Activation of the CMV-IE Promoter by Hyperthermia In Vitro and In Vivo: Biphasic Heat Induction of Cytosine Deaminase Suicide Gene Expression

Abstract

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.

Real-time Transcriptional Profiling of Cellular and Viral Gene Expression during Lytic Cytomegalovirus Infection

“…It can induce growth arrest by interacting with CDK2 and CDK4 and interacts with the heterodimer E2F-DP to inhibit cell growth [66], [67]. To date, little is known about its function in CMV infection, however, the MIEP promoter of CMV contains a binding site for this transcription factor [68].…”

Pharmacokinetics of combined gene therapy expressing constitutive human GM-CSF and hyperthermia-regulated human IL-12

“…We also noted that heat induction of hGM-CSF expression is more obvious in Hep3B cells than in A549 cells, suggesting cell type dependence. Recently, the stimulating effect of heat stress on CMV promoter activity has been studied [21,22]. Although the possible mechanisms might be complex, a considerable homology to the heat stress element core consensus (GA–TCC) within 18 bp elements in IE enhancer might be the most reasonable explanation [21,23].…”

“…Recently, the stimulating effect of heat stress on CMV promoter activity has been studied [21,22]. Although the possible mechanisms might be complex, a considerable homology to the heat stress element core consensus (GA–TCC) within 18 bp elements in IE enhancer might be the most reasonable explanation [21,23]. Heat stress might regulate CMV-IE activity directly and indirectly through heat-activated transcription factors.…”

“…Heat stress might regulate CMV-IE activity directly and indirectly through heat-activated transcription factors. Heat stress inducing various transcriptional factors, including those activating the CMV-IE promoter, has been reported [21,22]. Therefore, the cell type dependence might reflect the high specificity of the signaling pathway and transcription factors.…”

Marine Viruses: the Beneficial Side of a Threat

“…A recent study has demonstrated a high promoter activity of the WSSV IE gene ie1 in Sf9 insect cells [44]. Its promoter activity for gene expression in insect cells is stronger than that of the cytomegalovirus (CMV) IE promoter, which is widely used as a strong and constitutively active promoter [45,46]. [36] The unprecedented worldwide spread of highly pathogenic avian influenza viruses of the H5N1 strain is currently considered a global health concern that has provoked significant mortalities in poultry and wild bird's populations.…”