Okadaic acid induces DNA fragmentation via caspase-3-dependent and caspase-3-independent pathways in Chinese hamster ovary (CHO)-K1 cells.
DNA fragmentation is a hallmark of apoptosis that occurs in a variety of cell types; however, it remains unclear whether caspase-3 is required for its induction. To investigate this, we produced caspase-3 knockout Chinese hamster ovary (CHO)-K1 cells and examined the effects of gene knockout and treatment with caspase-3 inhibitors. Okadaic acid (OA) is a potent inhibitor of the serine/threonine protein phosphatases (PPs) PP1 and PP2A, which induce apoptotic cellular reactions. Treatment of caspase-3(-/-) cells with OA induced DNA fragmentation, indicating that caspase-3 is not an essential requirement. However, in the presence of benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe) fluoromethylketone (z-DEVD-fmk), DNA fragmentation occurred in CHO-K1 cells but not in caspase-3(-/-) cells, suggesting that caspase-3 is involved in OA-induced DNA fragmentation that does not utilize DEVDase activity. In the absence of caspase-3, DEVDase activity may play an important role. In addition, OA-induced DNA fragmentation was reduced but not blocked in CHO-K1 cells, suggesting that caspase-3 is involved in caspase-independent OA-induced DNA fragmentation. Furthermore, OA-induced cleavage of caspase-3 and DNA fragmentation were blocked by pretreatment with the wide-ranging serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride. These results suggest that serine proteases regulate DNA fragmentation upstream of caspase-3.
Kitazumi
Ikuko
I
Bio Process Research and Development Laboratories, Kyowa Hakko Kirin Co., Ltd, Hagiwara, Takasaki, Gunma, Japan.
Maseki
Yoko
Y
Nomura
Yoshiko
Y
Shimanuki
Akiko
A
Sugita
Yumi
Y
Tsukahara
Masayoshi
M
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology*; D001483:Base Sequence; D016466:CHO Cells; D053148:Caspase 3 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D061945:Caspase Inhibitors; D006224:Cricetinae; D003412:Cricetulus; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D008969:Molecular Sequence Data; D019319:Okadaic Acid / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology
D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D018076:DNA, Complementary; D009842:Oligopeptides; D015842:Serine Proteinase Inhibitors; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; D019319:Okadaic Acid; D014107:Tosyllysine Chloromethyl Ketone; D014108:Tosylphenylalanyl Chloromethyl Ketone; D011065:Poly(ADP-ribose) Polymerases; D010447:Peptide Hydrolases; D053148:Caspase 3; C108711:DEVDase