The B subunit of
diphtheria toxin (DT) is responsible for interaction with receptor on the cell surface and translocation of the catalytically active A subunit across endosomal membrane into the cell cytosole. Receptor for DT and its B subunit is membrane-anchored precursor of
heparin-binding
epidermal growth factor-like
growth factor (pro-
HB-EGF), which under the action of
metalloproteases turns into soluble form (sHB-
EGF), which acts as a potent
mitogen for different cell types. Since free B subunit of DT has no catalytic activity it is considered to be nontoxic. However its influence on the cells in vitro remains to be investigated. The aim of this study was to examine the influence of
diphtheria toxin B subunit on viability of
diphtheria-sensitive cells using B subunit recombinant analogues. It was shown that
diphtheria toxin B subunit recombinant analogue at a concentration of 12.8 x 10(-7) M had a cytotoxic effect on the human histocytic
lymphoma cell line U937, which expresses a large amount of sHB-
EGF. Besides, the similar cytotoxic effect had a fusion
protein which consisted of a B subunit and an
enhanced green fluorescent protein (EGFP). However recombinant EGFP alone didnot influence the cell viability.
Annexin-V-FITC/PI staining demonstrated that maximal cytotoxic effect had been elicited after 48 hours of cultivation. Cytotoxic test with
trypan blue and
propidium iodide staining excluded the direct influence of investigated
proteins on the integrity of plasma membrane because of the ability of B subunit to pore formation. So, we offer a hypothesis that realization of cytotoxic effect of
diphtheria toxin B subunit and its derivative on the U937 cell culture occurs via inhibition of mitogenic activity of sHB-
EGF resulted in induction of apoptosis.