Increased adenoviral transduction efficacy in human laryngeal carcinoma cells resistant to cisplatin is associated with increased expression of integrin alphavbeta3 and coxsackie adenovirus receptor.
In our study, we investigated molecular mechanisms of increased adenoviral transduction efficacy in cisplatin-resistant human laryngeal carcinoma cells CA3ST as compared to parental cells HEp2. Using reverse transcription-PCR, the genes potentially implicated in adenoviral entry were screened. In cisplatin-resistant cells, only upregulation of alphavbeta3 integrin was detected, which was additionally confirmed by flow cytometry. Moderately increased expression of CAR was determined in cisplatin-resistant CA3ST cells using flow cytometry and measurement of wild-type adenovirus Ad5CMVbetagal attachment. In order to test the implication of alphavbeta3 integrin in transduction efficacy, 6 HEp2-derived alphavbeta3-expressing clones with graded expression of alphavbeta3 were isolated. To a certain degree of density, expression of alphavbeta3 positively correlated with Ad5CMVbetagal transduction efficacy (i.e., increased viral transduction), suggesting a role of alphavbeta3 in transduction efficacy. However, HEp2 clones with the highest alphavbeta3) expression were negatively correlated with transduction efficacy (i.e., decreased viral transduction). This was shown to be associated with downregulation of alphavbeta5 integrin, also involved in viral transduction, in clones with the highest alphavbeta3 expression. The implication of CAR in increased adenoviral transduction efficacy in cisplatin resistant CA3ST cells was further assessed by transduction experiments using adenoviral mutant Ad5FbDelta639 whose entry is only to a very small extent dependent on the presence of CAR. Indeed, Ad5FbDelta639 infected 2.5-fold more, in comparison to wild-type adenovirus, which infected 5-fold more efficiently resistant CA3ST cells than parental HEp2 cells, indicating that increased expression of CAR contributes to increased efficacy of adenoviral transduction. Thus, the data presented provide evidence that both alphavbeta3 integrin and CAR are involved in increased adenoviral transduction efficacy in cisplatin resistant CA3ST cells. These findings may have significant implications in human gene therapy using adenoviruses, especially in patients after unsuccessful cisplatin treatment.
Ambriović-Ristov
Andreja
A
Laboratory for Genotoxic Agents, Division of Molecular Biology, Ruder Bosković Institute, Zagreb, Croatia. andrea@rudjer.irb.hr
Gabrilovac
Jelka
J
Cimbora-Zovko
Tamara
T
Osmak
Maja
M
D000256:Adenoviridae / Q000235:genetics*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002277:Carcinoma / Q000378:metabolism* / Q000628:therapy; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology*; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D017931:DNA Primers / Q000737:chemistry; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D007822:Laryngeal Neoplasms / Q000378:metabolism* / Q000628:therapy; D011991:Receptors, Virus / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D015854:Up-Regulation; D001616:beta-Galactosidase / Q000378:metabolism
D000970:Antineoplastic Agents; C568771:CLMP protein, human; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D017931:DNA Primers; D039302:Integrin alphaVbeta3; D011991:Receptors, Virus; D001616:beta-Galactosidase; D002945:Cisplatin