Unique activity associated with non-insecticidal Bacillus thuringiensis parasporal inclusions: in vitro cell-killing action on human cancer cells

Abstract

Parasporal inclusion proteins from a total of 1744 Bacillus thuringiensis strains, consisting of 1700 Japanese isolates and 44 reference type strains of existing H serovars, were screened for cytocidal activity against human leukaemia T cells and haemolytic activity against sheep erythrocytes. Of 1684 B. thuringiensis strains having no haemolytic activity, 42 exhibited in vitro cytotoxicity against leukaemia T cells. These non‐haemolytic but leukaemia cell‐toxic strains belonged to several H‐serovars including dakota, neoleonensis, shandongiensis, coreanensis and other unidentified serogroups. Purified parasporal inclusions of the three selected strains, designated 84‐HS‐1‐11, 89‐T‐26‐17 and 90‐F‐45‐14, exhibited no haemolytic activity and no insecticidal activity against dipteran and lepidopteran insects, but were highly cytocidal against leukaemia T cells and other human cancer cells, showing different toxicity spectra and varied activity levels. Furthermore, the proteins from 84‐HS‐1‐11 and 89‐T‐26‐17 were able to discriminate between leukaemia and normal T cells, specifically killing the former cells. These findings may lead to the use of B. thuringiensis inclusion proteins for medical purposes.

A Bacillus thuringiensis Crystal Protein with Selective Cytocidal Action to Human Cells

“…Bacterial Strains and Culture Media-The organisms used in this study was the soil isolate B. thuringiensis A1547, belonging to serovar dakota (7)(8)(9). The bacteria were grown on nutrient medium, containing 0.1% meat extract, 0.1% polypeptone, and 0.2% NaCl, pH 7.6, at 27°C for 8 days.…”

“…This, taken together with the mammalian cellrecognizing parasporal proteins reported previously (7)(8)(9)(10)(11), means the presence of a new distinct family of B. thuringiensis ␦-endotoxins that are highly cytotoxic to a wide range of mammalian cells but are non-hemolytic and non-insecticidal, in addition to the established insecticidal Cry and Cyt protein families (3). Each toxin protein has its specific and distinct target spectrum against mammalian cells, and some proteins are strictly specific to certain human cancer cells, as the toxin protein reported in this report.…”

“…Among the non-insecticidal B. thuringiensis isolates, we found isolates with a novel property, non-hemolytic but highly cytotoxic to a wide range of mammalian cells, including human cancer cells (7)(8)(9)(10)(11). The cytotoxic proteins were heterogeneous in cytotoxicity spectra, and some were active on a wide range of human cells, whereas the others killed a few specific cells.…”

Investigation of a NovelBacillus thuringiensisGene Encoding a Parasporal Protein, Parasporin-4, That Preferentially Kills Human Leukemic T Cells

“…Normal T cells were isolated from buffy coats supplied from Fukuoka Red Cross Blood Center (Fukuoka, Japan) by a method previously described. 8) Cells were cultured in RPMI1640 medium (Nissui Pharmaceuticals, Tokyo) containing 10% fetal bovine serum and kanamycin (30 mg/ml).…”

“…7) Recent extensive efforts to screen Cry proteins for biological activities other than insecticidal toxicity have led to the discovery of a unique activity, in vitro cytotoxicity preferential for certain human cancer cells. 8) Mizuki et al 9) obtained the first cry gene encoding the Cry31Aa protein, designated parasporin, defined as the B. thuringiensis and related bacterial parasporal proteins capable of preferentially killing cancer cells. This was followed by the identification of two more proteins, Cry46Aa 10) and Cry41Aa, 11) with cytotoxic activities against cancer cells (see N.…”