Photochemical internalization of transgenes controlled by the heat-shock protein 70 promoter.
Photochemical internalization (PCI) is a targeting technique that facilitates endosomal escape of macromolecules, such as transgenes, in response to photochemical treatment with endosome/lysosome-localized photosensitizers, such as disulfonated meso-tetraphenylporphine (TPPS(2a)). In gene therapy this leads to enhanced transgene expression. Moreover, photochemical treatment generally activates transcription of stress-response genes, such as heat-shock proteins (HSPs), via stimulation of corresponding promoters. Therefore, we used HSP70 (HSPp; a promoter from the HSP family gene) and investigated whether the PCI stimulus could also activate HSPp and thereby stimulate transcription (expression) of the HSPp-controlled transgene internalized via PCI. Using human colorectal carcinoma and hepatoma cell lines in vitro, we showed that TPPS(2a)-based photochemical treatment enhances expression of cellular HSP70, which correlated with a photochemically enhanced expression (approximately 2-fold, at PCI-optimal doses) of the HSPp-controlled transgene integrated in the genome. Furthermore, PCI enhanced expression of the HSPp-controlled episomal transgene delivered as a plasmid. However, in plasmid-based transfection, PCI-mediated enhancement with HSPp did not exceed the enhancement achieved with the constitutive active CMV promoter. In conclusion, we demonstrated that the PCI-relevant treatment initiates HSP70 response and that the HSP70 promoter can be used in combination with PCI, leading to PCI-enhanced expression of the HSPp-controlled transgene.
Prasmickaite
Lina
L
Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway. linap@ulrik.uio.no
Hellum
Marit
M
Kaalhus
Olav
O
Høgset
Anders
A
Wagner
Ernst
E
Berg
Kristian
K
D045744:Cell Line, Tumor; D004705:Endocytosis; D015316:Genetic Therapy / Q000379:methods; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D010777:Photochemistry; D010957:Plasmids / Q000008:administration & dosage / Q000493:pharmacokinetics; D011166:Porphyrins / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D014162:Transfection / Q000379:methods*; D019076:Transgenes; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
D018840:HSP70 Heat-Shock Proteins; D011166:Porphyrins; C425082:di(4-sulfonatophenyl)diphenylporphine