A novel 11C-labeled thymidine analog, [11C]AZT, for tumor imaging by positron emission tomography
© Tahara et al. 2015
Received: 15 April 2015
Accepted: 18 August 2015
Published: 4 September 2015
Nucleoside analogs labeled with positrons, such as 11C and 18F, are considered valuable in visualizing the proliferative activity of tumor cells in vivo using positron emission tomography (PET). We recently developed the 11C-labeled thymidine analogs [11C]zidovudine ([11C]AZT) and [11C]stavudine ([11C]d4T) via the Pd(0)-Cu(I) co-mediated rapid C–C coupling reaction. In this study, to examine whether [11C]AZT and [11C]d4T might be useful for visualization of tumors in vivo, we performed PET imaging, tissue distribution studies, and metabolite analysis in tumor-bearing mice.
Mice bearing tumors (rat glioma C6 and human cervical adenocarcinoma HeLa cells) were injected with 50 MBq of [11C]AZT or [11C]d4T, and PET was performed immediately thereafter. After PET imaging, the radioactivity in several tissues, including tumor tissues, was measured using a γ-counter. In addition, radioactive metabolites in plasma, bile, intestinal contents, and tumor were analyzed using thin layer chromatography (TLC). Cellular uptake of [11C]AZT in C6 was measured in the presence or absence of non-labeled thymidine (0.1 mM).
In PET studies, C6 and HeLa tumors in mice were clearly visualized using [11C]AZT. Time-activity curves using [11C]AZT showed that the accumulation of radioactivity in tumors plateaued at 10 min after injection and persisted for 60 min, while most of the radioactivity in other tissues was rapidly excreted into the urine. In various tissues of the body, tumor tissue showed the highest radioactivity at 80 min after injection (five to six times higher uptake than that of blood). Compared with tumor tissue, uptake was lower in other proliferative tissues such as the spleen, intestine, and bone marrow, resulting in a high tumor-to-bone marrow ratio. Cellular uptake of [11C]AZT in C6 cells was completely blocked by the application of thymidine, strongly indicating the specific involvement of nucleoside transporters. In contrast, the time-activity curve of [11C]d4T in the tumor showed transient and rapid excretion with almost no obvious tumor tissue accumulation.
Tumors can be detected by PET using [11C]AZT; therefore, [11C]AZT could be useful as a novel PET tracer for tumor imaging in vivo.
KeywordsAZT d4T PET Thymidine analog Tumor imaging
Thymidine analog nucleoside reverse transcriptase inhibitors (NRTIs) such as zidovudine (AZT) and stavudine (d4T) suppress the replication of human immunodeficiency virus (HIV) and are now used in the treatment of acquired immunodeficiency syndrome (AIDS) [1, 2]. Although AZT was the first anti-HIV drug to be approved worldwide, it was originally designed as an antitumor agent due to its prevention of DNA elongation by inhibiting the incorporation of thymidine into the DNA of cancer cells [3, 4].
Increased cell proliferative activity is a prominent feature of tumor cells. The capacity to non-invasively measure this proliferative activity is a potent tool in the clinical detection and diagnosis of tumors. In order to measure tumor proliferative activity using positron emission tomography (PET), several thymidine analogs have been labeled with positron-emitting nuclei, such as 11C and 18F [5–11]. One of the most promising PET tracers for proliferation imaging is 4′-[methyl-11C]thiothymidine ([11C]4DST), which is resistant to degradation by thymidine phosphorylase, and can be incorporated into DNA . Although [11C]4DST has shown high accumulation in tumors in mouse models and in patients [5, 12, 13], concomitant accumulation occurred in normal tissues, such as the bone marrow, spleen, thymus, and intestine, because of their relatively high proliferative activity.
Silica-gel RP-18 F254s TLC plates were purchased from Merck (Darmstadt, Germany) and 1.0-ml syringes were purchased from Terumo (Tokyo, Japan). Two AZT metabolites, 3′-Azido-3′-deoxythymidine 5′-monophosphate sodium salt and 3′-Azido-3′-deoxythymidine β-d-glucuronide sodium salt were purchased from Carbosynth Limited (Berkshire, UK) and Toronto Research Chemicals (Toronto, Canada), respectively. All other chemicals were purchased from Wako (Tokyo, Japan) or Nacalai Tesque (Kyoto, Japan) and were of analytical grade.
Synthesis of [11C]AZT, [11C]d4T and [11C]4DST
[11C]Carbon dioxide was produced by a 14N(p,α)11C reaction using a CYPRIS HM-12S cyclotron (Sumitomo Heavy Industries, Tokyo, Japan), and was then converted into [11C]CH3I by LiAlH4 reduction followed by HI treatment, using an original automated synthesis system for 11C-labeling in RIKEN CLST. The obtained [11C]CH3I was used for the Pd(0)-Cu(I) co-mediated rapid [11C]methylation with precursors 3′-azido-2′,3′-dideoxy-5-(tributylstannyl)-uridine, 2′,3′-didehydro-2′,3′-dideoxy-5-(tributylstannyl)-uridine and 5-tributylstannyl-4′-thio-2′-deoxyuridine to generate the PET tracers [11C]AZT, [11C]d4T and [11C]4DST, respectively (Fig. 1) .
All experimental protocols were approved by the Animal Care and Use Committee of RIKEN Kobe Institute (MAH24-03) and were performed according to the guidelines for animal experiments published by the National Institutes of Health . Rat glioma C6 and human cervix adenocarcinoma HeLa cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum and 100 μg/ml of penicillin-streptomycin. Five-week-old female BALB/c-nu/nu mice were obtained from CLEA Japan (Tokyo, Japan) and were held for 1 week prior to the experiment. Tumor-bearing mice were established by subcutaneous injection of tumor cells into the shoulder of mice (1 × 106 cells for C6 and 5 × 106 cells for HeLa), as described previously . The mice were used for imaging experiments when the tumors had grown to approximately 5 to 10 mm in diameter.
For examination of tracer accumulation in the inflammatory area, mice were injected 0.03 ml of turpentine oil into the right thigh and were conducted to the PET imaging 3 days after the administration .
A small animal PET scanner (microPET Focus220, Siemens Medical Systems, Erlangen, Germany) was used for the imaging studies of the mice. A venous indwelling catheter was inserted into the tail vein for probe injection before PET scanning. Animals were kept on a heating pad to maintain a body temperature of 37 °C. The probes ([11C]AZT, [11C]d4T and [11C]4DST at a concentration of 50 MBq/0.1 ml) were injected under 1.5 % isoflurane anesthesia, and dynamic PET scans (6 × 10 s, 6 × 30 s, 11 × 60 s, and 15 × 180 s) were performed for 60 min, immediately after probe injection. For inflammation imaging with [18F]FDG (5 MBq/0.1 ml), mice fasted for 4 h before injection were kept for 50 min in awake state and then were anesthetized with 1.5 % isoflurane to image during 60–80 min after the injection. Emission data were acquired using a 3D list-mode method, and PET images were reconstructed using the filtered back projection algorithm. The maximum-intensity-projection images were displayed using ASIPro software (Concorde Microsystems, Knoxville, USA). The uptake of the probes in tissues is shown as standardized uptake value (SUV).
A concentration of 50 MBq of probes in 100 μl of saline was injected into tumor-bearing mice via the tail vein. The mice were sacrificed under deep anesthesia with 3.0 % isoflurane at 80 min after the injection, and the blood, urine, brain, heart, lungs, liver, kidneys, spleen, pancreas, quadriceps muscles, ovaries, duodenum, intestinal contents, bone, and tumors were then collected, and their volumes and weights were promptly measured. The radioactivity in the various tissues, whole blood (WB), plasma, and urine samples was measured using a γ-well counter (Wizard 1480; PerkinElmer, Waltham, USA). The results are expressed as %ID/g .
Thin layer chromatography
Metabolite analysis using thin layer chromatography (TLC) was performed for plasma, bile, intestinal contents, and tumor samples that were obtained at 30 or 60 min after probe injection. The blood sample was collected by cardiac puncture using a heparinized syringe under deep anesthesia with 3.0 % isoflurane. Subsequently, the blood was centrifuged at 16,000×g at 4 °C for 2 min, and the plasma (approximately 100 μl) was vortexed with an equal volume of acetonitrile. After centrifugation at 16,000×g at 4 °C for 2 min, the supernatant was collected as the TLC sample. To prepare TLC samples from the liver and tumor, weighed tissues were added to an equal volume (w/v) of distilled water, and were homogenized and centrifuged. The supernatants were vortexed with three volumes (v/v) of acetonitrile and were then centrifuged. The supernatants were collected and used as TLC samples. Aliquots of the acetonitrile-treated supernatants (2.0 μl) were then spotted on a TLC plate Silica-gel 60 RP-18 and were developed with the solvent (CH3CN/phosphate buffer (pH 6.8) = 15:85) until the front line reached 7 cm from the origin. The TLC plate was then placed on an imaging plate (BAS-SR2040; Fuji Photo Film Co., Tokyo, Japan) for 1 h, and the exposed imaging plate was scanned with a fluoro-image analyzer (FLA-7000IR; Fuji Photo Film). The proportion of each [11C]AZT metabolite was calculated based on the radioactivity in each metabolite (photostimulable luminescence/mm2).
[11C]AZT cell uptake study with thymidine
For the uptake study, C6 cells were seeded on 6-well plates at a density of 2 × 105 cells/well for 24 h. Cellular uptake was initiated by adding [11C]AZT (27 nM = 1.4 MBq/well) in the presence or absence of non-labeled thymidine (0.1 mM), and the cells were incubated at 37 °C for 30 min. Uptake was terminated by adding ice-cold phosphate-buffered saline, and the cells were washed three times with this buffer. The cells were lysed with buffer (25 mM Tris-HCl, pH 7.4, 2.5 mM EDTA, 1 % Triton X-100), and the radioactivity of the lysate was measured using a γ-well counter. Cellular protein concentrations were determined by the Coomassie blue method  using bovine serum albumin as the standard. All experiments were performed in triplicate.
Data are expressed as means ± SD groups were compared using one-way or repeated measure (RM) two-way analysis of variance (ANOVA), and the Bonferroni method was used for the post hoc multiple comparison procedure with a significant level of p < 0.05.
PET imaging with [11C]AZT
Tissue distribution of [11C]AZT in tumor-bearing mice
Comparison of the accumulation of thymidine analogs in normal proliferative tissues of a C6 glioma-bearing mouse by PET analysis
Metabolite analysis of [11C]AZT
In vitro [11C]AZT uptake study using tumor cells
In this study, we successfully determined whether it was possible to measure the proliferative activity of tumors in a tumor-bearing mouse model by PET scanning using either of two novel PET ligands, [11C]AZT and [11C]d4T. The results revealed the usefulness of [11C]AZT for in vivo tumor imaging.
In previous studies, tumor imaging of thymidine analogs was used in order to assess their impact on cell proliferation [5–11]. Recently, Toyohara et al. reported that [11C]4DST was useful in imaging of tumor cell proliferation in vivo in mice and humans, because it was a substrate of the enzyme involved in the phosphorylation of thymidine but was resistant to degradation [5, 12, 13]. However, in those PET studies, [11C]4DST was also highly accumulated in normal proliferative tissues such as the bone marrow, spleen, and duodenum, which made it difficult to distinguish tumors from normal proliferative cells. In the present PET study of a mouse tumor model, as shown in Fig. 5, although the maximal SUV of [11C]AZT in tumor tissue was less than that of [11C]4DST, the accumulation of [11C]AZT in tumor tissue was much higher than that in the abovementioned normal proliferative tissues. In addition, [11C]AZT showed a higher tumor-to-bone marrow ratio than [11C]4DST. The mechanism that underlies this difference is unknown, but it may be related to the AZT substrate specificity of enzymes in the thymidine-phosphorylation pathway (thymidine kinase and thymidylate kinase), which are described in a later paragraph. Based on these data, [11C]AZT is expected to be useful for detecting tumors in bone or bone metastasis. Further studies are needed to validate this possibility using bone metastasis model animals.
Previous in vitro and in vivo studies have demonstrated that AZT is metabolized to AZT-β-d-glucuronide (AZT-G) by UDP-glucuronosyltransferase in hepatocytes and that it is excreted mainly into the urine in rats [20, 21], dogs , monkeys [20, 22], and humans [20–22]. In the present TLC analysis, although very little metabolism and degradation of [11C]AZT occurred in vivo in mice, we did observe three hydrophilic metabolites of [11C]AZT, two of which were observed in the bile (metabolite b and c, Fig. 6). Combined with the previous results [19–21], this finding suggests that the metabolite b found in the bile may be AZT-G because its R f value showed the same position that of AZT-G (Fig. 6a, b). AZT-G in bile was observed at 60 min after the injection but not at 30 min while much amount of metabolite c was observed in bile and intestinal contents after 30 min at the lower position of AZT-G in TLC. Therefore, it is possible to consider that low liver background uptake may be derived by faster production of metabolite c, which excreted into urine or intestinal contents via the bile bladder and a little and delayed production of AZT-G in liver. Since the metabolite c could not identify in the present study, so it would need further study to clarify the form.
Although AZT is known to be phosphorylated to 5′-monophosphate by enzymes in the thymidine-phosphorylation pathway (thymidine kinase and thymidylate kinase), little AZT is converted to 5′-di- and 5′-tri-phosphate forms in humans , rats , and mice . Therefore, the one major metabolite (metabolite a) that was observed in tumor tissues (Fig. 6) may be the 5′-monophosphate form of [11C]AZT (AZT-P). In Fig. 6a and b, TLC analysis with unlabeled compounds indicated that the R f value of AZT-P closely resemble that of metabolite a. Furthermore, these data suggest that [11C]AZT was converted to AZT-P after uptake in tumor cells but not in the blood.
Nucleoside transporters are involved in the cellular uptake of nucleoside analog antiviral drugs, as well as of nucleosides. In the present study, the addition of thymidine completely blocked the cellular uptake of [11C]AZT by C6 tumor cells, strongly indicating the specific involvement of nucleoside transporters (Fig. 7). In previous studies, several other transporters, including Na+-dependent concentrative nucleoside transporters (CNTs: human (h) /rat (r) CNT1 and hCNT3) [26, 27], Na+-independent equilibrative nucleoside transporters (ENTs), h/rENT2 , organic anion transporters (OATs) (h/rOAT1-3 and hOAT4) [28–32], and the organic cation transporter rOCT1 , were reported to be involved in AZT cellular uptake. Although the profile of nucleoside transporters in tumors has not yet been confirmed, these transporters may also contribute to the tumor-specific capacities of [11C]AZT uptake. Further studies are needed to identify the transporter(s) involved in [11C]AZT uptake.
Some studies have attempted to visualize the localization of viruses, such as HIV, within the cell using fluorescent proteins or fluorochromes in order to reveal the processes of cell invasion and virus particle maturation [34, 35]. It is well known that NRTIs such as AZT and d4T suppress the replication of HIV by inhibiting the activity of its reverse transcriptase [1, 2], indicating that [11C]AZT or [11C]d4T could have a role as a PET probe for imaging virus localization and virus dynamics in vivo. Further studies involving PET experiments and virus-infected animals are necessary to clarify virus localization and dynamics in vivo.
We have assessed whether the proliferative activity of tumor cells can be visualized by PET analysis using [11C]AZT or [11C]d4T, and have demonstrated that [11C]AZT, but not [11C]d4T, is highly accumulated in proliferating tumor tissues compared to normal proliferating tissues. [11C]AZT might therefore be a useful PET probe for tumor imaging.
acquired immunodeficiency syndrome
human immunodeficiency virus
nucleoside reverse transcriptase inhibitor
positron emission tomography
thin layer chromatography
The authors thank Masahiro Kurahashi for operating the cyclotron, Emi Hayashinaka and Yasuhiro Wada for reconstructing the PET images, and Chihiro Yokoyama for advising on statistical analysis. This study was supported in part by research grants “Research Center Network for Realization of Regenerative Medicine” from Japan Science and Technology Agency (JST), Agency for Medical Research and development (AMED) and “Molecular Imaging Research Program” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government.
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- Mitsuya H, Weinhold KJ, Furman PA, St Clair MH, Lehrman SN, Gallo RC, et al. 3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-associated virus in vitro. Proc Natl Acad Sci U S A. 1985;82:7096–100.PubMed CentralView ArticlePubMedGoogle Scholar
- Furman PA, Fyfe JA, St Clair MH, Weinhold K, Rideout JL, Freeman GA, et al. Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase. Proc Natl Acad Sci U S A. 1986;83:8333–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Wagner CR, Ballato G, Akanni AO, McIntee EJ, Larson RS, Chang S, et al. Potent growth inhibitory activity of zidovudine on cultured human breast cancer cells and rat mammary tumors. Cancer Res. 1997;57:2341–5.PubMedGoogle Scholar
- Lynx MD, Kang BK, McKee EE. Effect of AZT on thymidine phosphorylation in cultured H9c2, U-937, and Raji cell lines. Biochem Pharmacol. 2008;78:1610–5.View ArticleGoogle Scholar
- Toyohara J, Okada M, Toramatsu C, Suzuki K, Irie T. Feasibility studies of 4'-[methyl-11C]thiothymidine as a tumor proliferation imaging agent in mice. Nucl Med Biol. 2008;35:67–74.View ArticlePubMedGoogle Scholar
- Seitz U, Wagner M, Vogg AT, Glatting G, Neumaier B, Greten FR, et al. In vivo evaluation of 5-[18F]fluoro-2'-deoxyuridine as tracer for positron emission tomography in a murine pancreatic cancer model. Cancer Res. 2001;61:3853–7.PubMedGoogle Scholar
- Smith G, Sala R, Carroll L, Behan K, Glaser M, Robins E, et al. Synthesis and evaluation of nucleoside radiotracers for imaging proliferation. Nucl Med Biol. 2012;39:652–65.View ArticlePubMedGoogle Scholar
- Vander Borght T, Labar D, Pauwels S, Lambotte L. Production of [2-11C]thymidine for quantification of cellular proliferation with PET. Int J Rad Appl Instrum A. 1992;42:103–4.View ArticleGoogle Scholar
- Conti PS, Alauddin MM, Fissekis JR, Schmall B, Watanabe KA. Synthesis of 2'-fluoro-5-[11C]-methyl-1-β-D-arabinofuranosyluracil ([11C]-FMAU): a potential nucleoside analog for in vivo study of cellular proliferation with PET. Nucl Med Biol. 1995;22:783–9.View ArticlePubMedGoogle Scholar
- Grierson JR, Shields AF. Radiosynthesis of 3'-deoxy-3'-[18F]fluorothymidine: [18F]FLT for imaging of cellular proliferation in vivo. Nucl Med Biol. 2000;27:143–56.View ArticlePubMedGoogle Scholar
- Alauddin MM, Conti PS, Fissekis JR. Synthesis of [18F]-labeled 2′-deoxy-2′-fluoro-5-methyl-1-β-D-arabinofuranosyluracil ([18F]-FMAU). J Label Compd Radiopharm. 2002;45:583–90.View ArticleGoogle Scholar
- Minamimoto R, Toyohara J, Seike A, Ito H, Endo H, Morooka M, et al. 4'-[Methyl-11C]-thiothymidine PET/CT for proliferation imaging in non-small cell lung cancer. J Nucl Med. 2011;53:199–206.View ArticlePubMedGoogle Scholar
- Toyohara J, Nariai T, Sakata M, Oda K, Ishii K, Kawabe T, et al. Whole-body distribution and brain tumor imaging with 11C-4DST: a pilot study. J Nucl Med. 2011;52:1322–8.View ArticlePubMedGoogle Scholar
- Zhang Z, Doi H, Koyama H, Watanabe Y, Suzuki M. Efficient syntheses of [11C]zidovudine and its analogues by convenient one-pot palladium(0)-copper(I) co-mediated rapid C-[11C]methylation. J Label Compd Radiopharm. 2014;57:540–9.View ArticleGoogle Scholar
- Guide for the care and use of laboratory animals 8th ed. Washington, DC: Government Printing Office; 2011. NIH publication 11–103
- Tanaka K, Shirotsuki S, Iwata T, Kageyama C, Tahara T, Nozaki S, et al. Template-assisted and self-activating clicked peptide as a synthetic mimic of the SH2 domain. ACS Chem Biol. 2012;7:637–45.View ArticlePubMedGoogle Scholar
- Witney TH, Pisaneschi F, Alam IS, Trousil S, Kaliszezak M, Twyman F, et al. Preclinical evaluation of 3-18F-fluoro-2,2-dimethylpropionic acid as an imaging agent for tumor detection. J Nucl Med. 2014;55:1506–12.View ArticlePubMedGoogle Scholar
- Kawasaki T, Marumo T, Shirakami K, Mori T, Doi H, Suzuki M, et al. Increase of 20-HETE synthase after brain ischemia in rats revealed by PET study with 11C-labeled 20-HETE synthase-specific inhibitor. J Cereb Blood Flow Metab. 2012;32:1737–46.
- Tahara T, Tanaka M, Nozaki S, Jin G, Onoe H, Watanabe Y. Decrease of hepatic δ-aminolevulinate dehydratase activity in an animal model of fatigue. Biochem Biophys Res Commun. 2007;353:1068–73.View ArticlePubMedGoogle Scholar
- Nicolas F, De Sousa G, Thomas P, Placidi M, Lorenzon G, Rahmani R. Comparative metabolism of 3'-azido-3'-deoxythymidine in cultured hepatocytes from rats, dogs, monkeys, and humans. Drug Metab Dispos. 1995;23:308–13.PubMedGoogle Scholar
- Resetar A, Spector T. Glucuronidation of 3'-azido-3'-deoxythymidine: human and rat enzyme specificity. Biochem Pharmacol. 1989;38:1389–93.View ArticlePubMedGoogle Scholar
- Good SS, Koble CS, Crouch R, Johnson RL, Rideout JL, de Miranda P. Isolation and characterization of an ether glucuronide of zidovudine, a major metabolite in monkeys and humans. Drug Metab Dispos. 1990;18:321–6.PubMedGoogle Scholar
- Lynx MD, Kang BK, McKee EE. Effect of AZT on thymidine phosphorylation in cultured H9c2, U-937, and Raji cell lines. Biochem Pharmacol. 2008;75:1610–5.PubMed CentralView ArticlePubMedGoogle Scholar
- Lynx MD, Bentley AT, McKee EE. 3'-Azido-3'-deoxythymidine (AZT) inhibits thymidine phosphorylation in isolated rat liver mitochondria: a possible mechanism of AZT hepatotoxicity. Biochem Pharmacol. 2006;71:1342–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Chow HH, Li P, Brookshier G, Tang Y. In vivo tissue disposition of 3'-azido-3'-deoxythymidine and its anabolites in control and retrovirus-infected mice. Drug Metab Dispos. 1997;25:412–22.PubMedGoogle Scholar
- Yao SY, Cass CE, Young JD. Transport of the antiviral nucleoside analogues 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine by a recombinant nucleoside transporter (rCNT) expressed in Xenopus laevis oocytes. Mol Pharmacol. 1996;50:388–93.PubMedGoogle Scholar
- Ritzel MW, Ng AM, Yao SY, Graham K, Loewen SK, Smith KM, et al. Recent molecular advances in studies of the concentrative Na+-dependent nucleoside transporter (CNT) family: identification and characterization of novel human and mouse proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib). Mol Membr Biol. 2001;18:65–72.View ArticlePubMedGoogle Scholar
- Yao SY, Ng AM, Sundaram M, Cass CE, Baldwin SA, Young JD. Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes. Mol Membr Biol. 2001;18:161–7.View ArticlePubMedGoogle Scholar
- Wada S, Tsuda M, Sekine T, Cha SH, Kimura M, Kanai Y, et al. Rat multispecific organic anion transporter 1 (rOAT1) transports zidovudine, acyclovir, and other antiviral nucleoside analogues. J Pharmacol Exp Ther. 2000;294:844–9.PubMedGoogle Scholar
- Morita N, Kusuhara H, Sekine T, Endou H, Sugiyama Y. Functional characterization of rat organic anion transporter 2 in LLC-PK1 cells. J Pharmacol Exp Ther. 2001;298:1179–84.PubMedGoogle Scholar
- Takeda M, Khamdang S, Narikawa S, Kimura H, Kobayashi Y, Yamamoto T, et al. Human organic anion transporters and human organic cation transporters mediate renal antiviral transport. J Pharmacol Exp Ther. 2002;300:918–24.View ArticlePubMedGoogle Scholar
- Hasegawa M, Kusuhara H, Endou H, Sugiyama Y. Contribution of organic anion transporters to the renal uptake of anionic compounds and nucleoside derivatives in rat. J Pharmacol Exp Ther. 2003;305:1087–97.View ArticlePubMedGoogle Scholar
- Chen R, Nelson JA. Role of organic cation transporters in the renal secretion of nucleosides. Biochem Pharmacol. 2000;60:215–9.View ArticlePubMedGoogle Scholar
- Georgi A, Mottola-Hartshorn C, Warner A, Fields B, Chen LB. Detection of individual fluorescently labeled reovirions in living cells. Proc Natl Acad Sci U S A. 1990;87:6579–83.PubMed CentralView ArticlePubMedGoogle Scholar
- Elliott G, O'Hare P. Live-cell analysis of a green fluorescent protein-tagged herpes simplex virus infection. J Virol. 1999;73:4110–9.PubMed CentralPubMedGoogle Scholar