Expression of the bifunctional suicide gene CDUPRT increases radiosensitization and bystander effect of 5-FC in prostate cancer cells

Abstract

Purpose: To test the hypothesis that, with 5-fluorocytosine (5-FC) treatment, the co-expression of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) can lead to greater radiosensitization and bystander effect than CD-expression alone.

Methods and materials: R3327-AT cell lines stably expressing CD or CDUPRT were generated. The 5-FC and 5-FU cytotoxicity, and the radiosensitivity with/without 5-FC treatment, of these cells were evaluated under both aerobic and hypoxic conditions. The bystander effect was assessed by apoptosis staining and clonogenic survival. The pharmacokinetics of 5-FU and 5-FC metabolism was monitored in mice bearing CD- or CDUPRT-expressing tumors using 19F MR spectroscopy (MRS).

Results: CDUPRT-expressing cells were more sensitive to 5-FC and 5-FU than CD-expressing cells. CDUPRT-expression further enhanced the radiosensitizing effect of 5-FC, relative to that achieved by CD-expression alone. A 25-fold lower dose of 5-FC resulted in the same magnitude of radiosensitization in CDUPRT-expressing cells, relative to that in CD-expressing cells. The 5-FC cytotoxicity in co-cultures of parental cells mixed with 10-20% CDUPRT cells was similar to that in 100% CDUPRT cells. 19F MRS measurements showed that expression of CDUPRT leads to enhanced accumulation of fluorine nucleotide (FNuc), relative to that associated with CD-expression alone.

Conclusion: Our study suggests that CDUPRT/5-FC strategy may be more effective than CD/5-FC, especially when used in combination with radiation.

Figure 1

Protein expression detected by western blot (A), fluorescent microscopy (B, upper panel, ×200 magnification) and analyzed by flow cytometry (B, lower panel). Western blotting was performed with anti-yeast CD antibody. Fluorescence images were overlaid with the phase-contrasted images. In flow cytometric analysis, the gate (dashed line) was set to 0.56% mDsRed positive in parental cells. The mDsRed positive ratios in CD, CDUPRT cells according to the gate were showed above the bars.

Figure 2

Expression of CD or CDUPRT sensitizes R3327-AT cells to 5-FU (A) and 5-FC (B) under aerobic (closed symbols) or hypoxic condition (open symbols). Surviving fractions of parental (□, ■), CD (○, ●) and CDUPRT (Δ, ▲) cells after a 24 hr exposure to 5-FU or 5-FC are shown as a function of drug dose. Data are presented as the mean ± SE from three repeated experiments.

Figure 3

The bystander effects in CD (A) and CDUPRT (B) cells after a 24 hr 5-FC treatment. CD or CDUPRT cells were mixed with parental R3327-AT cells in various ratios and exposed to 5-FC (50 µg/ml to CD cells and 5 µg/ml to CDUPRT cells) under aerobic (solid bars) or hypoxic (open bars) condition, and cell survivals were determined by colony formation assay. The mean ± SE of three repeated experiments are shown.

Figure 4

Bystander effect in CD and CDUPRT cells after a 48 hr 5-FC treatment. Apoptosis is used as an endpoint and assessed by anti-AnnexinV-FITC staining. In flow cytometric analysis (4A), a rectangle gate was set with unstained parental cells. The numbers in the rectangle show the Annexin-V-FITC positive ratios. Under fluorescence microscopy (4B), phase-contrasted (upper row), mDsRed (middle row) and FITC (lower row) images from same field were acquired. See details in text.

Figure 5

Radiosensitizing effect of 5-FC on CD and CDUPRT cells under aerobic (A) and hypoxic (B) conditions, and radiosensitization of bystander cells co-cultured with CD or CDUPRT cells under aerobic (C) and hypoxic (D) conditions. See details in text. The average values of surviving fractions of three independent experiments (± SE) are shown.

Figure 6

Pharmacokinetics of 150 mg/kg 5-FU and 150mg/kg 5-FC given i.v. 10–120 min after administration in (A) CD expressing tumors treated with 5-FU (n=3), (B) CDUPRT expressing tumors with 5-FU treatment (n=4) (C) CD tumors treated with 5-FC (n=3), and (D) CDUPRT tumors treated with 5-FC (n=3). Results show the mean ± SD of the ratio of 5-FU/NaF (□), 5-FC/NaF (□) and FNuc/NaF (●). Representative in vivo ¹⁹F MR spectra of CD and CDUPRT tumors at 60 min after injection with 5-FU or 5-FC are shown alongside the pharmacokinetic plots, respectively. The chemical shift of 5-FU is normalized to 0 ppm, 5-FC at 1 ppm and FNuc between 4.8–5 ppm.