Recombinant Apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion

Exp Cell Res. 2003 Sep 10;289(1):36-46. doi: 10.1016/s0014-4827(03)00188-5.

Abstract

Apoptin, a protein derived from chicken anemia virus, induces apoptosis in human transformed or tumor cells but not in normal cells. When produced in bacteria as a recombinant fusion with maltose-binding protein (MBP-Apoptin), Apoptin forms a distinct, stable multimeric complex that is remarkably homogeneous and uniform. Here, using cytoplasmic microinjection, we showed that recombinant MBP-Apoptin multimers retained the characteristics of the ectopically expressed wild-type Apoptin; namely, the complexes translocated to the nucleus of tumor cells and induced apoptosis, whereas they remained in the cytoplasm of normal, primary cells and exerted no apparent toxic effect. In normal cells, MBP-Apoptin formed increasingly large, organelle-sized globular bodies with time postinjection and eventually lost the ability to be detected by immunofluorescence analysis. Costaining with an acidotrophic marker indicated that these globular structures did not correspond to lysosomes. Immunoprecipitation studies showed that MBP-Apoptin remained fully antibody-accessible regardless of buffer stringency when microinjected into tumor cells. In contrast, MBP-Apoptin in normal cells was only recoverable under stringent lysis conditions, whereas under milder conditions they became fully shielded with time on two epitopes spanning the entire protein. Further biochemical analysis showed that the long-term fate of Apoptin protein aggregates in normal cells was their eventual elimination. Our results provide the first example of a tumor-specific apoptosis-inducing aggregate that is essentially sequestered by factors or conditions present in the cytoplasm of healthy, nontransformed cells. This characteristic should reveal more about the cellular interactions of this viral protein as well as further enhance its safety as a potential tumor-specific therapeutic agent.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / immunology
  • Antineoplastic Agents / therapeutic use*
  • Apoptosis / drug effects*
  • Apoptosis / physiology
  • Capsid Proteins / immunology
  • Capsid Proteins / therapeutic use*
  • Carrier Proteins / pharmacology
  • Carrier Proteins / therapeutic use
  • Epitopes / immunology
  • Humans
  • Immune System / drug effects
  • Immune System / immunology
  • Lysosomes / drug effects
  • Lysosomes / metabolism
  • Macromolecular Substances
  • Maltose-Binding Proteins
  • Neoplasms / drug therapy*
  • Neoplasms / immunology
  • Neoplasms / metabolism
  • Recombinant Fusion Proteins / immunology
  • Recombinant Fusion Proteins / pharmacology
  • Recombinant Fusion Proteins / therapeutic use
  • Tumor Cells, Cultured

Substances

  • Antineoplastic Agents
  • Capsid Proteins
  • Carrier Proteins
  • Epitopes
  • Macromolecular Substances
  • Maltose-Binding Proteins
  • Recombinant Fusion Proteins
  • VP3 protein, Chicken anemia virus