Bcl-2 protein in 518A2 melanoma cells in vivo and in vitro

Luba Benimetskaya, Kanyalakshmi Ayyanar, Noah Kornblum, Daniela Castanotto, John Rossi, Sijian Wu, Johnathan Lai, Bob D. Brown, Natalia Popova, Paul Miller, Marilyn McMicken, Yin Chen, C. A. Stein

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)4940-4948
Number of pages9
JournalClinical Cancer Research
Volume12
Issue number16
DOIs
StatePublished - Aug 15 2006

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Fingerprint Dive into the research topics of 'Bcl-2 protein in 518A2 melanoma cells in vivo and in vitro'. Together they form a unique fingerprint.

Cite this