Hypoxia-selective Radiosensitization of Mammalian Cells by Nitracrine,an Electron-affinic DNA Intercalator

Summary

The radiosensitizing ability of the 1-nitroacridine nitracrine (NC) is of interest since it is an example of a DNA intercalating agent with an electron-affinic nitro group. NC radiosensitization was evaluated in Chinese hamster ovary cell (AA8) cultures at 4°C in order to suppress the rapid metabolism and potent cytotoxicity of the drug. Under hypoxic conditions, submicromolar concentrations of NC resulted in sensitization (SER = 1·6 at 1 µmol dm^?3). Sensitization was also seen under aerobic conditions but a concentration more than 10-fold higher was required. In aerobic cultures NC radiosensitization was independent of whether cells were exposed before and during, or after, irradiation. Postirradiation sensitization was not observed under hypoxic conditions. The time dependence of NC uptake and the development of radiosensitization were similar (maximal at 30 min at 4°C under hypoxia) suggesting that sensitization, unlike cytotoxicity, is due to unmetabolized drug. NC is about 1700 times more potent as a radiosensitizer than misonidazole. This high potency is adequately accounted for by the electron affinity of NC (E(1) value at pH 7 of ?275mV versus NHE) and by its accumulation in cells to give intracellular concentrations approximately 30 times greater than in the medium. However, concentrations of free NC appear to be low in AA8 cells, presumably because of DNA binding. If radiosensitization by NC is due to bound rather than free drug, it suggests that intercalated NC can interact very efficiently with DNA target radicals. This is despite a binding ratio in the cell estimated as less than 1 NC molecule/400 base pairs under conditions providing efficient sensitization. This work suggests a new approach in the search for more effective clinical radiosensitizers, and poses questions on the means by which intercalated drugs can interact with DNA damage.     

Adriamycin: a possible indirect radiosensitizer of hypoxic tumor cells.

Multicellular spheroids grown in vitro provide a convenient model of nodular tumors for experimental tumor therapy studies. Adriamycin was found to inactivate cells grown as spheroids less efficiently than single cells, presumably due to enhanced cellular resistance analogous to the increased radioresistance observed when these cells are grown in close contact. Spheroid growth was retarded by a minimally toxic (0.005 mug/ml) chronic level of adriamycin; irradiation and exposure to that drug concentration were not found to be synergistic. Large adriamycin concentrations (0.5 mug/ml) present during radiation exposure produced a marked "radiosensitization," presumably due to the drug-inhibitng cellular oxygen consumption and thus permitting reoxygenation of the previously hypoxic spheroid cells.     

The effect of actinomycin D on the survival and recoverability of irradiated human melanoma cells

The presence of actinomycin D during gamma-irradiation of human melanoma cells leads to an increased radiosensitivity under aerobic and hypoxic conditions. This interaction of both treatment modalities depends upon the drug dose. Incubation of cells prior or after irradiation with essentially non-cytotoxic amounts of actinomycin D under normal conditions has only little effect on the shape of the radiation dose-response-curve, irrespectively of the time interval between drug treatment and irradiation. However, high doses of actinomycin D given immediately after irradiation increase the radiosensitivity of human cells. The drug does not effect recovery from sublethal or potentially lethal radiation damage.     

Effect of combined treatment with cisplatin and irradiation on the survival of human melanoma cells

During a combined treatment of human melanoma cells with Cisplatin and irradiation under aerobic conditions, interaction between the two treatment modalities is observed. The presence of Cisplatin during irradiation leads to an increased bending, pretreatment with Cisplatin to an increased initial steepness of the survival curve irrespective of the time interval and the Cisplatin dose. Post-treatment with Cisplatin, however, does not influence the shape of the survival curve. Simultaneous treatment of human melanoma cells with Cisplatin and irradiation in hypoxia does not result in the radiosensitization seen under aerobic conditions.     

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.     

Interaction of paclitaxel (Taxol®) and irradiation

AIM: Optimal dose and schedule of paclitaxel in combined drug-irradiation treatment could not be determined for most of tumors yet. The aim of this study was to compare in vitro cytotoxicity and radiosensitizing abilities as a function of single paclitaxel (Taxol) exposure in tumor and fibroblastic cells using different drug incubation irradiation intervals. MATERIAL AND METHOD: A lung-carcinoma (SK-LU-1), glioblastoma (U-138 MG) and rodent fibroblast cell line (HyB14FAF28) were used. The clonogenic assay was applied for survival investigation. alpha beta-values were calculated using the linear-quadratic model (log S = -alpha D - beta D2). Cytotoxicity of Taxol was examined at 0 to 50 microM. Combined Taxol-radiotherapy exposure was accomplished with 10 microM Taxol plus 10 Gy irradiation (RT) following after a 1-hour and 9-hour interval. For controls cells were exposed to Cremophor EL/ethanol (CEL/eth) and a phosphate buffered saline (PBS). RESULTS: Single Taxol exposure (10 microM) resulted in 0.54/0.50/0.84 (3-hours incubation) and 0.094/0.48/0.82 (15-hours incubation) survival of SK-LU-1, U-138 MG and HyB14FAF28 cells, respectively. Taxol concentrations from 2 to 50 microM only had cytotoxic effect in tumor cells. Single dose RT (10 Gy) led to cell survival of 0.0006/0.006/0.03. The diluent CEL/eth also showed cytotoxic activity. Taxol plus RT led to cell survival of 0.00025/0.0014/0.042 (1 hour) and 0.0004/0.0019/0.04 (9 hours) without significant difference between chosen time intervals. alpha beta-values showed great variation lacking evidence for definite radiosensitization. alpha increase after Taxol and alpha decrease after CEL/eth exposure were detected. CONCLUSIONS: The data presented demonstrate a potential beneficial effect, described as co-operation, by combining Taxol and RT in human tumor cells and rodent fibroblasts. High intrinsic alpha components of the tumor cells as well as CEL/eth's antagonizing actions could be likely to disturb and influence paclitaxel's abilities leading to radiosensitization.     

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.     

Repair of DNA strand breaks at hyperthermic temperatures in Chinese hamster ovary cells.

The repair of DNA double-strand breaks was measured by pH 7.2 filter elution in cells incubated at 25-45 degrees C either before or after X-irradiation. Exposure to 45 degrees C for 15 minutes immediately prior to X-irradiation significantly increased both the half-time for DNA double-strand break closure and the number of DNA double-strand breaks remaining in nuclear DNA 180 minutes after irradiation. Exposure to temperatures between 41 and 45 degrees C immediately after X-irradiation accelerated DNA double-strand break closure and resulted in no increase in the number of DNA double-strand breaks remaining in the cell's genome 180 minutes after irradiation. The results indicate either that the radiosensitization produced by the administration of hyperthermic temperatures before and after irradiation result from two characteristically different molecular mechanisms, or that neither the rate of DNA strand break closure nor the number of DNA strand breaks remaining in nuclear DNA after irradiation accurately predict hyperthermic radiosensitization. These conclusions assume that no DNA strand breaks are below the resolution of this DNA damage assay and that a comparison between cytotoxicity and DNA repair after exposure to high radiation doses is valid.     

依他硝唑纳米粒对乏氧肿瘤细胞辐射增敏作用的研究

目的 制备出具有生物活性并可控制释放的依他硝唑纳米粒,探讨其对乏氧人乳腺癌细胞(MCF-7)和人子宫颈癌细胞(HeLa)的辐射增敏作用。方法 采用复乳溶剂挥发法制备聚乳酸-聚羟基乙酸共聚物(PLGA)包裹的依他硝唑纳米粒,高效液相色谱分析纳米粒的载药率、包封率和模拟体外释药,透射电镜研究纳米粒的形态,激光衍射粒度分析仪检测纳米粒的粒径分布。经乏氧处理的MCF-7和HeLa细胞与依他硝唑纳米粒和药物单体共培养,采用平板克隆形成实验检验其辐射增敏作用。结果 成功制备依他硝唑纳米粒,呈光滑球形,粒径分布在90～190 nm之间,载药率为1.66％,包封率为18.02％,模拟体外释药曲线呈双相,即在爆发释放之后为缓慢释放。同依他硝唑纳米粒和药物单体共培养的乏氧MCF-7和HeLa细胞克隆形成能力照射后明显降低,依他硝唑纳米粒作用更为显著。结论 具有生物活性的依他硝唑从纳米粒中以可控的方式被释放,有效地增加了乏氧肿瘤细胞的辐射敏感性,为辐射增敏剂的临床应用提供了一种新的给药方式。     

Enhancement of SR 2508 (etanidazole) radiosensitization by buthionine sulphoximine at low-dose-rate irradiation.

SR 2508 (etanidazole) (1 mM) or buthionine sulphoximine (BSO, 50 microM) or both drugs together did not radiosensitize oxic V79 Chinese hamster cells irradiated at either an acute dose rate (2.35 Gy/min) or at a low dose rate (0.117 Gy/min). BSO pretreatment (15 h at 37 degrees C) depleted cellular glutathione (GSH) to less than or equal to 1% of control level and radiosensitized hypoxic cells at both dose rates with an enhancement ratio (ER) of 1.2. SR 2508 alone radiosensitized hypoxic cells equally at both dose rates with an ER of 1.5. However, ER values of 2.2 and 2.5 were obtained with 1 mM SR 2508 in GSH-depleted cells at acute and low dose rate, respectively, with no significant difference between the two, i.e. there is no dose rate dependence for this potentiation. Since BSO increases SR 2508 radiosensitization and the combined BSO + SR 2508 treatment is extremely cytotoxic to hypoxic cells, our results suggest that combining BSO with SR 2508 will be useful in brachytherapy as well as external-beam therapy if the toxicity from both drugs in vivo is less than the gain in radiosensitization achieved.     

Chromosome aberrations detected by FISH and correlation with cell survival after irradiation at various dose-rates and after bromodeoxyuridine radiosensitization

PURPOSE: To determine whether measurement of chromosome aberrations by fluorescence in situ hybridization (FISH) predicts cell survival after irradiation at different dose-rates and after radiosensitization by bromodeoxyurdine (BrdU) in a lung carcinoma cell line. MATERIALS AND METHODS: The human lung carcinoma cell line SW1573 was irradiated at high dose-rate (HDR: 0.8 Gy min-1) or at pulsed low dose-rate (p-LDR: average dose-rate 1 Gy h-1) with or without radiosensitization by bromodeoxyuridine (BrdU). Cell survival was determined by clonogenic assay. Chromosome aberrations (colour junctions) were measured by whole-chromosome FISH of chromosome 2 and 18 and were scored according to the PAINT method. RESULTS: Clear radiosensitization by BrdU was observed both after HDR and p-LDR irradiation. Chromosome 18 was more radiosensitive than chromosome 2. There was a good correlation between induction of colour junctions and cell survival both after HDR and p-LDR irradiation and after radiosensitization by BrdU. CONCLUSIONS: Determination of chromosome aberrations by FISH can predict cell survival after different dose-rates and after radiosensitization by BrdU     

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.     

Survival of synchronized human NHIK 3025 cells irradiated aerobically following a prolonged treatment with extremely hypoxic conditions

Purpose : To investigate whether radiation survival of cells irradiated aerobically in the oxygen-sensitive restriction point in late G1 is dependent on where in the cell cycle the cells first were rendered hypoxic. Materials and methods : Human cervix carcinoma, NHIK 3025 cells, were synchronized and rendered hypoxic while in early-, midor late G1 or in early G2. Cell-cycle progression during the treatment was monitored by flow cytometry, and cell survival following either hypoxia alone or hypoxia with subsequent reoxygenation and irradiation was measured by the ability of the cells to form macroscopic colonies. Results : During prolonged hypoxia, all surviving cells accumulated in an oxygen-sensitive restriction point in late G1. Cells rendered hypoxic in G2 initiated DNA synthesis following reoxygenation and irradiation several hours later than cells rendered hypoxic in G1. Radiation survival of cells accumulated in the oxygensensitive restriction point was independent of where in the cell cycle the cells first were rendered hypoxic. The hypoxia-treated cells had lower radiation survival probability than untreated cells in late G1. Conclusions : Although cells accumulated in the oxygen-sensitive restriction point from different parts of the cell cycle are not biologically identical, they are radiobiologically similar. The radiosensitizing effect of prolonged hypoxia was not merely due to cell-cycle redistribution.     

Pentoxifylline Enhances Tumor Oxygenation and Radiosensitivity in Rat Rhabdomyosarcomas during Continuous Hyperfractionated Irradiation

PURPOSE: To examine the influence of the hemorrheologic agent pentoxifylline (PTX) on tumor oxygenation and radiosensitivity. MATERIAL AND METHODS: Tumor oxygenation in rat rhabdomyosarcomas R1H after PTX administration (50 mg/kg body weight) was measured using interstitial pO(2) probes (Licox CMP system and Eppendorf pO(2)-Histograph). Tumors were irradiated with (60)Co gamma-irradiation using single doses (15 and 30 Gy), conventional fractionation (60 Gy/30 fractions/6 weeks), and continuous hyperfractionation (54 Gy/36 fractions/18 days) in combination with PTX or an equivalent volume of physiological saline. Radiation effects were determined by tumor growth delay (2V(o)), and by partial and complete tumor remission. RESULTS: PTX increased tumor oxygenation for up to 45 min after administration of the drug. Single doses of 15 and 30 Gy of irradiation, when combined with PTX, produced little radiosensitization of the R1H tumors as indicated by dose-modifying factors (DMFs) of 1.11 and 1.04, respectively. In conventional fractionated irradiation with PTX, a DMF of 1.10 was obtained only. However, in continuous hyperfractionated irradiation with 18 x 50 mg/kg of PTX, the DMF with respect to tumor growth delay was found to be 1.37. Local tumor control was not influenced by PTX. In vitro studies identified R1H cells as p53 wildtype and showed a G1 arrest in response to irradiation. When 2 mM PTX was given prior to irradiation, it did not improve radiosensitivity of R1H cells as measured by clonogenic survival assays. CONCLUSION: PTX effectively enhances tumor oxygenation and radiosensitivity of R1H rhabdomyosarcomas, especially during continuous hyperfractionated irradiation. Given to rats as an adjuvant to fractionated irradiation, PTX does not enhance acute or late skin reactions or tumor metastasis. No radiosensitization was observed in vitro, when oxygen was not limiting. The observed radiosensitization by PTX is caused mainly by improved tumor oxygenation.     

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。     

Effect of growth rate of monolayer cells on survival after fractionated irradiation]

This study was performed to determine the effect of the growth rate of monolayer cells on survival following fractionated irradiation. HeLa and RMUG cells that had different radiosensitivities and growth rates (Do value: 2.3 vs 1.5 Gy, doubling time: 17 vs 46 hours) were irradiated with 2 Gy every day. The fractions surviving after fractionated irradiation were compared with those given single doses. The dose modifying factor for fractionated irradiation was larger in RMUG than HeLa: 1.7 and 1.2, respectively. Two clones from ADGU cells that had the same radiosensitivity but different growth rates were also given fractionated irradiation, but there was no difference in surviving fractions. When recovery following two split doses was determined in each type of cell, the results of the fractions surviving after fractionated irradiation were correlated only with recovery between split doses. These suggest that the growth rates of monolayer cells may not modify the fractions surviving after fractionated irradiation, and the monolayer cell system is not suitable for determining the effect of growth rates on survival following fractionated irradiation.     

Chemical agents that promote chromatin compaction radiosensitize tumour cells

Purpose : Previous studies indicated that cells whose chromatin is naturally compacted at the time of radiation are hypersensitive to radiation-induced killing, primarily by single-hit inactivation. Some chemicals that are known to promote chromatin compaction in interphase cells are here investigated for their radiosensitizing potential. Materials and methods : Okadaic acid (OA), a protein phosphatase inhibitor, fostriecin (FC), a topoisomerase II inhibitor and trichostatin A (TSA), a histone deacetylase inhibitor, were reported to promote chromatin compaction in mammalian cells. Asynchronous populations of HT-29 (human colon carcinoma) cells were exposed to various concentrations of OA, FC and TSA for various times before irradiation with various doses of Cs-137 n -rays and toxicity and radiosensitization were measured. Induced chromatin compaction was visualized by electron microscopy (EM). Histone 1 (H1) and histone 3 (H3) phosphorylation was measured by Western blotting, whole-cell fluorescence microscopy and confocal microscopy. Results : OA and FC produced significant radiosensitization at 2 Gy after short (2 h) exposures. These chemical treatments also produced increased phosphorylation of H3 and increased chromatin compaction as measured by EM. A 2-h exposure of cells to TSA had no effect on cell radiosensitivity, histone phosphorylation or chromatin condensation. However, a 16-h exposure to TSA produced significant radiosensitization, histone phosphorylation and chromatin condensation, presumably by secondary mechanisms. Conclusions : These data are consistent with the hypothesis that compacted chromatin is a hypersensitive target for radiation killing. Furthermore, the modulation of chromatin conformation by drugs selectively in tumour cells might radiosensitize tumours whose cells are intrinsically radioresistant.     

Chemical agents that promote chromatin compaction radiosensitize tumour cells.

PURPOSE: Previous studies indicated that cells whose chromatin is naturally compacted at the time of radiation are hypersensitive to radiation-induced killing, primarily by single-hit inactivation. Some chemicals that are known to promote chromatin compaction in interphase cells are here investigated for their radiosensitizing potential. MATERIALS AND METHODS: Okadaic acid (OA), a protein phosphatase inhibitor, fostriecin (FC), a topoisomerase II inhibitor and trichostatin A (TSA), a histone deacetylase inhibitor, were reported to promote chromatin compaction in mammalian cells. Asynchronous populations of HT-29 (human colon carcinoma) cells were exposed to various concentrations of OA, FC and TSA for various times before irradiation with various doses of Cs-137 gamma-rays and toxicity and radiosensitization were measured. Induced chromatin compaction was visualized by electron microscopy (EM). Histone 1 (H1) and histone 3 (H3) phosphorylation was measured by Western blotting, whole-cell fluorescence microscopy and confocal microscopy. RESULTS: OA and FC produced significant radiosensitization at 2 Gy after short (2 h) exposures. These chemical treatments also produced increased phosphorylation of H3 and increased chromatin compaction as measured by EM. A 2-h exposure of cells to TSA had no effect on cell radiosensitivity, histone phosphorylation or chromatin condensation. However, a 16-h exposure to TSA produced significant radiosensitization, histone phosphorylation and chromatin condensation, presumably by secondary mechanisms. CONCLUSIONS: These data are consistent with the hypothesis that compacted chromatin is a hypersensitive target for radiation killing. Furthermore, the modulation of chromatin conformation by drugs selectively in tumour cells might radiosensitize tumours whose cells are intrinsically radioresistant.     

Die Strahlenempfindlichkeit für verzögerten Reproduktivtod (DRD) nach Einzel- oder Splitdosisbestrahlung

BACKGROUND: The growth kinetics of tumors after irradiation (Figure 1) is defined by cells surviving with delayed reproductive death (DRD). The prediction of radiation sensitivity of locally recurrent tumor growth is among other factors dependent on the knowledge of the impact of fractionated irradiation on these surviving cells with DRD. Short recovery effects can be estimated in vitro by comparing the difference of the medians of the distributions of clone sizes, the median clone sizes difference (MCD) after single and split exposure irradiation of the progenitor cells. MATERIALS AND METHODS: CHO-cells of a sub clone of the line T71 have a spontaneous cell loss rate of < 5%. The cells were irradiated a) by a single exposure 3 hours after synchronization and b) by a fractionated irradiation of half the exposure of a 200-kVp radiation exposure 3 hours and 6 hours after synchronization, respectively. Clone survival was determined (Figure 2). As function of dose and incubation time the distributions of clone sizes and the MCD were determined by tapping the clone cells in microscopic projections. RESULTS: The radiation sensitivity El of the DRD can be defined as the proportional factor of the linear relationship between the MCD on one side and the dose K x the cell division factor m on the other side. EI is dependent on the age of the cells during irradiation (Figure 3) and the cell line (Table 1). The slope of the dually logarithmic growth curve of the cell population is: s = 1 - El.K. Experimentally El was found to be equal for single and split dose irradiation (Figures 4 and 5) and amounted to El = 0.065 with Sd = +/- 0.004.--Literature analysis for the mathematical estimation of El.K (Figure 6) was based on reports of measurements of the local tumor recurrence growth of carcinomas and sarcomas of rodents and pulmonary metastases of sarcomas in humans, respectively, after fractional irradiation. We obtained values of 0 < or = El.K < or = 0.77 (Table 2). Values for El are independent of the dose and lie considerably below data derived from in-vitro measurements of different cell cultures. CONCLUSIONS: Since recurrence kinetics of tumors are determined by the radiation sensitivity El of the DRD, El can be used for estimating the kinetics of tumor recurrence. As lately described, MCD is linearly proportional to the micro-nucleus frequency [12]. Determinations of the micro-nucleus frequencies in tumor cell biopsies pre and post radiation onset offer the option for developing a fast predictive assay. Organ malformations of embryos after exposition to ionizing radiation can be mathematically deduced by DRD to the partial cell mortality.     

Cytokinetic and cytotoxic responses of HeLa cells to the combination of hyperthermia and radiation

Colony formation and cell cycle changes of HeLa cells were studied after the combination of hyperthermia and 60Co irradiation. Cells were heated by immersion in a water bath. The cell cycle progression was monitored using flow cytometry. The dose survival curve of asynchronous growing cells, with immediate postirradiation thermal treatment at 43 degrees C for 60 min, showed significant enhancement of radiosensitivity. When the sequence of heat and irradiation was varied, the greatest decrease in cell survival was obtained when irradiation was given immediately before or after thermal treatment. Flow cytometry analysis showed that irradiation of exponentially growing cells with 5 Gy induced a G2 block, and that heat treatment immediately after 5 Gy exposure caused a progression delay of the cells from G2 + M to G1 phase. Furthermore, the cellular DNA histogram at 48 hours after 5 Gy exposure, immediately before heating, had more cells in G2 + M phase as compared with cells exposed to 5 Gy alone or thermal treatment at 5 hours before or after 5 Gy exposure. These results suggest that hyperthermia immediately after irradiation inhibits some of the cells accumulated in G2 + M phase from traversing into G1 phase. This inhibition may be related to the synergistic cell-killing by hyperthermia and radiation.