Detection of increased 64Cu uptake by human copper transporter 1 gene overexpression using PET with 64CuCl2 in human breast cancer xenograft model.

UNLABELLED: Copper is an essential cofactor for a variety of biochemical processes including oxidative phosphorylation, cellular antioxidant activity, and elimination of free radicals. The copper transporter 1 is known to be involved in cellular uptake of copper ions. In this study, we evaluated the utility of human copper transporter 1 (hCTR1) gene as a new reporter gene for (64)Cu PET imaging. METHODS: Human breast cancer cells (MDA-MB-231) were infected with a lentiviral vector constitutively expressing the hCTR1 gene under super cytomegalovirus promoter, and positive clones (MDA-MB-231-hCTR1) were selected. The expression of hCTR1 gene in MDA-MB-231-hCTR1 cells was measured by reverse transcription polymerase chain reaction, Western blot, and (64)Cu uptake assay. To evaluate the cytotoxic effects induced by hCTR1 expression, the dose-dependent cell survival rate after treatment with cisplatin (Cis-diaminedichloroplatinum (II) [CDDP]) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion. Small-animal PET images were acquired in tumor-bearing mice from 2 to 48 h after an intravenous injection of (64)Cu. RESULTS: The hCTR1 gene expression in MDA-MB-231-hCTR1 cells was confirmed at the RNA and protein expression and the cellular (64)Cu uptake level. MTT assay and trypan blue dye exclusion showed that the cell viability of MDA-MB-231-hCTR1 cells decreased more rapidly than that of MDA-MB-231 cells after treatment with CDDP for 96 or 72 h, respectively. Small-animal PET imaging revealed a higher accumulation of (64)Cu in MDA-MB-231-hCTR1 tumors than in MDA-MB-231 tumors. With respect to the biodistribution data, the percentage injected dose per gram of (64)Cu in the MDA-MB-231 tumors and MDA-MB-231-hCTR1 tumors at 48 h after (64)Cu injection was 2.581 ± 0.254 and 5.373 ± 1.098, respectively. CONCLUSION: An increase in (64)Cu uptake induced by the expression of hCTR1 gene was demonstrated in vivo and in vitro, suggesting the potential use of hCTR1 gene as a new imaging reporter gene for PET with (64)CuCl2.

Effective microPET imaging of brain 5-HT(1A) receptors in rats with [(18) F]MeFWAY by suppression of radioligand defluorination.

INTRODUCTION: [(18) F]MeFWAY has been developed for imaging the serotonin 1A receptors in the brain. The purpose of this study were to verify the metabolic stability of [(18) F]MeFWAY, to measure the degree of defluorination of [(18) F]MeFWAY in vivo, to investigate methods of inhibition of defluorination of [(18) F]MeFWAY, and to assess the efficacy of [(18) F]MeFWAY in rat brains in vivo. METHODS: MicroPET experiments in rats were conducted to confirm the distribution of radioactivity in the brain. Nondisplaceable binding potential (BP(ND) ) in the hippocampus and frontal cortex were also analyzed. Miconazole and fluconazole were tested for the ability to suppress defluorination of [(18) F]MeFWAY. We conducted a blockade and displacement experiment by treating with WAY-100635. RESULTS: In vitro stability tests showed that MeFWAY was very stable in serum for 6 h, but PET revealed that authentic [(18) F]MeFWAY underwent significant defluorination in vivo. In vitro inhibition study against decreasing parent activity in liver microsomes, miconazole and fluconazole suppressed metabolic elimination of MeFWAY. However, in the PET study, fluconazole showed more potent inhibitory activity than miconazole. In the suppression of metabolizing enzymes using fluconazole, radioactivity in skull was dramatically decreased by 81% (compared with 69% with miconazole) and it was coupled with an increase in brain uptake. Moreover, BP(ND) in hippocampus was 5.53 and 2.66 in frontal cortex. The blockade and displacement study showed the specificity of [(18) F]MeFWAY to 5-HT(1A) receptors. CONCLUSION: In the rat brain, [(18) F]MeFWAY microPET showed skull uptake due to defluorination in vivo. We can effectively overcome this drawback with fluconazole.

Gamma camera and optical imaging with a fusion reporter gene using human sodium/iodide symporter and monomeric red fluorescent protein in mouse model.

PURPOSE: Multimodality imaging contributes to the activation of translational research by compensating for its weak points. Herein, we developed a noninvasive dual-reporter gene system for nuclear and optical imaging. MATERIALS AND METHODS: We constructed a fusion reporter vector concurrently expressing the human sodium/iodide symporter (hNIS) and monomeric red fluorescent protein (mCherry), and evaluated the function of this fusion reporter system under in vitro and in vivo conditions. RESULTS: The expression of hNIS/mCherry fusion gene was confirmed in transfected cells using reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. As the numbers of cells increased, the fluorescence and 125I uptake increased in the hNIS/mCherry-transfected cells, and a high correlation between fluorescence intensity and radioactivity was noted. The fluorescence intensities and radioactivity signals were also well-correlated in HT-29-hNIS/mCherry tumors (R2=0.9304) in in vivo fluorescence and gamma camera imaging. CONCLUSIONS: The dual-reporter imaging method using hNIS and mCherry genes reflected tumor extent as well as viable cell numbers, and correlated well with one another. This suggests that the hNIS/mCherry dual-reporter system can be a useful tool for multi-modal imaging.

Application of bioluminescence imaging to therapeutic intervention of herpes simplex virus type I - Thymidine kinase/ganciclovir in glioma.

Lentiviral vector containing the HSV1-tk and firefly luciferase (fLuc) gene was infected into C6 and C6-TL expressing HSV1-tk and fLuc gene was generated. C6-TL showed higher [(125)I]IVDU uptake than C6. The survival rate of C6-TL decreased more rapidly with increasing GCV dose and was well correlated with fLuc activity. The images of microPET clearly demonstrated higher uptake of [(18)F]FHBG into the C6-TL tumor. Inhibition of tumor growth was observed in C6-TL tumor-bearing mice treated with GCV through tumor size measurement and bioluminescence imaging. The therapeutic effect of HSV1-tk/GCV system can be monitored using bioluminescent imaging and tumor size measurement.

Induction of G2 arrest and apoptosis of raji cells by continuous low-dose beta irradiation with 188Re-perrhenate.

Continuous irradiation with exponentially reducing beta-rays induces cell death, known as apoptosis. The aim of this study was to investigate the G2 arrest and apoptosis caused by the beta-ray emitted by the radioisotope (188)Re. Doses of 0.4 Gy (3.7 MBq), 4 Gy (37 MBq), and 40 Gy (370 MBq), were added to Blymphoma Raji cells, and cell viability, apoptosis, and DNA cell-cycle changes were assayed. (188)Re showed time- and dose-dependent effects on cell viability and on cell apoptosis and necrosis. At a (188)Re dose of 0.4 Gy, G(2) cell-cycle arrest was observed after 16 hours, and 4,6-diamidino-2-phenylindole (DAPI) staining indicated a slow, time-dependent increase in apoptotic bodies. At a (188)Re dose of 40 Gy, DNA fragmentation was observed at 2 hours, indicative of early damage in the nucleus. In summary, our results showed that continuous irradiation with low-dose beta-rays induced G(2) arrest and progressive apoptosis, which may be characteristic mechanisms of radionuclide therapy.