We investigated the ability of camptothecin (CPT), an inhibitor of topoisomerase (topo) I, and etoposide (VP-16), an inhibitor of topo II, to potentiate X-radiation response and to inhibit the repair of potentially lethal damage (PLDR) and sublethal damage (SLDR) in confluent cultures of a radioresistant (Sk-Mel-3) and a radiosensitive (HT-144) human melanoma cell line. CPT or VP-16 were present both during irradiation and during the subsequent delayed plating period allowed for repair of X-radiation damage. When the direct toxicities of CPT or VP-16 were corrected for, we found that a dose of either drug that killed approximately 15% of the clonogenic cells potentiated the effects of radiation differentially on the cell lines. CPT and VP-16 inhibited the increase in survival brought about by delayed plating of HT-144 but not Sk-Mel-3 cells. In both cell lines, CPT inhibited SLDR but not PLDR. VP-16 also inhibited SLDR in both cell lines, however, in contrast with CPT, it also inhibited PLDR in HT-144 cells. Our results therefore suggest that either topo I and II are both implicated in the repair of X-radiation damage, or that the lesions formed by CPT and VP-16 with DNA are able to impair the processing of X-radiation repair. In addition, we found that in the absence of the topo inhibitors, the two cell lines repaired similar amounts of PLD from an isosurvival level. Sk-Mel-3, however, repaired significantly increased SLD from an isosurvival level (about three-fold, p < 0.05) compared with HT-144.

Three human cell lines (glioma, melanoma and fibroblast) were evaluated for responses to low dose-rate irradiation (LDRI) (0.88 and 0.41 cGy per min) alone or with concurrent heating (41 degrees C) during irradiation. In order to avoid cell cycle redistribution cells were held in plateau phase. The results show that the lowest LDRI gave maximum sparing in the glioma and the fibroblast cell lines while both dose rates achieved approximately the same effect in the melanoma line. The melanoma line was the most heat sensitive and showed the greatest thermal enhancement ratio (TER). For all cell lines TER was greatest at the lowest dose-rate, and in the melanoma the heat plus LDRI curve gave lower survival than the high dose-rate irradiation survival curves. These data show that concurrent mild hyperthermia combined with LDRI used in brachytherapy can enhance the effectiveness of clinical brachytherapy treatments. In addition, this effect was largest in the most resistant cell line indicating the potential for using this combination to overcome radioresistance in brachytherapy.

Two human melanoma cell lines, one radioresistant (SK-MEL-3) and one radiosensitive (HT-144), and a normal human fibroblast line (AG1522) were evaluated for thermoradiosensitization of low-dose-rate irradiation by concurrent mild hyperthermia (39-41 degrees C). None of the cell lines expressed chronic thermotolerance during heating at 39-41 degrees C. The SK-MEL-3 cells were the most heat sensitive, while AG1522 and HT-144 cells had the same sensitivity at 39 and 40 degrees C but HT-144 cells were more sensitive at 41 degrees C. All cell lines expressed thermal enhancement of radiosensitivity with heating during irradiation which increased with heating temperature. The SK-MEL-3 cells, which were the most resistant to radiation and demonstrated the greatest repair of sublethal damage (SLD) during low-dose-rate irradiation, had the greatest thermal enhancement of radiosensitivity, while the HT144 cells, which were the most sensitive and expressed little repair of SLD during low-dose-rate irradiation, had the smallest thermal enhancement of radiosensitivity. These data show that concurrent mild hyperthermia during low-dose-rate irradiation may be most efficacious in radiation-resistant tumor cells which express resistance through an enhanced capacity for repair of SLD.

We investigated the modification of etoposide (i.e. VP-16)-induced cell killing by hyperthermia in a radioresistant human melanoma (Sk-Mel-3) and a human normal (AG1522) cell line. VP-16, a DNA topo II poison, was given as a 1 h exposure at variable doses up to 35 microM; hyperthermia was given either before or following VP-16 treatment. Hyperthermic treatment comprised one of the following: 41 degrees C for 8 h, 42 degrees C for 2 h or 45 degrees C for 15 min. Hyperthermia preceding VP-16 treatment reduced the cytotoxicity of the latter; the reduction of VP-16 cytotoxicity was directly proportional to the severity of the hyperthermic treatment. For a particular combination of hyperthermic dose and VP-16 concentration, generally similar responses were seen in both cell lines. There were no effects on VP-16 cytotoxicity when both Sk-Mel-3 and AG1522 cells were heated at 41 degrees C for 8 h following treatment with VP-16. However, heating both cell lines at 45 degrees C for 15 min following VP-16 treatment again reduced the amount of cytotoxicity associated with VP-16. In addition, we found that a preceding exposure to 45 degrees C, 15 min heating did not affect either cellular accumulation or efflux of [3H]VP-16 in both cell lines. This suggested that the reduction in VP-16 cytotoxicity observed under those conditions was not due to a modification of VP-16 transport. We found no differences between the catalytic activities of topo II extracted from nuclei of Sk-Mel-3 and AG1522 cells that were either heated at 45 degrees C for 15 min or that were not subjected to such treatment. These results therefore suggested that the substantial reduction of cytotoxicity seen when 45 degrees C, 15 min heating preceded VP-16 treatment was also not due to an effect on topo II catalytic activity. Our results therefore demonstrate that hyperthermia, given either before or after VP-16, can actually reduce the amount of VP-16 cytotoxicity and that this can occur without any overt changes in VP-16 accumulation and efflux or in topo II catalytic activity.

Four human cell lines (one fibroblast, two melanoma and one glioma) were evaluated for their responses to hyperthermia and thermalradiosensitization. For mild hyperthermia (40-42 degrees C), there was little to no chronic thermotolerance development during protracted heating for up to 72 h. In addition, there was no significant thermotolerance for polymerase inactivation during mild hyperthermia. For high temperature hyperthermia, polymerase beta was more thermal sensitive than aphidicolin sensitive polymerase alpha + delta + epsilon, (termed polymerase alpha) but during mild hyperthermia ther relative sensitivities were reversed. Polymerase beta was resistant to mild hyperthermia and polymerase alpha was very sensitive. Within each cell line there was a correlation between polymerase alpha inactivation and the degree of radiosensitization (TER) and amongst the cell lines the most radiation resistant cell line had less polymerase alpha inactivation than the most sensitive cell line for similar values of TER's. These data indicate that, amongst the cell lines, radiosensitivity and polymerase alpha sensitivity may influence TER and that for a given cell line, or possibly tumour, polymerase alpha inactivation may have potential as an indicator to determine TER for mild hyperthermia treatments in radiosensitization to low dose rates.