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E.-M. Demmerath et al.
vented by PGE2 (Figure 3A). Altogether, the data point towards a dose-dependent protective effect of PGE2 that is, however, restricted to certain forms of cell stress.
Unexpectedly, long-term culture of CD34+ cells revealed a toxic effect of PGE2 itself when it was used at high con- centrations for longer than two days. Numbers and viabil-
ity of CD34+ cells were strongly reduced when they were cultured in the presence of 50 mM PGE2 for up to eight days (Figure 3B and C). To unravel the paradoxical finding that PGE2 can both protect from and induce apoptosis, we analyzed regulation of BCL-2 family members on mRNA level. After 4 h of PGE2 treatment, CD34+ cells showed
AB
CD
EF
Figure 2. Epidermal growth factor (EGF) does not protect human CD34+ from apoptosis induced by sublethal irradiation. (A) Cord blood-derived human CD34+ cells were treated for 24 and 48 hours (h) with etoposide (1 μg/mL) and different doses of human EGF (hEGF). Apoptosis was measured using AnnexinV/7AAD and specific apop- tosis was calculated. Bars represent means±Standard Error of Mean (SEM) of 5 independent experiments. (B) Cord blood-derived human CD34+ cells were cultured for 24 and 48 h in the presence of human EGF and/or cetuximab at indicated concentrations and specific apoptosis was determined. Bars represent means±SEM of 4 inde- pendent experiments. (C) Human CD34+ cells were treated with both etoposide and cetuximab, and specific apoptosis was determined 24 h later. Bars represent means±SEM of 3 independent experiments. (D-F) Human CD34+ cells were transplanted into sublethally irradiated Rag2−/−γc−/− mice. Four weeks later, mice were irradi- ated with 3 Gy or left untreated. Mice that were irradiated received daily injections of human and/or murine EGF. Eight days after irradiation, mice were sacrificed and % human CD45+ cells was determined in bone marrow (D) and spleen (E). In addition, the proportion of CD34+ immature cells was determined within the human cell population (F). Bars represent means±SEM of 4-7 animals from 5 independent experiments. Mann-Whitney test, *P≤0.05; **P≤0.01; ***P≤0.001).
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haematologica | 2019; 104(4)