Inhibition of apoptosis in serum starved porcine embryonic fibroblasts.
In nuclear transplantation, serum starvation is a general method to synchronize donor cells at the quiescent stage (G(0)) of the cell cycle. However, serum starvation during culture of mammalian cells may induce cell death, especially through apoptosis, thus contributing to the low efficiency of nuclear transplantation. This study was performed to characterize apoptosis during serum starvation and to determine the effects of apoptosis inhibitors such as a protease inhibitor [alpha(2)-macroglobulin (MAC)] and antioxidants [N-acetylcysteine (NAC), glutathione (GSH)] on serum starved porcine embryonic fibroblasts (PEF). PEF, collected from day 25-30 porcine fetuses, were cultured for 5 days in media containing 0.5% FBS to induce quiescence. Serum starved PEF showed typical morphology of apoptotic cells and stained for DNA fragmentation by TUNEL assay (26.7%). All apoptosis inhibitors tested in this study significantly (P < 0.05) reduced apoptosis of serum starved PEF, with antioxidants having better results (MAC: 7.4% vs. NAC: 1.0%, and GSH: 0.8%). Equally and importantly, the treatment with apoptosis inhibitors did not change the proportion of G(0)/G(1) stage cells. Therefore, the addition of MAC and antioxidants during serum starvation of PEF reduces apoptosis of quiescent fibroblasts and may contribute to increasing the efficiency of nuclear transplantation by improving the quality of donor nuclei.
Lee
Chang-Kyu
CK
Department of Animal Science, Texas A&M University, College Station, TX 77843-4458, USA.
Piedrahita
Jorge A
JA
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects*; D016166:Free Radical Scavengers / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D013552:Swine; D000511:alpha-Macroglobulins / Q000494:pharmacology
D000975:Antioxidants; D016895:Culture Media, Serum-Free; D016166:Free Radical Scavengers; D011480:Protease Inhibitors; D000511:alpha-Macroglobulins; D000111:Acetylcysteine