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¹²³I-ITdU–Mediated Nanoirradiation of DNA Efficiently Induces Cell Kill in HL60 Leukemia Cells and in Doxorubicin-, ß-, or -Radiation–Resistant Cell Lines

Nuclear Medicine Clinic, Universität Ulm, Ulm, Germany

Correspondence: For correspondence or reprints contact: Sven N. Reske, MD, Universität Ulm, Klinik für Nuklearmedizin, Robert-Koch-Strasse 8, D-89081 Ulm, Germany. E-mail: sven.reske{at}uniklinik-ulm.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION References

 
Resistance to radiotherapy or chemotherapy is a common cause of treatment failure in high-risk leukemias. We evaluated whether selective nanoirradiation of DNA with Auger electrons emitted by 5-¹²³I-iodo-4'-thio-2'-deoxyuridine (¹²³I-ITdU) can induce cell kill and break resistance to doxorubicin, ß-, and -irradiation in leukemia cells. Methods: 4'-thio-2'-deoxyuridine was radiolabeled with ^123/131I and purified by high-performance liquid chromatography. Cellular uptake, metabolic stability, DNA incorporation of ¹²³I-ITdU, and the effect of the thymidylate synthase (TS) inhibitor 5-fluoro-2'-deoxyuridine (FdUrd) were determined in HL60 leukemia cells. DNA damage was assessed with the comet assay and quantified by the olive tail moment. Apoptosis induction and irradiation-induced apoptosis inhibition by benzoylcarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.fmk) were analyzed in leukemia cells using flow cytometry analysis. Results: The radiochemical purity of ITdU was 95%. Specific activities were 900 GBq/µmol for ¹²³I-ITdU and 200 GBq/µmol for ¹³¹I-ITdU. An in vitro cell metabolism study of ¹²³I-ITdU with wild-type HL60 cells demonstrated an uptake of 1.5% of the initial activity/10⁶ cells of ¹²³I-ITdU. Ninety percent of absorbed activity from ¹²³I-ITdU in HL60 cells was specifically incorporated into DNA. ¹²³I-ITdU caused extensive DNA damage (olive tail moment > 12) and induced more than 90% apoptosis in wild-type HL60 cells. The broad-spectrum inhibitor of caspases zVAD-fmk reduced ¹²³I-ITdU–induced apoptosis from more than 90% to less than 10%, demonstrating that caspases were central for ¹²³I-ITdU–induced cell death. Inhibition of TS with FdUrd increased DNA uptake of ¹²³I-ITdU 18-fold and the efficiency of cell kill about 20-fold. In addition, ¹²³I-ITdU induced comparable apoptotic cell death (>90%) in sensitive parental leukemia cells and in leukemia cells resistant to ß-irradiation, -irradiation, or doxorubicin at activities of 1.2, 4.1, 12.4, and 41.3 MBq/mL after 72 h. This finding indicates that ¹²³I-ITdU breaks resistance to ß-irradiation, -irradiation, and doxorubicin in leukemia cells. Conclusion: ¹²³I-ITdU–mediated nanoirradiation of DNA efficiently induced apoptosis in sensitive and resistant leukemia cells against doxorubicin, ß-irradiation, and -irradiation and may provide a novel treatment strategy for overcoming resistance to conventional radiotherapy or chemotherapy in leukemia. Cellular uptake and cell kill are highly amplified by inhibiting TS with FdUrd.

Key Words: Auger radiation • nuclear targeting • treatment • DNA nanoirradiation • chemoresistance • radioresistance

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION References

 
Chemoresistance and radioresistance are major obstacles to successful induction of long-term survival in high-risk leukemia. Inhibition of tumoral DNA synthesis with antimetabolites targeting specific key enzymes of thymidine metabolism or DNA synthesis is an effective, safe, and well-established strategy for antineoplastic chemotherapy. However, most neoplasms ultimately develop resistance to radiotherapy or chemotherapy, resulting in progressive, uncontrolled proliferation of neoplastic cells and finally patient death. Therefore, cytotoxic drugs with the potential to kill resistant cancer cells are needed.

In this study, we report extremely high cytotoxicity for selective ultra-short-ranged Auger electron–mediated nanoirradiation delivered by 5-¹²³I-iodo-4'-thio-2'-deoxyuridine (¹²³I-ITdU), metabolically incorporated into DNA. Auger electrons are known to generate clusters of high ionization density, inducing base damage, single-strand breaks, double-strand breaks, and multiple damaged sites (1–3). The low energy of Auger electrons, ranging from a few electron volts up to 100 keV (4), and their short pathlength of only several nanometers in water, limit dense ionizations and extensive DNA damage within a sphere of a few nanometers around the emission center (5), whereas cytosolic or extracellular decays of Auger emitters are 10–100 times less radiotoxic (6).

The radiolabeled thymidine analog ¹²³I-ITdU (Fig. 1) was used as a molecular carrier for the Auger electron–emitting radiohalogen ¹²³I into DNA. ¹²³I-ITdU was selected because it is specifically incorporated into nuclear DNA through the thymidine salvage pathway (7–9), has no selectivity for mitochondrial DNA, and is metabolically more stable than other 5-halogeno–substituted deoxyuridines (3,8–10).

View larger version (22K): [in this window] [in a new window]   FIGURE 1.  Thymidine de novo and salvage pathways. Thymidine de novo pathway is blocked by FdUrd, considerably enhancing flux of thymidine and 123I-ITdU through salvage pathway and, thus, 123I-ITdU incorporation into DNA.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION References

 
Chemicals
Chemicals and solvents were purchased from Sigma-Aldrich and Merck, or otherwise as indicated. All reagents and solvents were of the highest commercially available grade and used without further purification. No-carrier-added sodium ¹³¹I-iodide was obtained from Amersham Biosciences, and no-carrier-added sodium ¹²³I-iodide was purchased from Zyklotron AG. The 5-trimethylstannyl precursor of ITdU and the standard compound ITdU were synthesized according to previously reported methods (8,13–15).

Synthesis of ¹²³I-ITdU and ¹³¹I-ITdU
Fifteen microliters of chloramine-T (0.5 mg/mL in 66% methanolic solution) were added to a reaction vial containing 35 µL of no-carrier-added ^123/131I-NaI (1,200 MBq in 0.05 M NaOH) and 0.14 mg of precursor in 60 µL of buffer (1 M H₃PO₄, pH 2, in 40% methanolic solution). The reaction was stopped after 15 min by adding 25 µL of 0.1 thiosulfate solution in 50% methanolic solution. The radiochemical yield was 95%. ^123/131I-ITdU was purified via semipreparative high-performance liquid chromatography (HPLC) (sample loop, 200 µL; eluent, MeOH/H₂O 30/70 v/v; flow, 2 mL/min; column, LiChrospher 100 RP-18 [Agilent Technologies], 5 µm, 250 x 8 mm). For isolation of the product, the fraction between 10 and 13 min was collected, and 100 µL of a 10 mM methionine solution were added. The product volume of about 5 mL was reduced by evaporation at 80°C at reduced pressure (nitrogen flow and vacuum). The product was redissolved in 100 µL of water for injection and incubated for 15 min with anion exchange beads (Amberlite IRA-958, 13-45 mesh, acrylic resin, strong basic, Cl^– form). The supernatant was removed, the resin washed with another 100 µL, and the combined solutions passed through a sterile filter. The overall radiochemical yield was 20%. For production of high-activity doses of ¹²³I-ITdU, 37 µL of chloramine-T solution were added to a mixture of 140 µL of buffer, 4.7 µL of precursor solution (48 mg of buffer per milliliter) and 82 µL of ¹²³I-NaI (5 GBq in 0.05 M NaOH). At a reaction time of 15 min, the total volume was injected onto a C4 reversed-phase column (sample loop, 500 µL; eluent, EtOH/H₂O 10/90 v/v; flow, 1 mL/min; column, Kromasil 100-5µ C4 [Eka Chemicals], 250 x 4 mm). The peak fraction was made isotonic using a 10% NaCl solution and further diluted with isotonic saline to the desired activity concentration. For this procedure, overall radiochemical yield was 80%. Quality control for each procedure was conducted using HPLC (eluent, MeOH/H₂O 20/80 v/v; flow, 1 mL/min; column, LiChrospher 100 RP-18, 5 µm, 250 x 4 mm).

Cell Lines
The human myeloid leukemic cell line HL60 and the human lymphoblastic leukemia T cells CEM were grown in RPMI 1640 (Gibco BRL) containing 10% fetal calf serum (Biochrom); 10 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), pH 7.3 (Biochrom); 100 U of penicillin per milliliter (Gibco); 100 µg of streptomycin per milliliter (Gibco); and 2 mM L-glutamine (Biochrom). HL60^gammaR, a variant of HL60 that is resistant to a radiation-absorbed dose of up to 10 Gy (⁶⁰Co, 0.4 Gy/min) (16), and HL60^betaR, a variant of HL60 that is resistant to a radiation-absorbed dose of up to 10 Gy applied by ß-irradiation (⁹⁰Y, 87.6 MBq/100 mL, incubation for 24 h), were generated for more than 12 mo (16). To preserve the resistance of these cell lines, HL60^gammaR was irradiated with -irradiation (⁶⁰Co) and HL60^betaR was irradiated with ß-irradiation (⁹⁰Y) every 4 wk by applying a radiation-absorbed dose of 10 Gy. CEM^DoxoR, a variant of CEM resistant toward doxorubicin, was generated by continuous culture in doxorubicin (0.1 µg/mL) for more than 12 mo. For the experiments, CEM^DoxoR cells were washed and cultured for 2 wk in the absence of doxorubicin (17). All cell lines were mycoplasma-free.

Cellular Uptake and DNA Incorporation of ¹²³I-ITdU
HL60 cells (1 x 10⁶/mL) were preincubated in the absence or presence of FdUrd (0.01 nmol/L) for 1 h before incubation with 0.5 MBq of ¹²³I-ITdU, and the cells were incubated at 37°C with 5% CO₂ for 2 h and 24 h. After incubation, the medium was removed and the cells were washed 3 times with ice-cold phosphate-buffered saline (PBS). After washing, the cells were harvested and collected by centrifugation. The radioactivity of the cell pellet was measured in a -scintillation counter (Auto--5003; Canberra Packard).

DNA Uptake
Cultured HL60 cells were harvested and seeded at a concentration of 1 x 10⁶ into 24-well plates. HL60 cells (1 x 10⁶/mL) were preincubated in the absence or presence of FdUrd (0.01 nmol/L) for 1 h before incubation with 0.5 MBq of ¹²³I-ITdU, and the cells were incubated at 37°C with 5% CO₂ for 2 h and 24 h. After incubation, the medium was removed and the cells were washed 3 times with ice-cold PBS. After washing, the cells were centrifuged for 5 min at 300g, and the cell pellets were resuspended in 200 µL of PBS. DNA was extracted using the DNeasy Tissue Kit from Qiagen. In brief, cell pellets resuspended in 200 µL of PBS were incubated with 4 µL of RNase (100 mg/ mL; Qiagen) for 2 min at room temperature to get RNA-free genomic DNA. Then, 20 µL of proteinase K and 200 µL of buffer AL from the DNeasy Tissue Kit were added to the sample, mixed thoroughly in a vortex mixer, and incubated at 70°C for 10 min. After incubation, 200 µL of 96% ethanol were added to the sample and mixed thoroughly in a vortex mixer. Then, the mixture was pipetted into the DNeasy Mini spin column, placed in a 2-mL collection tube, and centrifuged at 6,000g for 1 min. After centrifugation, the flow-through and collection tubes were discarded. The DNeasy Mini spin column was placed in a new collection tube, 500 µL of buffer AW1 (DNeasy Tissue Kit) were added, and the column was centrifuged for 1 min at 6,000g. After centrifugation, the flow-through and collection tubes were discarded. The DNeasy Mini spin column was placed in a new collection tube, and 500 µL of buffer AW2 (DNeasy Tissue Kit) were added and centrifuged for 3 min at 20,000g to dry the DNeasy membrane. After centrifugation, the flow-through and collection tubes were discarded. The DNeasy Mini spin column was placed in a clean 1.5-mL microcentrifuge tube, and 200 µL of buffer AE (DNeasy Tissue Kit) were pipetted directly onto the DNeasy membrane, followed by incubation for 1 min at room temperature and then centrifugation for 1 min at 6,000g to elute the DNA. Radioactivity in the DNA was measured in a -scintillation counter (Auto--5003).

Metabolic Stability of ¹²³I-ITdU
For in vitro stability of ¹²³I-ITdU, 10⁶ HL60 cells were incubated with a 0.5 MBq/mL concentration of ¹²³I-ITdU in the absence or presence of FdUrd (0.01 nmol/L). The extent of ¹²³I-ITdU deiodination was determined by reversed-phase HPLC analysis of cell supernatant aliquots after 2 h and 21 h of incubation. Analytic HPLC system 1100 from Agilent was used with a quaternary low-pressure gradient pump, a 1-channel UV-VIS detector using 290 nm, and a final NaI detector–type GABI from Raytest for detection of -radiation. The radiochemical yields of all reaction products were obtained as the relative peak area fraction of the sum of all areas in the radiochromatogram. Complete elution was checked by analyzing the same sample amount choosing a column bypass.

Measurement of DNA Damage (Comet Assay)
DNA damage (DNA breaks) was measured by the alkaline comet assay as previously described (18). In brief, 10 µL of cell suspension (10,000 cells) from treated and untreated cells were resuspended in 120 µL of low-melting agarose at 37°C. One hundred microliters of this suspension were spotted onto a microscope slide and covered with a cover slide. After the slide had been kept on ice for 5 min in the dark, the cover slide was removed and the slide was incubated overnight in lysis buffer containing 2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid, 10 mM Tris, and 1% Na-laurylsarcosinate, pH 10. Thereafter slides were preincubated in electrophoresis buffer (300 mM NaOH and 1 mM ethylenediaminetetraacetic acid, pH > 13) for 25 min, and electrophoresis was performed for 25 min (25 V, 300 mA). The slides were neutralized in 0.4 M Tris, pH 7.5, washed in distilled water, fixed in 100% ETOH for 5 min, and air-dried overnight. Analysis after staining with ethidium bromide (20 µg/mL) was performed on a fluorescence microscope using a charge-coupled-device camera connected to a personal computer and analysis software. Relative DNA breakage was expressed as the olive tail moment, which was determined by measuring the fluorescence intensity of the tail and nucleus using Kinetic Imaging Komet 5.0 software (BFI Optilas).

Apoptosis Induction of ¹²³I-ITdU
Leukemia cells (2 x 10⁵ cells in 1 mL) were incubated with a 0.0041, 0.012, 0.041, 0.12, 0.41, 1.2, 4.1, 12.4, or 41.3 MBq/mL concentration of ¹²³I-ITdU, ¹³¹I-ITdU, or sodium ¹²³I-iodide or were treated with a 0.01, 0.1, 1, 10, or 100 µg/mL concentration of nonradioactive ¹²⁷I-ITdU as a control in a 150-mL flask or 96-well plates for 72 h. Activities of ¹³¹I and ¹²³I were applied in a 1-mL volume. After irradiation, apoptosis was quantified by flow cytometry as previously described (19). In brief, to determine apoptosis, the cells were lysed with Nicoletti buffer containing 0.1% sodium citrate plus 0.1% Triton X-100 (Union Carbide Corp.) and propidium iodide (50 µg/mL), as described by Nicoletti et al. (19). The percentage of apoptotic cells was measured by hypodiploid DNA (subG1) or forward scatter/side scatter analysis. Propidium iodide–stained nuclei or the forward scatter/side scatter profile of cells was analyzed by flow cytometry (FACSCalibur; Becton Dickinson).

Inhibition of Irradiation-Induced Apoptosis by z-VAD.fmk
Caspase activation was inhibited as previously described (20). In brief, the broad-spectrum tripeptide inhibitor of caspases benzoylcarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.fmk; Enzyme Systems Products) was used at a concentration of 50 µmol/L. HL60 cells were preincubated with z-VAD.fmk 1 h before ¹²³I-ITdU incubation. After 72 h, the percentage of apoptotic cells was measured by hypodiploid DNA (subG1) or forward scatter/side scatter analysis. Propidium iodide–stained nuclei or the forward scatter/side scatter profile of cells was analyzed by flow cytometry (FACSCalibur).

Dose-Effect Analysis
For densely ionizing radiation (Auger electron emitters), an exponential model appropriately describes the relationship between radioactivity concentration C (MBq/mL) and the number of surviving cells N (5); that is, , with N₀ being the number of cells surviving the procedure as described above and describing the sensitivity against the applied radiolabeled compound or treatment.

Statistical Analysis
The percentage of specific cell death was calculated as 100 x (experimental dead cells [%] – spontaneous dead cells in medium [%])/(100% – spontaneous dead cells in medium [%]). Data are given as the mean of triplicates. Similar results were obtained in 3 independent experiments.

Nonlinear curve fitting for the dose-effect analysis was performed using GraphPad Prism (GraphPad Software), version 4.03, for Windows (Microsoft). To compare different experiments, the ratio of the sensitivity parameter was calculated applying error propagation.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION References

 
Radiosynthesis of No-Carrier-Added ^123/131I-ITdU
Total radiochemical yields were 20% or 80%, depending on the desired final total product volume. Radiochemical purities were more than 95%. Specific activities were 900 GBq/µmol for ¹²³I-ITdU and 200 GBq/µmol for ¹³¹I-ITdU.

Cellular/DNA Uptake and Cellular Metabolism of ¹²³I-ITdU
Cellular uptake of ¹²³I-ITdU/10⁶ cells into HL60 cells was about 1.5% of the incubation activity in the medium (Fig. 2A). After 24 h, 90% of internalized activities from ¹²³I-ITdU in HL60 cells were specifically incorporated into DNA (Fig. 2A). However, after the uptake of ¹²³I-ITdU into HL60 cells, ¹²³I-ITdU was mostly deiodinated within 24 h (Fig. 2B). Deiodination of ¹²³I-ITdU was not detectable after incubation of ¹²³I-ITdU in culture medium in the absence of HL60 cells after 24 h.

View larger version (18K): [in this window] [in a new window]   FIGURE 2.  (A) Cellular uptake and DNA incorporation of 123I-ITdU. HL60 cells were incubated with 0.5 MBq of 123I-ITdU per milliliter at time points as indicated. (B) In vitro stability of 123I-ITdU. HL60 cells were incubated with 0.5 MBq of 123I-ITdU per milliliter. Extent of 123I-ITdU deiodination was determined by reversed-phase HPLC analysis of cell supernatant aliquots at time points as indicated. (C) Cellular uptake and DNA incorporation of 123I-ITdU with and without TS inhibitor FdUrd. HL60 cells were incubated with 0.5 MBq of 123I-ITdU per milliliter in absence (123I-ITdU) or presence (123I-ITdU/FdUrd) of 0.01 nmol of FdUrd per liter at time points as indicated. (D) In vitro stability of 123I-ITdU. HL60 cells were incubated with 0.5 MBq of 123I-ITdU per milliliter in absence or presence of 0.01 nmol of FdUrd per liter. Extent of 123I-ITdU deiodination was determined by reversed-phase HPLC analysis of cell supernatant aliquots at time points as indicated.

¹²³I-ITdU–Induced DNA Damage
After the application of a 12.4 or 41.3 MBq/mL concentration of ¹²³I-ITdU, metabolically incorporated into the DNA of HL60 cells, a strong induction of DNA damage was found as measured by the Comet assay (Fig. 3A) and quantified by the fluorescence intensity of the tail and nucleus (Fig. 3B) after 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, and 7 h.

View larger version (17K): [in this window] [in a new window]   FIGURE 3.  123I-ITdU–induced DNA damage in HL60 cells. HL60 cells were incubated with 41.3 MBq of 123I-ITdU or 12.4 MBq of 123I-ITdU per milliliter or were left untreated (control). After time points as indicated, alkaline electrophoresis (comet assay) was performed. (A) DNA damage measured by comet assay. (B) DNA damage quantified by fluorescence intensity of tail and nucleus, olive tail moment, at time points as indicated.

View larger version (14K): [in this window] [in a new window]   FIGURE 4.  Apoptosis induction of 123I-ITdU (Auger electron emitter bound to DNA) in HL60 cells, 123I-iodide (Auger electron emitter not bound to DNA), and 131I-ITdU (ß-particle emitter bound to DNA). Percentages of specific cell death were analyzed after 72 h. (A) HL60 cells were incubated with indicated concentrations of 123I-ITdU, 123I, or 131I-ITdU. (B) 127I-ITdU–induced apoptosis in HL60 cells. HL60 cells were incubated with nonradioactive 127I-ITdU at concentrations as indicated. (C) Inhibition of 123I-ITdU–induced apoptosis by z-VAD.fmk in HL60 cells. HL60 cells were incubated with indicated concentrations of 123I-ITdU in absence or presence of 50 µM z-VAD.fmk.

View larger version (8K): [in this window] [in a new window]   FIGURE 5.  Apoptosis induction of 123I-ITdU with and without FdUrd. HL60 cells were incubated with 0.5 MBq of 123I-ITdU per milliliter in absence or presence of 0.01 nmol of FdUrd per liter. After 2 h and 24 h, percentages of specific cell death were analyzed.

Overcoming ß-Irradiation, -Radiation, and Doxorubicin Resistance with ¹²³I-ITdU

View larger version (13K): [in this window] [in a new window]   FIGURE 6.  123I-ITdU–induced apoptosis in HL60 or CEM leukemia cells resistant to ß-irradiation, -irradiation, or doxorubicin. Percentages of apoptotic cell death were analyzed after 72 h. (A) 123I-ITdU–induced apoptosis in HL60betaR, HL60gammaR, and sensitive parental HL60 leukemia cells. HL60, HL60betaR, and HL60gammaR were incubated with indicated concentrations of 123I-ITdU. (B) 123I-ITdU–induced apoptosis in CEMDoxoR and parental sensitive CEM leukemia cells. CEM and CEMDoxoR were incubated with indicated concentrations of 123I-ITdU.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION References

In the present study, ¹²³I-ITdU was used as the molecular carrier of the Auger electron–emitting radionuclide ¹²³I. ¹²³I-ITdU is metabolically incorporated into DNA via the thymidine salvage pathway (7,8). This targeting approach seems promising because ENT1-type nucleoside transporters and key enzymes of the thymidine salvage pathway, major determinants of cellular ¹²³I-ITdU metabolism, are overexpressed in leukemic cells and many malignant tumors (23–27).

Metabolic stability is a critical requirement for incorporation of C-5 halogenated thymidine analogs into DNA. We thus followed the strategy to substitute the 4'-oxygen for sulfur in the IdUrd molecule to generate the thymidine analogs ¹²³I-ITdU and ¹³¹I-ITdU, with a stabilized glycosidic bond (7,8). In addition, TS-mediated dehalogenation of 5-iodo-2'-deoxyuridinemonophosphate as well as other 5-iodo- or 5-bromo-substituted deoxyuridine monophosphates is of major concern (28,29). TS catalyzed the facile dehalogenation of 5-iodo- and 5-bromo-2'-deoxyuridinemonophosphate to give the natural substrate of TS, 2'-deoxyuridinemonophosphate (29). Accordingly, we found a rapid and extensive dehalogenation of ¹²³I-ITdU mediated by HL60. As described for IdUrd (30), the TS inhibitor FdUrd could completely block TS-mediated dehalogenation of ¹²³I-ITdU in a nontoxic concentration and increase substrate flux through the thymidine salvage pathway as indicated by the 18-fold increased DNA uptake of ¹²³I-ITdU, resulting in roughly 20-fold apoptosis induction. Because TS is overexpressed in many leukemic cells and malignant tumors (31–33), a potential therapeutic use of ¹²³I-ITdU would almost certainly require a combination of ¹²³I-ITdU with FdUrd or a similar TS inhibitor.

An increased radiotoxicity of cells retained in S-phase after FdUrd pretreatment was observed by Perillo-Adamer et al. (34) in glioblastoma cell lines. However, Kassis et al. (35) found that FdUrd pretreatment does not seem to enhance the therapeutic potential of ¹²⁵I-ITdU. The observation that the radiotoxicity increase was most pronounced after prolonged incubation with ¹²³I-ITdU, thus probably targeting more cells during S-phase, might suggest that the state of cells in the cell cycle is probably relevant for the full evolution of the cytotoxic effect of Auger electron–emitting radionucleosides.

Considerably strong apoptosis and caspase-3, -8, and -9 and poly(adenosine diphosphate ribose) polymerase cleavage was observed after treatment with the DNA-bound Auger electron emitter ¹²³I-ITdU, in contrast to the DNA-bound ß-particle emitter ¹³¹I-ITdU, the nonradioactive DNA-bound ¹²⁷I-ITdU, and the Auger electron emitter ¹²³I-iodide not bound to DNA, demonstrating the high radiotoxicity of the DNA-bound Auger electron emitter ¹²³I-ITdU. Triggering of apoptotic cell death by a pharmacodynamic effect of ¹²³I-ITdU can be excluded, because the amount of ¹²³I-ITdU given to the incubation medium was 4–5 orders of magnitude below the cell culture infective dose (>100 µmol/L) required to reduce Vero cells by 50% over a 4-d period (13). Therefore, the cytotoxic effect of ¹²³I-ITdU could be attributed to Auger electron emitters bound to DNA and not to the -radiation component of ¹²³I or the pharmacodynamic effect of the tracer amounts of ¹²⁷I-ITdU. In addition, the 150-times-higher toxicity of ¹²³I-ITdU, compared with ¹²³I-iodide, distributed in the extracellular space clearly emphasizes the selectivity of the Auger electron–based DNA targeting approach. In line with this observation, a higher relative biologic efficacy of ¹²⁵I incorporated in DNA through radiolabeled thymidine analogs was observed, as compared with ¹²⁵I distributed in the cytoplasm (6).

Resistance to radiotherapy or chemotherapy is a common cause of treatment failure in cancer patients and has been linked to upregulated DNA repair pathways (36). It has been shown, however, that bulky DNA lesions, induced by Auger electrons, are only rarely or not at all repaired and preferentially trigger apoptotic cell death (5). Efficient induction of apoptotic signaling and cell death provide evidence that Auger electron emitters bound to DNA generated extensive, only rarely repairable DNA damage even in cells resistant to conventional ß- or -irradiation or doxorubicin, used as a model for resistance to cytotoxic chemotherapy.

We have previously shown cross-resistance of -irradiation–resistant CEM^gammaR cells to doxorubicin, cisplatinum, cyclophosphamide, and etoposide (37). Efficient apoptosis induction by nanoirradiation of DNA in multidrug-resistant CEM^DoxoR and in HL60^betaR or HL60^gammaR, resistant to ß-radiation or -radiation, indicates the potential of nanoirradiation to overcome resistance to diverse resistance mechanisms, provided that functional integrity of executioner caspases is maintained.

	CONCLUSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION References

 
These findings provide evidence that ultraselective nanoirradiation of DNA through Auger electron–carrying metabolic substrates offers an extremely effective strategy for inducing cell death and breaking resistance to more conventional types of irradiation or chemotherapy.

	ACKNOWLEDGMENTS

 
This work was supported by grant KFO120 from Deutsche Forschungsgemeinschaft (German Research Foundation) and grant DJCLS H04/05 from Deutsche Jose Carreras-Leukämie Stiftung. This work is dedicated to Professor L. E. Feinendegen.

	FOOTNOTES

 
COPYRIGHT © 2007 by the Society of Nuclear Medicine, Inc.

	References

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION References

 

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