PMC

Dose–response of 5-FU treatment on hRL expression in 293T cells. Cells containing either the reporter construct under control of the CMV promoter or the 1.7-kb hTERT promoter were treated with 1 μmol/L 5-FU, 2-fold serial dilutions of 5-FU (1:2, 1:4, 1:8, 1:16, 1:32, 1:64 μmol/L), or no 5-FU. RL activity was quantified by luminometry. Results for each sample are shown as a percentage of RL activity seen in untreated 293T cells using the CMV promoter. 1.7 T = 1.7-kb hTERT promoter.

Relative expression of the reporter gene constructs in hTERT-positive and hTERT-negative cell lines. The trimodality reporter construct was placed under control of either the constitutively active CMV promoter or 1 of 3 truncated hTERT promoters. These 4 constructs were transfected into 2 hTERT-positive cell lines (293T and MCF7) and 2 hTERT-negative cell lines (U-2 OS and Saos). The relative expression of each of the 3 reporter genes was determined for each promoter construct. hRL and tTK activities are quantified for each cell line as a percentage of the activity expressed from the CMV construct. mRFP1 activity is described for each cell line as high, medium (med), low, or absent (nil), as compared with high activity of the CMV construct.

mRFP1 activity in MCF7 (hTERT-positive) and Saos (hTERT-negative) cell lines. (A) RT-PCR and TRAP assays of MCF7 and Saos cells after transfection with 1 of 4 promoter constructs. RT-PCR: β-Actin is a control for cDNA quality and is present in all cell samples but is absent in the water negative controls (Ø). hTERT is expressed in transfected MCF7 cells, but not in Saos cells. TRAP: Telomerase activity, is expressed in transfected MCF7 cells but not Saos cells. (B) Fluorescence microscopy of mRFP1 expression in transfected MCF7 and Saos cells. mRFP1 signal is seen in both MCF7 and Saos cells when expressed from the constitutively active CMV promoter. When expressed from the 3 hTERT promoters, mRFP1 signal is seen only in the hTERT-expressing MCF7 cells. CMV = CMV promoter; 1.7 T = 1.7-kb hTERT promoter; 0.5 T = 0.5-kb hTERT promoter; 0.2 T = 0.2-kb hTERT promoter.

hTERT expression, telomerase activity, and TRF lengths in cell lines. Results of gene expression analysis by RT-PCR, telomerase activity measurement by TRAP assay, and TRF length measurement by Southern blotting are shown for the 4 cell lines used in this study. RT-PCR: β-Actin is a control for cDNA quality and is present in all cell lines but is absent in the water negative control (Ø). hTERT is expressed in 293T and MCF7 cell lines. TRAP: 293T and MCF7 cell lines express telomerase activity, apparent as a ladder of TRAP products. Telomerase activity is appropriately abolished by heat pre-treatment of the sample. TRF lengths: Mean TRF lengths of 293T and MCF7 cells are in the range of 4–6 kb. Mean TRF lengths of U-2 OS and Saos cells extend above 15 kb, characteristic of the ALT phenotype.

Repression of hTERT expression in 293T cells due to 1 μmol/L of 5-FU can be measured noninvasively by reporter gene activity. (A) RT-PCR and TRAP assays of transfected 293T cells with and without 5-FU treatment. RT-PCR: β-Actin is a control for cDNA quality and is present in all cell samples but is absent in the water negative control (Ø). hTERT is normally highly expressed in 293T cells but, after 5-FU treatment, hTERT expression is reduced ~95%, as measured by ImageQuant quantification software. TRAP: Telomerase activity is normally expressed in 293T cells but is absent after 5-FU treatment. (B) Fluorescence microscopy of mRFP1 activity in 293T cells with and without 5-FU treatment. High mRFP1 signal is seen in both treated and untreated cells when expressed from the constitutively active CMV promoter. When expressed from the 3 hTERT promoters, high mRFP1 signal is seen in untreated cells but is nearly absent in 5-FU-treated cells. (C) Bioluminescent imaging of RL activity in 293T cells with and without 5-FU treatment. Low-level signal at the edge of some 5-FU-treated sample wells is due to light refraction artifact, not reporter activity. (D) Quantitation of RL activity by luminometry in 293T cells with and without 5-FU treatment. Results for each sample are shown as a percentage of RL activity seen in untreated 293T cells using the CMV promoter. Error bars represent SE. (E) Quantitation of tTK activity by TK assay in 293T cells with and without 5-FU treatment. CMV = CMV promoter; 1.7 T = 1.7-kb hTERT promoter; 0.5 T = 0.5-kb hTERT promoter; 0.2 T = 0.2-kb hTERT promoter; Tx = treatment; P/s/cm²/sr = photons s⁻¹·cm⁻²·steradian⁻¹.