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Examining BCL-2 Family Function with Large Unilamellar Vesicles
Published on: October 5, 2012
Bcl-2 protein in 518A2 melanoma cells in vivo and in vitro
Luba Benimetskaya1, Kanyalakshmi Ayyanar, Noah Kornblum
1Albert Einstein-Montefiore Cancer Center, Department of Oncology, Montefiore Medical Center, Bronx, New York, USA.
Purpose:
Bcl-2 is an apoptotic protein that is highly expressed in advanced melanoma. Several strategies have been employed to target the expression of this protein, including G3139, an 18-mer phosphorothioate oligodeoxyribonucleotide targeted to the initiation region of the Bcl-2 mRNA. This compound has recently completed phase III global clinical evaluation, but the function of Bcl-2 as a target in melanoma has not been completely clarified. To help resolve this question, we have permanently and stably down-regulated Bcl-2 protein and mRNA expression in 518A2 cells by two different technologies and evaluated the resulting clones both in vitro and in vivo.
Experimental Design:
518A2 melanoma cells were transfected with plasmids engineered to produce either a single-stranded antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA or a short hairpin RNA also targeted to the Bcl-2 mRNA. In vitro growth, the apoptotic response to G3139, and the G3139-induced release of cytochrome c from isolated mitochondria were evaluated. Cells were then xenografted into severe combined immunodeficient mice and tumor growth was measured.
Results:
In vitro, down-regulation of Bcl-2 expression by either method produced no change either in the rate of growth or in sensitivity to standard cytotoxic chemotherapeutic agents. Likewise, the induction of apoptosis by G3139 was entirely Bcl-2 independent. In addition, the G3139-induced release from isolated mitochondria was also relatively independent of Bcl-2 expression. However, when xenografted into severe combined immunodeficient mice, cells with silenced Bcl-2, using either technology, either failed to grow at all or grew to tumors of low volume and then completely regressed. In contrast, control cells with "normal" levels of Bcl-2 protein expression expanded to be large, necrotic tumors.
Conclusions:
The presence of Bcl-2 protein profoundly affects the ability of 518A2 melanoma cells to grow as human tumor xenografts in severe combined immunodeficient mice. The in vivo role of Bcl-2 in melanoma cells thus differs significantly from its in vitro role, and these experiments further suggest that Bcl-2 may be an important therapeutic target even in tumors that do not contain the t14:18 translocation.
Insights
Bcl-2 protein is crucial for melanoma tumor growth in vivo, but not in vitro. Silencing Bcl-2 prevented tumor formation in mice, highlighting its potential as a therapeutic target.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Genetics
Background:
- Bcl-2 is an apoptosis-inhibiting protein overexpressed in advanced melanoma.
- Targeting Bcl-2 expression is a strategy for melanoma treatment.
- The precise role of Bcl-2 in melanoma progression requires further elucidation.
Purpose of the Study:
- To investigate the role of Bcl-2 in melanoma cell growth and response to therapy.
- To stably down-regulate Bcl-2 expression in 518A2 melanoma cells using two distinct technologies.
- To evaluate the in vitro and in vivo consequences of Bcl-2 down-regulation.
Main Methods:
- 518A2 melanoma cells were transfected with antisense oligonucleotides or short hairpin RNA targeting Bcl-2 mRNA.
- In vitro assessments included cell growth, apoptosis induction, and mitochondrial cytochrome c release.
- Tumorigenicity was evaluated by xenografting modified cells into severe combined immunodeficient mice.
Main Results:
- In vitro, Bcl-2 down-regulation did not alter cell growth or sensitivity to chemotherapy.
- Apoptosis induction by G3139 and cytochrome c release were largely Bcl-2 independent in vitro.
- In vivo, silenced Bcl-2 cells failed to grow or regressed, while control tumors grew significantly.
Conclusions:
- Bcl-2 expression is critical for the in vivo growth of 518A2 melanoma xenografts.
- The in vivo function of Bcl-2 in melanoma differs substantially from its in vitro role.
- Bcl-2 represents a promising therapeutic target in melanoma, irrespective of t14:18 translocation status.
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