The effect of DMSO was not limited to the Nbk construct or to the Nbk-transfected cell clone, rather a highly similar picture with respect to increased expression and increased apoptosis was seen in SKM13-CD95L cells stably transfected with CD95L (Figure 1, C and D). Enhanced apoptosis became also clearly visible under the light microscope as rounded, detached cells (Figure 1, E and F). Therefore, the transgene itself was not critical for the effect of DMSO, and a selective stabilization of exclusively the Nbk product was largely ruled out.
The DMSO effect also occurred after transient transfection of pTRE-Nbk in SKM13-Tet-On cells. After Dox treatment for 48 h, apoptosis induction and protein expression of Nbk was significantly enhanced by DMSO in SKM13-Tet-On (Figure 2A). To rule out that the effect was restricted to SK-Mel-13 melanoma cells, we investigated its effect also in another melanoma Tet-On cell line (Bro-Tet-On). As in SKM13-Tet-On, DMSO clearly enhanced expression of Nbk in Bro-Tet-On after transient transfection (Figure 2B). The only weak apoptotic response in Bro cells is in parallel with previous findings of a high apoptosis resistance of this cell line (5).
To investigate whether the effect of DMSO was related to an activation of the CMV promoter that drives the expression of rtTA, we investigated a melanoma cell line stably transfected with a pIRES/mbcl-2 construct (MelHO/Bcl-2). In the pIRES expression vector, transgene expression is driven by a constitutive CMV promoter. As seen for the nonactivated TRE promoter, and in contrast to the effects of DMSO on the activated TRE promoter, it remained without any effect on the exogeneously encoded transgene expression by the CMV promoter in the pIRES setting (Figure 3A). Thus, it may be largely ruled out that the effect of DMSO results from enhanced expression of the transactivator.
To further investigate whether the effect of DMSO was limited to melanoma cells or may also be applied to cells of other origin, we performed transient transfection experiments with the pTRE-Nbk plasmid in HeLa Tet-On cells. As depicted in Figure 3B, Dox-mediated gene expression in HeLa-Tet-On was significantly enhanced after 24 and 48 h of Dox treatment, as compared with cells that received Dox without DMSO, possibly indicating that the effects of DMSO may be generally applied to the used Tet-On system.
Polar solvents as DMSO can be used for enhancing skin permeability. Various effects of DMSO on cell membranes have been described, including cleansing and disruption of cation transports, as well as oxidant and antioxidant activities (18,19). DMSO may also oxidize doxycycline (20), but it remains speculative whether an oxidation of Dox affects its binding to Tet transactivators.
Besides the original Tet-On regulation system applied here, several modified Tet expression systems are available, which differ in the pTRE element (10,21,22) or in the responsible transactivator (23,24). If the effect of DMSO is based on its interaction with the VP16 activation domain to enhance its potency, transactivators carrying modified activation domains or those from other transcription factors (25,26,27,28) may be unresponsive or less responsive. Subsequent studies may address these questions and may finally provide the mechanism of how DMSO interferes with the tetracycline expression system.
In conclusion, our data reports for the first time synergistic effects between DMSO and Dox in Tet-On cells with regard to induction of the transgene. As DMSO is widely used in stock solutions of drugs applied in analytical assays, our findings may be considered when applying such drugs in combination with the Tet-On system. Finally, this new, easily applicable technique may further increase the effectiveness of the Tet-On system, and our findings may be proven for their usefulness in clinical settings when applying the Tet-On systems in therapeutic trials.
M.O. was a recipient of a scholarship from the Deutsche Forschungsgemeinschaft (DFG; no. GRK 276/3). This study was further supported by the Deutsche Krebshilfe/Mildred-Scheel-Stiftung (grant no. 10-1434-Eb2).
Competing Interests Statement
The authors declare no competing interests.
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