Bujard, H.

Gossen, M.

Nucleic Acids Research

English

PubMed

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©1993 Oxford University Press Nucleic Acids Research, 1993, Vol. 21, No. 18 4411-4412Anhydrotetracycline, a novel effector for tetracyclinecontrolled gene expression systems in eukaryotic cellsManfred Gossen and Hermann Bujard*Zentrum fur Molekulare Biologie der Universitat Heidelberg, Im Neuenheimer Feld 282, D-69120Heidelberg, GermanyReceived July 5, 1993; Accepted August 5, 1993Tetracycline (Tc) controlled gene expression has beendemonstrated in a variety of eukaryotic systems such as tobacco(1, 2, 3), Saccaromyces cerevisiae (4), Saccaromyces pombe (5)and Dictyostelium discoidum (6). In these systems the repressorof the E.coli TnlO tetracycline (Tc) resistance operon (7) isutilized to interfere with transcription initiation of RNApolymerase II or HI by binding to operators properly placedwithin a promoter region. This interference is abolished by Tc.By contrast, we have converted the Tet repressor into anefficient tetracycline controlled transcriptional transactivator (tTA)whose function can be controlled by Tc. In double-stable humanHeLa cell lines such as the XI cells (8), which contain both anexpression unit for tTA and a luciferase gene controlled by atTA dependent promoter, gene activity can be regulated over upto five orders of magnitude depending on the Tc concentrationin the medium (8).The general applicability of Tc controlled systems (e.g. intransgenic situations) will largely depend on the effector'stoxicity, its chemical and biological half-life time, etc. Thus,although Tc has excellent general pharmacological properties itsvery low but distinct cytotoxicity to mammalian cells may notbe negligible under all circumstances. On the other hand amongthe numerous well studied tetracyclines and their derivatives theremay be compounds with superior properties in Tc regulatedsystems. One such example is anhydrotetracycline (ATc) whichbinds the Tet repressor more efficiently than Tc (10" M"1 vs.3X109 M~", ref. 9) and which has a lower antibiotic activitytowards E.coli (10). Moreover, it was suggested that ATc actsat a different level than Tc, which interferes with the translationalprocess (10). Here we describe the properties of ATc as aneffector in our tTA dependent regulatory system. We incubatedXI cells at different concentrations of either Tc or ATc andmonitored the luciferase activity produced under these conditions.Our results show that ATc is much more effective than Tc ininactivating tTA and that it completely abolishes tTA mediatedluciferase activity at concentrations as low as 3 ng/ml (Figurela). In good correlation with the relative affinities of Tc and ATctowards the Tet repressor (9), this concentration is ten fold belowthat required for Tc. More importantly, the concentration at whichthe prolonged presence of ATc begins to affect the growth rateof HeLa cells in culture (ATc cone. > 3 /ig/ml; Figure 2) is morethan thousand fold above the effective concentration. Finally, theslope of the dose response curve indicates that like Tc, ATc iswell suited to attenuate gene expression at intermediate levelsof induction.These properties together with the high functional stability ofATc in cell culture make ATc a most attractive alternative effectorfor Tc-controlled gene expression in higher eukaryotic systems.ACKNOWLEDGEMENTSWe thank W.Hillen for helpful discussions. This work wassupported by the Deutsche Forschungsgemeinschaft (SFB 229)and by the Fonds der Chemischen Industrie Deutschlands.REFERENCES1. Gatz.C. and Quail.P.H. (1988) Proc. Natl. Acad Sd. USA 85, 1394-1397.2. Gatz.C, Kaiser,A. and Wendenburg.R. (1991) Mol. Gen. Genet. 227,229-237.,01 ,1 1 10 100 1000concentration of the antibiotic (ng/ml)Figure 1 Dose response of anhydrotetracycline (ATc) and tetracycline (Tc) onthe activity of luciferase in the HeLa cell line XI. HeLa XI cells grown in mediumcontaining 100 ng/ml Tc were seeded with a density of about 10 000 cells/35mm culture dish and incubated with the antibiotics at the concentrations indicated.After .wo days the antibiotic containing medium was renewed. After two furtherdays the cells were harvested and luciferase activity and protein content of theextracts were determined. The antibiotics used were tetracycline hydrochloride(dashed line) from Sigma Chemical Co., St Louis (stock 1 mg/ml in H2O, stableat 4°C for at least 2 weeks when light protected), and andydrotetracyclinehydrochloride (continuous line) from Janssen Chimica, Geel (stock 0.1 mg/mlin H2O, stable at 4°C for at least 2 weeks when light protected). Data shownare from two independent experiments. Cell culture, luciferase assays and proteindeterminations were carried out as described previously (8).• To whom correspondence should be addressed4412 Nucleic Acids Research, 1993, Vol. 21, No. 183. Gatz.C. Frohberg.C. and Wendenburg.R. (1992) Plant J. 2, 397-404.4. Dingermann.T., Frank-Stoll.U., Wemer.H., Wissmann.A., Hillen.W.,Jacquet.M. and Marschalek.R. (1992) EMBO J. 11, 1487-1492.5. Faryar.K. and Gatz.C. (1992) Curr. Genet. 21, 345-349.6. Dingermann.T., Wemer.H, Schlhz.A., Zundorf.I., Nerke.K., Knecht, D.and Marschlek.R. (1992) MoL Cell. Biol. 12, 40384045.7. Hflkn.W. and Wissmaim.A. (1989) In, Saenger.W. and Hemenarm.U. (eds),Protein—Nucleic Acid Interaction. Topics in Molecular and StructuralBiology. Macmillan Press, London Vol 10, pp. 143 — 162.8. Gossen.M. and Bujard.H. (1992) Proc Nad. Acad Sci. USA 89, 5547-5551.9. Degenkolbj., Takahashi.M., EUested.G. and Hillen.W. (1991) Antimicrob.Agents Chemother. 35, 1591-1595.10. Oliva.B., Gordon.G., McNicholas.P., Ellested.G. and Chopra,I. (1992)Antimicrob. Agents Chemother. 36, 913—919.11. Mossman.T. (1983) / Immunol Methods 65, 55 -63 .0,04V/0 ,01 ,1 1 10concentration of the antibiotic100Figure 2. Cytotoxic effect of tetracydine and anhydrotetracychne on HeLa cells.The cytotoxicity of elevated concentrations of the antibiotics on HeLa cells weredetermined according to Mossman (11). Cultures of 1000 cells/dish in 96 wellplates were incubated for 3 days with concentrations of the antibioacs indicatedand growth was analysed by the Ml l-assay. Values given are the mean of fourexperiments.

Anhydrotetracycline, a novel effector for tetracycline controlled gene expression systems in eukaryotic cells

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Anhydrotetracycline, a novel effector for tetracycline controlled gene expression systems in eukaryotic cells

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