Cell cycle synchronization of porcine fetal fibroblasts by serum deprivation initiates a nonconventional form of apoptosis.
The success of somatic nuclear transfer depends upon the cell cycle stage of the donor nucleus and the recipient cytoplast. Recently, we established efficient cell cycle synchronization protocols for porcine fetal fibroblasts and found that serum withdrawal leads to cell death. Here, we examined whether the specific cell death induced by serum deprivation follows the conventional apoptotic pathway in porcine fibroblasts. Terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling analysis revealed that serum deprivation induced DNA fragmentation in a concentration and time dependent manner. Semiquantitative RT-PCR and Western blotting revealed activation of cell death-related genes Bak and Bax of the Bcl-2 family. However, electrophoretic analysis of genomic DNA from serum deprived cells did not provide evidence for the internucleosomal DNA cleavage which is characteristic of conventional apoptosis. Thus, serum deprivation triggers initial steps in the apoptotic pathway, but does not lead to the typical oligonucleosome-sized DNA ladder. These findings contribute to a better understanding of apoptotic pathways and aid to define essential parameters of the donor nucleus for successful somatic cloning.
Kues
Wilfried A
WA
Department of Biotechnology, Institute for Animal Science, Neustadt, Germany.
Carnwath
Joseph W
JW
Paul
Dieter
D
Niemann
Heiner
H
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D003593:Cytoplasm / Q000502:physiology / Q000648:ultrastructure; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004249:DNA Damage / Q000187:drug effects*; D017931:DNA Primers; D005333:Fetus; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D020287:In Situ Nick-End Labeling; D053652:Nuclear Transfer Techniques; D009707:Nucleosomes / Q000648:ultrastructure; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine
D016895:Culture Media, Serum-Free; D017931:DNA Primers; D009707:Nucleosomes; D004247:DNA