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.
METHODS: Paraffin-embedded tissue from 38 lymphomas (17 Hodgkin's, 14 Burkitt's, four T cell and 3 B cell non-Hodgkin's lymphomas) and 14 nasopharyngeal carcinomas (NPC) were studied, with 12 reactive lymph nodes and tonsils as normal control. EBER in situ hybridisation was performed to confirm EBV association in the tumour cells. A nested polymerase chain reaction (PCR) protocol was employed using two pairs of consensus primers which flanked a 105-bp deletion in the type A virus. U2 region encoding for EBNA-2 was chosen as the target of amplification, with cell lines B95.8 and AG876 serving as positive controls for types A and B virus, respectively.
RESULTS: All cases showed presence of type A virus, consistently detected with nested PCR protocol but not with single step PCR. There was no type B virus or mix infections detected.
CONCLUSIONS: Nested PCR technique has successfully increased the sensitivity of EBV subtype detection, and type A virus is the prevalent strain associated with human diseases in Malaysia.