The goal of the Minority Medical Student Award Program (MMSAP) is to increase the number of medical students in hematology from under-represented minority groups by introducing them to hematology in their early years of medical school. ASH News Daily is pleased to share a summary of the research conducted by current MMSAP participant Nadine Thompson in the summer of 2008. Her research mentor was Steven Grant, MD, of Virginia Commonwealth University.

Background

The goal of my project was to investigate the role of pro- and anti-apoptotic signaling molecules in the mitochondrial apoptosis pathway of myeloma and leukemia cells treated with GX15-070 and flavopiridol. When cells are presented with a death signal, such as genomic instability or oncogene activation, they normally undergo apoptosis through the mitochondrial signal transduction pathway. The cascade begins with the activation of sensitizer BH3-only molecules that inhibit anti-apoptotic molecules like Bcl-2, Bcl-xl, and Bcl-w. These anti-apoptotic Bcl-2 proteins are normally bound to activator BH3-only proteins and pro-apoptotic proteins, Bax and Bak preventing apoptosis from occurring. Once Bcl-2 proteins are bound by sensitizer BH3-only molecules, Bax and Bak are free to oligomerize, forming a pore-like complex on the surface of the mitochondria, through which cytochrome c is released into the cytosol of the cell, causing cleavage of a number of caspase molecules and inducing the cell to undergo apoptosis.

The mitochondrial signaling transduction pathway is a promising target for therapeutic intervention. My role was to characterize the effects of GX15-070 and flavopiridol on several molecules involved in the mitochondrial apoptosis pathway in human multiple myeloma and leukemia cells.

Method

Laboratory techniques employed during this project included cell treatment, flow cytometry, spectrophotometry, and Western Blot analysis. U266 and U937 cells, cultured at a density of 3-5 x 105, were treated with increasing single and combination concentrations of GX15-070 and flavopiridol. U266 and U937 cells were treating with .5 µm and .75 µm of GX15-070, as well as 75 nM and 100 nM of flavopiridol.

Following treatment of U266 and U937 cell lines, flow cytometry was performed at 24 hours to measure the degree of cell death occurring with varying concentration of each drug. Cell lysates were then collected, and spectrophotometry was performed to determine the total protein content of each sample obtained for each condition. Western Blot analysis was then performed to assess protein levels of various molecules involved in the mitochondrial apoptosis pathway. The proteins that were analyzed during Western Blot experiments included the sensitizer and activator BH3-only proteins, Bcl anti-apoptotic molecules, Bax, Bak, cytochrome c, apoptosis-inducing factor (AIF), and caspases 3, 8, and 9.

Outcomes

Flow cytometry results indicate that cell viability decreased with increasing concentrations of GX15-070 and flavopiridol. Furthermore, these results showed a synergistic increase in cell death when both drugs were administered simultaneously. Western Blot analysis revealed that the level of sensitizer BH3-only protein, Bad, remained constant in both cell lines, while the BMF increased with flavopiridol and decreased with GX15-070. Surprisingly, sensitzer BH3-only proteins, Puma and Noxa, decreased with increased cell death. Bik levels increased significantly with increased killing using the combination agents. Most of the anti-apoptotic Bcl-2 proteins did not change with increasing concentrations of both drugs, except for Bcl-xl, which decreased with GX15-070 and increased with flavopiridol. Cytochrome c and AIF level, however, increased in the cytosol with increased apoptosis. Western Blot analysis detected Bax at a higher molecular weight, possibly indicating that Bax has oligomerized with increasing drug concentration. This phenomenon was not detected during Western blot analysis of Bak. Activator BH3-only proteins, Bid and Bim, decreased significantly in U937 cell when both drugs were combined during treatment. PARP and caspases 3, 8, and 9, however, show increased cleavage activity with increased apoptosis.

Conclusion

The next step toward completing this project will be to confirm these results with additional experiments, followed by immunoprecipitation experiments to investigate protein-protein interaction. It is hoped that this work will serve as the basis for a new approach to myeloma and leukemia therapy involving the rational combination of flavopiridol and obatoclax.

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