Mechanism of radiosensitization by hyperthermia (> or = 43 degrees C) as derived from studies with DNA repair defective mutant cell lines.

All biochemical and cytogenetic data on radiosensitization by heat treatment at and above 43 degrees C indicate that inhibition of DNA repair plays a central role. There are several DNA repair pathways involved in restoration of damage after ionising irradiation and the kinetics of all of them are affected by heat shock. This, however, does not imply that the inhibition of each of these pathways is relevant to the effect of heat on cellular radiosensitivity. The current review evaluates the available data on heat radiosensitization in mutant or knockout cell lines defective in various DNA repair proteins and/or pathways. The data show that thermal inhibition of the non-homologous end-joining pathway (NHEJ) plays no role in heat radiosensitization. Furthermore, limited data suggest that the homologous recombination pathway may also not be a major heat target. By deduction, it is suggested that inhibition of base damage repair (BER) could be the crucial step in radiosensitization by heat. While a lack of mutant cell lines and redundancy of the BER pathway have hampered efforts toward a conclusive study, biochemical and correlative evidence support this hypothesis.

Hyperthermic radiosensitization: mode of action and clinical relevance.

PURPOSE: To provide an update on the recent knowledge about the molecular mechanisms of thermal radiosensitization and its possible relevance to thermoradiotherapy. SUMMARY: Hyperthermia is probably the most potent cellular radiosensitizer known to date. Heat interacts with radiation and potentiates the cellular action of radiation by interfering with the cells' capability to deal with radiation-induced DNA damage. For ionizing irradiation, heat inhibits the repair of all types of DNA damage. Genetic and biochemical data suggest that the main pathways for DNA double-strand break (DSB) rejoining, non-homologous end-joining and homologous recombination, are not the likely primary targets for heat-induced radiosensitization. Rather, heat is suggested to affect primarily the religation step of base excision repair. Subsequently additional DSB arise during the DNA repair process in irradiated and heated cells and these additional DSB are all repaired with slow kinetics, the repair of which is highly error prone. Both mis- and non-rejoined DSB lead to an elevated number of lethal chromosome aberrations, finally causing additional cell killing. Heat-induced inhibition of DNA repair is considered not to result from altered signalling or enzyme inactivation but rather from alterations in higher-order chromatin structure. Although, the detailed mechanisms are not yet known, a substantial body of indirect and correlative data suggests that heat-induced protein aggregation at the level of attachment of looped DNA to the nuclear matrix impairs the accessibility of the damaged DNA for the repair machinery or impairs the processivity of the repair machinery itself. CONCLUSION: Since recent phase III clinical trials have shown significant benefit of adding hyperthermia to radiotherapy regimens for a number of malignancies, it will become more important again to determine the molecular effects underlying this success. Such information could eventually also improve treatment quality in terms of patient selection, improved sequencing of the heat and radiation treatments, the number of heat treatments, and multimodality treatments (i.e. thermochemoradiotherapy).

Role of DNA-PK subunits in radiosensitization by hyperthermia.

Thermal radiosensitization is thought to result from inhibition of repair of radiation-induced DNA damage, DNA double-strand breaks in particular. Since the DNA-dependent protein kinase (DNA-PK) complex plays a major role in the nonhomologous end-joining of DSBs, it has been suggested that inactivation of this complex as a whole or of its individual subunits by heat might be involved in radiosensitization by heat. To test this hypothesis further, the ability of heat to enhance the radiosensitivity of cells proficient or deficient in either Ku80 or the DNA-PK catalytic subunit (DNA-PKcs) was investigated. In cells of two Ku80-deficient and two DNA-PKcs-deficient and double-strand break-deficient cell lines, the extent of radiosensitization by heat was not reduced compared to that in both their isogenic gene-complemented counterparts as well as to that in their parental cells. Thus radiosensitization by hyperthermia can be obtained irrespective of the Ku80 or DNA-PKcs status in cells. Therefore, Ku80 or DNA-PKcs and hence nonhomologous DSB end-joining do not play a crucial role in the enhancement of cellular radiosensitivity by hyperthermia.

Nuclear matrix as a target for hyperthermic killing of cancer cells.

The nuclear matrix organizes nuclear DNA into operational domains in which DNA is undergoing replication, transcription or is inactive. The proteins of the nuclear matrix are among the most thermal labile proteins in the cell, undergoing denaturation at temperatures as low as 43-45 degrees C, i.e. relevant temperatures for the clinical treatment of cancer. Heat shock-induced protein denaturation results in the aggregation of proteins to the nuclear matrix. Protein aggregation with the nuclear matrix is associated with the disruption of many nuclear matrix-dependent functions (e.g. DNA replication, DNA transcription, hnRNA processing, DNA repair, etc.) and cell death. Heat shock proteins are believed to bind denatured proteins and either prevents aggregation or render aggregates more readily dissociable. While many studies suggest a role for Hsp70 in heat resistance, we have recently found that nuclear localization/delocalization of Hsp70 and its rate of synthesis, but not its amount, correlate with a tumor cell's ability to proliferate at 41.1 degrees C. These results imply that not only is the nuclear matrix a target for the lethal effects of heat, but it also is a target for the protective, chaperoning and/or enhanced recovery effects of heat shock proteins.

Rationale for different approaches to combined melphalan and hyperthermia in regional isolated perfusion.

The addition of hyperthermia (HT) to regional isolated perfusion (RIP) with Melphalan theoretically has two advantages. Firstly, heat can selectively kill cells in poorly vascularised areas that are usually not reached by the drug. Secondly, in vitro data have revealed that the effect of Melphalan is enhanced at temperatures 39-45 degrees C. However, for the simultaneous application of Melphalan and HT, as it is given in most institutes, both normal and tumour tissues within the volume are treated with both modalities. It is unclear whether--for the same heat dose--the cytotoxicity of Melphalan is enhanced more in tumour tissue than in normal tissues. As the applied dose of Melphalan in RIP is selected on maximum acceptable toxicity, any enhancement of toxicity is undesired. Indeed, Melphalan application at temperatures > 41 degrees C has resulted in unacceptable toxicity. In most institutes, the hyperthermia dose is reduced in comparison to application as a single-modality treatment, to allow simultaneous combination without unacceptable toxicity. In this review, the rationale for two different approaches is summarised which may make it possible to improve the benefit from the theoretical advantage of the use of HT in RIP. It is meant to stimulate discussion as a possible first step in the design of new treatment protocols.

Reduction of cellular cisplatin resistance by hyperthermia--a review.

Resistance to cisplatin (cDDP) is a major limitation to its clinical effectiveness. Review of literature data indicates that cDDP resistance is a multifactorial phenomenon. This provides an explanation why attempts to reverse or circumvent resistance using cDDP-analogues or combination therapy with modulators of specific resistance mechanisms have had limited success so far. It therefore provides a rationale to use hyperthermia, an agent with pleiotropic effects on cells, in trying to modulate cDDP resistance. In this review the effects of hyperthermia on cDDP cytotoxicity and resistance as well as underlying mechanisms are discussed. Hyperthermia is found to be a powerful modulator of cDDP cytotoxicity, both in sensitive and resistant cells. Relatively high heat doses (60 min 43 degrees C) seem to specifically interfere with cDDP resistance. The mechanism of interaction has not been fully elucidated so far, but seems to consist of multiple (simultaneous) effects on drug accumulation, adduct-formation and -repair. This may explain why hyperthermia seems to be so effective in increasing cDDP cytotoxicity, irrespective of the presence of resistance mechanisms. Therefore, the combination of hyperthermia and cDDP deserves further attention.

Mechanism of hyperthermic potentiation of cisplatin action in cisplatin-sensitive and -resistant tumour cells.

In this study, the mechanism(s) by which heat increases cis-diamminedichloroplatinum (cisplatin, cDDP) sensitivity in cDDP-sensitive and -resistant cell lines of murine as well as human origin were investigated. Heating cells at 43 degrees C during cDDP exposure was found to increase drug accumulation significantly in the cDDP-resistant cell lines but had little effect on drug accumulation in the cDDP-sensitive cell lines. DNA adduct formation, however, was significantly increased in all cell lines studied. Furthermore, ongoing formation of platinum (Pt)-DNA adducts after the end of cDDP treatment was enhanced and/or adduct removal was decreased in heated cells, resulting in relatively more DNA damage remaining at 24 h after the end of cDDP exposure. Correlation plots with survival revealed weak correlations with cellular Pt accumulation (r2 = 0.59) and initial Pt-DNA adduct formation (r2 = 0.64). Strong correlations, however, were found with Pt-DNA adducts at 6 h (r2 = 0.97) and 24 h (r2 = 0.89) after the incubation with the drug. In conclusion, the mechanism by which heat sensitizes cells for cDDP action seems to be the sum of multiple factors, which comprise heat effects on accumulation, adduct formation and adduct processing. This mechanism did not seem to differ between cDDP-sensitive and -resistant cells, emphasizing the potential of hyperthermia to reduce cDDP resistance.

Hyperthermia, thermotolerance and topoisomerase II inhibitors.

The cytoxicity of both intercalating (m-AMSA) and non-intercalating (VP16, VM26) topoisomerase II-targeting drugs is thought to occur via trapping DNA topoisomerase II on DNA in the form of cleavable complexes. First, analysis of cleavable complexes (detected as DNA double-strand breaks) by pulsed-field gel electrophoresis confirmed the correlation between cleavable complex formation and cytotoxicity of three topoisomerase-targeting drugs in HeLa S3 cells (the order of effects being VM26 > m-AMSA > VP16). In contrast to many antineoplastic agents, hyperthermic treatments were found to protect cells against the toxicity of all three topoisomerase II drugs. Hyperthermia treatment does not alter drug accumulation but reduces the ability of the drug-topoisomerase II complex to form the cleavable complexes. Nuclear protein aggregation induced by heat at the sites of topoisomerase II-DNA interaction may explain such an effect. In thermotolerant cells, the toxic effects of VP16 but not m-AMSA were reduced. For both drugs, however, the status of thermotolerance did not affect cleavable complex formation by the drugs. Thus, protection against VP-16 toxicity seems not to be associated with heat-induced activation of the P-gp 170 pump or altered topoisomerase II-DNA interactions. Rather, a protective (heat shock protein mediated?) mechanism against non-intercalating topoisomerase II drugs seems to occur at a stage after DNA-drug interaction. Finally, heat treatment before topoisomerase II drug treatment reduced toxicity and cleavable complex formation in thermotolerant cells to about the same extent as in non-tolerant cells, consistent with the presumption of nuclear protein aggregation being responsible for this effect.

Selective inhibition of repair of active genes by hyperthermia is due to inhibition of global and transcription coupled repair pathways.

Hyperthermia specifically inhibits the repair of UV-induced DNA photolesions in transcriptionally active genes. To define more precisely which mechanisms underlie the heat-induced inhibition of repair of active genes, removal of cyclobutane pyrimidine dimers (CPDs) was studied in human fibroblasts with different repair capacities and different transcriptional status of the adenosine deaminase gene, i.e. normal human cells, human cells carrying an inactive copy of the adenosine deaminase gene and xeroderma pigmentosum complementation group C fibroblasts. The results indicate that repair of active genes is impaired by inhibition of two repair pathways: (i) a global repair system involved in the repair of CPDs in potentially active genes; and (ii) the transcription-coupled repair pathway responsible for the accelerated repair of the transcribed strand. Since X-ray-induced DNA damage is also preferentially removed from the transcribed strand of active genes, selective inhibition of repair of radiation-induced DNA damage in active genes may play a key role in radiosensitization due to hyperthermia.

Cisplatin sensitivity and thermochemosensitisation in thermotolerant cDDP-sensitive and -resistant cell lines.

Development of thermotolerance is an important phenomenon that must be considered when thermochemotherapy with multiple heat treatments is used clinically. To study the effect of thermotolerance on cellular cisplatin (cDDP) sensitivity at 37 degrees C and 43 degrees C in cell lines with different cDDP sensitivities, two Ehrlich ascites tumour cell lines (one with high cDDP sensitivity and one with in vitro acquired cDDP resistance) were used. The results indicate that in both cell lines the state of thermotolerance per se did not affect the cDDP sensitivity at 37 degrees C. Thus, general elevations in 'all' heat shock protein levels as found in thermotolerant cells apparently do not influence cDDP sensitivity to a considerable extent. The sensitising effect of a (second) heat treatment given simultaneously with a cDDP treatment was less in thermotolerant cells. Thermal enhancement ratios (TERs) at the 10% survival level for heat doses of 43 degrees C for 30 min or 43 degrees C for 60 min were reduced by a factor of 1.6 and 2.1 in cDDP-resistant and -sensitive thermotolerant cells respectively, as compared with control cells. Thus, protection against heat damage in thermotolerant cells seems to be paralleled by diminished thermal chemosensitisation. Although the effect of thermotolerance on the cDDP-sensitising effect was less pronounced in the resistant cells, a modifying effect on the resistance factor was not achieved.

Hyperthermic potentiation of cisplatin toxicity in a human small cell lung carcinoma cell line and a cisplatin resistant subline.

A human small cell lung carcinoma cell line (GLC4) and its subline with in vitro acquired cisplatin (cDDP) resistance (GLC4-cDDP) were used to study the applicability of hyperthermia to interfere with acquired cDDP resistance. GLC4 and GLC4-cDDP did not differ in heat sensitivity (clonogenic ability). Both cell lines could be sensitized to cisplatin to a considerable extent, both at 42 and 43 degrees C. For 42 degrees C hyperthermia treatments up to 90 min no differences in TER between the cell lines were observed. Only prolonged (> or = 45 min) exposures to 43 degrees C hyperthermia sensitized the resistant cell line to a greater extent than the parent cell line, resulting in a reduction of the resistance factor from 3.6 (at 37 degrees C) to 1.7 (60 min 43 degrees C). The finding in this human system that for treatments up to 90 min, 43 degrees C heat is more suitable than 42 degrees C heat to reduce cDDP resistance, is in accordance with earlier findings with murine cells (Konings et al. 1993). Effects of heat, cisplatin and combined treatments on cell killing were not only measured with the clonogenic assay, but also with the microculture tetrazolium method (MTT assay), an assay of potential use in the clinic for rapid screening of cells obtained from patients. The data with the latter assay were comparable to those obtained with the clonogenic assay. However, its applicability to measure thermo-chemosensitization is limited due to its inability to measure more than one log of cell killing.

Cytotoxicity of flavonoids and sesquiterpene lactones from Arnica species against the GLC4 and the COLO 320 cell lines.

The cytotoxicity of 21 flavonoids and 5 sesquiterpene lactones, as present in Arnica species, was studied in GLC4, a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. Following continuous incubation, most flavonoids showed moderate to low cytotoxicity, as compared with the reference compound cisplatin (IC50 = 1.1 microM against GLC4 and 2.9 microM against COLO 320). Their IC50 values varied from 17 to > 200 microM. The most toxic compound was the flavone jaceosidin. Of the sesquiterpene lactones tested, helenalin, possessing both the reactive alpha-methylene-gamma-lactone moiety and a reactive alpha,beta-unsubstituted cyclopentenone ring, displayed the strongest cytotoxicity. For 2 h exposure, its IC50 value was 0.44 microM against GLC4 and 1.0 microM against COLO 320. COLO 320 was more sensitive than GLC4 for many flavonoids (especially for flavones), but more resistant to the cytotoxic effect of the sesquiterpene lactones bearing an exocylic methylene group fused to the lactone function.

Interaction between the cytostatic effects of quercetin and 5-fluorouracil in two human colorectal cancer cell lines.

Therapy with 5-fluorouracil (5-FU) as a single agent has only limited success in palliative treatment of cancer of the large bowel. In the current study, the effect of quercetin on the action of 5-FU in the human colorectal cancer cell lines COLO 320 DM and COLO 205 was evaluated using the MTT and clonogenic assays. Both assays were used in order to discriminate between cell growth inhibition and actual cell kill. As single agents as well as in combination, considerably higher concentrations of 5-FU and quercetin were required in the clonogenic assay to obtain detectable effects than in the MTT assay. In the MTT assay, but not in the clonogenic assay, a synergistic interaction between quercetin and 5-FU was found for COLO 320 DM cells. Our results therefore indicate that this interaction between quercetin and 5-FU mainly takes place at the level of growth inhibition. It is concluded that the cytostatic action of a combination of quercetin and 5-FU in COLO 320 DM cells is superior to that of the single agents.

Sensitizing for cis-diamminedichloroplatinum(II) action by hyperthermia in resistant cells.

cDDP-resistant Ehrlich ascites tumour (EAT) cells (ER cells) were tested for cellular content of total glutathione, heat sensitivity, cDDP sensitivity and synergistic effects of a combined treatment of heat and chemotherapy. In comparison with the non-resistant EAT cells (EN) the ER cells had an elevated level of glutathione. Treatment with D,L-buthionine-(S,R)-sulphoximine (BSO), resulting in almost complete depletion of cellular glutathione, did not cause drug sensitization. The ER cells were somewhat less heat sensitive compared with the EN cells. Heat chemosensitization was observed for the EN cells as well as for the ER cells. At 43 degrees C (but not at 42 degrees C) the thermal enhancement ratio (TER) for cDDP toxicity was significantly higher in the ER cells. The total number of cells killed by the combined treatment was less in the ER cells than in the EN cells. After analysing existing literature, combined with the current results, it is concluded that although cDDP-resistant cells can often considerably be chemosensitized by hyperthermia, in most cases the difference in cDDP sensitivity cannot be overcome totally. In those situations where cDDP-resistant cells are more sensitive to heat and also show a high TER, especially at clinically relevant temperatures, hyperthermia as added modality is indicated for clinical treatment.

Role of lipid peroxidation and DNA damage in paraquat toxicity and the interaction of paraquat with ionizing radiation.

Since the introduction of paraquat (PQ) as a herbicide in 1963, there have been many speculations concerning the critical lesion in PQ toxicity. Damage to membrane lipids might be an initial event leading to PQ-induced cell killing. The ability of PQ to induce lipid peroxidation was tested in liver homogenates of the mouse. Lipid peroxidation was indeed induced by PQ and shown to be dose dependent, starting to be significant at 2.5 mM. Subsequently, a possible correlation between lipid peroxidation and PQ-induced cell death was investigated in mouse fibroblasts (LM) and Ehrlich ascites tumour (EAT) cells using a clonogenic assay. It was found that in order to be cytotoxic PQ needs enzymatic activation (incubation at 37 degrees). In both cell lines, PQ-induced cell killing appeared to be dose dependent, starting at a dose of 0.5 mM. Supplementation of LM cells with the antioxidant vitamin E had no effect on PQ-induced cell killing and modification of the membranes of LM cells by incorporation of the polyunsaturated fatty acid 20:4 (arachidonic acid) did not sensitize the cells to PQ toxicity. PQ had no effect on the glutathione (GSH) level in EAT cells and complete GSH depletion by DL-buthionine-(SR)-sulfoximine could not sensitize the cells to PQ toxicity. In LM cells PQ-induced cell killing was enhanced after complete GSH depletion by DEM. This sensitization might, however, be attributed to the binding of DEM to proteins. From these results it seems unlikely that lipid peroxidation is the primary cause for PQ-induced cell killing. Another critical target in PQ toxicity is DNA. This possibility was investigated in EAT cells. PQ was found to induce DNA damage (detected by the alkaline unwinding assay) in the same dose range that caused cell death. A good correlation was obtained for cell killing after PQ treatment and DNA damage measured 2 hr after 37 degrees post-incubation. A proposed possible interaction between PQ and X-rays was also investigated. In EAT cells, X-ray-induced cell death was significantly enhanced by pre-incubation with PQ at doses of 0.5 mM and above. At the level of 10% survival an enhancement factor of 1.6 could be observed by treatment with 1 mM PQ when cell killing by PQ is not taken into account. Induction as well as processing of radiation-induced DNA damage seems to be unaffected by pre-incubation with PQ. The mechanism of radiosensitization by PQ is yet unclear.

Reduced DNA break formation and cytotoxicity of the topoisomerase II drug 4'-(9'-acridinylamino)methanesulfon-m-anisidide when combined with hyperthermia in human and rodent cell lines.

The interaction between hyperthermia and the anticancer drug 4'-(9'-acridinylamino)methanesulfon-m-anisidide (mAMSA) was studied in the human HeLa S3 and the rodent Ehrlich ascites tumor cell line. For both cell lines it was found that hyperthermia preceding the drug treatment reduced the extent of mAMSA induced DNA breakage as well as mAMSA cytotoxicity. Formation and resealing of mAMSA induced DNA break formation was found to be related to cytotoxicity. Hyperthermic protection for the action of mAMSA was found not to be a result of changed permeability for the drug. The data also do not support the possibility that heat has caused inactivation of the putative target enzyme of mAMSA, topoisomerase II. It is suggested that the hyperthermic protection for the mAMSA drug action is due to a hyperthermic alteration of the chromatin organization, especially at topoisomerase II target sequences that are found to be enriched in the nuclear matrix (P.N. Cockerill and W.T. Garrard. Cell, 44: 273-282, 1986). We show here that heat has caused an alteration of protein binding to the nucleus that seems related to the hyperthermic inhibition of mAMSA induced DNA break induction. It is concluded that preheating cells before treatment with mAMSA should not be used, at least not in this sequence, in cancer therapy.