Isolation of diphtheria toxin-sensitive mouse cells from a toxin-resistant population transfected with monkey DNA.
Diphtheria toxin (DTX)-sensitive mouse cells were isolated from a toxin-resistant thymidine kinase (TK)-negative L-M(TK-) mouse cell population that was transfected with DNA from highly toxin-sensitive monkey Vero cells. Sensitivity to DTX was screened by using a replica plate assay. The purified toxin-sensitive mouse cells were characterized with respect to their ability to bind, internalize, and translocate DTX into the cytosol. In contrast to the L-M(TK-) cells, these DTX-sensitive mouse cells were able to bind and internalize radioiodinated toxin into intracellular vesicles at 37 degrees C. Specific binding of radioiodinated toxin to their cell surface (at 4 degrees C) could not be demonstrated. However, the following evidence for functional receptors capable of binding DTX was obtained: (i) when the toxin-sensitive mouse cells were first allowed to bind DTX at 4 degrees C, followed by washing the cells and shifting the temperature to 37 degrees C (allowing cell surface-bound toxin to enter the cells), the cells were killed; (ii) when cells with surface-bound DTX were exposed briefly to an acidic medium (allowing the toxin to penetrate the plasma membrane directly), protein synthesis was inhibited; and (iii) when cells were incubated with DTX in the presence of the CRM 197, a nontoxic form of DTX with binding properties similar to native DTX, the cytotoxic effect of DTX was markedly decreased. The results demonstrate that the toxin-sensitive mouse cells are killed by a mechanism similar to that observed in naturally occurring toxin-sensitive cell lines. The data further suggest that the transfected mouse cells express functional receptors for DTX.
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
J G
JG
Naglich
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Eidels
D000818:Animals; D015139:Blotting, Southern; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000235:genetics*; D004167:Diphtheria Toxin / Q000378:metabolism / Q000494:pharmacology*; D004351:Drug Resistance; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007739:L Cells / Q000166:cytology / Q000187:drug effects; D051379:Mice; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection; D014709:Vero Cells
D004167:Diphtheria Toxin; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; D004247:DNA; D013937:Thymidine Kinase