Activation of the CMV-IE promoter by hyperthermia in vitro and in vivo: biphasic heat induction of cytosine deaminase suicide gene expression.
The cytomegalovirus-immediate early (CMV-IE) promoter is widely used as a strong and constitutively active promoter. Although the CMV-IE promoter does not harbor heat-responsive sequences, we determined its heat inducibility. We analyzed in vitro and in vivo heat responsiveness and possible mechanisms of heat induction of the CMV-IE promoter. We used transfected SW480 human colon carcinoma cells (SW480/CMVCD), expressing CMV-IE promoter-driven bacterial cytosine deaminase (CD) gene. These cells were heated at 42 degrees C. The SW480/CMVCD cells were also used for in vivo studies, in which tumor-bearing animals were treated with hyperthermia at 41.5 degrees C. As controls, SW480 (SW480/HSPCD) cells were used, in which CD expression is driven by the HSP70-promoter. In vitro, we observed a biphasic, up to 25-fold heat induction of CMV-IE-driven CD expression after hyperthermia in SW480/CMVCD cells. In vivo, we found a 2.5-fold induction of CD expression after hyperthermia in SW480/CMVCD tumor-bearing animals. The analysis of the CMV-IE promoter sequence revealed several transcription factor-binding sites, which mediate stress responsiveness. YB-1 and C/EBP-beta might mediate heat responsiveness of the CMV-IE promoter. These data point to limitations in heat-induction gene therapy studies, in which the CMV-IE promoter is used as control system. In addition, the CMV-IE promoter itself could well be used for construction of heat-inducible vectors.
Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany.
Dennis
D
Kobelt
Jutta
J
Aumann
Iduna
I
Fichtner
Ulrike
U
Stein
Peter M
PM
Schlag
Wolfgang
W
Walther
D000818:Animals; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics / Q000628:therapy; D043525:Cytosine Deaminase / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D004790:Enzyme Induction / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D006979:Hyperthermia, Induced; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism
D014157:Transcription Factors; D043525:Cytosine Deaminase