Figure 1.
Effects of genotoxic agents on L1 promoter activity in HepG2 cells.
L1 promoter activity 6 and 24 hours after treatment was evaluated by the luciferase reporter gene assay. Data are the average ratio ± the standard deviation (SD) of the L1 promoter activity in the presence of the test compound relative to that in the presence of the vehicle control from four independent experiments. Each experiment was carried out in duplicate (n = 8), except in the case of 0.5 µM benzo[a]pyrene and camptothecin and 0.1 and 5 µM vinblastine (at least two independent experiments in duplicate, n = 4 or more). Statistically significant differences as compared with the vehicle control for 6-hour and 24-hour treatments are indicated by *and #, respectively (*or #, p<0.05; **p<0.01; ***or ###, p<0.001).
Table 1.
Effects of various genotoxic agents on L1 promoter activity.
Figure 2.
Effects of cellular stress induced by chemicals on L1 promoter activity in HepG2 cells.
L1 promoter activity 6 and 24 hours after treatment was evaluated by the luciferase reporter gene assay. Data are represented as the average ratio (± SD) of the L1 promoter activity in the presence of the test compound relative to that in the presence of the vehicle control from at least three independent experiments (in duplicate; n = 6 or more) in the case of cyclosporin A, two independent experiments (in duplicate; n = 4) in the case of etomoxir and diethyl malate, and at least four independent experiments (in duplicate; n = 8 or more) in the case of exo1. The experimental data using diethyl malate and 500 µM etomoxir are shown only after 6 hours because of overt cytotoxicity after a 24-hour exposure (data not shown). Statistically significant differences as compared with the vehicle control for 6-hour and 24-hour treatments are indicated by *and #, respectively (*or #, p<0.05; ***or ###, p<0.001).
Table 2.
Effects of cellular stress induced by chemicals on L1 promoter activity.
Figure 3.
Effects of commercially available drugs on L1 promoter activity in HepG2 cells.
L1 promoter activity 6 and 24 hours after treatment was evaluated by the luciferase reporter gene assay. Data are represented as the average ratio (± SD) of the L1 promoter activity in the presence of the test compound relative to that in the presence of the vehicle control from two independent experiments (in quadruplicate; n = 8) for bezafibrate and fenofibrate and from two independent experiments (in duplicate; n = 4) for diflunisal, flufenamic acid, salicylamide, and sulindac. The experimental data using sulindac are shown only after 6 hours because overt cytotoxicity occurred after a 24-hour exposure (data not shown). Statistically significant differences as compared with the vehicle control for 6-hour and 24-hour treatments are indicated by *or #, respectively (#, p<0.05; **or ##, p<0.01; ***or ###, p<0.001).
Table 3.
Effects of drugs on L1 promoter activity.
Figure 4.
Effects of benzo[a]pyrene and merbarone on L1 promoter activity in HeLa cells.
L1 promoter activity 6 and 24 hours after treatment was evaluated by the luciferase reporter gene assay. Data are represented as the average ratio (± SD) of the L1 promoter activity in the presence of the test compound relative to that in the presence of the vehicle control from two independent experiments (in quadruplicate; n = 8). Statistically significant differences as compared with the vehicle control for 6-hour and 24-hour treatments are indicated by *and #, respectively (*p<0.05; ***or ###, p<0.001).
Figure 5.
A novel secreted Gaussia luciferase L1 retrotransposition assay system.
(A) A novel Gaussia luciferase cassette in a human L1. The mGLucI retrotransposition cassette was cloned into the 3′ UTR of L1RP in the antisense orientation. The cassette contains the GLuc gene interrupted by an antisense mini-intron with an EF-1α/eIF4g hybrid promoter (phEF1/peIF4g) and the TK poly(A) signal (TK pA). GLuc can be expressed only when retrotransposition occurs. (B) Coexpression in HEK293T cells of selected cDNA constructs, previously determined to alter retrotransposition levels in the EGFP reporter assay (right panel; [40]), similarly affect retrotransposition in the GLuc assay (left panel). Fifty µl of media were sampled from each well (four replicate wells for each construct) at the indicated hourly time points over the course of 10 days and tested at the conclusion of the experiment (RLU: Relative Light Units). EGFP assays were performed at approximately 115 hours and results were normalized to cotransfected 99-PUR-RPS-EGFP reporter construct and empty vector (pcDNA3). Data for the earliest time-points sampled are reproduced in Fig. S3. (C) Retrotransposition in HeLa cells is confirmed by PCR using primers that flank the intron of the GLuc reporter gene. The presence of a band of 149 bp is diagnostic for intron removal. A 283-bp band is amplified from the transfected 99-PUR-RPS-mGLucI or 99-PUR-JM111-mGLucI plasmid. (D) Immunofluorescence analysis of transfected and fixed HEK293T cells showing obvious expression of GLuc from retrotransposition events formed by 99-PUR-RPS-mGLucI but not 99-PUR-JM111-mGLucI. Constitutive luciferase expression from pCMV-GLuc is also shown (right), detected by α-Gaussia antibody.
Figure 6.
The dual secreted luciferase L1 retrotransposition assay.
(A) Timeline of dual secreted luciferase L1 retrotransposition assay. HeLa cells transfected with 99-PUR-RPS-mGLucI and pSV40-CLuc were exposed to a test compound and puromycin for 2 days. The cells were then exposed to the test compound alone for an additional 3 days. GLuc and CLuc luminescence in the medium and cellular ATP were measured with a luminometer. GLuc luminescence indicates L1 retrotransposition activity. CLuc luminescence was used for normalization as an internal control. Cell viability was evaluated by cellular ATP content. (B) Effect of JM111 (L1RP ORF1 mutant) on L1 retrotransposition activity. Either 99-PUR-RPS-mGLucI or 99-PUR -JM111-mGLucI containing two missense mutations in L1RP ORF1 and pSV40-CLuc were co-transfected. L1 retrotransposition activity is represented as normalized luminescence. ***p<0.001. (C, D) Effect of the reverse transcriptase inhibitor azidothymidine (AZT) on L1 retrotransposition activity (C) and cell viability (D). L1 retrotransposition activity and cell viability are indicated as the percentage of the vehicle control. Data in (B–D) are the mean (± SD) from four independent experiments and are expressed relative to each control. ***p<0.001.
Figure 7.
Effects of etomoxir, salicylamide, and WY-14643 on L1 retrotransposition.
The effects of L1 retrotransposition activity after treatment for 5 days are shown. Data are the mean (± SD) from four independent experiments and are expressed relative to the vehicle control. **p<0.01; ***p<0.001.