Cell-Surface Integrins and CAR Are Both Essential for Adenovirus Type 5 Transduction of Canine Cells of Lymphocytic Origin

Abstract

Adenoviruses are the most widely used vectors in cancer gene therapy. Adenoviruses vectors are well characterized and are easily manipulated. Adenovirus serotype 5 (Ad5) is the most commonly used human serotype. Ad5 internalization into host cells is a combined effect of binding of Ad5 fiber knob with the coxsackie virus and adenovirus receptor (CAR) and binding of RGD motifs in viral penton to cell surface integrins (αvβ3, αvβ5). Ad5’s wide range of host-cell transduction and lack of integration into the host genome have made it an excellent choice for cancer therapeutics. However, Ad5 has limited ability to transduce cells of hematopoietic origin. It has been previously reported that low or no expression of CAR is a potential obstacle to Ad5 infection in hematopoietic origin cells. In addition, we have previously reported that low levels of cell surface integrins (αvβ3, αvβ5) may inhibit Ad5 infection in canine lymphoma cell lines. In the current report we have examined the ability of an Ad5 vector to infect human (HEK293) and canine non-cancerous (NCF and PBMC), canine non-hematopoietic origin cancer (CMT28, CML7, and CML10), and canine hematopoietic origin cancer (DH82, 17–71, OSW, MPT-1, and BR) cells. In addition, we have quantified CAR, αvβ3 and αvβ5 integrin transcript expression in these cells by using quantitative reverse transcriptase PCR (q-RT-PCR). Low levels of integrins were present in MPT1, 17–71, OSW, and PBMC cells in comparison to CMT28, DH82, and BR cells. CAR mRNA levels were comparatively higher in MPT1, 17–71, OSW, and PBMC cells. This report confirms and expands the finding that low or absent expression of cell surface integrins may be the primary reason for the inability of Ad5-based vectors to transduce cells of lymphocytic origin and some myeloid cells but this is not true for all hematopoietic origin cells. For efficient use of Ad5-based therapeutic vectors in cancers of lymphocytic origin, it is important to address the defects in cell surface integrins.

Adenoviral vector with shield and adapter increases tumor specificity and escapes liver and immune control

“…Second, all viruses, independent of retargeting or not, infected murine cells, i.e., fibroblasts in the tumor stroma. We speculate that motifs like the RGD loop in the penton base might play a role, as fibroblasts have been reported to have low CAR but high levels of integrins 65 .…”

The RGD motif is involved in CD97/ADGRE5-promoted cell adhesion and viability of HT1080 cells

“…The quantitative real-time PCR analysis was performed using iQ SYBR Green Supermix [BioRad]. Complementary DNA is amplified by gene-specific primers Human αv integrin, 5′-GGCGATGGCGTAGATGACTT-3′, 5′-GGCGCTCCGATGAACACAT-3′, and human β5 integrin, 5′-TCAGATGGACTATCCGTCCCTT-3′, 5′-TTGTCCCAGGTATCAGGGCT-3′ 42 . The results were analyzed by LightCycler®96 software [Roche] and normalized to internal control GAPDH.…”

Unravelling Receptor and RGD Motif Dependence of Retargeted Adenoviral Vectors using Advanced Tumor Model Systems

“…While binding to integrins is not required for the cell association of the virion 17 , the RGD motif may contribute to fibroblast transduction by adenoviral vectors. Integrin expression is ubiquitous and particularly high on fibroblasts 18 . It is thus important to establish whether the association of the RGD motif with integrins is necessary for the infection process also when receptors other than CAR are being used for initial cell association.…”