Aerobic radiosensitization by SR 4233 in rodent and human cells: mechanistic and therapeutic implications.

Mammalian cells surviving exposure to the bioreductive, cytotoxic agent SR 4233 under hypoxic conditions are sensitized to X-irradiation under aerobic conditions (and in the absence of drug). Fits of both the single-hit, multi-target and linear-quadratic expressions to survival data, as well as direct measurement of surviving fractions after a dose of 2 Gy, indicate that the aerobic radiosensitization produced by SR 4233 can increase both the initial and final 'slopes' of the X-ray survival curve. The amount of radiosensitization produced, and whether the modification is principally in the slope or shoulder region of the survival curve, varies from cell line to cell line. Rodent cells are radiosensitized equally whether the drug treatment is given immediately before or after, the irradiation, but human cells are only sensitized for SR 4233 exposure administered before irradiation. Using rodent CHO cells, time-course experiments for SR 4233 and X-rays given in sequence, in which an interval of up to 2 h was interposed between the treatments, reveal different kinetics for the loss of radiosensitization depending on whether the hypoxic drug exposure was given before or after the aerobic irradiation. When SR 4233 treatment is given pre-irradiation, the radiosensitization effect persists for at least 2 h, but it does not when drug is given after irradiation. Taken together, the finding of a difference between rodent and human cells with respect to post-irradiation sensitization by SR 4233, and the differing time-course kinetics for this effect as a function of how the drug and radiation are sequenced, suggest that while SR 4233 behaves in a radiomimetic manner in most respects, there may be subtle differences in the nature of the lesions produced by the drug, the important cellular targets for this damage, and/or the cell's management of the damage.     

3-硝基三氮唑类衍生物体外放射增敏作用的生物学评价

探讨３－硝基三氮唑衍生物的放射增敏作用，并对其生物学效应作出评价。方法本文用克隆形成法研究了２２个３－硝基－１，２，４－三氮唑衍生物的体外放射增敏活性和乏氧、富氧细胞毒性。结果两个化合物的活性胜过ＭＩＳＯ，有三个化合物与ＭＩＳＯ和ＳＲ－２５０８相当，其余的其活性均较低。３－硝基三唑衍生物也像２－硝基咪唑类那样，其放射增敏活性与电子亲和性（用半波还原电位Ｅ１／２表示）有关。亲和性越大，即Ｅ１／２值越偏正，增敏活性越强。环核Ｎ１位和５位上取代基的结构和性质影响着化合物的Ｅ１／２、分配系数ｐ和细胞毒性。结论３－硝基三唑衍生物对乏氧和富氧（空气）细胞的毒性差别是很有限的，所以它们不是有效的乏氧调节细胞毒剂。但某些的放射增敏作用是值得深入研究的。     

Radiosensitization by Nickel Lapachol

The properties of interest in the radiosensitization of a metal complex, nickel lapachol, are compared with those of the 2-nitroimidazole, misonidazole. These very different compounds were found to be surprisingly similar in terms of their reduction potential (? 370 mV), enhancement ratios for killing of hypoxic Chinese hamster ovary cells by X-irradiation, and enhancement of DNA breaks in hypoxia. For nitroimidazoles, the sensitization depends on ‘electron affinity’, reduction of the nitro group; for nickel lapachol it is the metal which is necessary for reduction, yet the sensitization efficiencies are remarkably close. However, the metal complex has additional activities (some sensitization in aerobic cells; increased sensitization with preincubation) which are as yet unexplained but are assumed to be related to the nature of the naphthoquinone ligand, rather than to the reduction of the metal.     

Effect of actinomycin D on the survival and recovery capacity of irradiated connective tissue cells in the Chinese hamster

Simultaneous treatment of Chinese hamster fibroblasts with Actinomycin D and gamma rays under normal aerobic conditions leads to radiosensitization. In contrast to Actinomycin D pretreatment, modification to the effect of radiation is drug dose dependent in the case of post-treatment. No effect of Actinomycin D upon the shape of the radiation cell survival curve is seen under hypoxic conditions. Recovery from radiation damage in split dose experiments is not influenced by the drug.     

Photoreactivation of Ionizing-radiation-induced Damage in E. Coli

Since the cytokinesis-block micronucleus assay is very sensitive at low radiation doses, we used it to investigate the in vitro sensitizing effects of two new hypoxic cell sensitizers (KU-2285, a fluorinated 2-nitroimidazole and RP-170, a 2-nitroimidazole nucleoside analogue) at 1–3 Gy in comparison with etanidazole. Exponentially growing EMT6 cells were treated with the drugs under aerobic or hypoxic conditions for 40 min prior to and during irradiation, after which the drugs were removed and cytochalasin B (2 µg/ml) was added to the medium. The number of micronuclei in binucleate cells was counted after 42 h of culture. Under aerobic conditions the three compounds at 5 mm had no sensitizing effect. Under hypoxic conditions the sensitizer enhancement ratio (SER) at 5 mm was 3·8 for KU-2285, 3·2 for RP-170, and 2·3 for etanidazole, while the oxygen enhancement ratio was 2·9. When the cells were pretreated under hypoxic conditions with drugs at 5 mm but then irradiated under aerobic conditions, KU-2285 and RP-170 had a sensitizing effect whereas etanidazole did not. The sensitizers were also tested at 0·5 and 1 mm, and the SER values were compared with those obtained at high doses (15–30 Gy) using a colony assay. The SER at low doses was higher than that at high doses for 1 and 5 mm KU-2285 and 5 mm RP-170, while the SERs were similar for all concentrations of etanidazole and the lower concentrations of KU-2285 and RP-170. These results might suggest the potential usefulness of KU-2285 and RP-170 in clinical radiotherapy.     

In vitro and in vivo radiosensitizing effects of 2-nitroimidazole derivatives with sugar component

A new type of hypoxic cell sensitizer, 2-nitroimidazole substituted with an acyclic sugar analogue at the N-1 position of the imidazole ring (RK compounds) has been developed and tested on HeLa S3 and Chinese hamster V79 cells. As might be expected from their electron affinities, which are stronger than that of misonidazole, the abilities of RK compounds to sensitize hypoxic cells were correspondingly increased. One millimole of RK28 [1-(4'-hydroxy-2'-butenoxy)methyl-2-nitroimidazole] gave an enhancement ratio of 1.56 or 1.84 in comparison with 1.40 or 1.71 for the same concentration of misonidazole to HeLa S3 or V79 cells, respectively. RK28 showed slight cytotoxicity to aerobic HeLa S3 cells at a concentration of 5 mM after a three-hour exposure, whereas under hypoxic conditions, the agent was markedly cytotoxic. In vivo radiosensitization studies in ICR mice with Ehrlich ascites tumor indicated that RK28 produced an increase in DMF to hypoxic tumor cells with increased dose of the compound. Their DMF values were 1.63, 1.97 and 2.34 at 0.075, 0.15 and 0.3 mg/gbw of RK28, respectively. A dose of 0.3 mg/gbw of RK28 produced a DMF around 1.5 to two times greater than that resulting from the same dose of misonidazole. Pharmacokinetic studies of RK28 in ICR mice with Sarcoma-180 revealed a faster clearance from the serum and a slower decrease in the tumor than with misonidazole.     

Protection from radiation-induced DNA single-strand breaks by induction of nuclear metallothionein

Purpose : To examine the extent to which nuclear metallothionein protects from radiation-induced DNA damage under aerobic and hypoxic conditions. Materials and methods : A semiquantitative fluorescence image analysis method measured the nuclear content of metallothionein (MT) in ME180 and SiHa human squamous cervical carcinoma cell lines under normal growth conditions, and following MT induction by zinc. The extent of initial DNA damage following 60 Co irradiation under aerobic and hypoxic conditions was assessed using the alkaline comet assay. Results : Provided that cells were maintained at 37°C, most of the cellular content of MT was in the nucleus. Incubation at 4°C caused the rapid translocation of MT from the nucleus into the cytoplasm in both cell lines, with no net loss of cellular MT. Baseline nuclear MT levels were about four times greater in ME180 cells, and were much more readily induced by treatment with 100 μ m zinc acetate, compared with SiHa cells. Under aerobic conditions, MT induction by zinc resulted in no protection in either of the cell lines. Under hypoxic conditions, however, the number of DNA single-strand breaks in zinc-treated cells was reduced by ~40% in ME180, but not in SiHa cells, when compared with non-induced controls. Conclusions : Nuclear MT can exert a significant level of protection from radiation by a mechanism that involves competition with oxygen for DNA radical sites and/or scavenging of free radicals. Because increased MT levels have been reported in hypoxic micro-regions of some solid tumours, this protective mechanism might have clinical relevance.     

Hypoxia and etanidazole alter radiation-induced apoptosis in HL60 cells but not in MOLT-4 cells

Purpose : To examine how hypoxia influences ionizing irradiation-induced apoptosis in cultured mammalian cells and how a hypoxic cell radiosensitizer sensitizes apoptosis under hypoxic conditions. Materials and methods : Two cell lines derived from human lymphocytes, HL60 and MOLT-4, were exposed to 15 Gy X-rays under aerobic and hypoxic conditions. Etanidazole was used as a hypoxic cell radiosensitizer. The apoptotic morphological changes of nuclei and the induction of ladder-like DNA fragmentation were assessed by fluorescence microscopy and agarose gel electrophoresis, respectively. Results : In HL60 cells, apototic cell death and the activation of caspases 8, 9 and 3 were less induced under the hypoxic conditions than under the aerobic ones. Treatment of hypoxic cells with etanidazole enhanced X-ray-induced apoptosis and caspase activation. However, in MOLT-4 cells, neither hypoxia nor etanidazole influenced X-ray-induced apoptosis and caspase activation. In both cell lines, the frequency of X-ray-induced DNA double-strand breaks (DSB) under hypoxia was significantly smaller than that in aerobic conditions. Treatment of hypoxic cells with etanidazole enhanced them. Conclusion : These results suggested that X-ray-induced apoptosis in HL60 cells was initiated by DNA DSB and the treatment of hypoxic cells with etanidazole sensitized them through the enhancement of DSB induction, whereas X-ray-induced apoptosis in MOLT-4 cells occurred through damage other than to DNA.     

The Influence of Oxygen on the Induction of Radiation Damage in DNA in Mammalian Cells after Sensitization by Intracellular Glutathione Depletion

Summary

Treatment of mammalian cells with buthionine sulphoximine (BSO) or diethyl maleate (DEM) results in a decrease in the intracellular GSH (glutathione) and non-protein-bound SH (NPSH) levels. The effect of depletion of GSH and NPSH on radiosensitivity was studied in relation to the concentration of oxygen during irradiation. Single- and double-strand breaks (ssb and dsb) and cell killing were used as criteria for radiation damage. Under aerobic conditions, BSO and DEM treatment gave a small sensitization of 10–20 per cent for the three types of radiation damage. Also under severely hypoxic conditions (0·01 μm oxygen in the medium) the sensitizing effect of both compounds on the induction of ssb and dsb and on cell killing was small (0–30 per cent). At somewhat higher concentrations of oxygen (0·5–10 μm) however, the sensitization amounted to about 90 per cent for the induction of ssb and dsb and about 50 per cent for cell killing. These results strengthen the widely accepted idea that intracellular SH-compounds compete with oxygen and other electron-affinic radiosensitizers with respect to reaction with radiation-induced damage, thus preventing the fixation of DNA damages by oxygen. These results imply that the extent to which SH-compounds affect the radiosensitivity of cells in vivo depends strongly on the local concentration of oxygen.     

Hypoxia and etanidazole alter radiation-induced apoptosis in HL60 cells but not in MOLT-4 cells

PURPOSE: To examine how hypoxia influences ionizing irradiation-induced apoptosis in cultured mammalian cells and how a hypoxic cell radiosensitizer sensitizes apoptosis under hypoxic conditions. MATERIALS AND METHODS: Two cell lines derived from human lymphocytes, HL60 and MOLT-4, were exposed to 15 Gy X-rays under aerobic and hypoxic conditions. Etanidazole was used as a hypoxic cell radiosensitizer. The apoptotic morphological changes of nuclei and the induction of ladder-like DNA fragmentation were assessed by fluorescence microscopy and agarose gel electrophoresis, respectively. RESULTS: In HL60 cells, apototic cell death and the activation of caspases 8, 9 and 3 were less induced under the hypoxic conditions than under the aerobic ones. Treatment of hypoxic cells with etanidazole enhanced X-ray-induced apoptosis and caspase activation. However, in MOLT-4 cells, neither hypoxia nor etanidazole influenced X-ray-induced apoptosis and caspase activation. In both cell lines, the frequency of X-ray-induced DNA double-strand breaks (DSB) under hypoxia was significantly smaller than that in aerobic conditions. Treatment of hypoxic cells with etanidazole enhanced them. CONCLUSION: These results suggested that X-ray-induced apoptosis in HL60 cells was initiated by DNA DSB and the treatment of hypoxic cells with etanidazole sensitized them through the enhancement of DSB induction, whereas X-ray-induced apoptosis in MOLT-4 cells occurred through damage other than to DNA.     

Radiosensitization efficacy of KU-2285, RP-170 and etanidazole at low radiation doses: assessment by in vitro cytokinesis-block micronucleus assay.

Since the cytokinesis-block micronucleus assay is very sensitive at low radiation doses, we used it to investigate the in vitro sensitizing effects of two new hypoxic cell sensitizers (KU-2285, a fluorinated 2-nitroimidazole and RP-170, a 2-nitroimidazole nucleoside analogue) at 1-3 Gy in comparison with etanidazole. Exponentially growing EMT6 cells were treated with the drugs under aerobic or hypoxic conditions for 40 min prior to and during irradiation, after which the drugs were removed and cytochalasin B (2 micrograms/ml) was added to the medium. The number of micronuclei in binucleate cells was counted after 42 h of culture. Under aerobic conditions the three compounds at 5 mM had no sensitizing effect. Under hypoxic conditions the sensitizer enhancement ratio (SER) at 5 mM was 3.8 for KU-2285, 3.2 for RP-170, and 2.3 for etanidazole, while the oxygen enhancement ratio was 2.9. When the cells were pretreated under hypoxic conditions with drugs at 5 mM but then irradiated under aerobic conditions, KU-2285 and RP-170 had a sensitizing effect whereas etanidazole did not. The sensitizers were also tested at 0.5 and 1 mM, and the SER values were compared with those obtained at high doses (15-30 Gy) using a colony assay. The SER at low doses was higher than that at high doses for 1 and 5 mM KU-2285 and 5 mM RP-170, while the SERs were similar for all concentrations of etanidazole and the lower concentrations of KU-2285 and RP-170. These results might suggest the potential usefulness of KU-2285 and RP-170 in clinical radiotherapy.     

Gamma Radiation Response and Recovery Studies in Radiation Sensitive Mutants of Diploid Yeast

Summary

Cell killing and other deleterious biological effects of ionizing radiation are the result of chemical changes to critical targets, initiated at the time of exposure. Electron-affinic radiosensitizers act, primarily, by chemically modifying this radiation damage and its consequent biological expression, and such changes can be used to probe the nature of the cellular radiation target. According to a redox hypothesis of radiation modification, the molecular mechanism of electronic-affinic radiosensitization involves an oxidative interaction of the sensitizer with reactive, potentially damaging target radicals, which competes with reductive processes that restore the target to its undamaged state.

The effects have been compared of a series of hypoxic cell radiosensitizers on radiation-induced DNA damage and mammalian cell killing, in order to ascertain the nature of the critical radiation target site(s) involved. Sensitizer efficacy is determined by the ability to oxidize the radiation target and is found to increase exponentially with increasing electron affinity. The threshold redox potential, below which no sensitization occurs, corresponds to the oxidation potential of the target bioradical involved, and is characteristic, and useful in identification, of the particular radiation target.

Model product analysis studies of DNA base damage, inorganic phosphate release, single-strand breaks and incorporation of radioactively labelled sensitizer into DNA show a correspondence between the electronic-affinic radiosensitization of DNA damage and cell killing. A careful comparison of the radiosensitization of different DNA sites and cell killing indicates that the sugar-phosphate backbone of DNA, not the heterocyclic bases, is the DNA target site which mimics cell killing in its threshold redox potential and overall radiosensitization response. These results suggest that the enhancement by electron-affinic drugs of radiation damage to the DNA backbone (strand breaks) correlates strongly with, and is the most likely cause of, the radiosensitization of hypoxic cell killing.     

Effect of combined treatment with cisplatin and radiation on the survival of Chinese hamster cells

During combined treatment of Chinese hamster cells with cisplatinum and irradiation under aerobic conditions, there appear interactions between the two treatment modalities depending on the treatment sequence and the time interval. Treatment with cisplatinum followed by irradiation leads to a reduction of the shoulder of the survival curve with increasing time interval. Simultaneous treatment with cisplatinum and irradiation under aerobic or hypoxic conditions does not change the survival curve. Treatment with cisplatinum under aerobic conditions followed by irradiation in hypoxia does not lead to any interaction of both modalities independent of the time interval in contrast to subsequent irradiation under aerobic conditions. The specific sensitization of hypoxic cells by cisplatinum towards irradiation described in the literature could not be demonstrated with our cell line.     

Binding Mode of 2-amino-5-nitrothiazole (ANT) in Platinum Complexes,Trans-[PtCl2(ANT)2], Affects DNA Binding,Toxicity and Radiosensitizing Ability

Summary

The radiosensitizer 2-amino-5-nitrothiazole (ANT) can react with platinum to form many products because of the availability of two potential nitrogen donors as ligands for metals. Two of these complexes, both with two ANT molecules in the trans configuration, differ because of their linkages. In the first, Pt is bound to ANT via the amine group (A) and in the second via the thiazole ring nitrogen (R). Isomer R is a better radiosensitizer than A in hypoxic Chinese hamster ovary cells, giving enhancement ratios of 1·6 versus 1·15 with 100 µmol dm^?3 complex. The ring-bound isomer also exhibits higher toxicity than the amine bound in air and under conditions of hypoxia. Sensitization by isomer R is much higher under conditions of hypoxia than in air. In an assay to assess DNA binding, isomer R inhibited restriction enzyme cleavage of DNA but isomer A did not (up to 6 h at 300 µmol dm^?3). It appears from this study on two very similar complexes that the complex which exhibits stronger binding may be targetting the radiosensitizer to the DNA, resulting in greatly improved sensitization.     

不同浓度氧对放射所致肿瘤细胞凋亡影响的研究

目的：探讨不同浓度氧对放射所致肿瘤细胞凋亡的影响。方法：实验分为空气组、高氧组及低氧组。肿瘤细胞为LA-795小鼠自发性肺腺癌细胞，接种于T-739纯系小鼠右后肢皮下，待荷瘤肢直径达10mm时照射22Gy，HE染色及TdT末端标记法检测肿瘤细胞凋亡指数。结果：照射后LA-795肿瘤细胞发生细胞凋亡，在6h达高峰，以后逐渐下降。高氧可增加照射所诱导的凋亡；低氧吸入0.5min后照射对肿瘤细胞凋亡影响不大，其水平与空气组相近。结论：吸入高浓度氧后改吸低浓度氧的同时合并放疗的方法能够提高肿瘤细胞放射敏感性，这一现象可通过其增加肿瘤细胞凋亡的方式来解释。氧对细胞凋亡影响的作用机理尚待进一步研究。     

The Interaction Between Radiation and Complexes of Cis-Pt(II) and Rh(II): Studies at the Molecular and Cellular Level

Summary

A range of Rh(II) carboxylates and cis-Pt(II) complexes have been examined for their ability to increase the radiation sensitivity of aerobic and hypoxic V79 cells in vitro. The transition metal complexes sensitize in both air and nitrogen, with the greater effect generally occurring in nitrogen. The cis-Pt(II) complexes only show small levels of sensitization with dose modification factors (DMFs) of no more than 1·2. In contrast, the Rh(II) complexes can give DMFs of 2·0. Radiation chemical experiments show the transition metal complexes to have substantially lower redox potentials than metronidazole and, in addition, neither type of complex undergoes electron transfer reaction or adduct formation on interaction with radicals derived from DNA bases. Thus, the inorganic complexes do not operate by mechanisms similar to those occurring with electron affinic or stable free radical sensitizers. The increase in radiation sensitivity for cells treated with the Rh(II) carboxylates, but not the cis-Pt(II) complexes, is attributed to the ability of the Rh compounds to deplete intracellular thiols. Further, the efficiency of sensitization by the Rh(II) complexes and their ability to interact with cellular thiols depends upon the nature of the carboxylate ligand and follows the order butyrate > propionate > acetate > methoxyacetate. The differences between the carboxylates may be due to differences in drug uptake. A combination of the Rh(II) complexes with misonidazole given to hypoxic cells irradiated in vitro gives an additive response. However, it was not possible to demonstrate a similar effect in tumours in mice given the combination of Rh(II) methoxyacetate and the misonidazole analogue RSU 1070.     

Protection from radiation-induced DNA single-strand breaks by induction of nuclear metallothionein

PURPOSE: To examine the extent to which nuclear metallothionein protects from radiation-induced DNA damage under aerobic and hypoxic conditions. MATERIALS AND METHODS: A semiquantitative fluorescence image analysis method measured the nuclear content of metallothionein (MT) in ME180 and SiHa human squamous cervical carcinoma cell lines under normal growth conditions, and following MT induction by zinc. The extent of initial DNA damage following 60Co irradiation under aerobic and hypoxic conditions was assessed using the alkaline comet assay. RESULTS: Provided that cells were maintained at 37 degrees C, most of the cellular content of MT was in the nucleus. Incubation at 4 degrees C caused the rapid translocation of MT from the nucleus into the cytoplasm in both cell lines, with no net loss of cellular MT. Baseline nuclear MT levels were about four times greater in ME180 cells, and were much more readily induced by treatment with 100 microM zinc acetate, compared with SiHa cells. Under aerobic conditions, MT induction by zinc resulted in no protection in either of the cell lines. Under hypoxic conditions, however, the number of DNA single-strand breaks in zinc-treated cells was reduced by approximately 40% in ME180, but not in SiHa cells, when compared with non-induced controls. CONCLUSIONS: Nuclear MT can exert a significant level of protection from radiation by a mechanism that involves competition with oxygen for DNA radical sites and/or scavenging of free radicals. Because increased MT levels have been reported in hypoxic micro-regions of some solid tumours, this protective mechanism might have clinical relevance.     

Time Scale of Radiation Damage by 2,4-dinitrophenyl-aziridine (CB 1954) in Chinese Hamster Cells: A Rapid-mix Study

Summary

A rapid-mix device was used to study the time-scale of radiation sensitization of hypoxic cells by CB 1954, a monofunctional alkylating agent. It has an electron affinity (E¹₇–385 mV) similar to that of misonidazole but its effectiveness as a sensitizer occurs at a five-fold lower concentration under stationary-state conditions. In the rapid-mix study, the enhancement ratio (e.r.) value of 1 mmol dm^?3 CB 1954 rapidly rises to 1·75 within 120 ms, with no further rise by 500 ms. The e.r. obtained is lower than that observed under stationary-state conditions for a similar concentration. The data suggests that CB 1954 sensitizes by at least two independent mechanisms.     

Obituary

Summary

Using a cellular fast-mixing technique, the time course of radiation sensitization of hypoxic, V79 cells by various concentrations of RSU-1069 (0·25–2·5 mmol dm^?3) and misonidazole (2·5–50 mmol dm^?3) have been studied to distinguish between fast chemical processes and the much slower biochemical responses to ionizing radiation and the monofunctional alkylating action of RSU-1069. Under conditions of equi-concentration, misonidazole and RSU-1069 show similar radiosensitizing efficiencies for pre-irradiation contact times up to 1 s. The values of the sensitizer enhancement ratio of ～ 1·5 for both 2-nitroimidazoles (2·5 mmol dm^?3) is considerably less than that of 1·9–2·8 determined with misonidazole for a pre-irradiation contact time of 1 h under hypoxia. It is proposed that the enhanced radiosensitizing efficiency of RSU-1069 compared to that of misonidazole after long contact times involves, in part, the formation of ‘sub-toxic’ damage probably involving monofunctional and/or bifunctional action of RSU-1069 prior to irradiation.     

Radiobiological effects of hypoxia-dependent uptake of 64Cu-ATSM: enhanced DNA damage and cytotoxicity in hypoxic cells

Purpose

Hypoxia occurs frequently in cancers and can lead to therapeutic resistance due to poor perfusion and loss of the oxygen enhancement effect. ⁶⁴Cu-ATSM has shown promise as a hypoxia diagnostic agent due to its selective uptake and retention in hypoxic cells and its emission of positrons for PET imaging. ⁶⁴Cu also emits radiotoxic Auger electrons and β^- particles and may therefore exhibit therapeutic potential when concentrated in hypoxic tissue.

Methods

MCF-7 cells were treated with 0–10 MBq/ml ⁶⁴Cu-ATSM under differing oxygen conditions ranging from normoxia to severe hypoxia. Intracellular response to hypoxia was measured using Western blotting for expression of HIF-1α, while cellular accumulation of ⁶⁴Cu was measured by gamma counting. DNA damage and cytotoxicity were measured with, respectively, the Comet assay and clonogenic survival.

Results

⁶⁴Cu-ATSM uptake in MCF-7 cells increased as atmospheric oxygen decreased (up to 5.6 Bq/cell at 20.9% oxygen, 10.4 Bq/cell at 0.1% oxygen and 26.0 Bq/cell at anoxia). Toxicity of ⁶⁴Cu-ATSM in MCF-7 cells also increased as atmospheric oxygen decreased, with survival of 9.8, 1.5 and 0% in cells exposed to 10 MBq/ml at 20.9, 0.1 and 0% oxygen. The Comet assay revealed a statistically significant increase in ⁶⁴Cu-ATSM-induced DNA damage under hypoxic conditions.

Conclusion

The results support a model in which hypoxia-enhanced uptake of radiotoxic ⁶⁴Cu induces sufficient DNA damage and toxicity to overcome the documented radioresistance in hypoxic MCF-7 cells. This suggests that ⁶⁴Cu-ATSM and related complexes have potential for targeted radionuclide therapy of hypoxic tumours.