**6. Bystander effect can function bi-directionally**

Recent studies have shown an interplay between adjacent irradiated and non-irradiated cell populations. Thus, signals leading to damage in non-irradiated cells, sent by the irradiated ones, are answered by non-hit cells affecting in turn the directly-irradiated ones. Experiments performed on MM576 melanoma cells, the goal of which was to investigate the impact of modulating irradiation fields in a way to resemble the intensity-modulated radiotherapy technique (IMRT) on survival showed, that the mutual communication works in three different manners (Mackonis et al., 2007). The first type of this communication, the classic "bystander effect", occurs when irradiated cells growing in one part of the field damage the adjacent nonirradiated cells growing in another part of the field. The second type of communication, causes an increase in the survival of non-irradiated cells, when they are co-cultured with cells exposed to high doses (6-20 Gy) or even a lethal dose. One of the factors responsible for this process is, according to these authors, the eruption of "death-burst signals", which promotes proliferation of the non-irradiated cells, although the authors do not specify the chemical nature of these signals. The third type of communication causes increased survival of cells that have received a high dose of radiation, through signaling from neighboring cells exposed to low-dose in another part of the field (Mackonis et al., 2007). Also, the irradiation of human fibroblasts with low doses of alpha-particles resulted in an increased proliferation, reduction of the level of p53 and CDKN1 (p21Waf-) proteins and an increase in the level of the CDC2 kinase. The promitogenic effect was associated with an increase in the level of the TGFβ1-induced by reactive oxygen species (Iyer and Lehnert, 2002). Our recent study revealed bystander effect of the third type, similar to that described by the Mackonis, indicating the bilateral signaling of irradiated and non-irradiated cells (Widel et al., 2008, and unpublished). Using the transwell system of coincubated irradiated mouse lung cancer cells (LLC) with non-irradiated fibroblasts (NIH3T3) growing in inserts we studied the mutual interaction of cells in terms of micronuclei and apoptosis induction. The membrane of insert bottom with 0.4 µm pores separates both types of cells but enables free circulation of medium between them. LLC cells growing in 6-well plates were irradiated with doses of 2 and 4 Gy X-rays generated by a therapeutic accelerator (Clinac 600). Immediately after irradiation the inserts with non-irradiated (bystander) fibroblasts were

Intercellular Communication in Response to Radiation Induced Stress:

fibroblasts present in tumor tissue.

fractionated system (Fig. 3).

**7. Fractionated irradiation and bystander effect** 

Bystander Effects in Vitro and in Vivo and Their Possible Clinical Implications 345

The mechanism of this phenomenon requires clarification. It seems that the radioprotective bystander effect is a feature of normal fibroblasts. Indeed, the same effect, i.e. a significant reduction in the level of micronuclei and apoptosis in irradiated human melanoma Me45 cells was observed when these were co-incubated with normal human fibroblasts (Widel et al., unpublished). The progressive increase of micronuclei and apoptosis was paralleled by an increase of ROS; however, the ROS level in irradiated melanoma cells, which were cocultured with fibroblasts, was significantly diminished. Such a radioprotection was not observed in irradiated Me45 cells co-cultured with cells of the same line of melanoma (Widel et al, unpublished). We believe that the observed radio-protective effect of nonirradiated fibroblasts exerted on irradiated melanoma cells may result from signaling molecule(s) modifying the redox status of irradiated cells. Similar effect is likely to occur during cancer radiotherapy, causing some decrease of damage to cancer cells owing to

Experimental data on bystander effect mostly come from single-dose application experiments *in vitro*. However, there is a lack of knowledge, which would have potential clinical implication, e. g. whether bystander effect occurs during fractionated treatment. Mothersill and Seymour (2002a) performed experiments involving repeated treatment of bystander cells with medium collected from irradiated cells as well as involving repeated dose exposure of cells producing bystander signals, as a way of mimicking fractionated exposures. The recovery factor was defined as the surviving fraction of the cells receiving two doses (direct, or ICM) separated by an interval of 2 h divided by the surviving fraction of cells receiving the same dose in one exposure. The authors observed that fractionated bystander treatments removed the effect of dose sparing that is observed after conventional fractionated regime, during which cells can repair DNA damage. Using Me45 human melanoma cell line established at the Center of Oncology in Gliwice (Kramer-Marek et al, 2006) we compared frequency of apoptosis and micronuclei formation in directly irradiated and bystander cells after single doses (1.5 - 6 Gy) and after doses divided into 3 fractions given at consecutive days (3 x 0.5 Gy – 3 x 2 Gy). We used a transwell system of co-incubation which allows co-culturing the irradiated cells growing in wells with non- irradiated cells growing in inserts. This system to some extent resembles situation *in vivo*, due to prolonged contact of non-irradiated and irradiated cells. As a source of X-rays (6 MV) Clinac 600 therapeutic accelerator was used. Non irradiated control cells were-sham exposed. After irradiation, inserts with growing non irradiated cells were placed into wells with irradiated ones and co-incubated. Before irradiation medium in both, wells and inserts, was replaced by fresh aliquots. To observe the response of hit and bystander cells after the set time of incubation (0, 24 and 48 h), we performed microscopic analysis of micronuclei induction and apoptosis. The results obtained show that both single dose irradiation and fractionation of the dose into three fractions effectively induced bystander effect in malignant Me45 melanoma cells. However, fractionated irradiation at low doses (Fig. 2) appears to be much more effective in inducing micronuclei in directly hit and bystander cells, whereas higher apoptosis induction was clearly seen in hit, and especially in bystander cells, at all doses in

inserted into the wells and co-incubated for a desired time. Another set of irradiated LLC cells was incubated without cells in inserts, the latter filled with medium only. Micronuclei and apoptosis were scored in microscopic slides prepared from cells harvested at different timepoints. The results show that the irradiated cells induced apoptosis and micronuclei in bystander fibroblasts. For the first time we show the radioprotective effect of normal cells on irradiated cancer cells (the opposite bystander effect); thus the percentage of micronuclei and apoptosis in irradiated LLC cells co-incubated with NIH3T3 fibroblasts was significantly decreased in comparison with analogous levels in the irradiated LLC cells incubated without fibroblasts growing in inserts (Figure 1).

Fig. 1. Non-irradiated murine NIH3T3 fibroblasts co-cultured with irradiated Lewis lung carcinoma cells significantly diminish micronuclei (A) and apoptosis frequency (B) in irradiated (2 and 4 Gy) cancer cells compared with those irradiated and incubated without fibroblasts. Results are means ± standard deviation from three independent experiments (\*p<0.05, Student's t-Test).

inserted into the wells and co-incubated for a desired time. Another set of irradiated LLC cells was incubated without cells in inserts, the latter filled with medium only. Micronuclei and apoptosis were scored in microscopic slides prepared from cells harvested at different timepoints. The results show that the irradiated cells induced apoptosis and micronuclei in bystander fibroblasts. For the first time we show the radioprotective effect of normal cells on irradiated cancer cells (the opposite bystander effect); thus the percentage of micronuclei and apoptosis in irradiated LLC cells co-incubated with NIH3T3 fibroblasts was significantly decreased in comparison with analogous levels in the irradiated LLC cells incubated without

Fig. 1. Non-irradiated murine NIH3T3 fibroblasts co-cultured with irradiated Lewis lung carcinoma cells significantly diminish micronuclei (A) and apoptosis frequency (B) in irradiated (2 and 4 Gy) cancer cells compared with those irradiated and incubated without fibroblasts. Results are means ± standard deviation from three independent experiments

fibroblasts growing in inserts (Figure 1).

(\*p<0.05, Student's t-Test).

The mechanism of this phenomenon requires clarification. It seems that the radioprotective bystander effect is a feature of normal fibroblasts. Indeed, the same effect, i.e. a significant reduction in the level of micronuclei and apoptosis in irradiated human melanoma Me45 cells was observed when these were co-incubated with normal human fibroblasts (Widel et al., unpublished). The progressive increase of micronuclei and apoptosis was paralleled by an increase of ROS; however, the ROS level in irradiated melanoma cells, which were cocultured with fibroblasts, was significantly diminished. Such a radioprotection was not observed in irradiated Me45 cells co-cultured with cells of the same line of melanoma (Widel et al, unpublished). We believe that the observed radio-protective effect of nonirradiated fibroblasts exerted on irradiated melanoma cells may result from signaling molecule(s) modifying the redox status of irradiated cells. Similar effect is likely to occur during cancer radiotherapy, causing some decrease of damage to cancer cells owing to fibroblasts present in tumor tissue.
