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PhiC31 Integrase Induces a DNA Damage Response and Chromosomal Rearrangements in Human Adult Fibroblasts

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PhiC31 Integrase Induces a DNA Damage Response and Chromosomal Rearrangements in Human Adult Fibroblasts

Jian Liu et al. BMC Biotechnol.

Abstract

Background: PhiC31 integrase facilitates efficient integration of transgenes into human and mouse genomes and is considered for clinical gene therapy. However recent studies have shown that the enzyme can induce various chromosomal abnormalities in primary human embryonic cells and mammalian cell lines. The mechanisms involved are unknown, but it has been proposed that PhiC31 attachment sites in the host genome recombine leading to chromosomal translocations.

Results: We have studied possible effects of the PhiC31 integrase expression in human adult fibroblasts by karyotyping. All control cells were cytogenetically normal, whereas cells expressing PhiC31 integrase show chromosomal abnormalities confirming our previous results using primary embryonic fibroblasts. In order to study the early mechanisms involved we measured H2AX phosphorylation - a primary event in the response to DNA double-strand-breaks. Transient transfection with PhiC31 integrase encoding plasmids lead to an elevated number of cells positive for H2AX phosphorylation detected by immunofluorescence. Western blot analysis confirmed the upregulated H2AX phosphorylation, whereas markers for apoptosis as well as p53 and p21 were not induced. Cells transfected with plasmids encoding the Sleeping Beauty transposase remained cytogenetically normal, and in these cells less upregulation of H2AX phosphorylation could be detected.

Conclusion: In primary human fibroblasts expression of PhiC31 integrase leads to a DNA damage response and chromosomal aberrations.

Figures

Figure 1
Figure 1
Analysis of H2AX phosphorylation. a) Immunofluorescence analysis of NHDF cells transfected with pBabepuro alone (control) or in combination with pCMV-Int (integrase). Blue color is DAPI-staining, visualizing nuclear DNA. Red color is staining of phosphorylated H2AX using anti-γ-H2AX antibodies. b) Quantitation of the percentage of cells with upregulated H2AX phosphorylation. Each experiment was repeated 3 times, and 100 cells were counted in each sample. Error bars indicate standard error of the mean. Only in day 3 there is a significant difference between the three treatments (p < 0.05).
Figure 2
Figure 2
H2AX phosphorylation 3 days after transfection of NHDF cells. a) Immunofluorescence analysis. b) Western blotting analysis. Lanes: 1) pBabepuro 2) pBabepuro+pCMV-Int 3) pBabepuroInt 4) pBabepuro+ pCMV-mInt 5) pBabepuromInt 6) Colcemide control experiment causing DNA damage.
Figure 3
Figure 3
Analysis of H2AX phosphorylation using co-transfection of plasmids encoding EGFP and PhiC31 integrase or Sleeping Beauty (SB) transposase. a) Cells co-transfected with plasmids encoding EGFP (green) and integrase were immunostained using anti-γ-H2AX antibodies (red). b) H2AX immunostaining was quantified by ranking H2AX positive cells from 1 to 3, 3 being the very intensive nuclear staining and 1 being the vague but yet defined nuclear staining. Staining intensities were quantified in GFP positive cells.

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