Selective apoptosis induction in MCF-7 cell line by truncated minimal functional region of Apoptin.

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  • Additional Information
    • Source:
      Publisher: BioMed Central Country of Publication: England NLM ID: 100967800 Publication Model: Electronic Cited Medium: Internet ISSN: 1471-2407 (Electronic) Linking ISSN: 14712407 NLM ISO Abbreviation: BMC Cancer Subsets: MEDLINE
    • Publication Information:
      Original Publication: London : BioMed Central, [2001-
    • Subject Terms:
      Apoptosis*/drug effects ; Apoptosis*/genetics; Capsid Proteins/*genetics ; Capsid Proteins/*metabolism; Animals ; Capsid Proteins/isolation & purification ; Capsid Proteins/pharmacology ; Cell Line, Tumor ; DNA-Binding Proteins ; Gene Order ; Humans ; MCF-7 Cells ; Maltose-Binding Proteins/genetics ; Maltose-Binding Proteins/metabolism ; Microinjections ; Plasmids/genetics ; Protein Transport ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/isolation & purification ; Recombinant Fusion Proteins/metabolism ; Recombinant Fusion Proteins/pharmacology ; Viral Proteins
    • Abstract:
      Background: Chicken Anemia Virus (CAV) VP3 protein (also known as Apoptin), a basic and proline-rich protein has a unique capability in inducing apoptosis in cancer cells but not in normal cells. Five truncated Apoptin proteins were analyzed to determine their selective ability to migrate into the nucleus of human breast adenocarcinoma MCF-7 cells for inducing apoptosis.
      Methods: For identification of the minimal selective domain for apoptosis, the wild-type Apoptin gene had been reconstructed by PCR to generate segmental deletions at the N' terminal and linked with nuclear localization sites (NLS1 and NLS2). All the constructs were fused with maltose-binding protein gene and individually expressed by in vitro Rapid Translation System. Standardized dose of proteins were delivered into human breast adenocarcinoma MCF-7 cells and control human liver Chang cells by cytoplasmic microinjection, and subsequently observed for selective apoptosis effect.
      Results: Three of the truncated Apoptin proteins with N-terminal deletions spanning amino acid 32-83 retained the cancer selective nature of wild-type Apoptin. The proteins were successfully translocated to the nucleus of MCF-7 cells initiating apoptosis, whereas non-toxic cytoplasmic retention was observed in normal Chang cells. Whilst these truncated proteins retained the tumour-specific death effector ability, the specificity for MCF-7 cells was lost in two other truncated proteins that harbor deletions at amino acid 1-31. The detection of apoptosing normal Chang cells and MCF-7 cells upon cytoplasmic microinjection of these proteins implicated a loss in Apoptin's signature targeting activity.
      Conclusions: Therefore, the critical stretch spanning amino acid 1-31 at the upstream of a known hydrophobic leucine-rich stretch (LRS) was strongly suggested as one of the prerequisite region in Apoptin for cancer targeting. Identification of this selective domain provides a platform for developing small targets to facilitating carrier-mediated-transport across cellular membrane, simultaneously promoting protein delivery for selective and effective breast cancer therapy.
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    • Accession Number:
      0 (Capsid Proteins)
      0 (DNA-Binding Proteins)
      0 (Maltose-Binding Proteins)
      0 (MuB protein, Enterobacteria phage Mu)
      0 (Recombinant Fusion Proteins)
      0 (VP3 protein, Chicken anemia virus)
      0 (Viral Proteins)
    • Publication Date:
      Date Created: 20131023 Date Completed: 20140623 Latest Revision: 20240321
    • Publication Date:
      20240321
    • Accession Number:
      PMC4015422
    • Accession Number:
      10.1186/1471-2407-13-488
    • Accession Number:
      24144306