Increased adenovirus Type 5 mediated transgene expression due to RhoB down-regulation

Abstract

Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking.

Figure 1. Cell surface levels of CAR, integrin heterodimers αvβ3, αvβ5 and α3β1, integrin subunits α5, αv and β1 on HEp2 and CK2 cells.

Cells were detached by Versene and incubated with murine monoclonal antibodies or isotype-matched antibody as a negative control. After incubation with the secondary reagent (i.e. PE-conjugated anti-mouse antibody), labeled cells were analyzed by flow cytometry. (A). Representative histograms and (B) mean fluorescence intensities relative to HEp2 cells obtained in one of three independent experiments that provided similar results are shown.

Figure 2. Ad5-mediated transgene expression in cisplatin-resistant CK2 cells is higher than in parental HEp2 cell line.

Cells were plated in 96-well plates and, 24 hours later, infected for 1 hour at 37°C with two-fold serial dilutions of Ad5s. Twenty-four hours after infection, cells were stained for β-galactosidase expression. A similar difference was observed over a wide range of dilutions, from 2×10⁴ to 5000 pp/cell; however, only the transgene expression obtained with a MOI of 10⁴ pp/cell is presented. (A). Transgene expression in HEp2 and CK2 cells after transduction with Ad5wt, Ad5RGD4C, Ad5Δ639 and Ad5Δ639RGD4C. (B). Data shown in (A) represented as relative to HEp2 cells. The results presented are representative of three independent experiments with similar results ± standard deviation.

Figure 3. Ad5wt shows slightly higher attachment and internalization in cisplatin-resistant CK2 cells comparing to parental HEp2 cell line.

Cells were incubated with Ad5wt at MOI 1000 for 40 minutes on ice. To measure attachment, unbound viruses were removed and cells were scraped off and pelleted. To measure internalization, unbound viruses were removed and cells were incubated at 37°C for 40 minutes, trypsinized and pelleted. Total DNA (cellular plus viral) was extracted from cells and used as a sample for quantification of viral DNA by real-time PCR using a region within the hexon as the target sequence. The cell-derived DNA content per PCR reaction was normalized by a second real-time PCR assay targeting the GAPDH gene. The results presented are representative of three independent experiments with similar results ± standard deviation. Asterisks indicate significant differences (*, P<0.05; **, P<0.01).

Figure 4. Decreasing RhoB expression increases Ad5wt-mediated transgene expression.

HEp2 and CK2 cells were transfected with control or RhoB siRNA. Twenty-four hours after transfection cells were replated in 96-well plates for Ad5wt-mediated transgene expression measurement and 6-well plates for Western blot. Forty-eight hours after siRNA transfection, the level of RhoB was analyzed by Western blot and cells in 96-well plates were transduced for 1 hour at 37°C with two-fold serial dilutions of Ad5wt. Twenty-four hours after infection, cells were stained for β-galactosidase expression. The transgene expression obtained with an MOI of 10⁴ pp/cell is presented. (A) Forty-eight hours post-transfection the level of RhoB protein was analyzed by Western blot, i.e. at the time of Ad5wt transduction whose results are presented in C and D. Actin was used as a loading control. A representative blot of two independent experiments is presented. (B) Densitometric analysis of Western blot presented in (A). Data are presented as relative to RhoB expression in HEp2 cells that was set as 100%. (C) Ad5wt-mediated transgene expression in HEp2 and CK2 cells transfected with control or RhoB-specific siRNAs. (D). Data shown in (C) are presented as relative to control siRNA in HEp2 and CK2 cells, respectively. The results presented in (C) and (D) are representative of three independent experiments with similar results ± standard deviation.

Figure 5. Decreasing RhoB expression by siRNA transfection does not change Ad5wt attachment or internalization.

Forty-eight hours after transfection with control or RhoB-specific siRNAs, cells were incubated with Ad5wt at an MOI 1000 pp/cell for 40 minutes on ice. To measure attachment, unbound virus was removed and cells were scraped off and pelleted. To measure internalization, unbound viruses were removed and cells were incubated at 37°C for 40 minutes, trypsinized and pelleted. Total DNA (cellular plus viral) was extracted from cells and used as a sample for quantification of viral DNA by real-time PCR using a region within hexon as the target sequence. The cell-derived DNA content per PCR reaction was normalized by a second real-time PCR assay targeting the GAPDH gene. (A). Attachment of Ad5wt in HEp2 and CK2 cells transfected with control or RhoB-specific siRNAs presented as fold of HEp2. (B). Internalization of Ad5wt in HEp2 and CK2 cells transfected with control or RhoB-specific siRNAs presented as fold of HEp2. The results presented are representative of three independent experiments with similar results ± standard deviation. Asterisks indicate significant differences (*, P<0.05; **, P<0.01).

Figure 6. RhoB influences Ad5wt intracellular trafficking.

Confocal microscopy of attachment and internalization of fluorescently labeled Ad5wt at MOI 4×10⁴ pp/cell in HEp2 and CK2 cells. Cells were incubated with Alexa Fluor 488-labeled virions for 60 minutes on ice. Unbound virus was removed and cells were either fixed to measure attachment or fed with fresh medium and incubated at 37°C for 30 minutes before fixation to allow virus internalization. Overlay of virus (green), nucleus stained with DRAQ5 (blue) and phase-contrast is shown. Scale bar presents 20 µm.