Charles A. Vidair

p53-Dependent G1 arrest and p53-independent apoptosis influence the radiobiologic response of glioblastoma

PURPOSE Loss of the p53 tumor suppressor gene has been associated with tumor progression, disease relapse, poor response to antineoplastic therapy, and poor prognosis in many malignancies. We have investigated the contribution of p53-mediated radiation-induced apoptosis and G1 arrest to the well described radiation resistance of glioblastoma multiforme (GM) cells. METHODS AND MATERIALS Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Isogenic derivatives of glioblastoma cells differing only in their p53 status were generated using a retroviral vector expressing a dominant negative mutant of p53. Radiation-induced apoptosis was assayed by Fluorescence-activated cell sorter (FACS) analysis, terminal deoxynucleotide transferase labeling technique, and chromatin morphology. Cells were synchronized in early G1 phase and mitotic and labeling indices were measured. RESULTS Radiation-induced apoptosis of GM cells was independent of functional wild-type p53 (wt p53). Decreased susceptibility to radiation-induced apoptosis was associated with lower alpha values characterizing the shoulder of the clonogenic radiation survival curve. Using isogenic GM cells differing only in their p53 activity, we found that a p53-mediated function, radiation-induced G1 arrest, could also influence the value of alpha and clonogenic radiation resistance. Inactivation of wt p53 function by a dominant negative mutant of p53 resulted in a significantly diminished alpha value with no alteration in cellular susceptibility to radiation-induced apoptosis. The clonal derivative U87-LUX.8 expressing a functional wt p53 had an alpha (Gy-1) value of 0.609, whereas the isogenic clonal derivative U87-175.4 lacking wt p53 function had an alpha (Gy-1) value of 0.175. CONCLUSION We conclude that two distinct cellular responses to radiation, p53-independent apoptosis and p53-dependent G1-arrest, influence radiobiological parameters that characterize the radiation response of glioblastoma cells. Further understanding of the molecular basis of GM radiation resistance will lead to improvement in existing therapeutic modalities and to the development of novel treatment approaches.

Evaluation of a role for intracellular Na+, K+, Ca2+, and Mg2+ in hyperthermic cell killing

A dose of heat which renders 98% of a population of Chinese hamster ovary cells reproductively dead has no significant effect on their Na+, K+, or Mg2+ content by 28 h postheat. In contrast, the cellular Ca2+ content increases in a dose-dependent manner as observed at 22 h after heating for 15-35 min at 45 degrees C. However, the rates of both influx and efflux of Ca2+ were reduced by heating. Increasing the cellular Ca2+ content by incubating the cells in high extracellular Ca2+, either at the time of heating or for a period of 22 h following heat, does not potentiate the lethal effect of heat. Completely blocking the heat-induced increase in Ca2+ content by incubating the cells in medium containing a low Ca2+ concentration does not protect the cells. Therefore, we conclude that heat does not produce any significant changes in the Na+, K+, or Mg2+ content of cells and that the heat-induced increase in Ca2+ does not play an important role in hyperthermic cell killing.

Noninvolvement of the Heat-Induced Increase in the Concentration of Intracellular Free Ca2+ in Killing by Heat and Induction of Thermotolerance

Mouse C3H 10T1/2 cells exhibited a two- to threefold increase in the concentration of free Ca2+ during heating at 45 degrees C. The increase was maximal for a heat dose which was still in the shoulder region of the survival curve. The increase was fully reversible in heat-sterilized cells. By changing the concentration of extracellular Ca2+, it was possible to modulate the concentration of intracellular free Ca2+ in heated cells. Lowering the extracellular concentration to 0.03 mM reduced the baseline concentration of intracellular free Ca2+, and prevented it from increasing in heated cells to a level exceeding that of nonheated cells incubated in medium containing 2.0 or 5.0 mM Ca2+. Raising the concentration of extracellular Ca2+ to 15.0 mM raised the baseline, and resulted in a heat-induced increase in free Ca2+ which was twofold higher than that of cells heated in medium containing 2.0 or 5.0 mM Ca2+. An elevated concentration of intracellular free Ca2+ during and after heating did not potentiate thermal killing, nor did a reduced concentration during and after heating mitigate killing. Furthermore, the data argue against a heat-induced increase in free Ca2+ to some threshold level, which potentiates cell killing by some other parameter. In addition, cells heat-shocked in either 0.03 or 5.0 mM extracellular Ca2+, and then incubated in the same concentration for 12 h at 37 degrees C, developed quantitatively similar amounts of tolerance to a second heating. The data suggest that the concentration of intracellular free Ca2+ does not play a critical role in thermal killing or the induction and development of thermotolerance.

Mechanisms of mild hyperthermia-induced cytotoxicity in human prostatic carcinoma cells: Perturbation of cell cycle progression and DNA fragmentation

Abstract 1. 1.|We investigated the relationship between heat-induced perturbation of cell cycle progression and cytotoxicity at low temperature hyperthermia in three human cell lines human breast carcinoma cells (MCF-7) and human prostatic adenocarcinoma cells (DUT-145 and PC-3). 2. 2.|MCF-7 cells which were resistant to mild hyperthermia exhibited a G 1 block during heating at 41°C. In contrast, DUT-145 and PC-3 cells which were sensitive to mild hyperthermia failed to accumulate in G 1 -phase. Both DUT-145 and PC-3 cells progressed through S-phase and accumulated in the G 2 /M-phase. These results rule out any species-based generalization for heat sensitivity. 3. 3.|Electron micrographs and gel electrophoresis data show the accumulation of mitotic-like cells and DNA fragmentation during mild hyperthermia in prostatic carcinoma cells. 4. 4.|These results suggest that cell death is associated, at least partly with DNA fragmentation, perhaps apoptosis in prostatic carcinoma cells.

Maintenance of Intracellular Free Ca2+ Homeostasis Following Lethal Heat Shock

We have manipulated the extracellular Ca2+ concentration (1.8 mM in normal Dulbeccos modified Eagles medium) to test whether the resulting effect on intracellular free Ca2+ homeostasis was similar in heat-sterilized and nonheated mouse NIH-3T3 cells. The responsiveness of the intracellular free Ca2+ concentration to changes in the extracellular Ca2+ concentration was not affected by prior treatment of the cells with trypsin, or by the extracellular Ca2+ concentration during dye loading (indo-1, AM). Rather, the intracellular free Ca2+ concentration was dependent upon the ambient Ca2+ concentration during analysis by flow cytometry. When the extracellular Ca2+ concentration was decreased to 0.017 mM, either before or after a lethal heat shock, the intracellular free Ca2+ concentration (approximately 300 nM) decreased to a similar extent in both heated and control cells (to approximately 30-100 nM). Similarly, when the extracellular Ca2+ concentration was increased to 15.0 mM, either before or after a lethal heat shock, the intracellular free Ca2+ concentration exhibited a quantitatively similar increase in both heated and nonheated cells (to approximately 400-1000 nM). These data indicate that a lethal heat dose does not inhibit the intact cells ability to maintain intracellular free Ca2+ homeostasis.

Somatic Hybridization in Tomato

The reasons for subjecting the tomato eultivar, Lycopersicon esculentum, to numerous cell and tissue culture studies have been twofold: agronomic improvement of this important crop species and exploitation of tomatO’s abundant classical genetics for basic research purposes. Several years ago we began experiments utilizing protoplasts of Lycopersicon species, with the goals of developing methods to culture and regenerate plants, influencing transmission of genomes following cell fusion, and genetically analyzing plants resulting from protoplast or fusion product regeneration. At the time, little information was available about the requirements for successful culture and regeneration of members of the tomato genus. We looked to the facile protoplast culture and fusion of other Solanaceous species, Nicotiana and Petunia, as guides, as well as the arduous but effective methods for potato protoplasts developed by Shepard et al. (1980). Here we will summarize our knowledge of Lycopersicon protoplast culture and fusion and the analysis of regenerated plants.