Do heavy ions induce the bystander effect? : study to determine the induction of the bystander effect from Fe ion beam compared to X-rays in human keratinocytes

Abstract

The bystander effect is the observation that non-irradiated cells near a cell traversed by radiation express biological responses such as micronuclei formation and genomic instability. Most published studies of the bystander effect have been conducted using alpha particles, a high LET radiation. A few studies have been done with low LET radiation. This project studies the bystander effect from both low LET x-rays and high LET Fe particle beam irradiation. Using a transwell insert system, the bystander effect was studied in hTERT immortalized human keratinocytes. Cells are plated in a 6-well plate and in a companion permeable membrane insert. Cells in the 6-well plate are irradiated using conventional 250 kVp X-rays or 1000 MeV/nucleon Fe ion beam, LET of 151 keV/pm, from the NASA Space Radiation Laboratory at Brookhaven National Lab. After irradiation, inserts are immediately placed into the 6 well plate so that the irradiated and unirradiated cells are sharing medium but are not in contact. For both beams, frequency of micronuclei, chromatin bridges, and p21 wafl induction as well as cell cycle phase analysis were determined in both directly irradiated and bystander cells from 0.1 Gy to 5 Gy. From x-rays, a two-fold bystander effect at 24 h after irradiation with elevated p21' wafl induction and micronuclei was seen but in Fe ion irradiation, the p21 wafl bystander effect was delayed to 40-50 h after irradiation and no MN bystander effect was observed.

(cont.) Cell cycle analysis showed a slight G2 arrest in keratinocytes 5 h after x-rays but a strong G2 arrest until 40-50 h was seen after Fe ion irradiation. Bystander keratinocytes co-cultured with directly irradiated human fibroblasts AGO 1522 cells showed a two-fold p21 wafl and MN bystander effect 24 h after x-rays, and a potential 2-fold MN bystander effect 50 h after Fe ions. Bystander AGO1522 cells co-cultured with directly irradiated keratinocytes showed a two-fold MN bystander effect 24 h after x-rays, but no MN bystander response was seen at any time points studied from Fe ions. Striking differences in the bystander response were shown from the two radiation types, but the reason remains to be clarified.