RADIATION RESEARCH 150, 275-282 (1998) 0033-7587/98 $5.00 ?1998 by Radiation Research Society. All rights of reproduction in any form reserved. Increasing Radiosensitivity in the Course of FractionatedX Irradiation: The Effect of Contactwith Dead and Dying Cells Bozidar Djordjevic, Christopher S. Lange, Marvin Z. Rotman, Camilo Torres and Zihwa Zheng Department of Radiation Oncology, State University of New York, Health Science Center at Brooklyn, 450 Clarkson Avenue, Box 1212, Brooklyn, New York 11203 Djordjevic, B., Lange, C. S., Rotman, M. Z., Torres, C. and Zheng, Z. Increasing Radiosensitivity in the Course of Fraction- ated X Irradiation: The Effect of Contact with Dead and Dying Cells. Radiat. Res. 150, 275-282 (1998). We have measured survival after successive 2-Gy doses of X rays in HeLa cells and 1-Gy doses in cells of the nonimmortal- ized human fibroblast cell line AG15-22 under conditions where any effect of cell proliferation during multifraction X irradiation has been factored out. When HeLa cells in parallel series of (pseudo)hybrid spheroids (i.e. in agglomerates consisting of a mixture of supralethally irradiated HeLa feeder and viable HeLa cells) were exposed to n daily radiation doses and then tryp- sinized and exposed to the last dose, the surviving fraction at 2 Gy (SF2) declined exponentially from 0.55 ? 0.01 to 0.31 ? 0.01 after the fifth fraction. In monolayer HeLa cell cultures, the decline in SF2 was smaller but significant and was not influenced by the presence of feeder cells. Pure spheroids, composed entirely of viable HeLa cells, showed the same decline in SF2 as did mono- layer cells. The cumulative-effect curve (i.e. the product of SF2 values) was linear-quadratic with the quadratic term increasing in the order monolayer, pure spheroids, pseudohybrid spheroids. SF2" and Do Eff (deduced from the initial SF2) severely underesti- mated cumulative radiosensitivity. This cumulative effect is clearly associated with the proximity of lethally irradiated cells and might be explained by differential population shifts in the course of the multifraction regimen. Similarly, AG15-22 cells irradiated with daily 1-Gy doses of X rays showed a larger increase in radiosensitivity when in hybrid spheroids than when in pure spheroids. However, for the AG15-22 cells, SF1 was twofold lower for the former than for the latter condition and remained constant for both conditions rather than decreasing with increasing fraction number. This large radiosensitizing effect remains unexplained. o 1998 by Radiation Research Society INTRODUCTION The original aim of our multifraction irradiation studies was to obtain a more accurate radiation response than that available from several methods for measurement of the sur- viving fraction of cells at 2 Gy (SF2) (1-3). Multifraction irradiation was expected to produce an exponential survival curve which could be characterized by the effective Do (Do Ef), assuming that successive doses are of equal effective- ness (4, 5). Since the precision of determination of the SF depends on the number of colonies counted, for the same counting precision, the effect of 30 daily fractions of 2 Gy each given 5 days per week for 6 weeks would be known more precisely if one measured the effect of 5 fractions and raised that SF (and its errors) to the 6th power, rather than raising the SF2 (and its comparable errors) to the 30th power (6). This would also reduce the statistical fluctuations inher- ent in single measurements of SF2. As this study progressed, a phenomenon of equal importance emerged, namely the effects of the proximity of dead and dying cells on successive measurements of SF2. Consequently, we address in this report the question of the alleged constancy of SF2 through the multifraction regimen and the possible reasons for the deviation from it. For this purpose, we have used HeLa cells in spheroids (as an in vitro tumor model) and in monolayers and AG15-22 fibroblast cells in spheroids, all in a procedure where the effect of cell proliferation during the course of multifractionirradiation has been factored out. MATERIALS AND METHODS Cell Lines and Maintenance HeLa S3 cells have been maintained in this laboratory for more than a decadeand are periodically retrieved fromstocksfrozenin liquid nitro- gen to prevent genetic drift. Cultures were maintained in growth medium composed of Eagle's minimalessential medium (MEM) supplemented with antibiotics and 15% fetal bovine serum (Gibco). Cells were trypsinized twice weekly and reseededin fresh growth medium. Nonim- mortalized human foreskin fibroblast AG15-22cells were obtained from the CoriellCell Repositories, Camden, NJ, expanded in growth medium and stored in liquid nitrogen. For the present experiments, passages 12-15 were used,having a plating efficiency of about10%. Irradiation All irradiations were performed at room temperature with a Philips RT250 machine operating at 250 kVp, 15 mA, 50 cm FSD (HVL 0.39 mm Cu), at a dose rate of 2.7 Gy/min under conditions of full backscatter. The cells were irradiated in their Corning 25-cm2 culture flasks containing 5 ml of medium (2 mm deep), attached to the bottomas monolayers or as spheroids, or, for theirfinal dose, as cells in suspension. The dose was homogeneous within +1% both from top to bottom and over the area of the flasks.Both thermoluminescent and Frickeferrous sulfate dosimetry were performed. 275 Radiation Research Society is collaborating with JSTOR to digitize, preserve, and extend access to Radiation Research www.jstor.org ®