Because C. botulinum C2 toxin is a real binary toxin, studies of its effects on intact cells depend on the presence of both the binding (C2II) and enzyme component (C2I) (Reuner eta/., 1987; Wiegers et al., 1991 ; Ohishi et al., 1984; Ohishi and Yanagimoto, 1992; Li et al., 1994; Prepens et al., 1996).
The toxin components are usually applied at a C2I:C2II ratio of 1:2 (on a microgram basis) (Ohishi et al., 1980). C2II is only fully active in translocating C2I into intact cells when activated with trypsin (as described in Chapter 9). Both components are applied to the medium in a 1:100 dilution step, to minimize salt effects on the cells. Prediction of C2I and C2II should be performed in PBS or another appropriate buffer supplemented with 100|xg/ml of serum albumin.
Studies with C2 toxin revealed that cultured cells differ in their sensitivity to the toxin. While Vero (African green monkey kidney) cells are highly sensitive, with 100 % rounding up at 10 ng C2 toxin per ml after about 24 h of treatment, FL (human amnion) cells are much less sensi-different cells differ jn five and about 200-fold higher concentrations are needed for corn-sensitivity to C2 toxin p|ete rounding up of cells (Ohishi et al., 1984). To some extent, less effective concentrations of the toxin can be compensated for by an increase in the incubation time. Furthermore, cells are usually more sensitive to the toxin under FCS-free conditions. Therefore, to study the toxin's effects with rather insensitive cells or to save toxin, the culture medium can be changed to FCS-free medium when tolerated by the cells being studied. Otherwise the toxin is added for 30 to 60 min in FCS-free medium and, thereafter, the appropriate medium is added again. Even at very high concentrations of the toxin, the earliest onset of cytotoxic effects is usually 15 to 30 min after intoxication (Reuner et al., 1987). This delay is probably due to the process of uptake and translocation of the toxin into the cytosol. In most cells, increase in toxin treatment time for 48 h or longer results in detachment and degeneration of cells. In cell culture, individual cells appear to show different sensitivity towards the toxin. This is especially true at early phases of the intoxication process (1 to 2h after toxin addition). Whether this effect depends on the phase of the cell cycle remains to be clarified.
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