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First published online July 13, 2004
doi: 10.1242/10.1242/jcs.01241

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CPD-photolyase adenovirus-mediated gene transfer in normal and DNA-repair-deficient human cells

¹ Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 1374. Ed. Biomédicas 2, São Paulo, 05508-900, Brazil
² Laboratory of Genetic Instability and Cancer, UPR 2169, Institut Gustave-Roussy, 94805 Villejuif CEDEX 05, France

View larger version (43K): [in a new window]   Fig. 1. The Adphr-EGFP adenoviral vector. (A) The marsupial CPD-photolyase gene (gene phr) was fused with EGFP in its C-terminal. Thereafter, the phr-EGFP expression cassette, under the control of the cytomegalovirus (CMV) promoter and with the bovine growth hormone (BGH) poly(A) signal, was cloned in the pAdeno-X vector by standard recombinant technology at the indicated restriction sites. This plasmid was transfected into low passage HEK 293 cells for the production of recombinant viral particles containing the phr-EGFP gene. (B) The integrity of the phr-EGFP insert in the recombinant adenovirus vector: the presence of the sequences phr, EGFP and poly(A) signal regions from the virus vector were confirmed by a PCR reaction, with the positive controls pCY4Bphr, pEGFP-N1 and pAdeno-X, respectively.

View larger version (57K): [in a new window]   Fig. 6. Redistribution of phr-EGFP after PRL exposure in primary cells. (A) Normal human primary fibroblasts (198VI cell line) infected with Adphr-EGFP were local UV irradiated with 50 J/m2, immediately exposed to dark or PRL conditions and analyzed after different periods of time. In the representative photos analyzed by fluorescence microscopy, cells locally UV irradiated and kept in dark conditions display strongly-fluorescent foci of phr-EGFP in damaged sites (foci pattern A, indicated by blue arrows). Following PRL conditions, foci of phr-EGFP with less intense fluorescence (foci pattern B, indicated by yellow arrows) in comparison with cells kept in dark for the same period of time. (B) Normal human primary cells (198VI cell line), infected with Adphr-EGFP were locally UV irradiated with 50 J/m2 and immediately exposed to PRL during 2 hours. Four hours after local UV irradiation the cells were incubated with an anti-CPD. Two rows of representative fields in fluorescence microscopy are shown (60x objective).

View larger version (38K): [in a new window]   Fig. 2. Photorepair protects Adphr-EGFP-infected cells from UV irradiation. (A) Infection of primary cells with Adphr-EGFP vector. Normal human primary fibroblasts (198VI cell line) were infected with Adphr-EGFP (m.o.i. 1.6x105) and the fluorescence of EGFP was analyzed after 40 hours by fluorescence microscope (photos of representative fields; 60x objective) and flow cytometry of cell suspension (graphics). For the graphics of fluorescence, `counts' represents the number of cells and `FL1-H' the green fluorescence intensity. M1 corresponds to the percentage of EGFP fluorescence calculated (CellQuest Software, Becton Dickinson, USA). (B) Survival of XP12RO-SV cells after UV irradiation. Approximately 6.0x105 cells were mock-infected (dotted lines, squares) or infected with the Adphr-EGFP vector (m.o.i. 4x104; unbroken lines, triangles) and, after 40 hours, sub-cultivated in low density for analysis of clonogenic capacity. The cells were UV-irradiated at the indicated doses and immediately exposed to dark (closed symbols) or PRL (open symbols) conditions The bars indicate standard errors (s.d.; each sample in triplicate, two independent experiments).

View larger version (90K): [in a new window]   Fig. 3. CPD immunostaining in UV-irradiated 198VI and XP456VI cells. 5.0x104 cells were infected with Adphr-EGFP adenovirus and, 40 hours later, the cells were exposed to UV irradiation (10 J/m2). Different samples were maintained under dark or PRL conditions during 2 hours immediately after a period of 4 hours incubation. Thereafter, the cells were fixed and immunostained for CPD as described in the Materials and Methods. The cells were analyzed in fluorescence microscopy (Zeiss Axiovert S100, Hamamatsu 3CCD color camera). Representative fields of each sample for fluorescein and TRITC filters are shown.

View larger version (29K): [in a new window]   Fig. 4. Photorepair performed by the phr-EGFP recombinant enzyme prevents UV-induced apoptosis in XP12RO-SV and diploid human fibroblasts. (A) Apoptosis analysis by flow cytometry in UV-irradiated XP12RO-SV (XPA mutated) cells. Adphr-EGFP and mock-infected cells were UV-irradiated at the indicated doses and exposed immediately to PRL or dark conditions. The percentage of apoptosis was accessed 48 hours after UV irradiation by flow cytometry analysis. In the graphics presented, `counts' corresponds to the number of cells and `FL2-H' to the amount of DNA measured by P.I. fluorescence. M1 represents the percentage of cells in sub-G1 region of the cell cycle, that is apoptotic cells. (B) Photorepair performed by phr-EGFP prevents UV-induced morphological apoptotic markers in XP12RO cells. Mock and Adphr-EGFP-infected samples were UV-irradiated and treated as described in (A). The morphology of cells was analyzed 48 hours after UV-irradiation by fluorescence microscopy using acridine orange/ethidium bromide staining. Shaded bars represent control samples (UV=0 J/m2), black bars irradiated samples kept in the dark, and white bars, irradiated samples exposed to PRL. (C) Apoptosis analysis by flow cytometry in UV-irradiated human primary cells. 198VI (Normal), XP456VI (XPA mutated) and XP148VI (XPC mutated) were treated as described in (A), the samples being analyzed 72 hours after UV irradiation by flow cytometry. The frequency of subdiploid nuclei was calculated and plotted for the three cell lines employed. Shaded bars represent control samples (UV=0 J/m2), closed bars irradiated samples kept in dark, and open bars irradiated samples exposed to PRL. The data for each cell line represent the average of two independent experiments.

View larger version (30K): [in a new window]   Fig. 5. Investigation of foci of phr-EGFP in CPD-containing areas in primary cells. (A) Normal human primary fibroblasts (198VI cell line) were infected with Adphr-EGFP and, after 40 hours, locally UV irradiated with 50 J/m2. The formation of foci of phr-EGFP was analyzed after 2 minutes in culture by fluorescence microscopy. (B) Normal human primary fibroblasts (198VI cell line) infected with AdphrEGFP were incubated with anti-CPD, anti-XPC and anti-p62 1 hour after 50 J/m2 of local UV irradiation. Two rows of representative fields in fluorescence microscopy (60x objective) are presented for each colocalization experiment. Each photo contains a single cell/nucleus presenting independent foci of phr-EGFP in UV-irradiated areas.

View larger version (33K): [in a new window]   Fig. 7. Quantification of foci pattern A or B (as shown in Fig. 6A) in human primary cells locally UV irradiated. Normal (A; 198VI), XPA (B; XP456VI) and XPC mutated (C; XP148VI) human primary fibroblasts infected with Adphr-EGFP were locally UV irradiated with 50 J/m2. The cells were exposed to dark or PRL treatments immediately after local UV irradiation. These treatments were performed during different periods of time, but up to a maximum period of 2 hours, the samples then being kept in the incubator until analysis by fluorescence microscopy (see bar in legend). The number of cells with foci strongly-fluorescent (pattern A, black bars) or with a less intense fluorescence (pattern B, gray bars) was counted by fluorescence microscopy in different periods of time after local UV irradiation. We counted 100 for each sample, and the data thereby represent the average of three independent experiments.

View larger version (19K): [in a new window]   Fig. 8. Quantification of phr-EGFP fluorescence in foci sites in relation to non-irradiated areas. Normal (A; 198VI), XPA (B; XP456VI) and XPC mutated (C; XP148VI) human primary fibroblasts infected with Adphr-EGFP were locally UV irradiated with 50 J/m2. The cells were exposed to dark (black bars) or PRL (white bars) treatments immediately after local UV irradiation and then analyzed after the indicated periods (1 hour and 4 hours). The intensity of green fluorescent signal in UV-irradiated areas (foci) was quantified using the Molecular Analyst software (BioRad, CA) for a population of 50 cells (198VI and XP456VI cell lines) and 10 cells (XP148VI cell line) and compared to non-irradiated areas in the same nucleus.