The analyses of toxic and proapoptotic proteins require tightly regulated expression systems, and several systems have been developed to control gene expression in human cells. The tetracycline (Tet)-based regulatory strategies provide the advantage of high expression levels, strong inducibility, and the use of an inducer with welldescribed pharmacological kinetics and almost no pleiotropic effects in vivo. First developed by Gossen and Bujard in 1992, these are now the most frequently used mammalian inducible expression systems (1,2).

The Tet strategies apply elements from the Escherichia coli Tet resistance operon to control the expression of a gene of interest. In the Tet-Off® system, a Tet-controlled transactivator (tTA) is employed, which binds the Tet-responsive element (TRE) and induces gene expression in the absence of Tet or its derivative doxycycline (Dox). In contrast, in the Tet-On® expression system, binding of the reverse Tet-controlled transactivator (rtTA) and subsequent gene expression is induced by Dox. The transactivator tTA and its reverse mutant rtTA are chimeric proteins formed by fusion of the activation domain from herpes simplex virus VP16 and the Tet-controlled repressor protein from E. coli. Depending on the concentration of the drug, gene expression is induced in the Tet-On system (3,4).

For clinical settings, the Tet-On strategies are preferred, as the drug has to be given only as long as gene expression is needed. These systems have been applied successfully for in vitro research as well as for in vivo preclinical applications (5,6,7). In future, they may evolve as key technologies for successfully implementing gene therapies in clinical settings (8,9,10,11). For in vitro investigations, Tet-On cell lines can be generated by stable transfection of rtTA, which is then constitutively expressed.

Proapoptotic factors represent powerful tools in apoptosis-based therapeutic approaches for overcoming apoptosis resistance in tumor cells (12). Two proapoptotic genes (CD95L and Nbk/Bik) were used in the present study as examples to demonstrate gene expression by the Tet-On system and the subsequent cellular effects. Their proapoptotic potential in melanoma cells has been unequivocally demonstrated in previous work (5,13). Here we show DNA fragmentation, which is a final step in induced apoptosis (14).

By analyzing the proapoptotic effects of these factors, we found a new way for significantly increasing the Dox-dependent protein expression in melanoma Tet-On cells. It depends on the co-incubation with the commonly used organic solvent dimethyl sulfoxide (DMSO). As DMSO is also widely used for stock solutions of many drugs applied in analytical assays (15), care may have to be applied when analyzing their effects in combination with the Tet-On system. Of major importance however, our data supplies a new, easily applicable technique, which significantly increases the effectiveness of Dox-regulated protein expression in Tet-On melanoma cells.

The melanoma Tet-On cell lines SKM13-Tet-On and Bro-Tet-On were derived from melanoma cell lines SK-Mel-13 and Bro by stable transfection of the pTet-On plasmid (5). HeLa-Tet-On, which derived from cervix carcinoma cells (HeLa), was purchased from Clontech Laboratories (Palo Alto, CA, USA). The pTet-On plasmid encodes the rtTA transactivator under the control of a constitutive cytomegalovirus (CMV) promoter (Clontech Laboratories). Cells with inducible expression of proapoptotic genes (SKM13-Nbk and SKM13-CD95L) resulted from stable transfection of SKM13-Tet-On with pTRE-Nbk and pTRE-CD95L, respectively (5,13). Stable transfection of the melanoma cell line Mel-HO with the murine Bcl-2 cDNA cloned in pIRES (Clontech Laboratories) resulted in the cell clone MelHO/Bcl-2, constitutively overexpressing the Bcl-2 protein from a CMV promoter (16).

Cells were cultivated in Dulbecco's modified Eagle's medium (DMEM; 4.5 g/L glucose; Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS) and antibiotics (Biochrom AG, Berlin, Germany). For selection of SKM13-Tet-On, Bro-Tet-On, HeLa-Tet-On, and MelHO/Bcl-2, 400 µg/mL geneticin (Invitrogen) were added to the growth medium. For SKM13-Nbk and SKM13-CD95L, 400 µg/mL geneticin and 100 µg/mL hygromycin (Boehringer, Mannheim, Germany) were added. The pTRE promoter was activated in Tet-On cells by 2 µg/mL Dox (ICN Biochemicals, Aurora, OH, USA).

The plasmid pTRE-Nbk (13) was obtained by subcloning the human full-length cDNA of Nbk into the Tet-inducible vector pTRE2 (Clontech); pTRE-CD95L (5) resulted from subcloning the full-length murine CD95L cDNA into pTRE1 (Clontech). For transient transfection, melanoma cells were seeded in 6-well plates (2 × 10⁵ cells/well). At a confluence of 50%, cells were washed with serum-free Opti-MEM® medium (Invitrogen), followed by incubation with 0.1% DMRIE-C (Invitrogen), and 0.4 µg/mL of the respective pTRE construct in Opti-MEM at 37°C for 4 h. After transfection, Opti-MEM was replaced by growth medium ± Dox for 48 h, and cells were subjected to Western blot analysis or apoptosis assays.