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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.

Objective: Postoperative radiotherapy is standard treatment for patients with a glioblastoma multiforme (GBM). However, a GBM is radioresistant and almost always recurs, even after a high dose of radiation. A GBM is characterized by its extensive neo-angiogenesis, which can be attributed to the high levels of vascular endothelial growth factor (VEGF). The scope of this study is to investigate the VEGF secretion by GBM cells with different radiosensitivity after irradiation., Methods: Three human GBM cell lines (U251, U251-NG2 and U87) were irradiated with single doses of 0, 5, 10 and 20 Gy of gamma-rays from a (137)Cs source. VEGF levels in medium were measured by ELISA at 24, 48 and 72 h after radiation. Cell survival was measured by the XTT assay 7 days after irradiation., Results: Following single dose radiation, the VEGF levels showed a dose dependent increase in U251, U251-NG2 and U87 glioma cells. Both base-line and radiation-enhanced VEGF levels were about 10-fold higher in U87 compared to U251 and U251-NG2 cells. In addition, in the XTT assay, the U87 was more radioresistant than both U251 and U251-NG2 cell lines (dose modifying factor (DMF) = 1.6 and 1.7 resp)., Conclusion: Irradiation enhanced VEGF secretion in all three tested glioma cell lines (up to eight times basal levels). It is tempting to associate the radiation-enhanced VEGF secretion with an increased angiogenic potential of the tumor, which may be a factor in radioresistance.

Purpose: Mismatch repair (MMR) proficiency has been reported to either increase or decrease radioenhancement by 24-h incubations with gemcitabine. This study aimed to establish the importance of MMR for radioenhancement by gemcitabine after short-exposure, high-dose treatment and long-exposure, low-dose treatment., Methods and Materials: Survival of MMR-deficient HCT116 and MMR-proficient HCT116 + 3 cells was analyzed by clonogenic assays. Mild, equitoxic gemcitabine treatments (4 h, 0.1 microM vs. 24 h, 6 nM) were combined with gamma-irradiation to determine the radioenhancement with or without recovery. Gemcitabine metabolism and cell-cycle effects were evaluated by high-performance liquid chromatography analysis and bivariate flow cytometry., Results: Radioenhancement after 4 h of 0.1 microM of gemcitabine was similar in both cell lines, but the radioenhancement after 24 h of 6 nM of gemcitabine was reduced in MMR-proficient cells. No significant differences between both cell lines were observed in the gemcitabine metabolism or cell-cycle effects after these treatments. Gemcitabine radioenhancement after recovery was also lower in MMR-proficient cells than in MMR-deficient cells., Conclusion: Mismatch repair proficiency decreases radioenhancement by long incubations of gemcitabine but does not affect radioenhancement by short exposures to a clinically relevant gemcitabine dose. Our data suggest that MMR contributes to the recovery from gemcitabine treatment.

The role of DNA repair mechanisms in the cellular response to low dose rate (LDR) irradiation was studied with the aim to gain insight in the process of sublethal damage (SLD) repair. Chinese hamster cell lines mutated in either DNA single strand break (ssb) repair or DNA double strand break (dsb) repair by non homologous end joining (NHEJ) and homologous recombination (HR), or showing an AT-like phenotype, were irradiated in plateau-phase either at high dose rate (HDR, 3.3 Gy/min) or at pulsed low dose rate (p-LDR, average 1 Gy/h). Cell survival after irradiation was assessed using the clonogenic assay. A change in sensitivity when the dose rate was decreased was observed for all parental cell lines and the DNA ssb repair mutant. No difference in cell survival after p-LDR versus. HDR irradiation was observed for the two NHEJ mutants, the AT-like mutant and the HR mutant. Based on these results we conclude that single strand break repair does not play a role in the dose rate effect. The AT like protein, functional NHEJ and XRCC3 are required for the dose rate effect.

Purpose: To determine cross-resistance to anti-tumor treatments in 2',2'difluorodeoxycytidine (dFdC, gemcitabine)-resistant human tumor cells., Methods and Materials: Human lung carcinoma cells SW-1573 (SWp) were made resistant to dFdC (SWg). Sensitivity to cisplatin (cDDP), paclitaxel, 5-fluorouracil (5-FU), methotrexate (MTX), cytarabine (ara-C), and dFdC was measured by a proliferation assay. Radiosensitivity and radioenhancement by dFdC of this cell panel and the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000 were determined by clonogenic assay. Bivariate flowcytometry was performed to study cell cycle changes., Results: In the SWg, a complete deoxycytidine kinase (dCK) deficiency was found on mRNA and protein level. This was accompanied by a 10-fold decrease in dCK activity which resulted in the >1000-fold resistance to dFdC. Sensitivity to other anti-tumor drugs was not altered, except for ara-C (>100-fold resistance). Radiosensitivity was not altered in the dFdC-resistant cell lines SWg and AG6000. High concentrations (50-100 microM dFdC) induced radioenhancement in the dFdC-resistant cell lines similar to the radioenhancement obtained at lower concentrations (10 nM dFdC) in the parental lines. An early S-phase arrest was found in all cell lines after dFdC treatment where radioenhancement was achieved., Conclusions: In the dFdC-resistant lung tumor cell line SWg, the deficiency in dCK is related to the resistance to dFdC and ara-C. No cross-resistance was observed to other anti-tumor drugs used for the treatment in lung cancer. Sensitivity to ionizing radiation was not altered in two different dFdC-resistant cell lines. Resistance to dFdC does not eliminate the ability of dFdC to sensitize cells to radiation.

Sensitization by bromodeoxyuridine (BrdUrd) and hyperthermia (HT) on cell reproductive death induced by ionizing radiation was analyzed using the linear-quadratic [S(D)/S(0)=exp(-(alphaD + betaD2)]] model. Plateau-phase human lung tumor cells (SW-1573) and human colorectal carcinonoma cells (RKO) were treated with BrdUrd, radiation and HT. LQ-analysis was performed at iso-incubation dose and at iso-incorporation level of BrdUrd. and at iso-HT doses and iso-survival levels after HT. Clonogenic assays were performed 24 h after treatment to allow repair of potentially lethal damage (PLD). In SW cells BrdUrd. HT or the combination significantly increased the alpha-parameter (factor 2.0-5.7), without altering the beta-parameter. In RKO cells sensitization with BrdUrd increased both a (factor 1.4) and beta (factor 1.3) while HT only influenced beta (factor 2.1-4.0). The combination did not further increase the a and beta. The results indicate that BrdUrd has its main effect on the parameter alpha, dominant at clinically relevant radiation doses but that HT can affect both a and beta. The addition of BrdUrd and HT provides a method to enhance the efficacy of radiotherapy.

Bromodeoxyuridine (BrdUrd)-induced radiosensitization of two different tumour cell lines was compared at equal levels of thymidine replacement. Human lung carcinoma cells (SW-1573) and human colorectal carcinoma cells (RKO) were grown for 48 h in the presence of respectively 1 microM BrdUrd and 4 microM of BrdUrd in order to obtain equal levels of BrdUrd into the DNA. In SW cells the level of thymidine replacement by BrdUrd was 6.7+/-0.5% and in RKO cells this was 7.1+/-0.8. Cell survival after irradiation with single doses up to 8 Gy, was determined with clonogenic assay. The magnitude of BrdUrd-induced radiosensitization was determined by analyzing radiation-dose survival curves with the linear-quadratic formula [S(D)/S(0)=exp-(alphaD+betaD2)]. In the SW cells BrdUrd radiosensitization led to a significant increase of the linear parameter, alpha, determining the initial slope of the survival curves, by a factor of about 2. In the RKO cells BrdUrd increased the value of alpha by a factor 1.4. This suggests that repair of potentially lethal damage (PLD) is inhibited. In both cell lines the quadratic term, beta, strongly influencing the high dose region of the survival curves, was not altered by sensitization by BrdUrd. The increase of alpha is of interest for clinical applications as BrdUrd sensitizes tumour cells after low doses of radiation.

Radiosensitization of exponentially growing and plateau phase Chinese hamster V79 cells by incorporation of halogenated pyrimidines (HP) was investigated for different culture conditions that influenced repair. For this purpose cells were grown for 72 h with 0, 1, 2 and 4 microM of chloro-(CldUrd), bromo- (BrdUrd) or iodo-deoxyuridine (IdUrd) and were subsequently irradiated with gamma-rays from a 197Cs source, either in exponential growth or in plateau-phase. Cell survival after irradiation was determined by clonogenic assay. In exponentially growing cultures thymidine-replacement in the DNA of the cells after incubation with 4 microM of CldUrd, BrdUrd and IdUrd was 22.3, 32.7 and 12.7%, respectively. In plateau-phase cultures the percentage thymidine replacement in the DNA of the cells after incubation during growth with 4 microM CldUrd, BrdUrd and IdUrd was 27.5, 33.8 and 10.7%, respectively. Linear-quadratic analyses of the radiation survival curves were performed. In exponentially growing cells a marked increase by a factor 2-3 of the value of alpha was obtained. The beta term significantly increased only in cells which were grown in the presence of BrdUrd and which were trypsinized and replated immediately after irradiation. In plateau-phase cells which were trypsinized and plated immediately after irradiation both alpha and beta increased up to a factor 2-3 with increasing incorporation of halogenated pyrimidines. In plateau phase cells which were allowed to repair potentially lethal damage (PLD) for 6 h and subsequently trypsinized and plated, alpha increased by a factor 3-4. In these latter conditions changes in beta were smaller. In exponentially growing cells in which repair was allowed after irradiation by plating prior to the treatment, the alpha values decreased for all the HP drugs tested as compared to the alpha of cells plated immediately after irradiation. In contrast, delay of plating for plateau phase cells yielded increased alpha values not only when compared with the alpha of plateau phase cells plated immediately after treatment but also when compared with the alpha value of radiosensitized exponentially growing cells. The increase of alpha might be interpreted as an enhancement in the expression of PLD. The larger contribution of fixation of PLD might be due to initial DNA damage and/or to inhibition of PLD repair resulting from incorporation of HP. The increase of beta might be attributed to enhanced interaction or to fixation of sublethal damage (SLD). In view of clinical applications of HP it is of interest that sensitization is not abolished in plateau-phase cells.