Cell Cyle Effects Contribute to Multicellular Radiosensitization

In the current project on effects of heavy ion irradiation in planar multilayer cell cultures, a “multicellular sensitization” was observed, i. e., multilayers were more radiosensitive than monolayers when treated under identical conditions [1]. This phenomenon was observed in multilayers, but also in multicell spheroids of SiHa and WiDr human tumor cells using plateau region or Bragg peak carbon ions or conventional x-rays for irradiation. The observation is in contrast to the “contact effect” or “multicellular resistance” commonly seen in small size multicellular tumor spheroids [2]. Interestingly, incubation of SiHa monoand multilayers with the topoisomerase-II inhibitor etposide revealed a multicellular chemoresistance [1] which was in accordance with data from the literature [3]. Since the percentage of cells in the S-phase was much lower in multilayers than in monolayers, and since the proportion of G0/G1-phase cells showed the opposite behavior [1], we suspected this difference in cell cycle distribution to be, at least in parts, responsible for the multicellular radiosensitization. One approach to challenging this hypothesis was to seed monolayers in Petri dishes with a regular or a high cell density. Flow cytometry analysis of these cultures at day 5 after plating showed that the cell cycle distribution of dense monolayers was in between that of regular monolayers and multilayers (Tab. 1). Standardized colony forming assays were carried out on these three types of SiHa cell cultures with and without heavy ion irradiation. The resulting clonogenic survival curves are shown in Fig. 1. It can be clearly recognized that the multicellular radiosensitization in multilayers can be approximated by a similar effect in high density monolayers with heavy ion irradiation in both the plateau region (Fig. 1a) and in the extended Bragg peak (Fig. 1b). Corresponding experiments following an etoposide treatment resulted in survival curves of dense monolayers which are very similar to those of multilayers, and which indicated a multicellular chemoresistance under those conditions (data not shown). These findings suggest that cell cycle dependencies of radiation effects in multicellular culture systems have not been considered to a sufficient extent in a number of previous studies, and that they should be reconsidered in a comprehensive way. Furthermore, the data imply that the dependency on cell cycle phase of heavy ion irradiation still is to be investigated in a systematic manner. Table 1: Cell cycle distribution in SiHa cells at day 5 in culture (values: means ± SD)