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The cytotoxicity of cisplatin, applied alone or in combination with hyperthermia, to mouse mammary adenocarcinoma cells (M8013S) was studied with the cells either treated in medium [Eagle's minimum essential medium (MEM), supplemented with 10% foetal bovine serum, 100 IU/ml penicillin, 200 mM glutamine and 0.35 g/l NaHCO3] or in Hank's balanced salt solution (HBSS) without serum. To study the role of platinum uptake by the M8013 cells in cytotoxicity, uptake was determined under conditions similar to those used in the survival experiments. Our results show that hyperthermia (30 min at 43 degrees C) enhances the toxicity of cisplatin. Enhanced toxicity by heat treatment is not observed with the cells in HBSS. The thermal enhancement of effects of cisplatin to cells in MEM with serum is clearly related to an enhanced uptake of cisplatin. A novel observation is that in order to obtain a considerable thermal enhancement of the cytotoxic effect of cisplatin, the exposure of the cells to the drug is required not only during the hyperthermic treatment but the exposure has to be maintained for at least 2 h after hyperthermia. These same conditions are also required for enhanced uptake of cisplatin. The present results may indicate that cisplatin has to be bound to some serum component in order to facilitate an 'active' uptake. Hyperthermia leads to a considerable intracellular accumulation of cisplatin, relative to the extracellular concentration. This accumulation takes place during exposure to cisplatin but after heat treatment

The dependence of parameters of the linear-quadratic (LQ) model on cell proliferation kinetics of tumors in relation to potentially lethal damage (PLD) and its repair is evaluated. The influence of sensitizing agents on these parameters during fractionated radiotherapy is assessed. Suggestions for scheduling of radiation combined of with sensitizing agents are derived. The parameters alpha and beta of the linear-quadratic model for dose dependence of cell reproductive inactivation, derived from experimental and clinical data, are evaluated to assess their dependence on cell proliferative state, on PLD repair and on the action of various sensitizing agents. PLD contributes to the linear as well as to the quadratic component of the LQ model. PLD is less effectively repaired in proliferating (P) cells than in clonogenic (G0) cells of the quiescent (Q) cell compartment. PLD is influenced by various agents applied during, as well as after irradiation. The parameters alpha and beta are affected differently by the proliferative state of cells, by some of the sensitizing agents, and by radiation quality. The relative fractions of P cells and Q cells can change during fractionated treatments. If recruitment is effective, the fraction of G0 cells decreases in the latter part of a treatment schedule. PLD from subsequent radiation doses is then repaired less and the effectiveness of radiation combined with sensitizing agents may be enhanced. The analyses using the LQ model show differences in PLD and its repair between P cells and G0 cells in tumors. If due to recruitment the compartment of clonogenic G0 cells diminishes during treatment, the combination of radiation with sensitizing agents and the application of high-LET radiation should be scheduled to take this factor into account. For poorly differentiated tumors with high labeling indices (LI), benefit from combined treatments is expected from early in the course of fractionated radiotherapy. Well differentiated tumors with low LI are suggested to benefit most from irradiation combined with sensitizing agents in the latter part of a treatment schedule. New methods are required to assess the clonogenic G0 cells in the Q cell compartment and to monitor recruitment of these cells into the P cell compartment.

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

The induction of chromosome exchanges was investigated in SW-1573 human lung tumour cells radiosensitized with iododeoxyuridine (IdUrd) and irradiated with gamma-rays. Following treatment chromosome 2 and X were analyzed using fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries. The yield of chromosome exchanges involving chromosome 2 was higher than those involving chromosome-X. On the basis of the DNA content the relative involvement of the X-chromosome in exchange frequencies after 2 Gy was much higher than of chromosome 2. After 4 Gy the relative involvement of both chromosomes in exchanges is approximately equal. After radiosensitization, increased chromosome exchange frequencies are observed in both studied chromosomes. For the total chromosome exchange frequencies the sensitizer enhancement ratio (SER) at 2 Gy is 1.8 and 1.3 for chromosome 2 and X respectively. The SER at 4 Gy for total exchange frequencies is 1.6 and 1.9 chromosome 2 and X respectively. For reciprocal exchanges at 2 Gy higher SER values and at 4 Gy lower SER values were observed for both chromosomes

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

The combination of local hyperthermia (HT) with thermostable liposomal daunorubicin (DaunoXome, DX) was investigated to assess targeted drug delivery to experimental tumors. Female Wag/Rij rats bearing solid R-1 rhabdomyosarcomas received i.v. injections of 10 or 15 mg/kg of DX or free-Daunorubicin (f-Dau). After a 1-h interval, HT (60 min at 43 degrees C) was applied. Pharmacokinetics were studied in relation to tumor growth time (TGT), i.e., the time for tumors to reach their original volumes. Pharmacokinetic studies revealed that DX accumulation in tumor tissue was similar to f-Dau. A 5.4-fold increase (P = 0.0084) in tumor drug delivery was observed when DX was combined with HT, whereas liposomes remained stable. For f-Dau, HT had an additional effect on TGT at both drug doses tested (9.6 and 6.2 days, respectively, for 10 mg/kg, P = 0.0092; 17.7 and 13.7, respectively, for 15 mg/kg, P = 0.0431). For DX, HT significantly enhanced TGT of DX in the lower dose (17.1 and 6.4 days, respectively, P = 0.0005), whereas tumors did not regrow at day 25 after DX + HT in the higher dose. Unfortunately, after this time interval, the animals died of late toxicity, probably not related to HT. These results indicate that HT promotes extravasation of DX into tumor tissue and enhances its effectiveness. The finding that HT-induced drug release from the liposomes was not responsible for enhanced antitumor activity provides a rationale for further investigation of thermostable liposomes in conjunction with HT