title: Bcl-2 protein in 518A2 melanoma cells in vivo and in vitro.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
authors.affiliation: Albert Einstein-Montefiore Cancer Center, Department of Oncology, Montefiore Medical Center, Bronx, New York, USA.
authors.forename: Luba
authors.initials: L
authors.lastname: Benimetskaya
authors.forename: Kanyalakshmi
authors.initials: K
authors.lastname: Ayyanar
authors.forename: Noah
authors.initials: N
authors.lastname: Kornblum
authors.forename: Daniela
authors.initials: D
authors.lastname: Castanotto
authors.forename: John
authors.initials: J
authors.lastname: Rossi
authors.forename: Sijian
authors.initials: S
authors.lastname: Wu
authors.forename: Johnathan
authors.initials: J
authors.lastname: Lai
authors.forename: Bob D
authors.initials: BD
authors.lastname: Brown
authors.forename: Natalia
authors.initials: N
authors.lastname: Popova
authors.forename: Paul
authors.initials: P
authors.lastname: Miller
authors.forename: Harilyn
authors.initials: H
authors.lastname: McMicken
authors.forename: Yin
authors.initials: Y
authors.lastname: Chen
authors.forename: C A
authors.initials: CA
authors.lastname: Stein
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001483:Base Sequence; D045304:Cytochromes c / Q000378:metabolism; D016373:DNA, Antisense / Q000235:genetics; D015536:Down-Regulation; D020868:Gene Silencing; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D051379:Mice; D008813:Mice, Inbred ICR; D016513:Mice, SCID; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D013873:Thionucleotides / Q000235:genetics / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays
chemical_list: D016373:DNA, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013873:Thionucleotides; C408162:oblimersen; D045304:Cytochromes c