November
30, 2011
Fall
2011
R50
Pool, RNA, Bcl-2
Abstract
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 abstract over the dopamine transporter (DAT).
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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