Figure 6

Fast proliferating breast cancer cells with mutant p53 and a strong necrosis response towards ablative γ-irradiation release factors that stimulate monocyte migration. (A) Breast cancer cell lines used. Tumor subtype and receptor status have previously been reported [29]. p53 mutational status was determined by cDNA sequencing. HCC1806 gave no p53 PCR product, but the mutation in codon 256 was reported before [30]. (B) Growth curve analysis in the presence of 2.5% or 10% FCS. (C) Induction of necrosis in response to irradiation. Cells were irradiated as indicated ± 50 μM zVAD-fmk. PS externalization and plasma membrane integrity were determined as in Figure 2. Double positive cells were considered necrotic. Means ± s.d. of triplicates are shown. (D) Fast proliferating cells reveal a stronger necrosis response towards ablative γ-irradiation. Pearson correlation analysis of doubling times (Figures 1C and 6B) and necrosis (Figures 2A,B and 6C) was performed. The percentage of total necrotic cells or primary necrotic cells was employed. (E) THP-1 cell migration. Cell-free supernatants were collected 4 days after irradiation and applied to transwell assays as in Figure 3. Means ± s.d. of quadruplicates are given. (F) p53 mutant cell lines with a strong necrosis response towards ablative γ-irradiation release monocyte migration factors. Pearson correlation analysis of transmigration (Figures 3A and 6E) and necrosis (Figures 2A,B and 6C) 4 days after irradiation (0 Gy, 2 Gy, daily 2 Gy, or 20 Gy) was performed for p53 mutant cells. The percentage of total or primary necrotic cells was used as in (D). (G) Conclusions. Ablative γ-irradiation induces a strong necrotic response in fast proliferating breast cancer cell lines. The concomitant release of nucleotides stimulates monocyte migration and chemokinesis. In p53 wildtype, hormone receptor positive MCF7 cells this is impaired due to irradiation-induced upregulation of CD39, which degrades extracellular nucleotides.