Natesan Senthilkumar, Kataria J M, Dhama K, Bhardwaj N, Sylvester A
Molecular Biology Laboratory, Division of Avian Diseases, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122 (UP), India.
J Gen Virol. 2006 Oct;87(Pt 10):2933-2940. doi: 10.1099/vir.0.82085-0.
The anti-neoplastic effect of chicken anemia virus VP3 protein (apoptin) was investigated in vitro in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblast (CEF) cells and in RSV-induced tumours of specific-pathogen-free (SPF) chicks in vivo. The apoptin gene was cloned in the pVAX expression vector and in vitro expression of the recombinant vector pVAX-CAV-VP3 was confirmed. Two groups of SPF chicks, each containing ten chicks, were used. Chicks in groups I and II were inoculated with RSV at 1 day old. Group I served as the control, receiving pVAX vector without insert, and group II received recombinant vector pVAX-CAV-VP3 containing the apoptin gene, on day 10. An in vitro study confirmed that apoptin induced apoptosis in RSV-transformed CEF cells, which was demonstrated by observation of the characteristic changes of apoptosis using the indirect immunofluorescence technique and acridine orange/ethidium bromide staining. In vivo study also indicated that apoptin induced apoptosis and caused tumour regression by an intratumoral-delivery method. Apoptotic changes, such as nuclear condensation, fragmentation of the chromatin and formation of apoptic bodies in the tumour cells, were demonstrated by histopathology and acridine orange/ethidium bromide staining. No apoptotic changes were seen in the tumours of the control group. The results of the present study showed that apoptin had an anti-neoplastic effect in vivo and in vitro in RSV-induced tumours. The anti-neoplastic effect is due to apoptin-induced apoptosis. Further improvements in the dose, delivery method and delivery frequency of the apoptin-expressing recombinant vector could help to develop apoptin as an anti-neoplastic drug.
在体外，研究了鸡贫血病毒VP3蛋白（凋亡素）对劳氏肉瘤病毒（RSV）转化的鸡胚成纤维细胞（CEF）的抗肿瘤作用；在体内，研究了其对RSV诱导的无特定病原体（SPF）雏鸡肿瘤的作用。将凋亡素基因克隆到pVAX表达载体中，并证实了重组载体pVAX-CAV-VP3的体外表达。使用两组SPF雏鸡，每组十只。第I组和第II组雏鸡在1日龄时接种RSV。第I组作为对照，接受不含插入片段的pVAX载体，第II组在第10天接受含有凋亡素基因的重组载体pVAX-CAV-VP3。体外研究证实，凋亡素可诱导RSV转化的CEF细胞凋亡，通过间接免疫荧光技术和吖啶橙/溴化乙锭染色观察凋亡的特征性变化得以证明。体内研究还表明，凋亡素通过瘤内给药方法诱导凋亡并导致肿瘤消退。通过组织病理学和吖啶橙/溴化乙锭染色证实了肿瘤细胞中的凋亡变化，如核浓缩、染色质碎片化和凋亡小体形成。对照组肿瘤未观察到凋亡变化。本研究结果表明，凋亡素在RSV诱导的肿瘤体内和体外均具有抗肿瘤作用。抗肿瘤作用归因于凋亡素诱导的凋亡。进一步改进表达凋亡素的重组载体的剂量、给药方法和给药频率，可能有助于将凋亡素开发为一种抗肿瘤药物。
