The effect of continuous wave ultrasound exposures on the cytotoxicity of adriamycin has been studied. It has been found that 2.6 MHz, 2.3 Wcm-2 (spatial average) ultrasound can enhance the cell killing potential of adriamycin both in suspensions of single V79 chinese hamster fibroblast cells and in spheroids formed from these cells. The ratio of the slopes of the survival curves for single cell suspensions is 1.5. For spheroids, the growth delay is increased by 1.3 days by simultaneous ultrasound exposure. Flow cytometric studies of the intracellular concentration of adriamycin following ultrasound exposure reveals that this is increased when compared with that measured when the cells are only exposed to adriamycin. Evidence is presented to suggest that this is a non-thermal effect of ultrasound.

Two methods involving labelling cells with bromodeoxyuridine (BrdUrd) have been used to study by flow cytometry the effect of hyperthermia (43 degrees C for up to 1 h) on Chinese hamster V79 cells. One method involved the use of an antibody to BrdUrd after pulse-labelling the cells either before or at time intervals after treatment. In the second method, the cells were incubated continuously in BrdUrd after heat treatment, and the components of the cell cycle were then visualized by staining with a combination of a bis-benzimidazole and ethidium bromide. All three methods showed that heating at 43 degrees C stopped DNA synthesis which, at 37 degrees C, subsequently recovered reaching the normal rate 8-12 h later. The cells in S phase at the time of treatment then progressed to G2 where they were further delayed. Cells heated in G1. after the recommencement of synthesis, progressed around the cycle, albeit slower than in unheated cells. The difference between the cells in G1 and S phases at the time of treatment may account for the greater sensitivity of S phase cells to hyperthermia.

The effect of combined ultrasound and heat treatments on Chinese hamster multicellular spheroids of varying size was investigated using growth rate, single cell survival and ultrastructural damage as endpoints. Ultrasonic irradiation at 37 degrees C had no effect on the growth rate of 200-730 microns spheroids. Similarly there was no effect on the growth rate of 350 microns spheroids when irradiated during a 60 min exposure to 41.5 degrees C. However, spheroids of 200-700 mm diameter showed growth delay when held at 43 degrees C for 1 h. The effect was enhanced with concomitant ultrasound irradiation but was not dependent on spheroid size. When 200 and 400 microns spheroids held at 43 degrees C for 60 min were irradiated with different ultrasonic intensities a dose-dependent decrease in surviving fraction and a dose-dependent increase in growth delay was obtained. When surviving fraction was plotted as a function of growth delay a good correlation was obtained, suggesting that the combination of heat and ultrasound irradiation does not produce cytostasis in the surviving cells of either 200 or 400 microns spheroids. At the ultrastructural level increased cytoplasmic vacuolation was the only result of ultrasonic irradiation at 37 degrees C. Exposure to 43 degrees C for 60 min was required to elicit thermal damage. This took the form of membrane evagination at the spheroid surface, vacuolation of the cytoplasm, grouping of organelles around the periphery of the nucleus, and fragmentation of the nucleolus. These effects were enhanced with concomitant ultrasonic irradiation but other features were also noted, viz. disaggregation of polyribosomes, dilation of the rough endoplasmic reticulum and blebbing of the nuclear membrane. Damage was independent of spheroid size. These results are in agreement with previous data obtained from single-cell studies. Indicating that there is a non-thermal, non-cavitational component to the cell killing in multicellular spheroids resulting from combined heat and ultrasound treatment.

No abstract

No abstract