of 15% acrylamide solution was prepared. • 3. The gel was poured and the plates were sealed on top with isopropyl alcohol. • 4. 4 mL of stacking gel was prepared and added to the gels. • 5. Combs were placed in the stacking gel to create wells. • 6. The gels were given 1 hour to polymerize. • 7. The protein extracts were prepared. There were 3 samples of each nutrient condition. • 5x loading dye was added to the samples. • Samples were heated for 5 minutes at 95°C. • 8. The extracts were loaded onto the gels which were run for 2.5 hours.

8. Procedure cont. • 9. The proteins were transferred from the gels to nitrocellulose membranes using a transfer apparatus. • 10. Membranes were stained with Ponceau-S dye and then washed. • 11. Membranes were blocked for an hour with 5% milk in TBST. • 12. 10 mL of each primary antibody was prepared. The antibodies were hybridized to the membranes in the cold room overnight. • 13. Unbound antibodies were removed and the membranes were washed. • 14. The secondary antibodies were placed on the membrane for an hour. Then the secondary antibodies were removed and the membranes were washed. • 15. ECL, a chemiluminescence reagent, was added to the membranes which were taken to a dark room and developed. • 16. For a control, the blots were stripped with a stripping buffer. Then the β-actin antibody was added to the blots and the same procedure for detection which was previously described was performed.

9. Diagrams • This image depicts gel electrophoresis.Note the wells in the gel for protein loading. • This image is intended as a reference point and is not identical to this experiment’s procedure.

10. Diagrams cont. This image depicts protein transfer. This image is intended as a reference point and is not identical to this experiment’s procedure.

11. Photographs 1.5mm plates with polymerizing gels. Vaseline seal on bottom. The gels with wells before protein loading. All photographs were taken by me. Running the gels. Bubbles show current. The nitrocellulose membranes between the first and second antibodies.

12. Variables • Independent Variables- • the apoptotic protein being tested for, either Bcl-XL or Bax • the nutrient condition under which the cells of the extracts were cultured • Dependent Variable- the expression of the proteins in the three nutrient conditions • Control- βactin was used as a loading control • Constants- the amounts of antibodies and solutions used, the materials used, temperatures, the location used for experimentation

13. Scans of Bcl-XL and Bax Blots Scans show a western blot analysis of the expression of Bcl-XL and Bax under replete (R), serum and oxygen limitation (SO), and serum and oxygen limitation with fatty acid supplementation (SO+FA).

14. Tables for Bax Levels of Cleaved Bax in Three Different Nutrient Conditions Statistical Analysis for Bax

15. Tables for Bcl-XL Levels of Bcl-XL in Three Different Nutrient Conditions Statistical Analysis for Bcl-XL

16. Bax Graph

17. Bcl-XL Graph

18. Conclusion • Hypothesis- The dual hypothesis was that… • Cells under lipid deprivation die through the mechanism of apoptosis. • If both proteins are involved, then higher levels of the pro-survival protein Bcl-XL will be found in both the replete and limited serum and oxygen with fatty acids conditions while higher levels of the pro-apoptosis protein Bax will be found in the limited serum and oxygen condition. • It was found that cleaved Bax is involved in the lipid rescue phenotype and Bcl-XL is not. The involvement of Bax in the lipid rescue phenotype supports the idea of apoptosis as the death mechanism. Therefore, the hypothesis was supported. • Applications • Increased knowledge of the phenotype this experiment focused on is helpful in determining which areas of tumors to target when developing new cancer treatments. • The unexpected result which suggests that cleaved Bax is involved in the lipid rescue phenotype could help in the development of artificially cleaved Bax as a more effective initiator of apoptosis.

19. Conclusion cont. • Possible Sources of Error • Non-specific binding particularly of Bax antibodies may have made it appear as though Bax was involved when it was not. • The occurs because antibodies bind to the nitrocellulose membrane instead of the proteins on the membrane. • However, steps were taken to prevent this, the blocking of the membranes in 5% milk in TBST. • Improvements and Further Experimentation • Other members of the Bcl-2 family such as Bcl-2 could be tested in a western blot with extracts of cells cultured under the same conditions used in this experiment. • It would be interesting to determine which other pro-apoptotic proteins are involved in the lipid phenotype.

20. Acknowledgements • Thank you to my teacher Mrs. Giannantonio, Simon Lab at the Abramson Family Cancer Research Institute, and most of all to Dr. Gina Young. • Thank you to all sources used. • Adams, J., & Cory, S. (1998, August 28). The Bcl-2 protein family: Arbiters of cell survival. Science, 281. Abstract retrieved from http://www.ncbi.nlm.nih.gov/‌pubmed/‌9735050 • Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (Eds.). (2002). Molecular biology of the cell (4th ed.). New York, NY: Garland Science. (Original work published 1983) • Bensaccount (2009, September 12). SDS-PAGE Electrophoresis [Digital Diagram]. Retrieved from Wikipedia database. • Campell, N. A., Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., & Jackson, R. B. (Eds.). (2008). AP edition biology (8th ed.). San Francisco, CA: Pearson Benjamin Cummings. • Cao, X., Deng, X., & May, W. S. (2003, October 1). Cleavage of Bax to p18 Bax accelerates stress-induced apoptosis, and a cathepsin-like protease may rapidly degrade p18 Bax. Blood Journal, 102. • Cell signaling technology. (2011). [Brochure]. Beverly, MA: New England BioLabs. • DeBarardinis, R. J., Sayed, N., Ditsworth, D., & Thompson, C. B. (2008). Brick by brick: Metabolism and tumor cell growth. Current Opinion in Genetics and Development, 18(1), 54-61. • The Intrinsic Mitochondrial Apoptosis Pathway] [Digital Diagram]. (n.d.). Retrieved from AbDserotec database. • Jürgensmeier, J., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D., & Reed, J. (1998, April 28). Bax directly induces release of cytochrome c from isolated mitochondria. Proceedings of the National Academy of Sciences of the United States of America, 95. Abstract retrieved from http://www.ncbi.nlm.nih.gov/‌pubmed/‌9560217 • Key, J. P. (1997). MODULE s5 - “t” TEST [Math Resource]. Retrieved January 31, 2011, from Research Design in Occupational Education database. • Marzo, I., Brenner, C., Zamzami, N., Jürgensmeier, J., Susim, S., Vieira, H., . . . Xie, Z. (1998, September 25). Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science, 281. Abstract retrieved from http://www.ncbi.nlm.nih.gov/‌pubmed/‌9748162 • Pierce, R. (2011, September 12). Standard deviation [Math Resource]. Retrieved January 31, 2011, from Math is Fun - Maths Resources database. • Vander Heiden, M., Li, X., Gottleib, E., Hill, R., Thompson, C., & Colombini, M. (2001, June 1). Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane. The Journal of biological chemistry, 276. Abstract retrieved from http://www.ncbi.nlm.nih.gov/‌pubmed/‌11259441 • Weinberg, R. A. (Ed.). (2006). The biology of cancer. New York, NY: Garland Science. • Young, R. M. (under revision). Dysregulated mTORC1 renders cells dependent on serum lipids for survival under tumor-like stress. Cell Metabolism. • Thank you to my audience for listening. I will now take any questions you may have.