A new pyrimidine-specific reporter gene: a mutated human deoxycytidine kinase suitable for PET during treatment with acycloguanosine-based cytotoxic drugs

Abstract

In this article, we describe a series of new human-derived reporter genes based on human deoxycytidine kinase (dCK) suitable for clinical PET.

Methods: Native dCK and its mutant reporter genes were tested in vitro and in vivo for their phosphorylation of pyrimidine- and acycloguanosine-based radiotracers including 2'-deoxy-2'-fluoroarabinofuranosylcytosine, 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (FEAU), penciclovir, and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) and clinically applied antiviral and anticancer drugs.

Results: Cells transduced with dCK mutant reporter genes showed high in vitro and in vivo uptake of pyrimidine-based radiopharmaceuticals ((18)F-FEAU) comparable to that of herpes simplex virus type-1 thymidine kinase (HSV1-tk)-transduced cells. These mutants did not phosphorylate acycloguanosine-based radiotracers ((18)F-FHBG) or antiviral drugs (ganciclovir). Furthermore, the mutants displayed suicidal activation of clinically used pyrimidine-based prodrugs (cytarabine, gemcitabine).

Conclusion: The mutants of human dCK can be used as pyrimidine-specific PET reporter genes for imaging with (18)F-FEAU during treatment with acycloguanosine-based antiviral drugs. Additionally, the prosuicidal activity of these reporters with pyrimidine-based analogs will allow for the safe elimination of transduced cells.

FIGURE 1

Assessment of reporter gene expression in vitro. (A) Subcellular localization of native-dCK/GFP, dCKDM/GFP, dCKTM/GFP, and ΔdCK/GFP reporter proteins. (B) Western blot analysis of dCK and mutant protein expression in nontransduced and transduced cell populations using human dCK and GFP- and β-actin–specific monoclonal antibodies. (C) ³H-FAC uptake (K_i) in nontransduced and transduced U87 cells is listed on abscissa. Values are mean ± SD, n = 3. N/T = nontransduced.

FIGURE 2

Radiotracer accumulation in transduced cells in vitro. Radiotracer uptake (K_i) of ³H-FEAU, ¹⁴C-FIAU, and ³H-penciclovir in nontransduced and transduced U87 cells is listed on abscissa. Values are mean ± SD, n = 3, P < 0.05. N/D = not done; N/T = nontransduced; PCV = penciclovir.

FIGURE 3

Small-animal PET of reporter gene expression. (A) Coronal small-animal PET images through xenografts placed subcutaneously over shoulders are shown: nontransduced and transduced U87 xenografts, including dCK, dCKDM, HSV1-tk, and HSV1-R176Qsr39tk. ¹⁸F-FEAU and ¹⁸F-FHBG images at 2 h after radiotracer administration obtained on consecutive days are shown for same animal. All images were adjusted to same color scale. (B) Image-based measurements of ¹⁸F-FEAU and ¹⁸F-FHBG at 2 h after radiotracer administration in same animals, expressed as percentage injected dose per cubic centimeter of tissue (%ID/cm³). Values are mean ± SD, n = 5. N/T = nontransduced.

FIGURE 4

Small-animal PET/micro-CT of dCKDM/GFP-expressing T cells: colocalization of ¹⁸F-FEAU accumulation in small-animal PET images with tumor regions in micro-CT images. Representative tumor-bearing animals from Pz1+dCKDM/GFP+ treated (A) and control nontreated (B) groups are shown. High ¹⁸F-FEAU accumulation was detected on day of T-cell administration in region corresponding to tumor foci on micro-CT image in Pz1+dCKDM/GFP+ treated animal. No ¹⁸F-FEAU accumulation was observed in region corresponding to tumor foci in nontreated control animal. Heart and blood vessels were enhanced by iodinated lipids. Immunofluorescent analysis revealed presence of GFP-positive (green, stained with anti-GFP antibody) T cells infiltrating hPSMA-positive (red, stained with anti-hPSMA antibody) tumor in lung from representative Pz1+dCKDM/GFP+ T-cell–treated (C) and –nontreated tumor-bearing (D) animals. Nuclei were stained with 4,6-diamidino-2-phenylindole (blue). Magnification, ×10. Quantitative analysis of ¹⁸F-FEAU-accumulation (E) and region-of-interest–to–background ratios (F) in treated and nontreated groups is presented. Data are expressed as percentage injected dose per cubic centimeter of tissue (%ID/cm³). Values are mean ± SD, n = 5. BGR = background; ROI = region of interest, marked with arrow.