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 journalArticle

21 Citations (Scopus)

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

Fingerprint

Melanoma
SCID Mice
Proteins
Messenger RNA
Neoplasms
Mitochondria
Growth
Technology
Initiator Codon
Antisense Oligonucleotides
Oligodeoxyribonucleotides
Cytotoxins
Cytochromes c
In Vitro Techniques
Tumor Burden
Heterografts
Small Interfering RNA
Plasmids
Research Design
Down-Regulation

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Benimetskaya, L., Ayyanar, K., Kornblum, N., Castanotto, D., Rossi, J., Wu, S., ... Stein, C. A. (2006). Bcl-2 protein in 518A2 melanoma cells in vivo and in vitro. Clinical Cancer Research, 12(16), 4940-4948. https://doi.org/10.1158/1078-0432.CCR-06-1002

Bcl-2 protein in 518A2 melanoma cells in vivo and in vitro. / Benimetskaya, Luba; Ayyanar, Kanyalakshmi; Kornblum, Noah; Castanotto, Daniela; Rossi, John; Wu, Sijian; Lai, Johnathan; Brown, Bob D.; Popova, Natalia; Miller, Paul; McMicken, Marilyn; Chen, Yin; Stein, C. A.

In: Clinical Cancer Research, Vol. 12, No. 16, 15.08.2006, p. 4940-4948.

Research output: Contribution to journalArticle

Benimetskaya, L, Ayyanar, K, Kornblum, N, Castanotto, D, Rossi, J, Wu, S, Lai, J, Brown, BD, Popova, N, Miller, P, McMicken, M, Chen, Y & Stein, CA 2006, 'Bcl-2 protein in 518A2 melanoma cells in vivo and in vitro', Clinical Cancer Research, vol. 12, no. 16, pp. 4940-4948. https://doi.org/10.1158/1078-0432.CCR-06-1002
Benimetskaya L, Ayyanar K, Kornblum N, Castanotto D, Rossi J, Wu S et al. Bcl-2 protein in 518A2 melanoma cells in vivo and in vitro. Clinical Cancer Research. 2006 Aug 15;12(16):4940-4948. https://doi.org/10.1158/1078-0432.CCR-06-1002
Benimetskaya, Luba ; Ayyanar, Kanyalakshmi ; Kornblum, Noah ; Castanotto, Daniela ; Rossi, John ; Wu, Sijian ; Lai, Johnathan ; Brown, Bob D. ; Popova, Natalia ; Miller, Paul ; McMicken, Marilyn ; Chen, Yin ; Stein, C. A. / Bcl-2 protein in 518A2 melanoma cells in vivo and in vitro. In: Clinical Cancer Research. 2006 ; Vol. 12, No. 16. pp. 4940-4948.
@article{fb23eff0443844f8b0cb56f6bf3b52bb,
title = "Bcl-2 protein in 518A2 melanoma cells in vivo and in vitro",
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.",
author = "Luba Benimetskaya and Kanyalakshmi Ayyanar and Noah Kornblum and Daniela Castanotto and John Rossi and Sijian Wu and Johnathan Lai and Brown, {Bob D.} and Natalia Popova and Paul Miller and Marilyn McMicken and Yin Chen and Stein, {C. A.}",
year = "2006",
month = "8",
day = "15",
doi = "10.1158/1078-0432.CCR-06-1002",
language = "English (US)",
volume = "12",
pages = "4940--4948",
journal = "Clinical Cancer Research",
issn = "1078-0432",
publisher = "American Association for Cancer Research Inc.",
number = "16",

}

TY - JOUR

T1 - Bcl-2 protein in 518A2 melanoma cells in vivo and in vitro

AU - Benimetskaya, Luba

AU - Ayyanar, Kanyalakshmi

AU - Kornblum, Noah

AU - Castanotto, Daniela

AU - Rossi, John

AU - Wu, Sijian

AU - Lai, Johnathan

AU - Brown, Bob D.

AU - Popova, Natalia

AU - Miller, Paul

AU - McMicken, Marilyn

AU - Chen, Yin

AU - Stein, C. A.

PY - 2006/8/15

Y1 - 2006/8/15

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=33748349447&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33748349447&partnerID=8YFLogxK

U2 - 10.1158/1078-0432.CCR-06-1002

DO - 10.1158/1078-0432.CCR-06-1002

M3 - Article

VL - 12

SP - 4940

EP - 4948

JO - Clinical Cancer Research

JF - Clinical Cancer Research

SN - 1078-0432

IS - 16

ER -