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Mallinckrodt Institute of Radiology [M. W., A. L. C., M. R. L., L. A. M., C. J. A.], and Department of Radiation Oncology [R. P. V.], Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 64Cu-TETA-OC, in which TETA = 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid, is a somatostatin analogue that has been shown to have applications for PET imaging and targeted radiotherapy of cancer (5, 6, 7)
. The structures of DTPA-OC and TETA-OC are shown in Fig. 1
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To investigate this hypothesis further, we chose to evaluate the subcellular distribution of 64Cu-TETA-OC compared with 111In-DTPA-OC, because radiolabeled somatostatin analogues are known to be internalized (14) . Other investigators have observed the localization of 111In to the nuclei of tumor cells after incubation with 111In-DTPA-OC (3 , 4) , and a comparison of 64Cu-TETA-OC with this agent is warranted. Most importantly, determining the extent of the nuclear localization of 64Cu from 64Cu-TETA-OC will be a first step in determining whether the delivery of 64Cu radiopharmaceuticals to the nuclei of tumor cells has implications for cancer therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TETA-OC was obtained from Mallinckrodt, Inc. and labeled with [64Cu]cupric acetate as described previously (5) . Briefly, 1–11 mCi (37–407 MBq) of [64Cu]cupric acetate in 100–250 µl of 0.1 M ammonium acetate (pH 5.5) were added to 1 µg of TETA-OC in 100–250 µl of 0.1 M ammonium acetate (pH 5.5). The reaction mixture was incubated at room temperature for 30–60 min, and then 64Cu-TETA-OC was purified with a C18 SepPak cartridge. DTPA-OC (1 µg; Mallinckrodt, Inc.) was labeled with 111InCl3 [1–9 mCi (37–333 MBq)] as described previously (5) . Radiochemical purity was determined by radio-thin layer chromatography as described previously (5) .
Internalization Assay.
The amount of cell membrane-bound 64Cu-TETA-OC was determined using modifications of the procedure of Zinn et al. (16)
. AR42J cells were seeded in three 12-well plates containing Hams F12K with 20% fetal bovine serum and were incubated at 37°C until 
80% confluency. Cells were washed with HBSS (pH 7.2), and then internalization medium (30 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% BSA in DMEM) was added to each well, and the mixture was incubated for 10 min at room temperature. For determination of nonspecific internalization, one set of wells was incubated with unlabeled TETA-OC (2 µg/10 µl) on a shaker at 37°C for 10 min to block somatostatin receptors. 64Cu-TETA-OC (300,000 cpm/10 µl; specific activity = 440 µCi/µg; 4 pmol peptide) was added to all of the wells and was incubated with rocking at 37°C for 15 min or 1, 4, or 24 h, respectively. At each time point, radioactive media were aspirated, and the plate was washed twice with HBSS (pH 7.2). To collect the surface-bound fraction, each well was treated with 20 mM sodium acetate-HBSS (pH 4.0) and was incubated with rocking at 37°C for 10 min followed by a second 20-mM sodium acetate-HBSS (pH 4.0) wash without incubation and pooled with the first rinse. After removal of the surface-bound fraction, trypsin was used to dislodge the pellet. All of the fractions were counted for radioactivity on the gamma counter. The percentage internalized was the amount of activity in the final cell pellet, corrected for activity in the blocked fractions and background activity.
Isolation of AR42J Nuclei after Administration of 64Cu-TETA-OC or 111In-DTPA-OC.
111In-DTPA-OC or 64Cu-TETA-OC was added to AR42J cells (1 x 107 cells/flask). To maintain a molar ratio of somatostatin receptors:SSTR2 ligand that was at least 10:1, we added 0.15–0.20 pmol of cold DTPA-OC or TETA-OC. This was based on a Bmax (number of receptors as determined by Scatchard analysis of 244.4 fmol/mg of membrane protein (17)
. After incubation times of 1, 4, and 24 h, the cells were pelleted and resuspended in CSK buffer [0.5% Triton X-100, 300 mM sucrose, 100 mM NaCl, 1 mM EGTA, 2 mM MgCl2, and 10 mM PIPES (pH 6.8)] and were incubated on ice for 2 min. Cell lysates were centrifuged at 560 x g for 5 min at 4°C, and the supernatant was discarded. The nuclear pellet was resuspended in 1 ml of CSK buffer without Triton X-100, and centrifuged at 560 x g at 4°C for 5 min. The supernatant was discarded and the postwash nuclear pellet was counted in the gamma counter. Aliquots of nuclei were assayed qualitatively for purity by fluorescence microscopy after staining with a 1:10 dilution of FITC (30 µg/ml) and propidium iodide (70 µg/ml). Micrographs were obtained at x100, x200, and x1000 magnification. The yield of nuclei was determined by counting the initial cell number and the collected nuclei using a Coulter Counter. The percentage in the cell nucleus was determined by the gamma counts in the pure nucleus divided by gamma counts associated with whole cells, and was corrected for the yield of nuclei.
Subcellular Fractionation.
111In-DTPA-OC or 64Cu-TETA-OC (0.15–0.20 pmol) was added to AR42J cells (1 x 107 cells/flask) as described above. Cell disruption and subcellular fractionation were performed using a modification of the procedure described by Morton et al. (18)
. Briefly, the cell pellets were resuspended in homogenization buffer [0.25 M sucrose, 10 mM HEPES-KOH (pH 7.3), containing 1 mM EDTA and 1 mM Pefabloc-SC] and then were disrupted by nitrogen cavitation (200 psi, 10 min). Nuclei and nonlysed cells were pelleted by centrifugation (400 x g for 20 min, 4°C). The supernatant was then centrifuged at 3,000 x g for 20 min at 4°C to pellet mitochondria. The postmitochondrial supernatants were collected and layered over a 37.5% (v/v) Percoll gradient. The gradient was centrifuged at 60,000 x g for 30 min at 4°C and fractions of 1.0 ml were collected from the bottom of the gradient. Enzymatic assays for LDH (cytosol) and ß-hexosaminidase (lysosomes) were performed on each fraction and on the mitochondrial pellets. Enzyme assays were also done on nuclei isolated as described above. Mitochondrial pellets and Percoll gradient fractions were counted for radioactivity in the gamma counter.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Internalization of 64Cu-TETA-OC in AR42J Cells.
We previously showed that 64Cu-TETA-OC and other 64Cu-labeled somatostatin analogues are taken up by AR42J cells (19)
. Here, experiments were performed to determine the amount of AR42J cell-associated activity that was membrane-bound versus internalized from 15 min to 24 h postadministration of 64Cu-TETA-OC (Fig. 2)
. Internalization of 64Cu-TETA-OC (4 pmol) by AR42J cells (1 x 106) increased over time. Internalization was significantly higher at 24 h (6.89 ± 1.42% of total activity added per 1 x 106 cells) than at 4 h (3.57 ± 0.16%; P < 0.005) and 1 h (1.45 ± 0.26%; P < 0.001). The percentage of cell-associated activity that was surface bound ranged from 6.86 ± 0.33% to 16.55 ± 0.85%, with the amount of surface-bound activity increasing at 24 h, indicating that the majority of 64Cu-TETA-OC is internalized. Blocking studies were performed at all time points, and in all cases less than 1% of the 64Cu-TETA-OC was cell associated when 2 µg of unlabeled TETA-OC was added to the wells, demonstrating that internalization was receptor mediated.
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Uptake of 111In-DTPA-OC, 64Cu-TETA-OC and [64Cu]Cupric Acetate in AR42J Nuclei.
The isolation of AR42J nuclei by centrifugation techniques yielded high purity nuclei as demonstrated by fluorescence staining with FITC and propidium iodide, which stain for CSK and nuclei, respectively. As seen in Fig. 3
, the nuclei isolated by our protocol showed no detectable cytoskeletal debris. In addition, enzyme assays were performed on purified nuclei to determine the contamination from lysosomes, cytosol, mitochondria, or plasma membrane. The overall contamination from the other organelles was 12%, with the majority of the contamination being lysosomal (data not shown).
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. The uptake of 111In in the nucleus increased significantly over the time of incubation (1 h, 1.0 ± 0.35 (n = 3); 4 h, 2.4 ± 0.4 (n = 4); 24 h, 6.0 ± 1.1 (n = 5); P < 0.002). 64Cu from 64Cu-TETA-OC in the cell nuclei showed a 13-fold increase from 1 to 24 h, whereas the amount of 111In from 111In-DTPA-OC showed a 6-fold increase over the same time period. The nuclear uptake of 111In from 111In-DTPA-OC was significantly less than 64Cu from 64Cu-TETA-OC at all time points (P < 0.05).
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. Interestingly, in the control pulse-chase experiment, levels of 64Cu from [64Cu]cupric acetate decreased significantly from 4 to 24 h postadministration (20.6 ± 8.7 to 5.4 ± 1.9; P < 0.05) rather than increased as in the case of 64Cu-TETA-OC.
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shows the typical profile of cytosolic and lysosomal enzyme markers, respectively, and 64Cu activity of the Percoll gradient for AR42J cells incubated with 64Cu-TETA-OC for 4 h. After a 4-h incubation, there was minimal 64Cu associated with the fractions containing higher concentrations of cytosolic enzymes; however, there was a significant amount of 64Cu activity associated with the lysosomal fractions of the Percoll gradient. Because of the difficulties in isolating high purity lysosomal fractions without significant loss of the organelle, we were unable to conduct reliable quantitative experiments. Fig. 7
demonstrates that the 64Cu from 64Cu-TETA-OC associated with the mitochondrial fraction increased over time, similar to uptake in the cell nucleus.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our interest in 64Cu-TETA-OC was initially to design a PET imaging agent for SSTR2-positive tumors (5
, 7)
, because of the superior imaging capabilities of PET versus 
scintigraphy. Concurrent studies evaluating 64Cu-TETA-OC for therapy showed that the agent inhibited tumor growth with low toxicity in a CA20948 tumor-bearing rat model (6)
, which was consistent with our finding using 64Cu-labeled mAb 1A3 in tumor-bearing hamsters (8)
. Like 64Cu-BAT-2IT-1A3, 64Cu-TETA-OC is internalized in tumor cells, and elucidating the subcellular distribution might help provide an understanding of whether therapeutic efficacy might be linked with localization in specific tumor cell organelles.
Our hypothesis for both 111In-DTPA-OC and 64Cu-TETA-OC is that dissociation of the radiometal occurs inside cells, which is followed by trafficking of the radiometal to the cell nucleus. This occurs to a much greater extent for 64Cu than for 111In, most likely because 111In is more stably chelated to DTPA in vivo than 64Cu is to TETA as demonstrated by in vivo metabolism studies with 111In-DTPA-OC and 64Cu-TETA-OC (9 , 21) . As a metal ion, In(III) may possibly be mimicking the behavior of Fe(II), which binds nonspecifically to exposed regions of DNA (22) .
The role of Cu(II) binding to the DNA and/or other structures in the nucleus has been widely reported. Copper ion has been suggested to play an important role in the maintenance of nuclear matrix organization and DNA folding (10
, 11)
. Chiu et al. have found that treatment of isolated nuclei with micromolar levels of Cu(II) not only binds nuclear matrix-associated DNA to matrix proteins but also enhances the production of additional DNA-protein cross-linking, as well as DNA double-strand breaks on subsequent irradiation (12
, 13)
. Studies by George et al. (10)
suggest that cell killing or malfunction at the nuclear level that is induced by ionizing radiation is caused by the reduction of Cu(II) to Cu(I) and also by specific hydroxyl radical attack on proteins or DNA at a copper site. More recently, Trumbore et al. (23)
reported that, when concentrations of copper that are an order of magnitude lower than DNA base concentrations are present in different DNAs, ranging from small, double-stranded oligonucleotides to high-molecular-weight DNA from natural sources, irradiation induces changes in conformation from a right- to a left-handed DNA helix. Taken together, interactions of radiolytic intermediates in the region of the nuclear matrix could have significant biological consequences if changes in DNA conformation adversely affect transcription or translation processes.
Although the above mentioned studies suggest that copper binding to structures in the nucleus may enhance radiation damage to DNA, these studies do not directly compare with the studies presented here. The majority of the above-mentioned studies were performed using isolated nuclei or isolated DNA, not whole cells. The amount of copper added to the isolated nuclei in the study by Chiu et al. was 1–1000 µM (13) . In the studies with isolated DNA, the amount of copper used was also in the µM range. In the studies performed here, the concentration of 64Cu-TETA-OC in the wells containing intact AR42J cells was approximately 4 nM. Additional studies are warranted to determine whether the localization of copper radionuclides to tumor cell nuclei enhances cell killing, as are microdosimetry experiments to determine absorbed doses to the cell nuclei.
Our studies demonstrating the translocation of 64Cu from 64Cu-TETA-OC to the nuclei of tumor cells are consistent with the hypothesis that there are binding sites for copper in the cell nucleus as described above. 64Cu-TETA-OC was not designed to target the cell nucleus, although it was assumed that the somatostatin receptor ligand would be internalized because of previous studies with 111In-DTPA-OC (14)
. The instability of the 64Cu-TETA moiety under biological conditions (9)
is likely the cause of 64Cu translocating to the nucleus. This hypothesis is supported by the similarity in 64Cu uptake in the nucleus when 64Cu-TETA-OC is delivered, either continuously over 24 h (Fig. 4)
or in a 4-h pulse followed by removal of the ligand from the media (Fig. 5)
. It was previously shown that 111In-DTPA-OC is internalized via receptor-mediated endocytosis and that the ligands then accumulate in lysosomes (14)
. There is no known pathway for somatostatin receptor ligands to accumulate in the cell nucleus. The fact that 64Cu continues to localize to the cell nucleus after the removal of 64Cu-TETA-OC from the media suggests the 64Cu may be translocating to another protein in the lysosomes and then migrating to the nucleus and/or the mitochondria. In another study, the amount of 111In from 111In-DTPA-OC increased in the nucleus over time, and it was suggested that for therapeutic purposes, a continuous infusion of 111In-DTPA-OC over a long period of time might improve therapy (3)
. Our data suggest that 111In also translocates to the nucleus after a 4-h pulse, although not to the extent that occurs with 64Cu-TETA-OC. However, a slow continuous infusion of 111In-DTPA-OC may not substantially improve therapy, because our experiments demonstrated that a 24-h versus a 4-h exposure to 111In-DTPA-OC did not increase nuclear uptake of the radiometal to a large extent.
As a control, the nuclear localization of a non-receptor-mediated tracer, [64Cu]cupric acetate, was also performed in AR42J cells. 64Cu from [64Cu]cupric acetate was taken up more rapidly in the nucleus, with 14% taken up at 1 h postadministration. Under conditions in which the cells were continuously incubated with [64Cu]cupric acetate, the nuclear localization leveled out at 20% by 4 h. One of the goals of this study was to better understand mechanisms of uptake of 64Cu tracers into cell nuclei. Mechanisms may depend on how the 64Cu is taken up into cells, whether by a general mechanism most likely involving the Ctr1 copper transporter (most likely in the case of [64Cu]cupric acetate), or whether the initial uptake of the 64Cu tracer is receptor-mediated, as in the case of 64Cu-TETA-OC. It is noteworthy that, in the pulse-chase experiment, levels of 64Cu from [64Cu]cupric acetate decreased significantly from 4 to 24 h postadministration (20.6 ± 8.7 to 5.4 ± 1.9, respectively; P < 0.05) rather than increased as in the case of 64Cu-TETA-OC. The data suggest that the redistribution mechanism or the kinetics of 64Cu from 64Cu-TETA-OC, is different from those for 64Cu from [64Cu]cupric acetate. Additional studies to elucidate the mechanisms of nuclear uptake of 64Cu from receptor- versus transporter-mediated uptake are warranted.
Data presented here also show that an accumulation of 64Cu activity occurs in mitochondria in a fashion similar to that in nuclei, although to a lesser extent (Fig. 7)
. Mitochondria are the energy-producing centers of the cell, and many cell functions are bioenergy-requiring processes. Mechanisms for delivery of copper to the mitochondria have been elucidated in yeast (24)
and mammalian tissues and cells (25)
via the Cox17p chaperone protein, which delivers copper to cyctochrome c oxidase, the terminal complex of mitochondrial and bacterial respiratory chains. To the best of our knowledge, the presence of this mitochondrial copper chaperone has not been verified in mammalian tumor cell lines. Damage to the mitochondria in cells has also been implicated in cell death (26)
. In the present studies, the administration of 64Cu-TETA-OC resulted in substantial accumulation of 64Cu in the mitochondria of AR42J cells. This uptake may have implications for cytotoxicity resulting from mitochondrial DNA damage or disruption of critical structures or functions in that organelle.
The cited examples of copper binding to structures in the cell nucleus suggest that copper plays a role in the radiation chemistry and biology of DNA. Data presented here show an increase in 64Cu localization to the cell nucleus and mitochondria after the addition of 64Cu-TETA-OC to intact AR42J cells over time. These processes may induce cytotoxic responses resulting from radiochemical and radiobiological effects on nuclear structure and function, as well as on cellular respiration. Specifically designing copper radiopharmaceuticals to target the cell nucleus or mitochondria could have a significant impact on the field of targeted radiotherapy of cancer. Gaining a better understanding of the subcellular and subnuclear localization of copper and mechanisms of cell killing by copper radionuclides will also benefit the development of therapeutic radiopharmaceuticals. Future studies are planned to determine the intranuclear structure of 64Cu localization, as well as the chemical form of the 64Cu in the cell nucleus.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by National Cancer Institute Grant R01 CA64475 and in part by Department of Energy Training Grant DE F0101 NE23051 (to A. L. C.). The production of copper radionuclides at Washington University is supported by National Cancer Institute Grant R24 CA86307 (to Dr. Michael J. Welch). 
2 Present address: Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, MO 65211.
3 To whom requests for reprints should be addressed, at Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 South Kingshighway Boulevard, Campus Box 8225, St. Louis, MO 63110. Phone: (314) 362-8427; Fax: (314) 362-9940; E-mail: andersoncj{at}mir.wustl.edu
4 The abbreviations used are: DTPA, diethylenetriaminepentaacetic acid; TETA, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid; OC, octreotide; mAb, monoclonal antibody; BAT, bromoacetamidobenzyl-TETA; 2IT, 2-iminothiolane; CSK, cytoskeleton; LDH, lactate dehydrogenase; SSTR2, somatostatin receptor subtype 2; PET, positron emission tomography.
Received 4/25/03. Revised 7/21/03. Accepted 8/ 5/03.
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Abs, change in absorbance.