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, 5 (7), 1805-11

Utilization of Rad51C Promoter for Transcriptional Targeting of Cancer Cells

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Utilization of Rad51C Promoter for Transcriptional Targeting of Cancer Cells

Yan Cao et al. Oncotarget.

Abstract

Cancer therapy that specifically targets malignant cells with minimal or no toxicity to normal tissue has been a long-standing goal of cancer research. Rad51 expression is elevated in a wide range of cancers and Rad51 promoter has been used to transcriptionally target tumor cells, however, a large size of Rad51 promoter limits its application for gene therapy. To identify novel tumor-specific promoters, we examined expression levels of Rad51 paralogs, Rad51B, Rad51C, and Rad51D as well as Rad52 in a panel of normal and tumor cell lines. We found that Rad51C is significantly overexpressed in cancer cells. The expression was up-regulated by approximately 6-fold at the mRNA level and 9-fold at the protein level. Interestingly, the 2064 bp long Rad51C promoter fragment was approximately 300-fold higher in cancer cells than in normal cells. A construct containing Rad51C promoter driving diphtheria toxin A efficiently killed several types of cancer cells with very mild effect to normal cells. These results underscore the potential of targeting the homologous recombination pathway in cancer cells and provide a proof of principle that the Rad51C promoter fragment can be used to transcriptionally target cancer cells.

Figures

Figure 1
Figure 1. Rad51C transcripts are upregulated in cancer cells
A. Quantitative analysis of mRNA expression of Rad51B, Rad51C, Rad51D and Rad52 in normal and cancer cells. Exponentially growing cells were harvested for total RNA extraction. The transcript levels were determined using real time RT-PCR followed by analysis using delta CT method [29]. Then the relative expression level of Rad genes in different types of cells was normalized to that in HCA2 cells. All experiments were repeated at least 9 times. Error bars represent s.d. B. Statistical analysis of expression of Rad51B, Rad51C, Rad51D and Rad52 in normal and cancer cells. Man Whitney U test was employed to examine significance. * The upregulation of Rad51C in cancer cells was statistically significant (PMWU=0.037).
Figure 2
Figure 2. Rad51C protein levels are elevated in cancer cells
A. Representative Western blot showing expression levels of Rad51C in normal and cancer cell lines. B. Quantitative analysis of Rad51C protein levels in normal and cancer cells. The Western blot results were quantified with ImageJ software. All experiments were repeated at least three times. Error bars indicate s.d. C. The Man Whitney U test shows that the elevation of Rad51C protein expression in cancer cell lines is statistically significant. ** PMWU=0.0006.
Figure 3
Figure 3. Rad51C promoter is hyperactive in cancer cells
A. Diagram of Rad51C reporter construct. B. The activity of Rad51C promoter is strongly enhanced in cancer cells. Cells were transfected with pRad51C-luciferase. The ratio of luciferase activity to GFP+ cells was used as the measure of Rad51C promoter activity. All experiments were repeated more than three times. Error bars indicate s.d. C. The elevation of Rad51C promoter activity was highly significant in cancer cells. ** PMWU=0.0006.
Figure 4
Figure 4. pRad51C-DTA selectively targets cancer cells with minimal toxicity to normal cells
A. A diagram of pRad51C-DTA. B. Survival of cells transfected with pRad51C-DTA. Zero, 0.05 μg or 0.1 μg of pRad51C-DTA was transfected into cells using electroporation. At 72 hours post transfection, cells were harvested and cell number was counted on a Millipore Muse machine. The survival rate was calculated as described16. All experiments were repeated more than three times. Error bars represent s.d. C. Inhibitory effect on luciferase activity in cancerous cell lines by pRad51C-DTA. Zero, 0.05 μg or 0.1 μg of pRad51C-DTA was cotransfected with 1 μg SV40-luciferase to the 14 cell lines using Fugene. At day 3 post transfection, cells were harvested and luciferase activity was analyzed in cell lysates. All experiments were repeated at least three times. Error bars represent s.d.

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