Hyperthermic killing and hyperthermic radiosensitization in Chinese hamster ovary cells: effects of pH and thermal tolerance.

To quantitatively relate heat killing and heat radiosensitization, asynchronous or G1 Chinese hamster ovary (CHO) cells at pH 7.1 or 6.75 were heated and/or X-irradiated 10 min later. Since no progression of G1 cells into S phase occurred during the heat and radiation treatments, cell cycle artifacts were minimized. However, results obtained for asynchronous and G1 cells were similar. Hyperthermic radiosensitization was expressed as the thermal enhancement factor (TEF), defined as the ratio of the D0 of the radiation survival curve to that of the D0 of the radiation survival curve for heat plus radiation. The TEF increased continuously with increased heat killing at 45.5 degrees C, and for a given amount of heat killing, the amount of heat radiosensitization was the same for both pH's. When cells were heated chronically at 42.4 degrees C at pH 7.4, the TEF increased initially to 2.0-2.5 and then returned to near 1.0 during continued heating as thermal tolerance developed for both heat killing and heat radiosensitization. However, the shoulder (Dq) of the radiation survival curve for heat plus radiation did not manifest thermal tolerance; i.e., it decreased continuously with increased heat killing, independent of temperature, pH, or the development of thermotolerance. These results suggest that heat killing and heat radiosensitization have a target(s) in common (TEF results), along with either a different target(s) or a difference in the manifestation of heat damage (Dq results). For clinical considerations, the interaction between heat and radiation was expressed as (1) the thermal enhancement ratio (TER), which is the dose of X rays alone divided by the dose of X rays combined with heat to obtain an isosurvival, e.g., 10(-4), and (2) the thermal gain factor (TGF), the ratio of the TER at pH 6.75 to the TER at pH 7.4. Since low pH reduced the rate of development of thermal tolerance during heating at low temperatures, low pH enhanced heat killing more at 42-42.5 degrees C than at 45.5 degrees C where thermal tolerance did not develop. Therefore, the increase in the TGF after chronic heating at 42-42.5 degrees C was greater than after acute heating at 45.5 degrees C, due primarily to the increase in heat killing causing an even greater increase in heat radiosensitization. These findings agree with animal experiments suggesting that in the clinic, a therapeutic gain for tumor cells at low pH may be greater for temperatures of 42-42.5 degrees C than of 45.5 degrees C.

Hyperthermic survival of Chinese hamster ovary cells as a function of cellular population density at the time of plating.

The survival of synchronous G1 or asynchronous Chinese hamster ovary cells in vitro to heat treatment may depend on the cellular population density at the time of heating and/or as the cells are cultured after heating. The addition of lethally irradiated feeder cells may increase survival at 10(-3) by as much as 10- to 100-fold for a variety of conditions when cells are heated either in suspension culture or as monolayers with or without trypsinization. The protective effect associated with feeder cells appears to be associated with close cell-to-cell proximity. However, when cells are heated without trypsinization about 24 hr or later after plating, when adaptation to monolayer has occurred, the protective effect is reduced; i.e., addition of feeder cells enhances survival much less, for example, about 2- to 3-fold at 10(-2)-10(-3) survival. Also, the survival of a cell to heat is independent of whether the neighboring cell in a microcolony is destined to live or die. Finally, if protective effects associated with cell density do occur and are not controlled, serious artifacts can result as the interaction of heat and radiation is studied; for example, survival curves can be moved upward, and thus changed in shape as the number of cells plated is increased with an increase in the hyperthermic treatment or radiation dose following hyperthermia. Therefore, to understand mechanisms and to obtain information relevant to populations of cells in close proximity, such as those in vivo, these cellular population density effects should be considered and understood.

Cytoplasmic microtubules in normal and transformed cells in culture: analysis by tubulin antibody immunofluorescence.

Monospecific antibody directed against bovine brain tubulin was used as an immunofluorescent probe to evaluate the distribution of microtubules in normal and transformed cells grown in tissue culture. The fluorescent staining pattern of transformed and nontransformed cells is significantly different and may be used in conjunction with other morphological features to identify transformants in mixed cell populations. Normal cells are flattened, elongated, and fibroblastic; they display numerous Colcemid-sensitive fluorescent cytoplasmic filaments, presumably microtubules. Transformed cells, however, are smaller, more polygonal in shape, and contain very few cytoplasmic tubules. During mistosis the cytoplasmic microtubule complex of normal cells completely disappears, but reappears after cell division. Treatment of transformed cells with dibutyry-adenosine 3':5'-cyclic monophosphate plus testosterone or theophylline restores the normal fibroblastic appearance of the cells and stimulates the assembly of numerous cytoplasmic microtubules. This study provides further evidence for two separate microtubule entities in cycling nontransformed cells: a cytoplasmic microtubule complex and the microtubules of the mitotic spindle. Although an interchange of tubulin dimers seems to exist between microtubules in the two systems, control of tubule assembly may be under separate constraints. Stimulation of cytoplasmic microtuble assembly in transformed cells by derivatives of adenosine 3':5'-cycle monophosphate suggests that impairment of the cytoplasmic microtubule complex in these cells may be due to suboptimal levels of adenosine 3':5'-cyclic monophosphate.