G.Peter Raaphorst

The effect of pH on cell lethality induced by hyperthermic treatment

Chinese hamster ovary (CHO) cells were subjected to hyperthermic treatment (41–45.5°C) in either acidic or alkaline medium. Consistent with previous reports, thermal sensitivity was increased as the range of the environmental pH level was decreased from 7.4–7.5 to 6.6–6.7. The increase in sensitivity was expressed as a decrease in the shoulder and an increase in the slope of the survival curve. Incubation at 37°C in acidic medium for 24 hours prior to hyperthermic treatment at 45.5°C neither enchanced nor diminished thermal sensitivity. However, thermal damage acquired during heating (42.5–45.5°C) in acidic or alkaline medium could be augmented by incubating the cells at 37°C in acidic medium after hyperthermic treatment. This augmentation of thermal damage was expressed as a further increase in the final slope of the survival curve. An Arrhenius plot of the rate of heat inactivation as a function of the inverse of absolute temperature indicated that heating in acidic medium increased the inactivation rate compared to the rate which occurred for cells heated in alkaline medium. However, the slope of the Arrhenius curve (i.e., activation energy) was fairly independent of the environmental pH level. When CHO cells were treated in alkaline medium, the Arrhenius curve changed slope at 43°C. But when cells were treated in acidic medium, the temperature at which the curve changed slope depended upon the duration of treatment in acidic medium after the hyperthermic exposure. Heating at 42°C or below resulted in the appearance of a thermotolerant population. The onset of thermotolerance occurred after 200–300 minutes of heating, regardless of the environmental pH level at the time of heating. However, the percentage of the population that expressed thermotolerance when cells were heated at a given temperature decreased when cells were heated in acidic medium. The most interesting observations were: 1 that heat treatments at low pH decreased the number of thermotolerant cells; and 2) that when cells were maintained at low pH after hyperthermic treatment, thermal damage was augmented. These observations may have important implications in the clinic.

Cell killing and the sequencing of hyperthermia and radiation.

Abstract Previous reports have indicated that the greatest cell killing occurs when heat and radiation exposure occur simultaneously. Our studies using Chinese hamster ovary cells comparing cell killing from simultaneous exposure to radiation (500 rad) and heat (either 42.0°C for 60 min or 42.5°C for 40 min) with cell killing from irradiation at 37°C between split heat treatments indicate that these two methods may be equally effective as long as the time between heat treatments remains relatively short (less than 10 min). However, a greater increase in survival with increasing time between split heat doses was observed at 42.0°C than at 42.5°C. While these studies may indicate a more practical clinical method for obtaining maximum interaction between heat and radiation, they do not indicate whether the increased therapeutic ratio that is obtained from in vivo studies when heat and radiation are administered simultaneously will be affected. Investigations comparing the effect of equivalent heat doses (chosen to achieve equal cell killing) at different temperatures on the enhancement of cell killing by radiation either during the heating interval or exactly 3 min after heating indicate that temperatures between 42.5 and 43.5°C are the most effective in enhancing X-ray induced cell killing. Thus, the choice of temperature for the therapeutic use of hyperthermia with irradiation may prove to be an important factor in the success of the treatment

Correlation between normal tissue complications and in vitro radiosensitivity of skin fibroblasts derived from radiotherapy patients treated for variety of tumors

PURPOSE To assess the relationship between fibroblast intrinsic radiosensitivity in vitro and late reactions of normal tissues in patients treated by definitive radiotherapy for variety of tumors. PATIENTS AND METHODS Ten patients were selected for this study. They were treated by radical radiotherapy for variety of tumors, including non-Hodgkins lymphoma, prostate, glottic larynx, anal canal, cervix, bladder, thyroid gland, and tonsil pillar. Five patients did not develop any significant late reactions (normally sensitive group, NS). The other five developed late complications in different normal tissues and organs that proved to be fatal in one patient (clinically hyper-sensitive group, HS). Fibroblast cultures were established from punch skin biopsy and radiosensitivity in vitro was measured. The survival fraction at 2 Gy (SF2) was calculated and compared between the two groups. RESULTS SF2 ranged between 0.10 and 0.38 with a mean of 0.24. The mean SF2 for each of the NS and the HS groups were 0.31 and 0.17, respectively. The non-parametric rank test of Mann-Whitney shows that the difference between the two groups is statistically significant (p = 0.01). CONCLUSION This study indicates that the in vitro radiosensitivity of skin fibroblasts is correlated with late complications in different organs and normal tissues following radiotherapy for variety of tumors. It also lends support to the existence of a common genetic component determining the radiosensitivity of cells targeted by the late effects of ionizing radiation.

THE EFFECT OF pH ON HYPERTHERMIC AND X RAY INDUCED CELL KILLING

Abstract Chinese hamster ovary cells in vitro were heated (42.5°C) before, during, or after X-irradiation (5 Gy). First, a slight degree of radioprotection was observed when cells were irradiated in acidic medimn compared to irradiation in alkaline medium; i.e., 10 Gy reduced survival to 0.0035 at pH 6.75 compared to 0.0010 at pH 7.45. Second, survival increased independent of pH as the time between irradiation and subsequent heat treatment was increased. Third, both heat treatments alone and combined heat and irradiation treatments in acidic medium produced more cell killing than treatments in alkaline medium; the amount of recovery from heat damage which subsequently interacted with radiation damage decreased as the pH was decreased from 7.40 to 6.70. Furthermore, inhibition of recovery was reflected primarily as an increase in the slope of the radiation survival curve; i.e., heating for 60 min 150 min before irradiation decreased the D 0 from 1.00 Gy for cells heated and irradiated under alkaline conditions ad to 0.70 Gy for cells treated under acidic conditions. The extravolation numbers were 5 and 6 for the respective curves.

Enhanced in vitro radiosensitivity of skin fibroblasts in two patients developing brain necrosis following AVM radiosurgery: a new risk factor with potential for a predictive assay

PURPOSE Radiosurgery is an effective treatment for arteriovenous malformations (AVM) with a low risk of developing brain necrosis. Models have been developed to predict the risk of complications. We postulated that genetic differences in radiosensitivity may also be a risk factor. METHODS AND MATERIALS Fibroblast cultures were established from skin biopsies in two AVM patients developing radiation necrosis. The results of clonogenic survival assays were compared to a parallel study with two groups of cancer patients treated with radiation: 1) patients without late side effects; 2) patients experiencing severe late sequelae. RESULTS The survival fraction at 2 Gy (SF2) of the 2 AVM patients was 0.17 (0.14-0.19) and 0.18 (0.14-0.22). The SF2s of the cancer patients ranged between 0.25-0.38 (mean = 0.31) for the control group, and between 0. 10-0.20 (mean = 0.17) for the hypersensitive group. The SF2s of the AVM patients who developed brain necrosis were comparable to that of the hypersensitive group (p = 0.85) but significantly lower than the control group (p = 0.05). CONCLUSION The two patients who developed radiation necrosis demonstrate increased fibroblast radiosensitivity. The SF2 of skin fibroblasts may potentially be used as a predictive assay to detect patients at risk for brain necrosis.

Cisplatin and low dose rate irradiation in cisplatin resistant and sensitive human glioma cells

PURPOSE Human glioma cell lines resistant (U373MGCP) and sensitive (U373MG) to cisplatin were used to evaluate the effect of cisplatin as a sensitizer to low dose rate irradiation (LDRI). METHODS AND MATERIALS A cisplatin resistant glioma cell line U373MGCP was developed by chronic exposure of parental U373MG cells to cisplatin. Plateau phase cells were treated with cisplatin, high dose rate (HDR) irradiation (1.12 Gy/min), LDRI (0.0088 Gy/min), or cisplatin concurrent with LDRI. Cell survival was determined by the colony forming assay. RESULTS Both cell lines showed increased resistance to radiation at LDR compared with HDR, with Dose Modifying Factors (DMF at 10% survival level) of 1.7 for U373MG and 2.5 for U373MGCP. The increased LDR sparing effect in the cisplatin resistant U373MGCP cells indicates increased repair proficiency. The resistant cell line showed a fourfold increase in resistance to cisplatin cytotoxicity at the 10% survival level compared with the parental U373MG cells. Cisplatin enhanced the response of both cell lines to LDRI. The DMFs were 1.2, 1.2, and 1.7, respectively, for the sensitive U373MG cell line given 1 microgram/ml, and the resistant cell line given 3 or 6 micrograms/ml cisplatin treatments concurrent with LDRI. CONCLUSIONS These data show that cisplatin can be an effective sensitizer to LDRI in both cisplatin resistant and sensitive glioma cell lines. However, in the resistant cell line, higher concentrations of cisplatin were necessary to achieve the same level of sensitization as in the sensitive cell line.

HYPERTHERMIA RADIOSENSITIZATION IN HUMAN GLIOMA CELLS COMPARISON OF RECOVERY OF POLYMERASE ACTIVITY, SURVIVAL, AND POTENTIALLY LETHAL DAMAGE REPAIR

PURPOSE DNA polymerase inactivation is compared to thermal radiosensitization and inhibition of damage recovery in human glioma cells. METHODS AND MATERIALS Two human glioma cell lines (U87MG and U373MG) were exposed to hyperthermia and irradiation. Hyperthermia was given at 43 degrees C and 45 degrees C and DNA polymerase alpha + delta + epsilon and beta activities were measured. Hyperthermia was given at various times before irradiation and the degree of radiosensitization and polymerase activity was assessed at various times after heating. In addition the ability of cells to undergo repair of potentially lethal radiation damage was assessed for cells irradiated at various times after heating. RESULTS Polymerase alpha + delta + epsilon and polymerase beta both recovered after heating but polymerase beta was faster and was complete in U373MG but not in the U87MG cell lines after 48 h incubation after heating (45 degrees C, 60 min). Incubation, between hyperthermia and irradiation resulted in a loss of radiosensitization and a loss of inhibition of repair of potentially lethal damage. These changes correlated well with recovery of polymerase beta but not with polymerase alpha + delta + epsilon. CONCLUSION The correlation of polymerase beta activity and thermoradiosensitization and its recovery indicate that polymerase beta may be one of the mechanisms involved in thermoradiosensitization.PURPOSE: DNA polymerase inactivation is compared to thermal radiosensitization and inhibition of damage recovery in human glioma cells. METHODS AND MATERIALS: Two human glioma cell lines (U87MG and U373MG) were exposed to hyperthermia and irradiation. Hyperthermia was given at 43 degrees C and 45 degrees C and DNA polymerase alpha + delta + epsilon and beta activities were measured. Hyperthermia was given at various times before irradiation and the degree of radiosensitization and polymerase activity was assessed at various times after heating. In addition the ability of cells to undergo repair of potentially lethal radiation damage was assessed for cells irradiated at various times after heating. RESULTS: Polymerase alpha + delta + epsilon and polymerase beta both recovered after heating but polymerase beta was faster and was complete in U373MG but not in the U87MG cell lines after 48 h incubation after heating (45 degrees C, 60 min). Incubation, between hyperthermia and irradiation resulted in a loss of radiosensitization and a loss of inhibition of repair of potentially lethal damage. These changes correlated well with recovery of polymerase beta but not with polymerase alpha + delta + epsilon. CONCLUSION: The correlation of polymerase beta activity and thermoradiosensitization and its recovery indicate that polymerase beta may be one of the mechanisms involved in thermoradiosensitization.

Comparison of radiosensitization by 41°C hyperthermia during low dose rate irradiation and during pulsed simulated low dose rate irradiation in human glioma cells

PURPOSE Long duration mild hyperthermia has been shown to be an effective radiosensitizer when given concurrently with low dose rate irradiation. Pulsed simulated low dose rate (PSLDR) is now being used clinically, and we have set out to determine whether concurrent mild hyperthermia can be an effective radiosensitizer for the PSLDR protocol. MATERIALS AND METHODS Human glioma cells (U-87MG) were grown to plateau phase and treated in plateau phase in order to minimize cell cycle redistribution during protracted treatments. Low dose rate (LDR) irradiation and 41 degrees C hyperthermia were delivered by having a radium irradiator inside a temperature-controlled incubator. PSLDR was given using a 150 kVp X-ray unit and maintaining the cells at 41 degrees C between irradiations. The duration of irradiation and concurrent heating depended on total dose and extended up to 48 h. RESULTS When 41 degrees C hyperthermia was given currently with LDR or PSLDR, the thermal enhancement ratios (TER) were about the same if the average dose rate for PSLDR was the same as for LDR. At higher average dose rates for PSLDR the TERs became less. CONCLUSIONS Our data show that concurrent mild hyperthermia can be an effective sensitizer for PSLDR. This sensitization can be as effective as for LDR if the same average dose rate is used and the TER increases with decreasing dose rate. Thus mild hyperthermia combined with PSLDR may be an effective clinical protocol.

The kinetics of cellular recovery in exponential and plateau growth phase human glioma cells following γ-irradiation

PURPOSE In this study the kinetics of recovery following irradiation was examined in a human glioma cell line. Specific objectives were: to determine whether recovery is mono- or biexponential in nature; to determine if recovery half-times are different in exponential and plateau growth phase cells; to compare recovery half-times as a function of dose or recovery levels; and finally, to compare the kinetics of sublethal damage recovery and potentially lethal damage recovery in plateau growth phase cells. METHODS AND MATERIALS U-87MG cells were irradiated in exponential and plateau growth phases and then subjected to incubation at 37 degrees C for various periods of time following or between doses prior to assaying for survival. Survival recovery curves were fit to a sum of exponential terms. RESULTS Potentially lethal damage recovery was monoexponential in both exponential and plateau growth phase cells and occurred at the same rate when isorecovery values were compared. Recovery half-times increased in an exponential manner within the observed dose range. Recovery between doses of radiation (sublethal damage recovery) proceeded at a slower rate than recovery following a single dose of radiation (potentially lethal damage recovery). CONCLUSIONS This study suggests that potentially lethal damage recovery is a saturated process and that the recovery half-time may increase in a linear-quadratic exponential function of dose similar to the absolute recovery level. In addition, if iso-recovery levels are compared, the recovery half-time is similar in rapidly and slowly proliferating cell populations.