Michael D. O'Hara

Mammalian cells adapted to growth at pH 67 have elevated HSP27 levels and are resistant to cisplatin

HSP27 levels are elevated in two Chinese hamster cell lines and in a human melanoma cell line adapted to growth at pH 6.7. The level of HSP72 is elevated in the melanoma cell line but not in the hamster cell lines adapted to growth at pH 6.7. HSC73 levels are not elevated in any of the adapted cell lines. Low pH adapted cells from all cell lines are resistant to cisplatin. It is proposed that elevated HSP27 levels in low pH-adapted cells may play a role in resistance to hyperthermia and resistance to cisplatin.

Thermal response and hyperthermic radiosensitization of scid mouse bone marrow CFU-C.

PURPOSE Scid mice are severely immunodeficient as a result of a defective recombinase system. Mice with the scid mutation have been shown to have an increased sensitivity to ionizing radiation, presumably as a result of an inability to repair DNA damage. Little is known of the impact of this mutation on the thermal response and on hyperthermic radiosensitization. This investigation established the thermal response (42-44 degrees C), patterns of thermotolerance development, and the impact of hyperthermia (60 min at 40 degrees C or 42 degrees C) on the radiation response of bone marrow colony forming unit-culture cells (CFU-C) in scid mice. METHODS AND MATERIALS Anesthetized scid mice (pentobarbital, 90 mg/kg) were killed by cervical dislocation and the nucleated marrow obtained from both tibia and femora by passing 2 ml of cold McCoys 5A medium supplemented with 15% fetal bovine serum through each bone. Single cell suspensions of nucleated marrow were heated in 12 x 75 mm sterile tissue culture tubes at a concentration of approximately 5 x 10(6) cells/ml. Radiation, when used, was delivered immediately prior to hyperthermia by a 137Cs irradiator (dose rate of 1.20 Gy/min). Colony forming unit-culture were cultured in semisolid agar in the presence of colony stimulating factor (conditioned medium from L929 cells) for 7 days. RESULTS The slope of the radiation dose-response curve for CFU-C in scid mice was biphasic, the Dos (+/- SE) were 0.29 +/- 0.03 Gy and 1.09 +/- 0.20 Gy, respectively. The Dos of the radiation dose-response curve for wild type marrow from CB-17 and Balb/c mice were 1.28 +/- 0.05 Gy and 1.47 +/- 0.15 Gy, respectively. The Dos of the hyperthermia dose-response curves for scid mice were 75 +/- 5, 10 +/- 1.4, and 4 +/- 0.2 min, respectively, for temperatures of 42 degrees, 43 degrees, and 44 degrees C. Thermotolerance development at 37 degrees C increased to a maximum at approximately 240 min after acute hyperthermia (15 min at 44 degrees C) and thereafter, decreased to control levels within 15 h. Thermotolerance did not develop in scid CFU-C during chronic hyperthermia at temperatures < 42.5 degrees C. Hyperthermia (60 min at 40 degrees or 42 degrees C) immediately after ionizing radiation did not significantly alter the terminal slope of the radiation dose-response curve of scid CFU-C (Do = 1.28 +/- 0.08 Gy). By contrast, hyperthermia following radiation of wild type CFU-C resulted in a decrease in the Do from 1.47 +/- 0.05 Gy (Balb/c, rad only) to 1.31 +/- 0.08 or 1.06 +/- 0.18 Gy for 60 min at 40 degrees or 42 degrees C, respectively. CONCLUSION These results show that the thermal response and the pattern of thermotolerance development of scid CFU-C were similar to that of wild type Balb/c CFU-C, but that hyperthermia given immediately after ionizing radiation did not alter the radiation response of scid CFU-C. The scid mutation does not increase hyperthermic sensitivity or change the pattern of thermotolerance development of scid mouse CFU-C, implying that the scid mutation is not involved with thermal response, but does render the already radiation-sensitive scid cells incapable of thermal radiosensitization.

Intrinsic thermal response, thermotolerance development and stepdown heating in murine bone marrow progenitor cells

Thermal response, thermotolerance development and stepdown heating (SDH) in the murine bone marrow granulocyte-macrophage (CFU-GM) progenitors were determined in vitro. Marrow was removed from femora and tibia, heated in McCoys 5A medium plus 15% FBS and cultured in soft agar in the presence of three different sources of colony stimulating factor. D0s (+/- SE) for survival curves of CFU-GM heated in vitro were 147 +/- 13, 71 +/- 9, 37 +/- 2, 19 +/- 0.7, 11 +/- 1, and 4.3 +/- 0.3 min, for temperatures of 41.8, 42, 42.3, 42.5, 43 and 44 degrees C, respectively. Arrhenius analysis showed inactivation enthalpies of 812 +/- 9 KJoules/mole (193 +/- 2 Kcal/mole) above, and 2142 +/- 157 KJoules/mole (509 +/- 37 Kcal/mole) below, an inflection at 42.5 degrees C. Thermotolerance development was evident during prolonged hyperthermia exposure at temperatures below 42.5 degrees C (chronic hyperthermia) as a change in the slope of the survival curves after approximately 110 min of heating. Thermotolerance development at 37 degrees C after exposure to temperatures of 43 degrees C or greater (acute hyperthermia) was assessed by fractionated heat treatments consisting of an initial heat treatment (15 min at 44 degrees C) followed by incubation at 37 degrees C and challenge with 15 min or 25 min at 44 degrees C. Maximum thermotolerance occurred after 210 and 330 min at 37 degrees C, respectively. The half-time for maximum thermotolerance development was 36 min. Depending on the amount of heat damage and the maximum amount of thermotolerance development, the decay of thermotolerance was complete after approximately 48-72 h at 37 degrees C. An exposure of 10 min at 44 degrees C before incubation at 40 or 41 degrees C (stepdown heating) reduced the slope of the 40 or 41 degrees C survival curves by inhibiting thermotolerance development that would have otherwise occurred. D0s were 100 +/- 19 and 45 +/- 5 min for 40 and 41 degrees C incubation preceded by 10 min at 44 degrees C, respectively. These studies indicate that whole-body or regional hyperthermia protocols designed either to treat solid tumours or to purge leukemic stem cells from marrow ex vivo should avoid inadvertent temperature elevations to large volumes of marrow. Although, marrow progenitors are capable of thermotolerance development during exposure to temperatures up to 42.3 degrees C, results suggest that conditions of stepdown heating may prevent thermotolerance development.