Ralph E. Durand

Cell killing, radiosensitization and cell cycle redistribution induced by chronic hypoxia☆

Some of the biological changes associated with extreme hypoxia at 37 degrees C (less than 10 ppm pO2) were examined in Chinese hamster V79 cells. Specifically, extreme hypoxia caused an initial decrease in plating efficiency to 55% in 4 hr after the onset of hypoxia. Beyond this time, the decline in plating efficiency was more gradual reaching 35% of control at 20 hr. Flow microfluorimetry (FMF) studies, in which cells are sorted on the basis of DNA content and then assayed for viability, demonstrated that mid S phase cells were most sensitive to chronic hypoxia, with surviving fraction equal to 2.5% at 20 hr. Furthermore, the viability of G1 and G2/M cells, after 20 hr of hypoxic storage, was also reduced to 20 and 7.6%, respectively. Hypoxia also caused alterations in the cell cycle distribution of initially asynchronous cells, as determined by dual parameter FMF measurements of both cellular DNA content and incorporated BudR. In particular, G2/M cells completed mitosis, while G1 cells showed little or no movement. Lastly, cells stored in chronic hypoxia displayed an enhanced radiosensitivity when compared to acutely hypoxic cells. Possible reasons for these observations are discussed.

Evaluation of Nitroheterocyclic Radiosensitizers Using Spheroids

Publisher Summary This chapter focuses on the studies regarding in vitro testing of sensitizers largely using multicell spheroids, and presents the optimistic and pessimistic views as to the future of hypoxic cell sensitizers in the clinical setting. Some nitroheterocycles show promise for enhancing the response of selected tumors to radiation. Use of the drugs in humans is predicated on the belief that the presence of hypoxic cells within some tumors limits the success of conventional radiotherapy; however, in many tumors, conclusive evidence supporting this prejudice is lacking. The development of new drugs may be self-limiting as nitroheterocycles that are more effective as sensitizers are generally more reactive at a biochemical level. The effects of the nitroheterocycles can be observed under controlled and quantifiable conditions, and the roles of growth inhibition, cytotoxicity, and radiosensitization can be separated. The correlations between chemical and biological activities of nitroheterocycles suggest that at least with compounds that are effective as sensitizers only through the property of nitro-group reactivity, sensitization and toxicity are likely to be inseparable. As with most predictions, this may or may not be accurate. Combinations of radiation and hypoxic cell sensitizers with other modalities such as hyperthermia and selected chemotherapeutic agents such as alkylating agents, and even radioprotectors may improve the therapeutic ratio.

Roles of thiols in cellular radiosensitivity

Cellular thiols appear to have two separable mechanisms for influencing cellular radiosensitivity: 1) a direct role, through radical scavenging and/or hydrogen donation processes, and 2) an indirect role, regulating the amount of oxygen (or other electron affinic sensitizer) able to reach the radiosensitive targets of the cell. The contribution of each is easily measured with multicell spheroids, using fluorescence activated cell sorting techniques for selective recovery of cells from any depth within spheroids (i.e., from areas with different oxygenation). We have found that the region in the spheroid over which the transition from aerobic to anoxic conditions occurs is highly dependent on cellular thiol levels. Combining thiol depletion by DL-buthionine-S, R-sulfoximine (BSO) and electron affinic radiosensitization using misonidazole resulted in a markedly potentiated response to radiation, which we interpret as being primarily a result of reoxygenation.

Effects of lucanthone on chinese hamster V-79 cells I. Interaction with radiation in monolayers and spheroids

Abstract Lucanthone has previously been shown to reduce the capacity of cells to accumulate and repair sublethal radiation damage, which suggests its potential as an adjuvant to clinical radiotherapy. Present results indicate that lucanthone was effective even for V-79 cells having a markedly enhanced shoulder on the radiation survival curve as a result of growth as multicell spheroids. Lucanthone also proved to be an effective cytotoxic agent in large spheroids. Unlike radiation toxicity, however, lucanthone toxicity was equal for cells from asynchronous monolayers or small spheroids. Thus, for equal levels of drug toxicity, the radiation-modifying effect of lucanthone was greater in the spheroid; the “interaction” was larger in the system which had a greater capacity for accumulation and repair of sublethal radiation damage. Direct extrapolation of this result to the clinic would suggest that lucanthone be used judiciously, and that it might be of value as an adjuvant to radiotherapy only in the unusual circumstance where the malignant tissue has a larger capacity for accumulation and repair of sublethal radiation damage than the surrounding normal tissue.

Cellular oxygen utilization and radiation response of V-79 spheroids.

Chinese hamster V-79-171 cells, when placed in stirred suspension cultures, spontaneously grow as spherical multicell aggregates (spheroids) which eventually contain many thousand tightly-packed cells. These spheroids thus provide a valuable model for the study of oxygen transport, since oxygen concentration at the spheroid periphery can be fairly precisely controlled in the stirred suspension, and the consequences of oxygen diffusion inward through the tissue-like mass can be determined experimentally and compared with theoretical calculations. As in human tumors, appearance of central necrosis can be correlated with severe hypoxia. Additionally, since severely hypoxic cells are considerably more resistant to radiation than are oxygenated cells, determination of the fraction of hypoxic cells by their radioresistance provides a noninvasive technique for monitoring respiratory processes. Using this approach, we have found an excellent correlation between the rate of cellular oxygen utilization by a single cell suspension and the net radio-sensitivity of multicell spheroids when treated with modulating agents, varying from direct control of metabolic activity by changing the ambient temperature, to less direct effects on cellular oxygen consumption induced by exposure of cells to nitroheterocyclic radiosensitizers, chemotherapeutic agents, anesthetics, antibiotics, and even cell growth factors including insulin. Our data from these studies indicate that, at least in the simplistic spheroid system where oxygen delivery to the internal cells is a direct function of the respiratory activity of the external cells, control of cellular oxygen utilization is a convenient and effective method of controlling the net radiosensitivity.

Combination adriamycin/misonidazole toxicity in V79 spheroids.

The internal, hypoxic cells of V79 spheroids are preferentially killed by misonidazole, whereas adriamycin is preferentially toxic to the external cells due to diffusion limitations. Evaluation of combination treatments with misonidazole and adriamycin was therefore undertaken in an attempt to optimize and equalize cell killing throughout these spheroids. A fluorescence-activated cell sorter was used to select cells as a function of intracellular adriamycin or Hoechst-33342 fluorescence (and, therefore, as a function of location in the spheroid) for subsequent viability determinations. As expected, preferential toxicity of each agent was easily demonstrable, and with prolonged treatment times with both agents, greater uniformity of killing was achieved throughout the spheroid. Additionally, some suggestion of enhanced adriamycin toxicity to aerobic cells of spheroids chronically exposed to misonidazole was observed, analogous to the increased radiation sensitivity previously reported for similar exposure conditions. These results demonstrate that combinations of chemotherapeutic agents can be chosen to have preferential activity against different subpopulations of cells within V79 spheroids. Presumably, a better understanding of the factors contributing to drug resistance will lead to the application of similar principles for human cancer therapy.

On the Mechanism for Enhancement of Tumor Radiation to Hyperbaric Oxygen in Sodium Pentobarbital Anesthetized Rodents

Pentobarbital anesthesia has been observed to increase markedly the effectiveness of respiration of oxygen at 3 atmospheres of pressure absolute to increase the response of early generation isotransplants of C3H mouse tumors to two-dose irradiation. A possible mechanism of this phenomenon is suppression of oxygen utilization by the pentobarbital and hence increasing mean pO2 and oxygen diffusion lengths. Measurements of QO2 of suspension of MCaIV and FSaII cells from freshly excised tumor tissue have been measured for cells suspended in PBS, Hanks buffered with HEPES +/- glutamate. The oxygen utilization by these tumor cells in vitro (when measured at congruent to 10 minutes after excision) is low, viz. 1 nmole/min/mg protein as compared with 6-9 nmoles/min/mg protein for established cell lines cultured in vitro. The suppression of QO2 by 2mM pentobarbital is less than 10%. This is a concentration of pentobarbital that is judged to be close to that which obtains in the tissues of the animals in the radiation response assays. Pentobarbital at .2mM did not change the cell survival characteristics of Chinese V79 cell spheroids irradiated in vitro. The results of these experiments do not indicate the suppression of oxygen utilization is an important contributor to the observed phenomenon of the increased response of tumors irradiated in mice respiring oxygen at high pressure. The role of hypothermia produced by the anesthesia is under further study.

The enhanced radiation response of in vitro tumor models to insulin.

Abstract The presence of insulin during the irradiation of plateau phase baby hamster kidney, Chinese hamster ovary, Ehrlich tumor, human oral carcinoma, human laryngealcarcinoma, and V-79 cells improved radiation response. Insulin was also found to enhance the radiation response of multiceli V-79 spheroids. The radiosensitizing effect of insulin is both time and concentration dependent. Additional studies indicate that glucagon-free insulin and the insulin mimicking tripeptide, glyeyl-L-histidyl-L-lysine-acetate, also sensitize plateau phase cultures of V-79 cells to radiation.

Low dose-rate irradiation of multicell spheroids☆

Abstract The effectiveness of irradiation at 88 rad/hr was studied in multicell spheroids of three sizes. Survival curves for all three spheroid populations (including a population initially containing a significant number of hypoxic cells) were nearly identical at this dose-rate. Attempts to determine the mechanism(s) responsible for the unexpected sensitivity of the large spheroids indicated that hypoxic cells were no longer detected after accumulation of 2130 rad. The possible role of an increased radiation killing of the hypoxic cells at the low dose-rate thus could not be evaluated.