RNA Aptamer Selection against Bcl-2 to Promote Apoptosis in Cancer Cells

Jessica Beardsley
November 30, 2011
Fall 2011
R50 Pool, RNA, Bcl-2

B-cell lymphoma 2 (Bcl-2) is an integral membrane protein found on the outer membrane of mitochondria, the intermembrane of endoplasmic reticulum and the nuclear envelop of most mammals (Yang et al. 1997).  Bcl-2 takes part in the complex apoptosis signaling pathway by preventing cell death without promoting cell proliferation (Chao and Korsmeyer 1998).  Bcl-2 regulates apoptosis by inhibiting c-Myc or p53 activated Bax/Bid proteins, which increase mitochondrial permeability and ultimately result in cell death (Yang et al. 1997).  Cancer cells often have high amounts of Bcl-2 due to an over-expressed BCL-2 gene, a proto-oncogene that can become activated into an oncogene (Gross 2001).  An abundance of Bcl-2 contributes to tumor initiation, progression, metastasis, and treatment resistance (Fernandez et al. 2002, Oltersdorf et al. 2005).
Bcl-2 specific inhibitors- including drugs such as Genasense- have been developed by researchers to promote apoptosis in cancerous cells with high levels of Bcl-2 ("CancerQuest | Oncogenes: Bcl-2" 2011).  In one study, a small molecule labeled YC137 was discovered and used to inhibit the anti-apoptic protein in breast cancer cells.  This was the first Bcl-2 inhibitor that was able to selectively kill cancer cells that over-expressed or relied on the protein for survival, but had no effect on the majority of primary cells.  Some cells did manage to develop resistance against YC137 by becoming less dependent on Bcl-2 for survival and decreasing Bcl-2 levels.  But these resistant breast cancer cells did become more sensitive to chemotherapy (Real et al. 2004).

Specific Aim 1: Selection of RNA aptamers against over-expressed Bcl-2 in cancer cells.

 Using a high affinity and specific binding RNA aptamer would be an ideal approach to studying the potentially therapeutic effects of Bcl-2 inhibitors on malignant tumor cells.  Prohibiting Bcl-2’s intervention of the cell death pathway will likely stimulate cancerous cells with high concentrations of the protein to undergo cell apoptosis.  A potential problem is that successful aptamers, unlike YC137, might bind to Bcl-2 in both cancerous and normal cells, encouraging apoptosis in important, primary cells.  But the possibility of finding a Bcl-2 aptamer could have a positive impact on the lives of those battling cancer by aiding in mutated cell death and allowing chemotherapy, radiotherapy, and hormone treatments to be more successful (Kimball 2011, Oltersdorf et al. 2005).  

Figure 1. The figure above is simplified version of the p53 cell apoptosis pathway, including specific aim one.  If Bcl-2 is inhibited, cancerous cells avoid survival and complete apoptosis.  There are several independent signal transduction pathways (caused by different stimuli) leading to cell death that include Bcl-2.  Adapted from “Nonsmall Cell Lung Cancer (2011).”

50 ug of recombinant, human Bcl-2 protein with a GST tag can be bought from the SignalChem.  The catalog number is H00006531-P01 and the company can be reached at 909-839-7620.  It is optimal to store the protein in 50 mM Tris-HCl buffer at a pH of around 7.5 at -70˚C for up to a year after the shipping date.

Here is the link to my old project proposal over DAT.
Here is the link to Progress Report 1.
Here is the link to my Final Manuscript over Bcl-2.

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