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Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
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Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

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Characterization and evaluation of apoptotic potential of double gene construct pVIVO.VP3.NS1

Shikha Saxena, G S Desai, G Ravi Kumar

    Indian Journal of Experimental Biology
    |June 5, 2015
    PubMed

    Insights

    Viral gene oncotherapy offers a promising cancer treatment alternative. This study demonstrates that a combined VP3 (apoptin) and NS1 (Non-structural protein 1) gene construct effectively induces apoptosis in cancer cells.

    Area of Science:

    • Oncology
    • Virology
    • Molecular Biology

    Background:

    • Viral gene oncotherapy is an emerging cancer treatment modality.
    • Conventional chemo and radiotherapy have limitations including genotoxic side effects and reliance on functional p53.
    • VP3 (apoptin) from chicken infectious anaemia virus and NS1 from Canine Parvovirus-2 show oncolytic potential.

    Purpose of the Study:

    • To clone and characterize a bicistronic vector expressing both VP3 and NS1.
    • To evaluate the combined oncolytic potential of VP3 and NS1.
    • To assess the apoptotic inducing capability of the dual gene construct in cancer cells.

    Main Methods:

    • Cloning of VP3 and NS1 genes into a bicistronic vector (pVIVO.VP3.NS1).
    • Confocal microscopy and flow cytometry to confirm gene expression.
    • Apoptosis assays including DNA fragmentation, TUNEL assay, and Hoechst staining.

    Main Results:

    • The pVIVO.VP3.NS1 construct successfully expressed both VP3 and NS1 proteins in HeLa cells.
    • Flow cytometry confirmed increased expression of VP3 and NS1 in transfected cells.
    • The dual gene construct significantly induced apoptosis in human cervical cancer cells (HeLa).

    Conclusions:

    • The bicistronic vector effectively expresses both VP3 and NS1.
    • The combined VP3 and NS1 construct demonstrates significant oncolytic potential by inducing apoptosis in cancer cells.
    • This dual gene approach holds promise for developing novel non-infectious cancer therapies.

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