Fitting the linear-quadratic model to detailed data sets for different dose ranges.

Survival curve behaviour and degree of correspondence between the linear-quadratic (LQ) model and experimental data in an extensive dose range for high dose rates were analysed. Detailed clonogenic assays with irradiation given in 0.5 Gy increments and a total dose range varying from 10.5 to 16 Gy were performed. The cell lines investigated were: CHOAA8 (Chinese hamster fibroblast cells), U373MG (human glioblastoma cells), CP3 and DU145 (human prostate carcinoma cell lines). The analyses were based on chi2-statistics and Monte Carlo simulation of the experiments. A decline of LQ fit quality at very low doses (<2 Gy) is observed. This result can be explained by the hypersensitive effect observed in CHOAA8, U373MG and DU145 data and an adaptive-type response in the CP3 cell line. A clear improvement of the fit is discerned by removing the low dose data points. The fit worsening at high doses also shows that LQ cannot explain this region. This shows that the LQ model fits better the middle dose region of the survival curve. The analysis conducted in our study reveals a dose dependency of the LQ fit in different cell lines.

Evaluation of adaptive responses to cisplatin in normal and mutant cell lines with mutations in recombination repair pathways.

Cell lines mutant in specific DNA repair pathways were used to determine if these pathways are involved in adaptive responses. For these studies, the effect of deficiencies in homologous recombination repair (HR) were studied in the parental AA8 and mutant (irs)ISF cell line pair and for deficiencies in the nonhomologous endjoining (NHEJ) pathway in the mouse MEF parental and Ku80 mutant cell line pair. The results showed that the XRCC3 mutation in the HR-deficient mutant inhibited adaptive responses to low doses of cisplatin and radiation. The parental lines showed transient adaptive responses to both low-dose cisplatin and radiation treatment. For the mouse MEF and the Ku80 cells, no adaptive responses were observed in either cell line. However, there was an initial transient sensitization response followed by partial recovery. Thus, it appears that the HR repair system may be involved in the adaptive response to cisplatin and radiation. For the NHEJ repair system the question could not be answered since no adaptive responses were evident in the parental line.

Response to pulsed dose rate and low dose rate irradiation with and without mild hyperthermia using human breast carcinoma cell lines.

The purpose of this study was to establish whether a pulsed dose rate (PDR) treatment of 1.5 Gy given every 3 h in combination with 41 degrees C mild hyperthermia or a continuous low dose rate (LDR) treatment with mild hyperthermia could radiosensitize two isogenic human breast carcinoma cell lines in comparison to pulsed dose rate or low dose rate irradiation alone. The radiation resistant cell line was derived from the parental cell line and was transfected to over-express DNA polymerase beta. The end-points assessed were the survival of the cells using the clonogenic assay, the amount of residual DSB(s) using the comet assay and gene expression of polymerase beta using RT-PCR. Results showed that the PDR and LDR treatments combined with mild hyperthermia caused significant radiosensitization when compared to PDR and LDR irradiation alone in terms of the clonogenic and comet assays with both cell lines. RT-PCR results showed that polymerase beta levels of expression were not elevated in response to these treatments, implying that this polymerase may not be involved in sub-lethal damage repair or thermal radiosensitization. These results suggest a potential clinical advantage when combining LDR or PDR with hyperthermia, since they indicate that hyperthermia is an effective radiosensitizer.

The evaluation of thermal cisplatin sensitization in normal and XP human cells using mild hyperthermia at 40 and 41 degrees C.

The effect of protracted mild hyperthermia treatment at 40 and 41 degrees C given, concurrently with cisplatin, was evaluated in human normal AG1522 and human mutant XPA cells. While mild hyperthermia itself for up to 6 hours showed little to no toxic effects, it did result in significant sensitization of response to cisplatin treatment. Sensitization for the normal and mutant cell line was comparable, indicating that nucleotide excision repair (NER) probably does not have a role in this process. For the 41 degrees C heating, thermotolerance developed and heating times greater than 4 hours resulted in protective effects from cisplatin cytotoxicity. This was not observed for heating at 40 degrees C for up to 6 hours.

Cisplatin sensitization by concurrent mild hyperthermia in parental and mutant cell lines deficient in homologous recombination and non-homologous endjoining repair.

The effect of mild hyperthermia on cisplatin sensitization was examined in two cell line pairs, CHO parental AA8 and irsISF, an XRCC3 mutant (deficient in homologous recombination repair), and mouse parental MEF and knockout Ku80 mutants (deficient in non-homologous endjoining repair). The results showed that mild hyperthermia 40, 41 and 42 degrees C given concurrently with cisplatin treatment caused significant sensitization. The degree of sensitization was comparable for the parental and mutant lines, indicating that these repair pathways were likely not involved in cisplatin thermal sensitization. The shorter concurrent treatments cause a larger sensitization than the longer treatments. The reasons for this are not clear, but thermotolerance may be a factor.

A comparison of response to cisplatin, radiation and combined treatment for cells deficient in recombination repair pathways.

The responses of cells with mutated DNA repair pathways were compared for cisplatin, radiation and combination treatments. The knockout of the nonhomologous endjoining (NHEJ) pathway resulted in increased radiation sensitivity, but no change in cisplatin response in the mouse cells and increased radiosensitivity but decreased cisplatin sensitivity in chicken cells. The mutation of the homologous recombination repair (HR) pathway through XRCC3 in CHO cells resulted in increased radiation and cisplatin sensitivity and to a lesser extent for the Rad54 knockout in the DT40 chicken cells. The combination treatments of cisplatin and radiation showed that inhibition of the HR repair pathway resulted in super additive effects while the inhibition of the NHEJ pathway in DT40 had no effect. In mouse cells the knockout of the NHEJ pathway resulted in reduced super additivity compared to the parental cell lines. These data show that radiation, cisplatin and combination treatment damage is affected differently by the various DNA repair pathways, which could have a range of effects on combination treatments in tumour cells expressing different levels of DNA repair in the various repair pathways.

Is DNA polymerase beta important in thermal radiosensitization?

Thermal radiosensitization was tested in a pair of mouse cells (MB+ wild-type and MB-, DNA polymerase beta knockout cells) and in human breast carcinoma cells (MCF7 wild-type and C716 transfected to give elevated DNA polymerase beta expression). Results showed that neither reducing DNA polymerase beta (involved in base excision repair) nor increasing it had any significant effect on thermal radiosensitization. The data indicated that polymerase beta was not involved in thermal radiosensitization, and since hyperthermia is known as a radiation damage repair inhibitor, other repair pathways might be involved and need to be explored.

Long duration mild temperature hyperthermia and brachytherapy.

Combining long duration mild temperature hyperthermia (LDMH) and low dose-rate (LDR) brachytherapy to enhance therapeutic killing of cancer cells was proposed many years ago. The cellular and tumour research that supports this hypothesis is presented in this review. Research describing LDMH interaction with pulsed brachytherapy and high dose-rate brachytherapy using clinically relevant parameters are compared with LDMH/LDR brachytherapy. The mechanism by which LDMH sensitizes LDR has been established as the inhibition of sublethal damage repair. The molecular mechanisms have been shown to involve DNA repair enzymes, but the exact nature of these processes is still under investigation. The relative differences between LDMH interactions with human and rodent cells are presented to help in the understanding of possible roles of LDMH in clinical application. The role of LDMH in modifying tumour blood flow and its possible role in LDR sensitization of tumours is also presented. The positive aspects of LDMH-brachytherapy for clinical application are sixfold; (1) the thermal goals (temperature, time and volume) are achievable with currently available technology, (2) the hyperthermia by itself has no detectable toxic effects, (3) thermotolerance appears to play a minor if any role in radiation sensitization, (4) TER of around 2 can be expected, (5) hypoxic fraction may be decreased due to blood flow modification and (6) simultaneous chemotherapy may also be sensitized. Combined LDMH and brachytherapy is a cancer therapy that has established biological rationale and sufficient technical and clinical advancements to be appropriately applied. This modality is ripe for clinical testing.

Comparison of human tumour cell responses to cisplatin and ZD0473 with and without irradiation.

Three pairs of human tumour cell lines, with one line of each pair resistant to cisplatin, were used to compare the effects of cisplatin and ZD0473 on cellular toxicity and radiosensitization. Whilst all three cell line pairs had one line that was resistant to cisplatin, for ZD0473 the lung tumour HTB56cp and cervical carcinoma ME180 cell lines did not express resistance to their HTB56 and SHA counterparts, respectively. Only the ovarian carcinoma line A2780cp showed resistance to ZD0473 compared to its counterpart A2780S. For radiosensitization both cisplatin and ZD0473 show additive and subadditive effects in the ovarian carcinoma lines, and additive and superadditive effects in the cervical carcinoma and lung tumour cell lines. In fact in the lung tumour cell lines ZD0473 appeared to be a more effective radiosensitizer than cisplatin.

Evaluation of recombination repair pathways in thermal radiosensitization.

Thermal radiosensitization has been shown to cause inhibition of repair of sublethal and potentially lethal damage and DNA DSBs. In this study we assessed thermal radiosensitization in mutants deficient in homologous recombinational (HR) repair and nonhomologous end joining repair (NHEJ). Using cells of the mouse wild-type embryo fibroblast cell line MEF and its Ku80(-/-) derivative that is deficient in NHEJ, we showed that thermal radiosensitization is the same in both cell lines. Further studies with cells of the wild-type CHO-AA8 cell line and its derivative IRS(ISF), which is deficient in HR, also showed comparable thermal radiosensitization in both cell lines. Further experiments using cells of chicken DT40 cell lines also showed comparable thermal radiosensitization between the wild-type HR mutant Rad54, the NHEJ mutant Ku70, and the double mutant Rad 54-Ku70. These results indicate that the HR and NHEJ pathways may not be targets for thermal radiosensitization.

Prediction of radiosensitivity by measurement of X-ray induced apoptosis in human blood using the comet assay.

BACKGROUND: The aim of this work was to determine whether levels of radiation-induced apoptosis in human peripheral leukocytes could be used as a predictor of radiosensitivity. MATERIALS AND METHODS: Peripheral blood was obtained from venous blood and exposed to 0-3 Gy of X-rays. Apoptosis levels were measured at 4, 24, 48 and 72 hours after exposure using the neutral comet assay. Intra-individual apoptotic response was measured using repeated blood samples from four healthy individuals. Inter-individual variation was investigated in whole blood, granulocytes and mononuclear cells from 8 radiotherapy patients (4 demonstrating a radiosensitive response and 4 demonstrating a normal response to radiation exposures). RESULTS: Amongst the four healthy individuals there was both inter- and intra-individual variation of about the same magnitude. However, when comparing the apoptotic response of the radiosensitive and normal patients, consistent trends were observed at all X-ray doses for all of the patients. CONCLUSION: This indicates that apoptosis has some potential as a predictive assay, however, large intra-individual variation exists. More studies are required to investigate the causes of intra-individual variation and how it might be minimized.

Adaptive responses in human glioma cells assessed by clonogenic survival and DNA strand break analysis.

PURPOSE: Human gliomas are known to be radioresistant and the aim was to determine if this resistance in part could be due to an adaptive response. MATERIALS AND METHODS: Human U-87MG glioma cells were used. Three different radiation regimens that could be related to clinical treatments were tested for their ability to cause an adaptive response. Cell survival and DNA double-strand breakage were the measured endpoints. RESULTS: All three regimens caused an adaptive response in terms of cell survival when given priming doses of radiation. The DNA double-strand break endpoint also showed fewer breaks when the adaptive response occurred. CONCLUSIONS: Using irradiation regimens that closely resembled clinical applications, in vitro data are presented that show an adaptive response in human glioma cells. This effect in part could be responsible for the radioresistance of human gliomas.

Examining the non-homologous repair process following cisplatin and radiation treatments.

PURPOSE: To investigate the extent of non-homologous end-joining (NHEJ) in the mechanism of cisplatin radiosensitization. MATERIALS AND METHODS: Ku80-deficient cells are deficient in the non-homologous DNA double-strand break repair process, while the wild-type MEF cells maintain full mammalian cell repair capabilities. Both cell lines were exposed to clinically applicable doses of cisplatin (1, 3 and 6 microg x ml(-1)) for 1 h immediately before exposure to 250 kV X-rays. Radiation responses were plotted for each cell line and for all doses of cisplatin to observe relative levels of radiosensitization. Split-dose experiments were also performed on each cell line to measure levels of sublethal damage repair. RESULTS: Radiosensitization was observed in the wild-type cells but not in the Ku80 cells when treated with a combination of 1 microg x ml(-1) cisplatin followed by X-rays, implying that the NHEJ pathway may play a large role in cisplatin radiosensitization. Concurrent administration of this cisplatin dose with radiation produced similar levels of radiosensitization. Conversely, 3 and 6 microg x ml(-1) cisplatin applied immediately before radiation revealed an increasing resistance to radiation in both cell lines--possibly due to resistant subpopulations of cells remaining after subsequent lethal doses of cisplatin. Further experiments revealed that the high concentrations of cisplatin did not alter cell cycle distribution. Finally, split-dose experiments revealed that the NHEJ pathway also plays a significant role in sublethal damage repair. CONCLUSIONS: The study reveals that clinically applicable doses of cisplatin treatment results in the radiosensitization of mammalian cells due to the inhibition of the operation of NHEJ.

Comparison of response to radiation, hyperthermia and cisplatin in parental and polymerase beta knockout cells.

The role of polymerase beta in response to radiation, cisplatin and hyperthermia was examined in a pair of mouse cell lines, comprising a normal parental line and a derivative with polymerase beta knockout. Cell survival was assessed using the colony survival assay. For irradiation, there was no difference in response between the two cell lines. Treatment with cisplatin for 1 h showed a large increase in resistance in the mutant cell line. The results with hyperthermia were more complex. The mutant was more resistant to 45 degrees C heating, but was slightly more heat sensitive than the wild type at 41 degrees C. Thus, in summary, while the knockout of polymerase beta did not alter radiation sensitivity, it did increase resistance to cisplatin and induced resistance to hyperthermia at higher temperatures (45 degrees C).

Comparison of repair and rejoining fidelity between two isogenic human ovarian carcinoma cell lines.

PURPOSE: The difference in radiosensitivity between two isogenic tumour cell lines was evaluated to determine whether factors such as sublethal and potentially damage repair, DNA double-strand break repair and fidelity of repair can be related to differences in radiosensitivity. MATERIALS AND METHODS: The cell lines used were the ovarian carcinoma A2780s and a radiation-resistant derivative A2780cp. Radiation response was measured in terms of cell survival, recovery of sublethal (SLD) and potentially lethal damage (PLD), induction of and recovery of DNA strand breaks, and fidelity of DNA repair using a cell-free plasmid assay. RESULTS: While A2780cp was more resistant to radiation than A2780s, it showed less ability for recovery of SLD and PLD. DNA strand-break induction was the same for both cell lines, while only at very high doses did A2780cp show greater DNA strand-break recovery than A2780s. Fidelity of rejoining DNA was significantly higher in the A2780cp cell line. CONCLUSION: The difference in radiosensitivity between these two cell lines was not related to recovery of PLD or SLD or to the induction of DNA damage. It appears that fidelity of DNA rejoining, which was significantly higher in the resistant cell line, may be related to the difference in radiosensitivity.

Response to pulsed dose rate irradiation with and without mild hyperthermia using tumour and normal cell lines.

To determine whether pulsed dose rate irradiation in combination with mild hyperthermia could radiosensitize cells in comparison to pulsed dose rate irradiation alone, human ovarian carcinoma (A2780s, cisplatin- and radiation-sensitive, and A2780cp, cisplatin- and radiation-resistant) and human fibroblast (AG1522) cell lines were used. Cells were irradiated in vitro using two fraction sizes, 0.53 Gy given every hour and 1.6 Gy given every 3h, with an overall average dose rate of 0.53 Gy/h. The data showed that 40 degrees C hyperthermia did not radiosensitize any of the cell lines for the 0.53 Gy every 1 h fractionation scheme. In addition, mild hyperthermia radiosensitized both carcinoma cell lines when using the 1.6 Gy fraction size for all doses tested in the A2780s and at higher doses in the A2780cp, but not the normal cell line. These results suggest a potential clinical advantage when using the 1.6 Gy fraction size with 40 degrees C mild hyperthermia, since hyperthermia radiosensitized the carcinoma cells but not the normal cells.

Evaluation of cell survival, DNA double strand breaks, and DNA synthesis during concurrent camptothecin and X-radiation treatments.

We evaluated cell survival, DNA double strand breaks (dsbs), and DNA synthesis following camptothecin (CPT) alone or concurrent CPT and X-radiation treatments in exponential-phase cultures of a radioresistant human melanoma cell line. Cell survival was measured by a clonogenic assay. DNA dsbs were measured by pulsed-field gel electrophoresis. DNA synthesis was measured by incorporation of (3)H-thymidine. We found that (i) concurrent CPT and X-radiation interacted additively, unlike previous results with plateau-phase cultures of these cells, which showed synergistic interaction; (ii) there were strong negative correlations (correlation coefficients of at least 0.82) between clonogenic surviving fractions and DNA dsbs following CPT alone or concurrent CPT and radiation treatments; and (iii) concurrent CPT and radiation (10 Gy) treatment did not completely inhibit DNA synthesis, even though addition of radiation to CPT did further decrease DNA synthesis (relative to CPT alone) at CPT concentrations below 20 microM. Our results suggest that during concurrent CPT and radiation treatment residual DNA dsb levels were good indicators of cell killing and that the absence of complete inhibition of DNA synthesis could at least in part explain the additive interaction between CPT and radiation.

The response of human breast tumour cell lines with altered polymerase beta levels to cisplatin and radiation.

MCF 7 (human breast carcinoma cells) and mutants transfected with the DNA polymerase beta gene were tested for response to cisplatin, radiation and combined treatments. The transfected cells showed a higher level of polymerase beta activity and were more resistant to radiation and cisplatin compared to the parental cell line. Further studies showed that for isosurvival treatments the mutant cells were more effective in sublethal radiation damage repair compared to the parental line. The combination of cisplatin with radiation showed effective radiosensitization which was less in the mutants compared to the parental line. In addition, the sequence of cisplatin before irradiation was more effective then cisplabn after irradiation. Pre-exposure to low levels of cisplatin for up to 24 h before irradiation showed a small significant adaptive response in one mutant line at 8 h and while similar trends were observed in the parental lines at earlier times they were not significant. In summary our data show that polymerase beta and thus base excision repair may play a role in cellular responses to cisplatin and radiation.

Equivalence of pulsed-dose-rate to low-dose-rate irradiation in tumor and normal cell lines.

To determine whether different fractionation schemes could simulate low-dose-rate irradiation, ovarian cells of the carcinoma cell lines A2780s (radiosensitive) and A2780cp (radioresistant) and AG1522 normal human fibroblasts were irradiated in vitro using different fraction sizes and intervals between fractions with an overall average dose rate of 0.53 Gy/h. For the resistant cell line, the three fractionation schemes, 0.53 Gy given every hour, 1.1 Gy every 2 h, and 1.6 Gy every 3 h, were equivalent to low dose rate (0.53 Gy/h). Two larger fraction sizes, 2.1 Gy every 4 h and 3.2 Gy every 6 h, resulted in lower survival than that after low-dose-rate irradiation for the resistant cell line, suggesting incomplete repair of radiation damage due to the larger fraction sizes. The survival for the sensitive cell line was lower at small doses, but then it increased until it was equivalent to that after low-dose-rate irradiation for some fractionation schemes. The sensitive cell line showed equivalence only with the 1.6-Gy fraction every 3 h, although 0.53 Gy every 1 h and 1.1 Gy every 2 h showed equivalence at lower doses. This cell line also showed an adaptive response. The normal cell line showed a sensitization to the pulsed-dose-rate schemes compared to low-dose-rate irradiation. These data indicate that the response to pulsed-dose-rate irradiation is dependent on the cell line and that compared to the response to low-dose-rate irradiation, it shows some equivalence with the resistant carcinoma cell line, an adaptive response with the parental carcinoma cell line, and sensitization with the normal cells. Therefore, further evaluation is required before implementing pulsed-dose-rate irradiation in the clinic.

Evidence for adaptive response and implication in pulse-simulated low-dose-rate radiotherapy.

PURPOSE: Pulsed-dose-rate (PDR) brachytherapy as a substitute for continuous low-dose-rate (LDR) brachytherapy has a number of clinical advantages. However, early results show that some cells can exhibit an adaptive response to radiation and in PDR where many pulses are given such an adaptive response may play an important role in the outcome. METHODS AND MATERIALS: Nine human cell lines (two normal fibroblast and seven tumor) were evaluated for an adaptive response. Cells were given either a single adapting dose before a challenge dose or given PDR sequences for which the average dose rate matched the LDR dose rate. Response was assessed using the colony survival assay. RESULTS: Five of the nine cell lines showed an adapting response to single small doses of radiation. Three of these cell lines were further investigated for adapting response to PDR and two of the three lines (one ovarian carcinoma and one glioma) showed an adaptive response which was dependent on pulse size and interval. CONCLUSION: The data show that an adaptive response can occur in human cells and that it can vary among cell lines. In addition, PDR sequences also produced an adaptive response which could have an affect on PDR therapy if such a response is found in tissues.