Evidence for pronounced bystander effects caused by nonuniform distributions of radioactivity using a novel three-dimensional tissue culture model

Abstract

A new in vitro multicellular cluster model has been developed to assess the impact of nonuniform distributions of radioactivity on the biological response of mammalian cells, and the relative importance of bystander effects compared to conventional radiation effects. Chinese hamster V79 cells are labeled with tritiated thymidine ([³H]dThd), mixed with unlabeled V79 cells, and centrifuged gently to form multicellular clusters about 1.6 mm in diameter. The short range of the ³H β particles effectively allows only self-irradiation of labeled cells and no cross-irradiation of unlabeled cells. The percentage of labeled cells is controlled precisely by varying the number of labeled cells mixed with unlabeled cells prior to assembling the cluster. The clusters are assembled in the absence or presence of 100 μM lindane, a chemical that is known to inhibit formation of gap junctions. After the clusters are maintained at 10.5°C for 72 h, the cells are dispersed and plated for colony formation. In the case of 100% labeling, the survival of cells in the cluster depends exponentially (SF = e^−A/1.8) on the cluster activity A (in kBq), and lindane has no effect on the response. A two-component exponential response is obtained for 50% labeling in the absence of lindane (SF = 0.33 e^−A/0.81 + 0.67 e^−A/11.8), and lindane has a marked effect on the response (SF = 0.33 e^−A/1.6 + 0.67 e^−A/41.6). These data suggest that bystander effects play an important role in the biological response of V79 cells when the ³H is localized in the cell nucleus and distributed nonuniformly among the cells. In contrast, bystander effects cannot be detected above traditional radiation effects (i.e. direct + indirect) when the ³H is localized in the cell nucleus and distributed uniformly among the cells. These results indicate that this multicellular cluster model is well suited for studying the effects of nonuniform distributions of radioactivity, including bystander and “hot-particle” effects. Furthermore, these results suggest that by-stander effects may play an important role in the prediction of the biological effects of radiopharmaceuticals used in medical diagnosis and treatment.

FIG. 1

Assembly of multicellular cluster of V79 cells in which 50% of the cells are radiolabeled with [³H]dThd.

FIG. 2

Survival of V79 cells after acute (◆) and chronic (◇) irradiation of multicellular clusters with ¹³⁷Cs γ rays. Irradiations were carried out at 10.5°C. The acute dose rate was ~1–1.7 Gy per minute. For chronic irradiation, the tubes containing clusters were placed at different distances from a 370-MBq ¹³⁷Cs source housed in a small stainless steel capsule. After the acute and chronic irradiation, the clusters were dismantled, cells were processed, and the surviving fraction was determined compared to cells from unirradiated control clusters. Representative standard deviations are indicated by the error bars. Solid curves represent least-squares fits to the linear-quadratic model.

FIG. 3

Survival of V79 cells as a function of cluster activity of [³H]dThd. Data are shown for experiments where 50% (●, ○) or 100% (▲, △) of the cells were radiolabeled in multicellular clusters which were maintained at 10.5°C for 72 h and then the surviving fraction was determined compared to unlabeled cells. Data from two independent experiments are plotted for each labeling condition and are differentiated by open and closed symbols. Representative standard deviations are indicated by the error bars.

FIG. 4

Chemotoxicity of lindane when V79 multicellular clusters were exposed to the chemical at 10.5°C for 72 h. Representative standard deviations for individual data points are shown. Data from three independent experiments are indicated by different symbols (○, □, △).

FIG. 5

Effect of lindane concentration on survival of V79 cells from multicellular clusters in which 50% of the cells are labeled with [³H]dThd. The surviving fraction increased steadily with increasing lindane concentration up to 100 μM, after which no additional protective effect was observed. Data from three independent experiments are indicated by three symbols (●, ■, ▲). Representative standard deviations are indicated by the error bars.

FIG. 6

Survival of V79 cells as a function of cluster activity of [³H]dThd when 50% of the cells were labeled. Multicellular clusters were maintained at 10.5°C for 72 h in the presence of (1) [³H]dThd (▼, ▽; data reproduced from Fig. 3); (2) [³H]dThd + 0.58% DMSO (■, □); or (3) [³H]dThd + 0.58% DMSO + 100 μM lindane (◆, ◇). Data from two independent experiments are plotted for each treatment condition and are differentiated as open and closed symbols. Representative standard deviations are indicated by the error bars. The short-dashed, long-short dashed, and solid curves represent least-squares fits of the data to Eq. (1) for cases 1, 2 and 3, respectively.

FIG. 7

Survival of V79 cells as a function of cluster activity of [³H]dThd when 100% of the cells were labeled. Multicellular clusters were maintained at 10.5°C for 72 h in the presence of (1) [³H]dThd (▼, ▽); (2) [³H]dThd + 0.58% DMSO (■, □); (3) [³H]dThd + 0.58% DMSO + 100 μM lindane (◆, ◇). Data from two independent experiments are plotted for each treatment condition and are differentiated as open and closed symbols. Representative standard deviations are indicated by the error bars. The short-dashed, long-short dashed, and solid curves represent least-squares fits of the data to Eq. (1) for cases 1, 2 and 3 respectively.

FIG. 8

Transfer of the fluorescent dye Lucifer yellow through gap junctions in V79 cells maintained as a monolayer culture at 10.5°C for 72 h.