The Presence of Human Coxsackievirus and Adenovirus Receptor Is Associated with Efficient Adenovirus-Mediated Transgene Expression in Human Melanoma Cell Cultures

Abstract

Adenovirus (AdV)-mediated gene expression of immune stimulators represents a valuable in vivo approach for gene therapy of human cancer. The expression level of the therapeutic gene is of crucial importance for the efficacy of this type of treatment. Entry of AdV is dependent on the primary adenovirus receptor CAR and the secondary AdV receptor identified earlier to be a member of the integrin family of surface molecules. We have analyzed 14 different human melanoma cell cultures from different stages together with one melanoma cell line for their AdV-mediated transduction and expression efficiency. Recombinant viruses at various concentrations were used for expression of the B7-1 costimulatory molecule under the control of different promoters and the expression levels of B7-1 were analyzed by flow cytometry. AdV-mediated IL-12 expression was measured using a commercial ELISA. Levels of transgene expression were compared with the expression levels of HCAR, the alpha(v)beta3 and alpha(v)beta5 integrins, and HLA class I. In 4 of 14 cell cultures tested, the presence of the primary virus receptor CAR was associated with the high transduction efficiency phenotype when using the B7-1- and IL-12-expressing viruses at a relatively low multiplicity of infection (MOI) of 50. Immunohistochemistry on cryosections from the original biopsies yielded a strong signal specific for CAR. In contrast, cell cultures expressing low or undetectable levels of CAR needed a 20- to 40-fold higher viral input to show comparable expression level of B7-1 or IL-12. Expression levels of the transgenes hardly varied when using different promoters and no association was observed with the presence or absence of HLA class I molecules or with the expression levels of integrins.

Vascular endothelial growth factor promoter-based conditionally replicative adenoviruses for pan-carcinoma application

“…29,[41][42][43] Furthermore, downregulation of CAR may be associated with a more malignant phenotype. 44 Due to variable expression of CAR on human primary cancer cells, the utility of Ad5 as a cancer gene therapy vector is compromised, limiting overall efficiency of cancer gene therapy including the use of CRAds.…”

Adenovirus-mediated p53 gene therapy in pediatric soft-tissue sarcoma cell lines: sensitization to cisplatin and doxorubicin

“…This variability could be at least partially attributed to the variability in CAR and integrin expression levels that are required for viral uptake into the cells. [76][77][78][79][80][81][82][83][84] To demonstrate suppression of cell growth in the sarcoma cell lines, lower doses of Ad-wtp53 were used so as not to produce an overt cytotoxic effect with massive cell death as seen in our previous experiments. Doses ranging from 0.1 to 0.4 PFU/cell (2-10 PFU/cell for RD) were used and a cell count was performed every 24 hours for 5 days.…”

A rapid and efficient method for purification of recombinant adenovirus with arginine–glycine–aspartic acid-modified fibers

“…After attachment, the virion is then internalized by endocytosis through the interaction of its penton base with α v β 3 and α v β 5 integrins on the host cell surface [3]. However, there is growing evidence that the expression of CAR is frequently suppressed in various types of cells, including tumor cells and in primary tumors [4,5], resulting in resistance to adenovirus infection [6][7][8] Unfortunately, technical difficulties may be encountered in production of certain modified adenovectors. We have encountered difficulties when producing certain adenovectors by this method, especially those with RGD-modified fibers or "sticky" virus.…”