Because accurate regulation of toxin gene expression is critical for safe and effective gene therapy applications, the authors have examined the regulation of
diphtheria toxin A (DTA) fragment expression in human
glioma cell lines using two transcriptional control systems derived from Escherichia coli: the
tetracycline (Tet) system and the
lactose (Lac) system. The Tet system includes a
tetracycline-controlled
transactivator (tTA), a tTA-responsive minimum human cytomegalovirus (hCMV) promoter controlling the expression of the DTA gene, and
tetracycline as an allosteric inhibitor. The Lac system includes the
lac repressor (lacR), a lacR-regulated Rous sarcoma virus-long terminal repeat (RSV-LTR) promoter controlling the expression of the DTA gene, and isopropyl-thio-beta-D-galactoside (
IPTG) as an allosteric inducer. Expression plasmids encoding either tTA or lacR were transfected into U-87MG and U-343MG
glioma cells along with the responsive DTA plasmid. Cell killing was monitored by the ability of the toxin to abolish
protein synthesis and was quantitated using a
luciferase reporter gene. In the Tet system,
tumor cell killing could be regulated by
tetracycline up to 120-fold. In contrast, only a twofold
IPTG-dependent regulation was obtained using the Lac system because of an incomplete repression of DTA expression in the uninduced state. Replacement of the RSV-LTR promoter with the
heavy metal-inducible
mouse metallothionein-1 promoter in the lacR-responsive unit, as well as the generation of a clonal
glioma cell line expressing lacR, did not significantly enhance regulation of DTA in the Lac system. In conclusion, this study demonstrates that the Tet system is of potential use in gene therapy applications in which regulated expression of a therapeutic gene is an important issue.