Photochemical treatment with endosomally localized photosensitizers enhances the number of adenoviruses in the nucleus.
BACKGROUND
In the present study the physical targeting technique photochemical internalization (PCI) has been used in combination with adenovirus. We have previously shown that PCI enhances transgene expression from AdhCMV-lacZ, and the aim of the present study was to further increase the understanding of photochemically mediated adenoviral transduction.


METHODS
Two colorectal carcinoma cell lines, WiDr and HCT116, were pre-incubated with the photosensitizer TPPS(2a) or methylene blue derivates (MBD) followed by infection with adenovirus and light exposure. Transgene expression was measured by flow cytometry. Real-time polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) were used to quantify the level of viral DNA in the nuclei. Real-time PCR was also used to measure the level of beta-galactosidase mRNA in samples infected with AdhCMV-lacZ.


RESULTS
Exposing TPPS(2a)-treated cells to light enhanced the quantity of viral DNA in the nucleus, the mRNA level of the transgene and the transgene expression compared to non-illuminated cells. The increased transgene expression was independent of the promoter used, but dependent on the time of light exposure and the cellular localization of the photosensitizer.


CONCLUSIONS
The enhanced transgene expression observed after photochemical treatment is most likely not a result of one event, but more an interplay between various mechanisms. An increased level of adenoviral DNA in the nucleus and a dependency of endosomal localization of the photosensitizer to obtain enhanced transgene expression suggested that endosomal rupture facilitated the transport of adenoviruses to the nucleus.
Engesaeter
Birgit Ø
BØ
Department of Tumor Biology, Rikshospitalet-Radiumhospitalet Health Enterprise, Montebello, N-0310 Oslo, Norway. birgite@labmed.uio.no
Tveito
Siri
S
Bonsted
Anette
A
Engebraaten
Olav
O
Berg
Kristian
K
Maelandsmo
Gunhild M
GM
D000256:Adenoviridae / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects* / Q000528:radiation effects* / Q000821:virology; D004279:DNA, Viral / Q000235:genetics; D011992:Endosomes / Q000378:metabolism*; D015870:Gene Expression / Q000528:radiation effects; D016679:Genome, Viral / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D045325:HCT116 Cells; D006801:Humans; D017319:Photosensitizing Agents / Q000378:metabolism / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014161:Transduction, Genetic; D019076:Transgenes; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics
D004279:DNA, Viral; D017319:Photosensitizing Agents; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase