Naslov
Changes in promoter activity in human laryngeal carcinoma cells resistant to vincristine are responsible for increased adenoviral transgene expression

Autori
Ambriović-Ristov, Andreja ; Majhen, Dragomira ; Brozović, Anamaria ; Buger, Tvrtko ; Osmak, Maja

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Abstracts of the 9th International Adenovirus Meeting / - Dobogóko, 2009, 60-60

Skup
International Adenovirus Meeting (9 ; 2009)

Mjesto i datum
Dobogókő, Mađarska, 26.04.2009. - 30.04.2009

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Nije recenziran

Ključne riječi
adenovirus; gene therapy; drug resistance

Sažetak
Human adenovirus serotype 5 (Ad5) offers great promise in cancer gene therapy where a transient high-level expression of toxic gene products is required. Ad5 infection occurs via binding of the fiber knob domain to coxsackie and adenovirus receptor (CAR), followed by endocytosis of the virion through interaction of penton base RGD (Arg-Gly-Asp) motif with cellular integrins. Although the success of Ad5-mediated gene therapy depends on the ability of Ad5 to enter cells it depends also on the promoter activity driving the transgene expression. Chemotherapy is regularly used for cancer treatment but often results in the development of drug resistance. Numerous molecular mechanisms that have been recognized as the cause of drug resistance could lead to differential expression of Ad5 receptors as well as to changes in activity of promoters that drive the expression of the therapeutic transgene from Ad5 vector. We have previously found increased Ad5 transgene expression in human laryngeal carcinoma (HEp2) cells resistant to anti-cancer drug cisplatin in comparison to parental HEp2 cells, due to the upregulation of CAR and α vβ 3 integrins, and we showed important role of α vβ 3 integrin in drug resistance. Since gene therapy might be an attractive approach for treating drug resistant tumors we investigated whether there are differences in adenovirus mediated transgene expression between HEp2 cells and corresponding, HEp2-derived, vincristine resistant cell line VK2. We measured the transgene expression from three different Ad5s: two wild type, Ad5wtRSVβ gal and Ad5wtCMVβ gal, differing only in promoter that drives transgene expression (β -galactosidase), and short fibered Ad5639RSVβ gal which infects cells independent of CAR. We observed 4-fold increase in Ad5wtRSVβ gal transgene expression in VK2 cells comparing to HEp2 cells, while for Ad5639RSVβ gal this difference was 10-fold. Surprisingly, Ad5wtCMVβ gal showed equal transgene expression in both HEp2 and VK2 cell line. We measured in both cell lines the expression of CAR, α vβ 3 and α vβ 5 integrins implicated in Ad5 entry, and found decreased expression of CAR, increased expression of α vβ 3 integrin and equal amount of α vβ 5 integrin in VK2 in comparison to HEp2 cells. Finally, in VK2 cells we found 2-fold lower amount of internalized virus as well as 2-fold decreased Ad5wtRSVβ gal attachment as compared to HEp2 cells. These results could be explained by decreased expression of CAR in VK2 cells, but it contradicts the difference in transgene expression. We hypothesized that this discrepancy between internalization/attachment and transgene expression is likely due to the different activity of RSV and CMV promoters in HEp2 and VK2 cells. In order to verify this hypothesis we compared the activity of these promoters after transient transfection of plasmids pRSVCAT and pCMVCAT in HEp2 and VK2 cells. In VK2 cells the activity of the RSV promoter was approximately 63-fold higher than in HEp2 cells, while we observed approximately 4-fold increased activity of CMV promoter in VK2 in comparison to HEp2 cells. Our results suggest that multiple changes occur during resistance development: (i) those that affect adenovirus entry and (ii) those that could influence promoter activity. Therefore, when evaluating the success of Ad5-mediated cancer gene therapy, especially for tumors treated with antitumor drugs where possible resistant mechanisms could have developed, one should measure not only cell surface CAR and/or integrin levels but also transgene expression driven by different promoters.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

Napomena
Rad je kao poster prezwentiran i na skupu Adenoviruses : basic biology to gene therapy, održanom od 23.-24.09.2008., Zadar, Hrvatska ; objavljen u Knjizi sažetaka / Dragomira Majhen ; Andreja Ambriović-Ristov (ur.) ; Zagreb : Croatian Microbiological Society, 2008. ; str. 21-21 ; ISBN 953-96567-6-1.

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