Radiosensitization by Hyperthermia: The Effects of Temperature, Sequence, and Time Interval in Cervical Cell Lines.

Cervical cancers are almost exclusively caused by an infection with the human papillomavirus (HPV). When patients suffering from cervical cancer have contraindications for chemoradiotherapy, radiotherapy combined with hyperthermia is a good treatment option. Radiation-induced DNA breaks can be repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Hyperthermia can temporarily inactivate homologous recombination. Therefore, combining radiotherapy with hyperthermia can result in the persistence of more fatal radiation-induced DNA breaks. However, there is no consensus on the optimal sequence of radiotherapy and hyperthermia and the optimal time interval between these modalities. Moreover, the temperature of hyperthermia and HPV-type may also be important in radiosensitization by hyperthermia. In this study we thoroughly investigated the impact of different temperatures (37-42 °C), and the sequence of and time interval (0 up to 4 h) between ionizing radiation and hyperthermia on HPV16+: SiHa, Caski; HPV18+: HeLa, C4I; and HPV-: C33A, HT3 cervical cancer cell lines. Our results demonstrate that a short time interval between treatments caused more unrepaired DNA damages and more cell kill, especially at higher temperatures. Although hyperthermia before ionizing radiation may result in slightly more DNA damage, the sequence between hyperthermia and ionizing radiation yielded similar effects on cell survival.

Increased uptake of doxorubicin by cells undergoing heat stress does not explain its synergistic cytotoxicity with hyperthermia.

Purpose: A proposed mechanism for the enhanced effectiveness of hyperthermia and doxorubicin (Dox) combinations is increased intracellular Dox concentrations resulting from heat-induced cell stress. The purpose of this study was to determine whether specific varied Dox and heat combinations produce measurable effects greater than the additive combination, and whether these effects can be attributed to heat-induced increases in intracellular Dox concentrations. Methods: HCT116, HT29 and CT26 cells were exposed to Dox and water bath heating independently. A clonogenic survival assay was used to determine cell killing and intracellular Dox concentrations were measured in HCT116 cells with mass spectrometry. Cells were exposed to heating at 42 °C (60 min) and 0.5 µg/ml of Dox at varying intervals. Synergy was determined by curve-fitting and isobologram analysis. Results: All cell lines displayed synergistic effects of combined heating and Dox. A maximum synergistic effect was achieved with simultaneous cell exposure to Dox and heat. For exposures at 42 °C, the synergistic effect was most pronounced at Dox concentrations <0.5 µg/ml. Increased intracellular concentrations of Dox in HCT116 cells caused by heat-stress did not generate a concomitant thermal enhancement. Conclusions: Simultaneous exposure of HCT116 cells to heating and Dox is more effective than sequential exposure. Heat-induced cell responses are accompanied by increased intracellular Dox concentrations; however, clonogenic survival data do not support this as the cause for synergistic cytotoxicity.

Enhancing radiosensitisation of BRCA2-proficient and BRCA2-deficient cell lines with hyperthermia and PARP1-i.

Poly(ADP-ribose)polymerase1 (PARP1) is an important enzyme in regulating DNA replication. Inhibition of PARP1 can lead to collapsed DNA forks which subsequently causes genomic instability, making DNA more susceptible in developing fatal DNA double strand breaks. PARP1-induced DNA damage is generally repaired by homologous recombination (HR), in which BRCA2 proteins are essential. Therefore, BRCA2-deficient tumour cells are susceptible to treatment with PARP1-inhibitors (PARP1-i). Recently, BRCA2 was shown to be down-regulated by hyperthermia (HT) temporarily, and this consequently inactivated HR for several hours. In this study, we investigated whether HT exclusively interferes with HR by analysing thermal radiosensitisation of BRCA2-proficient and deficient cells. After elucidating the equitoxicity of PARP1-i on BRCA2-proficient and deficient cells, we studied the cell survival, apoptosis, DNA damage (γ-H2AX foci and comet assay) and cell cycle distribution after different treatments. PARP1-i sensitivity strongly depends on the BRCA2 status. BRCA2-proficient and deficient cells are radiosensitised by HT, indicating that HT does not exclusively act by inhibition of HR. In all cell lines, the addition of HT to radiotherapy and PARP1-i resulted in the lowest cell survival, the highest levels of DNA damage and apoptotic levels compared to duo-modality treatments. Concluding, HT not only inhibits HR, but also has the capability of radiosensitising BRCA2-deficient cells. Thus, in case of BRCA2-mutation carriers, combining HT with PARP1-i may boost the treatment efficacy. This combination therapy would be effective for all patients with PARP1-i regardless of their BRCA status.

Enhancing synthetic lethality of PARP-inhibitor and cisplatin in BRCA-proficient tumour cells with hyperthermia.

BACKGROUND: Poly-(ADP-ribose)-polymerase1 (PARP1) is involved in repair of DNA single strand breaks. PARP1-inhibitors (PARP1-i) cause an accumulation of DNA double strand breaks, which are generally repaired by homologous recombination (HR). Therefore, cancer cells harboring HR deficiencies are exceptionally sensitive to PARP1-i. For patients with HR-proficient tumors, HR can be temporarily inhibited by hyperthermia, thereby inducing synthetic lethal conditions in every tumor type. Since cisplatin is successfully used combined with hyperthermia (thermochemotherapy), we investigated the effectiveness of combining PARP1-i with thermochemotherapy. RESULTS: The in vitro data demonstrate a decreased in cell survival after addition of PARP1-i to thermochemotherapy, which can be explained by increased DNA damage induction and less DSB repair. These in vitro findings are in line with in vivo model, in which a decreased tumor growth is observed upon addition of PARP1-i. MATERIALS AND METHODS: Survival of three HR-proficient cell lines after cisplatin, hyperthermia and/or PARP1-i was studied. Cell cycle analyses, quantification of γ-H2AX foci and apoptotic assays were performed to understand these survival data. The effects of treatments were further evaluated by monitoring tumor responses in an in vivo rat model. CONCLUSIONS: Our results in HR-proficient cell lines suggest that PARP1-i combined with thermochemotherapy can be a promising clinical approach for all tumors independent of HR status.

Prostate Cancer Patients with Late Radiation Toxicity Exhibit Reduced Expression of Genes Involved in DNA Double-Strand Break Repair and Homologous Recombination.

Severe late damage to normal tissue is a major limitation of cancer radiotherapy in prostate cancer patients. In a recent retrospective study, late radiation toxicity was found to relate to a decreased decay of γ-H2AX foci and reduced induction of DNA double-strand break repair genes. Here, we report evidence of prognostic utility in prostate cancer for γ-H2AX foci decay ratios and gene expression profiles derived from ex vivo-irradiated patient lymphocytes. Patients were followed ≥2 years after radiotherapy. Clinical characteristics were assembled, and toxicity was recorded using the Common Terminology Criteria (CTCAE) v4.0. No clinical factor was correlated with late radiation toxicity. The γ-H2AX foci decay ratio correlated negatively with toxicity grade, with a significant difference between grade ≥3 and grade 0 patients (P = 0.02). A threshold foci decay ratio, determined in our retrospective study, correctly classified 23 of 28 patients with grade ≥3 toxicity (sensitivity 82%) and 9 of 14 patients with grade 0 toxicity (specificity 64%). Induction of homologous recombination (HR) repair genes was reduced with increasing toxicity grade. The difference in fold induction of the HR gene set was most pronounced between grade 0 and grade ≥3 toxicity (P = 0.008). Notably, reduced responsiveness of HR repair genes to irradiation and inefficient double-strand break repair correlated with severe late radiation toxicity. Using a decay ratio classifier, we correctly classified 82% of patients with grade ≥3 toxicity, suggesting a prognostic biomarker for cancer patients with a genetically enhanced risk for late radiation toxicity to normal tissues after radiotherapy. Cancer Res; 77(6); 1485-91. ©2017 AACR.

Sensitizing thermochemotherapy with a PARP1-inhibitor.

Cis-diamminedichloroplatinum(II) (cisplatin, cDDP) is an effective chemotherapeutic agent that induces DNA double strand breaks (DSBs), primarily in replicating cells. Generally, such DSBs can be repaired by the classical or backup non-homologous end joining (c-NHEJ/b-NHEJ) or homologous recombination (HR). Therefore, inhibiting these pathways in cancer cells should enhance the efficiency of cDDP treatments. Indeed, inhibition of HR by hyperthermia (HT) sensitizes cancer cells to cDDP and in the Netherlands this combination is a standard treatment option for recurrent cervical cancer after previous radiotherapy. Additionally, cDDP has been demonstrated to disrupt c-NHEJ, which likely further increases the treatment efficacy. However, if one of these pathways is blocked, DSB repair functions can be sustained by the Poly-(ADP-ribose)-polymerase1 (PARP1)-dependent b-NHEJ. Therefore, disabling b-NHEJ should, in principle, further inhibit the repair of cDDP-induced DNA lesions and enhance the toxicity of thermochemotherapy. To explore this hypothesis, we treated a panel of cancer cell lines with HT, cDDP and a PARP1-i and measured various end-point relevant in cancer treatment. Our results demonstrate that PARP1-i does not considerably increase the efficacy of HT combined with standard, commonly used cDDP concentrations. However, in the presence of a PARP1-i, ten-fold lower concentration of cDDP can be used to induce similar cytotoxic effects. PARP1 inhibition may thus permit a substantial lowering of cDDP concentrations without diminishing treatment efficacy, potentially reducing systemic side effects.

Targeting DNA double strand break repair with hyperthermia and DNA-PKcs inhibition to enhance the effect of radiation treatment.

Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation.The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 µM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found.In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.

Dynamics of chromosomal aberrations, induction of apoptosis, BRCA2 degradation and sensitization to radiation by hyperthermia.

Hyperthermia can transiently degrade BRCA2 and thereby inhibit the homologous recombination pathway. Induced DNA-double strand breaks (DSB) then have to be repaired via the error prone non-homologous end-joining pathway. In the present study, to investigate the role of hyperthermia in genotoxicity and radiosensitization, the induction of chromosomal aberrations was examined by premature chromosome condensation and fluorescence in situ hybridisation (PCC-FISH), and cell survival was determined by clonogenic assay shortly (0-1 h) and 24 h following exposure to hyperthermia in combination with ionizing radiation. Prior to exposure to 4 Gy γ-irradiation, confluent cultures of SW­1573 (human lung carcinoma) and RKO (human colorectal carcinoma) cells were exposed to mild hyperthermia (1 h, 41˚C). At 1 h, the frequency of chromosomal translocations was higher following combined exposure than following exposure to irradiation alone. At 24 h, the number of translocations following combined exposure was lower than following exposure to irradiation only, and was also lower than at 1 h following combined exposure. These dynamics in translocation frequency can be explained by the hyperthermia-induced transient reduction of BRCA2 observed in both cell lines. In both cell lines exposed to radiation only, potentially lethal damage repair (PLDR) correlated with a decreased number of chromosomal fragments at 24 h compared to 1 h. With combined exposure, PLDR did not correlate with a decrease in fragments, as in the RKO cells at 24 h following combined exposure, the frequency of fragments remained at the level found after 1 h of exposure and was also significantly higher than that found following exposure to radiation alone. This was not observed in the SW­1573 cells. Cell survival experiments demonstrated that exposure to hyperthermia radiosensitized the RKO cells, but not the SW­1573 cells. This radiosensitization was at least partly due to the induction of apoptosis, which was only observed in the RKO cells and which may have been induced by BRCA2 degradation or different types of chromosomal aberrations. An important observation of this study is that the genotoxic effect of hyperthermia shortly after combined epxosure (to hyperthermia and radiation) is not observed at 24 h after treatment.

Hyperthermia Selectively Targets Human Papillomavirus in Cervical Tumors via p53-Dependent Apoptosis.

Human papillomavirus (HPV) is associated with cervical cancer, the third most common cancer in women. The high-risk HPV types 16 and 18 are found in over 70% of cervical cancers and produce the oncoprotein, early protein 6 (E6), which binds to p53 and mediates its ubiquitination and degradation. Targeting E6 has been shown to be a promising treatment option to eliminate HPV-positive tumor cells. In addition, combined hyperthermia with radiation is a very effective treatment strategy for cervical cancer. In this study, we examined the effect of hyperthermia on HPV-positive cells using cervical cancer cell lines infected with HPV 16 and 18, in vivo tumor models, and ex vivo-treated patient biopsies. Strikingly, we demonstrate that a clinically relevant hyperthermia temperature of 42 °C for 1 hour resulted in E6 degradation, thereby preventing the formation of the E6-p53 complex and enabling p53-dependent apoptosis and G2-phase arrest. Moreover, hyperthermia combined with p53 depletion restored both the cell-cycle distribution and apoptosis to control levels. Collectively, our findings provide new insights into the treatment of HPV-positive cervical cancer and suggest that hyperthermia therapy could improve patient outcomes.

Inhibition of homologous recombination by hyperthermia shunts early double strand break repair to non-homologous end-joining.

In S and G2 phase mammalian cells DNA double strand breaks (DSBs) can potentially be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Results of several studies suggest that these two mechanistically distinct repair pathways can compete for DNA ends. Because HR and NHEJ differ with respect to error susceptibility, generation of chromosome rearrangements, which are potentially carcinogenic products of DSB repair, may depend on the pathway choice. To investigate this hypothesis, the influence of HR and NHEJ inhibition on the frequencies of chromosome aberrations in G2 phase cells was investigated. SW-1573 and RKO cells were treated with mild (41 °C) hyperthermia in order to disable HR and/or NU7441/cisplatin to inactivate NHEJ and frequencies of chromosomal fragments (resulting from unrepaired DSBs) and translocations (products of erroneous DSB rejoining) were studied using premature chromosome condensation (PCC) combined with fluorescence in situ hybridization (FISH). It is shown here that temporary inhibition of HR by hyperthermia results in increased frequency of ionizing-radiation (IR)-induced chromosomal translocations and that this effect is abrogated by NU7441- or cisplatin-mediated inhibition of NHEJ. The results suggest that in the absence of HR, DSB repair is shifted to the error-prone NHEJ pathway resulting in increased frequencies of chromosomal rearrangements. These results might be of consequence for clinical cancer treatment approaches that aim at inhibition of one or more DSB repair pathways.

Relative biological effectiveness of high linear energy transfer α-particles for the induction of DNA-double-strand breaks, chromosome aberrations and reproductive cell death in SW-1573 lung tumour cells.

Ionizing radiation-induced foci (IRIF) of DNA repair-related proteins accumulated at DNA double-strand break (DSB) sites have been suggested to be a powerful biodosimetric tool. However, the relationship between IRIF induction and biologically relevant endpoints, such as cell death and formation of chromosome rearrangements is less clear, especially for high linear energy transfer (LET) radiation. It is thus not sufficiently established whether IRIF are valid indicators of biological effectiveness of the various radiation types. This question is more significant in light of the recent advancements in light ion-beam and radionuclide therapy. Dose-effect relationships were determined for the induction of DNA-DSBs, chromosome aberrations and reproductive cell death in cultured SW-1573 cells irradiated with γ-rays from a Cs-137 source or with α-particles from an Am-241 source. Values of relative biological effectiveness (RBE) of the high LET α-particles were derived for these effects. DNA-DSB were detected by scoring of γ-H2AX foci, chromosome aberrations by fragments and translocations using premature chromosome condensation and cell survival by colony formation. Analysis of dose-effect relations was based on the linear-quadratic model. Except for the survival curves, for other effects no significant contribution was derived of the quadratic term in the range of doses up to 2 Gy of γ-rays. Calculated RBE values derived for the linear component of dose-effect relations for γ-H2AX foci, cell reproductive death, chromosome fragments and colour junctions are 1.0±0.3, 14.7±5.1, 15.3±5.9 and 13.3±6.0, respectively. RBE values calculated at a certain biological effect level are 1, 4, 13 and 13, respectively. The RBE values derived from the LQ model are preferred as they are based on clinically relevant doses. The results show that with low LET radiation only a small fraction of the numerous DNA-DSBs yield chromosome damage and reproductive cell death. It is concluded that many of the chromosomal aberrations detected by premature chromosome condensation do not cause reproductive cell death. Furthermore, RBE values for DNA-DSB detectable by γ-H2AX foci shortly after irradiation, provide no information relevant to applications of high LET radiation in radiotherapy. The RBE values of chromosome aberrations assessed by premature chromosome condensation are close to the value for reproductive cell death. This suggests possible relevance to assess RBE values for radiotherapy with high LET ions.

Comparison of RBE values of high-LET α-particles for the induction of DNA-DSBs, chromosome aberrations and cell reproductive death.

BACKGROUND: Various types of radiation effects in mammalian cells have been studied with the aim to predict the radiosensitivity of tumours and normal tissues, e.g. DNA double strand breaks (DSB), chromosome aberrations and cell reproductive inactivation. However, variation in correlations with clinical results has reduced general application. An additional type of information is required for the increasing application of high-LET radiation in cancer therapy: the Relative Biological Effectiveness (RBE) for effects in tumours and normal tissues. Relevant information on RBE values might be derived from studies on cells in culture. METHODS: To evaluate relationships between DNA-DSB, chromosome aberrations and the clinically most relevant effect of cell reproductive death, for ionizing radiations of different LET, dose-effect relationships were determined for the induction of these effects in cultured SW-1573 cells irradiated with gamma-rays from a Cs-137 source or with α-particles from an Am-241 source. RBE values were derived for these effects. Ionizing radiation induced foci (IRIF) of DNA repair related proteins, indicative of DSB, were assessed by counting gamma-H2AX foci. Chromosome aberration frequencies were determined by scoring fragments and translocations using premature chromosome condensation. Cell survival was measured by colony formation assay. Analysis of dose-effect relations was based on the linear-quadratic model. RESULTS: Our results show that, although both investigated radiation types induce similar numbers of IRIF per absorbed dose, only a small fraction of the DSB induced by the low-LET gamma-rays result in chromosome rearrangements and cell reproductive death, while this fraction is considerably enhanced for the high-LET alpha-radiation. Calculated RBE values derived for the linear components of dose-effect relations for gamma-H2AX foci, cell reproductive death, chromosome fragments and colour junctions are 1.0 ± 0.3, 14.7 ± 5.1, 15.3 ± 5.9 and 13.3 ± 6.0 respectively. CONCLUSIONS: These results indicate that RBE values for IRIF (DNA-DSB) induction provide little valid information on other biologically-relevant end points in cells exposed to high-LET radiations. Furthermore, the RBE values for the induction of the two types of chromosome aberrations are similar to those established for cell reproductive death. This suggests that assays of these aberrations might yield relevant information on the biological effectiveness in high-LET radiotherapy.

Tumor microvasculature supports proliferation and expansion of glioma-propagating cells.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The identification of 'cancer stem cells' (CSC) has shed new light on the potential mechanism of therapy resistance of these tumors. Because these cells appear to be more resistant to conventional treatments, they are thought to drive tumor regrowth after therapy. Therefore, novel therapeutic approaches that target these cells are needed. Tumor cells interact with their microenvironment. It has been reported that close contact between CSCs and tumor microvascular endothelium in GBM is important for CSCs to preserve their undifferentiated state and self-renewal ability. However, our understanding of this interaction is still rudimentary. This is in part due to a lack of suitable in vitro models that accurately represent the in vivo situation. Therefore, we set up a co-culture system consisting of primary brain tumor microvascular endothelial cells (tMVECs) and glioma propagating cells (GPCs) derived from biopsies of GBM patients. We found that tMVECs support the growth of GPCs resulting in higher proliferation rates comparing to GPCs cultured alone. This effect was dependent on direct contact between the 2 cell types. In contrast to GPCs, the FCS-cultured cell line U87 was stimulated by culturing on tMVEC-derived ECM alone, suggesting that both cell types interact different with their microenvironment. Together, these results demonstrate the feasibility and utility of our system to model the interaction of GPCs with their microenvironment. Identification of molecules that mediate this interaction could provide novel targets for directed therapy for GBM.

G0 cell cycle arrest alone is insufficient for enabling the repair of ionizing radiation-induced potentially lethal damage.

The repair of ionizing radiation-induced potentially lethal damage (PLD) is suggested to be important for the clinical response to radiotherapy. PLD repair is usually studied in quiescent cultures prepared by growing cells to confluence with an accumulation of cells in G(0) phase of the cell cycle, but the biological pathways enabling PLD repair are still unknown. In this study, we examined whether the controlled expression of two different inducers of G(0) cell cycle arrest, the human tumor suppressor gene growth arrest specific 1 (GAS1) in murine fibroblasts and the forkhead transcription factor FOXO3a in human colon carcinoma cells, is sufficient to enable PLD repair. We found that GAS1 and FOXO3a induced a cell cycle arrest in G(0) phase with a concomitant reduction of proliferation of log-phase cells. In both cell systems, this cell cycle arrest in G(0) phase did not enable PLD repair in log-phase cells. Significant PLD repair was found in all confluent cultures that showed similar cell cycle distributions, while GAS1 and FOXO3a in confluent cells did not influence PLD repair. No differences were found in cell cycle re-entry after replating cells with different capacities for PLD repair. Our data suggest that the induction of G(0) cell cycle arrest and the reduction of proliferation are not sufficient to enable PLD repair.

Chromosome fragments have the potential to predict hyperthermia-induced radio-sensitization in two different human tumor cell lines.

Cellular radiosensitivity, assessed by loss of clonogenicity, has been shown to correlate with the number of radiation-induced chromosomal aberrations. Also an increased radiosensitivity by hyperthermia has been shown to correlate with an increase in chromosomal aberrations. Therefore, determination of the number of chromosomal aberrations might be used as an assay to predict the radiosensitivity of tumors pre-treated with hyperthermia at clinically relevant temperatures. The use of premature chromosome condensation combined with fluorescent in situ hybridisation (PCC-FISH) has been shown to be clinically applicable. Therefore, the use of chromosomal aberrations as determined with PCC-FISH for the prediction of hyperthermia-induced radio-sensitization in human tumor cells was investigated. Confluent cultures of SW-1573 (human lung carcinoma) and RKO (human colorectal carcinoma) cells were treated with 1 h 41 degrees C or 43 degrees C hyperthermia prior to gamma-irradiation. Clonogenic cell survival and induction of chromosomal aberrations (unrejoined chromosomal fragments and translocations), by PCC-FISH, were studied at 24 h after treatment. Pre-treatment with hyperthermia at 41 degrees C for 1 h enhanced the radiosensitivity of RKO cells but not of SW-1573 cells. Increasing the temperature to 43 degrees C for 1 h enhanced the radiosensitivity of SW-1573 cells. When radio-sensitization was observed, a significant increase in the number of unrejoined chromosomal fragments was found but the frequency of translocations was not increased. Hyperthermia-induced radio-sensitization is correlated with an increase in unrejoined chromosomal fragments. This suggests that determination of the number of chromosomal fragments after hyperthermia and radiation treatment might be used for the prediction of combined treatment response in cancer patients.

Effect of 41 degrees C and 43 degrees C on cisplatin radiosensitization in two human carcinoma cell lines with different sensitivities for cisplatin.

The effect of trimodality treatment consisting of hyperthermia, cisplatin and radiation was investigated in two cell lines with different sensitivities to cisplatin. Hyperthermia treatment was performed for 1 h at 41 degrees C and 43 degrees C in order to compare the effects of the two temperatures. Clonogenic assays were performed with cisplatin-sensitive SiHa human cervical carcinoma and cisplatin-resistant SW-1573 human lung carcinoma cell lines. Cells were treated with various combinations of hyperthermia, cisplatin and radiation. Radiation was performed after 1 h of simultaneous hyperthermia and cisplatin treatment. Cisplatin exposure was for 1 h or continuous without refreshment of the cisplatin-containing medium. SiHa cells were more sensitive to cisplatin than SW-1573 cells. Hyperthermia at 41 degrees C decreased survival in SW-1573 cells but was not cytotoxic in SiHa cells. Hyperthermia at 43 degrees C decreased survival dramatically in both cell lines with SiHa being the most sensitive. The addition of hyperthermia at 41 degrees C and 43 degrees C to cisplatin treatment led to enhanced cell kill in both cell lines compared with cisplatin alone. Radiosensitization was observed after continuous but not after 1 h of cisplatin treatment. Hyperthermia at 43 degrees C increased radiosensitivity whereas hyperthermia at 41 degrees C did not. A combination of 41 degrees C hyperthermia with continuous cisplatin treatment had an additive effect on SW-1573 cells but enhanced cisplatin radiosensitivity of SiHa cells. In SW-1573 cells trimodality treatment using 43 degrees C hyperthermia enhances cisplatin radiosensitivity. We conclude that hyperthermia at 43 degrees C enhances cisplatin-induced radiosensitization in both cisplatin-sensitive and -resistant cell lines. Hyperthermia at 41 degrees C was also able to increase cisplatin-induced radiosensitivity but only in the cisplatin-sensitive SiHa cell line.

Induction of the early response protein EGR-1 in human tumour cells after ionizing radiation is correlated with a reduction of repair of lethal lesions and an increase of repair of sublethal lesions.

The role of EGR-1 in potentially lethal damage repair (PLDR) was studied. Induction of the early response protein EGR-1 and survival after ionizing radiation of two human tumour cell lines after culturing for 48 h in serum-deprived medium was investigated. The glioblastoma cell line (Gli-6) and a lung carcinoma cell line (SW-1573) were selected as these cell lines differ considerably in the degree of PLD repair after radiation. In both cell lines induction of EGR-1 protein was observed between 30-120 min after treatment with 10 Gy in serum-deprived cultures. In cells growing in medium with normal serum no induction of EGR-1 was observed. No difference in EGR-1 expression levels between the two cell lines was detected. Linear-Quadratic analysis of the survival curves showed a much larger difference between the values of alpha after immediate and delayed plated cells of the cultures in normal serum as compared to cells cultured in serum-deprived medium. The cells cultured in serum-deprived medium showed much larger difference between the values of beta. This indicates that induction of EGR-1 is correlated with a reduction of repair of lethal lesions (PLDRalpha) and with an increase of repair of sublethal lesions (PLDRbeta).