Overcoming the radioresistance of prostate cancer cells with a novel Bcl-2 inhibitor.

Bcl-2 overexpression is an important mechanism underlying the aggressive behavior of prostate cancer cells and their resistance to radio- or chemotherapy. HA14-1, a recently discovered organic Bcl-2 inhibitor, potently induces apoptosis in various human cancer cells. Sequential exposure of radioresistant LNCaP (wild-type (wt) p53), LNCaP/Bcl-2 (wt p53) and PC3 (mutant p53) prostate cancer cells to a minimally cytotoxic concentration of 10 microM HA14-1 for 1 h followed by 1-6 Gy gamma radiation, resulted in a highly synergistic (combination index <1.0) induction of cell death as determined by an apoptosis assay at 72 h, and a clonogenicity assay at 12 days, after the initial treatment. The reverse treatment sequence did not cause a synergistic induction of cell death. When compared to individual treatments, cell death induced by the combined treatment was associated with dramatically increased reactive oxygen species (ROS) generation, c-Jun N-terminal kinase (JNK) activation, Bcl-2 phosphorylation, cytochrome c release, caspase-3 activation and DNA fragmentation. Exposure to either 200 microg/ml of the antioxidant alpha-tocopherol or 10 microM JNK inhibitor SP600125 before the combined treatment resulted in decreased activation of JNK and caspase-3 as well as decreased DNA fragmentation. However, treatment with the pancaspase inhibitor carbobenzoxyl-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone before the combined treatment inhibited apoptosis without affecting JNK activation, and this inhibitory effect was enhanced in the presence of alpha-tocopherol or SP600125. Taken together, our results indicate that HA14-1 potently sensitizes radioresistant LNCaP and PC3 cells to gamma radiation, regardless of the status of p53. ROS and JNK are important early signals that trigger both caspase-dependent and -independent cell death pathways and contribute to the apoptotic synergy induced by the combined treatments.

Thermotolerance in chicken red blood cells studied by 31P NMR spectroscopy.

Chicken red bloods cells (RBCs) were used as a model for cell survival following hyperthermia of differentiated cells, with rat RBCs serving as controls. The purpose was to investigate whether cells heated at 51.5 degrees C with or without prior heat shock exhibited discernible differences in phosphorus metabolites, intracellular pH or Mg2+ using 31P nuclear magnetic resonance spectroscopy. The biochemical differences that distinguished the heat-shocked from the non-heat-shocked chicken cells were: (1) a decrease in intensity of the low and wide (300 Hz) resonance that underlies the high resolution signals and which arises from the partially mobile membrane phospholipids, suggesting that membrane fluidity was decreased during the induction of thermotolerance; and (2) a time-dependent leftward shift of the peak representing the 4,6P of inositol pentaphosphate together with a broadening of all of the 31P peaks during heat-shock and heat challenge, which persisted after return to the control temperature. This is consistent with significant oxygen consumption in the heat-shocked but not the non-heat-shocked cells. We conclude that chicken RBCs are capable of specific metabolic and physiologic responses to heat shock, as expected in cells known to produce heat-shock proteins and to be capable of thermotolerance induction.

The effects of ionizing radiation on avian erythrocytes.

The effects of ionizing radiation were examined in terminally differentiated cells using nucleated chicken erythrocytes (RBCs) as the model. We used a hemolytic assay to score radiation damage to RBCs. Chicken RBCs received 0 to 100 Gy of radiation at dose rate of 10 Gy/min. Radiation-induced hemolysis occurred in a dose-dependent manner but not immediately after irradiation. Hemolysis became apparent at 24 h after treatment. A threshold for radiation dose response was observed. At doses below 30 Gy, hemolysis in irradiated samples was indistinguishable from that in nonirradiated controls. A total dose of 100 Gy was used for the split-dose experiments. The results showed that chicken RBCs were able to repair radiation damage and that the half-time for maximum recovery was approximately 30 min at 36 degrees C. Recovery from gamma radiation was also affected by the interfraction temperature.

Effects of hyperthermia on avian red blood cells.

Our primary goal was to develop a model for studying the effects of heat on terminally differentiated cells. Using nucleated chicken red blood cells (RBC) as the model, heat dose-response and thermotolerance were investigated. A new haemolytic assay was developed to score them. Heating chicken RBC for as long as 3 h at 51.5 degrees C resulted in only small amounts of haemolysis immediately after heating. When haemolysis was scored 1-2 days after heating, heat-induced haemolysis increased with heating temperature and duration. Heat shock of 30-90 min at 42.6 degrees C just before heat challenge of 40 min at 51.5 degrees C, or heat shock of 15 min at 43.1 degrees C with incubation of 0-2 h at 35-37 degrees C prior to the same heat challenge induced thermotolerance, but the levels of heat resistance achieved were different. Similar experiments were performed using potassium leakage as the endpoint. Potassium leakage was measurable immediately after heating, and it increased with heating duration, but there was no correlation between potassium leakage and haemolysis. Potassium leakage was not a suitable criterion for measuring thermotolerance.

Radiation factors and their influence on induction of oxygen resistance.

Shortly after gamma irradiation, flour beetles exhibited a decline in resistance to oxygen toxicity. Then, about 2 weeks after irradiation, the 50% lethal dose (LD50) exposure time in pure oxygen was much greater than that of nonirradiated beetles, and this enhanced resistance persisted for about 6 months. The magnitude of the enhancement was a function of dose, decreased with increasing age at irradiation, and was modified by radiation factors. Both dose protraction and dose fractionation reduced the development of oxygen resistance to approximately the same degree that it reduced acute radiation lethality. This suggests that both the initial sensitization and the later enhancement of resistance are correlated with the residual biological damage rather than with the physical dose or initial damage.

Radiation-enhanced resistance to oxygen: a possible relationship to radiation-enhanced longevity.

Shortly after gamma-irradiation, flour beetles (Oklahoma strain of Tribolium confusum) exhibited a decline in resistance to oxygen toxicity. Beginning about two weeks after irradiation, however, the LD50 exposure time in pure oxygen was much greater than that of nonirradiated beetles, and this enhanced resistance persisted for about 6 months. The magnitude of the enhancement was a function of dose, and decreased with increasing age at irradiation. These characteristics of a radiation-enhanced resistance to a stress are comparable to the characteristics of radiation-enhanced longevity in Tribolium, in that after irradiation mortality rate is less than that of controls for about 6 months, the magnitude of the effect is dose-dependent, and older beetles are refractory to the effect. Irradiation under anoxic conditions reduced the development of oxygen resistance to the same degree that it reduced acute radiation lethality, suggesting that the amount of biological damage is the critical factor. These results are discussed in terms of the "induced repair" theory of radiation-enhanced longevity of insects.

Repair studies utilizing a fly-pupariation model of postmitotic cell response.

Delay in pupariation of irradiated fly (and other insect) larvae results from a neuroendocrine disturbance and represents a dose-dependent response of differentiated postmitotic tissue. Many experimental manipulations (e.g. heating) may themselves upset timing, limiting usefulness of this phenomenon as a model of postmitotic tissue response. Doses greater than 20 Gy inhibit retraction of specific muscles at time of pupariation, leading to formation of elongate puparia. Degree of elongation (ratio of length: breadth) was also a function of dose. Peak sensitivity for elongation occurs later than that for delay, but both endpoints exhibit rapid kinetics for sparing effect of dose fractionations (sdf). With degree of elongation as endpoint, we demonstrated synergism between heat and radiation, and heat markedly inhibited sdf. Maintenance of irradiated larvae under wet conditions greatly prolonged the larval stage, and degree of elongation was reduced. This recovery, analogous to repair of potentially-lethal damage (PLD) in plateau-phase cell cultures, proceeds much more slowly than sdf.

Age and sensitivity to oxygen in the flour beetle, Tribolium confusum.

Sensitivity of Tribolium confusum males and females to pure oxygen at one atmosphere was measured as a function of adult age. Sensitivity to oxygen increased markedly with age; the reciprocal to the LD50 exposure time was a linear function of age. The sensitivity to oxygen increased more rapidly with age in males than in females; this may be related to the greater longevity of females in this species. Thus the LD50 exposure time represents an index of physiological age. Sublethal exposure to pure oxygen, unlike equivalent exposure to ionizing radiation, did not affect longevity. This suggests that the age-associated increase in oxygen sensitivity is a non-specific stress effect, rather than additivity of acute oxygen damage with cumulative atmosphere-induced lesions.