Indoleamine 2,3-dioxygenase (IDO) suppresses T cell responses by its action in catabolising tryptophan. It is important in maintenance of immune privilege in the placenta. We investigated the activity of IDO in the cornea, following corneal transplantation and the effect of IDO over-expression in donor corneal endothelium on the survival of corneal allografts. IDO expression was analysed and functional activity was quantified in normal murine cornea and in corneas following transplantation as allografts. Low levels of IDO, at both mRNA and protein levels, was detected in the normal cornea, up-regulated by IFN-c and TNF. Expression of IDO in cornea was significantly increased following corneal transplantation. However, inhibition of IDO activity in vivo had no effect on graft survival. Following IDO cDNA transfer, murine corneal endothelial cells expressed functional IDO, which was effective at inhibiting allogeneic T cell proliferation. Over-expression of IDO in donor corneal allografts resulted in prolonged graft survival. While, on one hand, our data indicate that IDO may augment corneal immune privilege, up-regulated IDO activity following cytokine stimulation may serve to inhibit inflammatory cellular responses. While increasing IDO mRNA expression was found in allogeneic corneas at rejection, over-expression in donor cornea was found to significantly extend survival of allografts.
Thymidylate synthase (EC 2.1.1.45) is a key enzyme for the de novo synthesis of DNA and as such a target for anticancer drug development. There is a need to develop noninvasive methods for assessing thymidylate synthase inhibition in tumors. The aim of this study was to assess the potential of 3 ¶-deoxy-3 ¶-[18 F]fluorothymidine ([ 18 F]FLT) positron emission tomography (PET) for early measurement of thymidylate synthase inhibition and to elucidate the cellular mechanisms involved. Radiation-induced fibrosarcoma-1 tumor-bearing mice were injected with a single i.p. dose of the thymidylate synthase inhibitor 5-fluorouracil (5-FU; 165 mg/kg) and imaged by [18 F]FLT-PET at 1 to 2 hours after treatment. Deoxyuridine, thymidine kinase 1 (cytoplasmic thymidine kinase; EC2.7.1.21), and ATP levels in excised tumors were measured. Cellular assays for membrane transport were also done. There was a 1.8-fold increase in the 60-minute [18 F]FLT tumor/heart radioactivity ratio in drug-treated mice compared with vehicle controls (P = 0.0016). Plasma and tumor deoxyuridine levels increased significantly but thymidine kinase and ATP levels were unchanged. Whole-cell assays implicated a (low level) functional role for the type-1 equilibrative nucleoside transporter (ENT). There was an increase in type-1 ENT-binding sites per cell from 49,110 in untreated cells to 73,142 (P = 0.03) in cells treated with 10 Mg/mL 5-FU for 2 hours, without a change in transporter affinity (P = 0.41). We conclude that [18 F]FLT-PET can be used to measure thymidylate synthase inhibition as early as 1 to 2 hours after treatment with 5-FU by a mechanism involving redistribution of nucleoside transporters to the plasma membrane. (Cancer Res 2006; 66(17): 8558-64)
Histone deacetylase inhibitors (HDACI) are emerging as growth inhibitory compounds that modulate gene expression and inhibit tumor cell proliferation. We assessed whether 3 ¶-deoxy-3 ¶-[18 F]fluorothymidine-positron emission tomography ([ 18 F]FLT-PET) could be used to noninvasively measure the biological activity of a novel HDACI LAQ824 in vivo. We initially showed that thymidine kinase 1 (TK1; EC2.7.1.21), the enzyme responsible for [18 F]FLT retention in cells, was regulated by LAQ824 in a drug concentration-dependent manner in vitro. In HCT116 colon carcinoma xenograftbearing mice, LAQ824 significantly decreased tumor [18 F]FLT uptake in a dose-dependent manner. At day 4 of treatment, [ 18 F]FLT tumor-to-heart ratios at 60 minutes (NUV60) were 2.16 F 0.15, 1.86 F 0.13, and 1.45 F 0.20 in vehicle, and 5 and 25 mg/kg LAQ824 treatment groups, respectively (P V 0.05). LAQ825 at 5 mg/kg also significantly reduced both TK1 levels and [18 F]FLT uptake at day 10 but not at day 2 (P V 0.05). [ 18 F]FLT NUV60 correlated significantly with cellular proliferation (r = 0.68; P = 0.0019) and was associated with druginduced histone H4 hyperacetylation. Of interest to [18 F]FLT-PET imaging, both TK1 mRNA copy numbers and protein levels decreased in the order vehicle >5 mg/kg LAQ824 > 25 mg/kg LAQ824, providing a rationale for the use of [ 18 F]FLT-PET in this setting. We also observed increases in Rb hypophosphorylation and p21 levels, factors that could have contributed to the alteration in TK1 transcription in vivo. In conclusion, we have shown the utility of [18 F]FLT-PET for monitoring the biological activity of the HDACI, LAQ824. Drug-induced changes in tumor [18 F]FLT uptake were due, at least in part, to reductions in TK1 transcription and translation. (Cancer Res 2006; 66(15): 7621-9)
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