Characterisation of the binding properties of Bacillus thuringiensis 18 toxin on leukaemic cells.
BACKGROUND
Various strains of Bacillus thuringiensis (Bt) have been found to produce parasporal proteins that are cytotoxic to human cancer cells. This study aims to establish the binding affinity of purified Bt 18 toxin for CEM-SS (T lymphoblastic leukaemia cell line), to determine if competition exists between the toxin and commercial anticancer drugs for the binding site on CEM-SS and to localise the binding site of the toxin on CEM-SS.


METHODS
In homologous competitive binding study, the purified toxin was labelled with biotin and allowed to compete with unlabelled toxin for binding sites on CEM-SS and its dissociation constant (Kd) was determined. Comparisons were made with CCRF-SB, CCRF-HSB-2 and MCF-7. In heterologous competitive binding study, biotinylated toxin competition was determined with two other Bt toxins (crude Btj and crude Bt 22) and anticancer drugs (cisplatin, doxorubicin, etoposide, navelbine and methotrexate). To localise the binding site under the confocal microscope, the biotinylated toxin was tagged with FITC-conjugated streptavidin.


RESULTS
Homologous competitive binding assays revealed decreasing binding affinity of Bt 18 toxin for CEM-SS, CCRF-SB, and CCRF-HSB-2 with Kd of 8.44 nM, 14.98 nM and 17.71 nM respectively. Kd for MCF-7 was not determined as the inhibitory concentration (IC50) was not reached. Heterologous competitive study showed little competition (< 30%) between biotinylated Bt 18 toxin and all test compounds used. Confocal microscopy revealed binding of toxin at the periphery of the cell.


CONCLUSIONS
It was postulated that purified Bt 18 toxin binds on the cell surface of CEM-SS and the mechanism of cell death may differ from that of Btj toxin, Bt 22 toxin and all five anticancer drugs used in this study, since it did not significantly compete with these compounds for the same binding site.
Wong
Rebecca S Y
RS
Division of Human Biology, School of Medical Sciences, International Medical University, No 126, Jalan 19/155B, Bukit Jalil, 57000 Kuala Lumpur, Malaysia.
Mohamed
Shar M
SM
Nadarajah
Vishna D
VD
Tengku
Ibrahim Azmi T
IA
D000970:Antineoplastic Agents / Q000494:pharmacology; D001413:Bacillus thuringiensis / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D001667:Binding, Competitive; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002945:Cisplatin / Q000494:pharmacology; D005260:Female; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology
D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D002945:Cisplatin