Figure 1

Cell proliferation in conditioned medium from irradiated cells and analysis of DNA. For a determination of the growth-promoting capacity, conditioned media from 96 h cell cultures were added to 24 h test cultures inoculated with 50 000 cells. Test cells were counted daily in combination with a clonogenic assay. (A). Parallel cultures were prepared for DNA analysis and the sub-G1 fraction (B) was determined from the DNA histograms (C). Cells were incubated in fresh medium (closed circles), or in conditioned medium from irradiated cells (CMX), that is, after irradiation with 8 Gy, 50 000 cells were seeded from which approximately 65 000 were present at 96 h. Alternatively, cells were grown in conditioned medium from 50 000 nonirradiated cells (CM) with a final density at 96 h of 2.5 × 10⁶. For a comparison, cells were also incubated at 42.5°C for 90 min (H), an isotoxic treatment that kills approximately 95% of the cells (Van Rijn et al, 2000). At variance with X-rays, hyperthermic cell death follows within a few hours and a couple of thousand cells were present in cultures with the surviving cells after the 96 h conditioning period. Except for 72 and 96 h with CMX, the clonogenic capacity of the test cell cultures was unaffected (data not shown).