Abstract
We have studied the specific targeting of leukaemic cells using the Chicken Anaemia Virus (CAV)-derived protein VP3 (Apoptin) linked to the protein transduction domain (PTD) from HIV TAT with the aim of using this strategy for in vitro purging. Apoptin is a 13.6 kDa protein which induces apoptosis specifically in cancer cells whilst leaving normal cells unaffected. Expression of Apoptin in normal cells results in its cytoplasmic localisation. In tumour cells Apoptin resides initially in the cytoplasm and subsequently translocates to the nucleus and induces apoptosis. Apoptin is phosphorylated both in vitro and in vivo in tumour cells but negligibly in normal cells at threonine 108. A gain-of-function point mutation (T108E) results in accumulation of Apoptin in the nucleus and the killing of normal cells, implying that phosphorylation is a key factor of the tumour-specific properties of Apoptin.
We have demonstrated that Apoptin induces apoptosis in a variety of human solid tumour cell lines, but not in normal fibroblast and epithelial cells. Apoptin induced apoptosis in HSC3 head and neck cancer cells acts through the mitochondrial pathway and was blocked (>75%) by shRNA against PUMA, a BH3 Only protein which induces Bax and BAK resulting in loss of mitochondrial membrane potential and release of cytochrome C. Furthermore, activation of the p53 family member, p73, substantially increased (5–10 fold for p73 β and γ) sensitivity of Saos2 tumour cells to Apoptin-induced killing.
For efficient protein delivery, Apoptin was fused to a TAT PTD and addition of this protein to normal and tumour cells resulted in the selective killing of tumour cells. To increase the stability and solubility of TAT-Apoptin we have fused it to the maltose binding protein (MBP), this modification significantly increases both yield and the solubility of Apoptin while retaining its biological function. Apoptin tumour specific toxicity was assessed in a range of leukaemic and solid tumour cell lines. Addition of MBP-TAT-Apoptin protein to HL60, K562 and Jurkat cells resulted in 50%, 55% and 75% cell death by apoptosis as judged by PARP cleavage, respectively, at day 4 as compared to MBP-TAT control whilst normal B cells, fibroblasts and epithelial cells are unaffected. Fluorescent microscopy demonstrated that MBP-TAT-Apoptin was rapidly internalised in almost 100% of cells within 24hrs in all cell types tested. Direct injection of Apoptin expressing Ad vectors also showed clear regression of established tumours in mice.
The cancer specific toxicity of Apoptin has potential value for a range of therapeutic applications such as purging of autologous bone marrow as used for the treatment of multiple myeloma and possibly direct treatment of leukaemias either alone or linked to antibodies for targeting of specific types of leukaemias.
Differential expression of cytokine transcripts in human epithelial ovarian carcinoma by solid tumour specimens, peritoneal exudate cells containing tumour, tumour-infiltrating lymphocyte (TIL)-derived T cell lines and established tumour cell lines