A novel inhibitor that protects apoptotic DNA fragmentation catalyzed by DNase gamma.
The internucleosomal cleavage of genomic DNA is the biochemical hallmark of apoptosis. DNase gamma, a Ca(2+)/Mg(2+)-dependent endonuclease, has been suggested to be one of the apoptotic endonucleases. We identified here 4-(4,6-dichloro-[1,3,5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396) as a novel and potent DNase gamma inhibitor using stable HeLa S3 transfectants of DNase gamma (HeLa-gamma cells). DR396 inhibited apoptotic DNA fragmentation in HeLa-gamma cells induced by staurosporine (STS) and in rat splenocytes exposed to gamma-ray irradiation in a dose-dependent manner. This compound potently and selectively inhibited DNase gamma activity with an IC(50) value of 3.2 microM. DR396 did not delay the apoptotic processes as judged by the morphological changes and the cleavage of a death substrate, poly(ADP-ribose) polymerase (PARP). Furthermore, the compound did not prevent apoptotic DNA fragmentation in Jurkat cells induced by anti-Fas antibody (Ab), which is catalyzed by caspase-activated DNase (CAD). These findings clearly indicate that DR396 exerts chemical knockdown effect of DNase gamma on cells, suggesting that the compound could be an attractive tool for understanding of the physiological significance of DNase gamma.
Sunaga
Satoshi
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki Noda, Chiba 278-8510, Japan.
Kobayashi
Takanobu
T
Yoshimori
Atsushi
A
Shiokawa
Daisuke
D
Tanuma
Sei-Ichi
S
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000528:radiation effects; D002384:Catalysis; D002460:Cell Line; D004247:DNA / Q000187:drug effects* / Q000528:radiation effects; D053938:DNA Fragmentation / Q000187:drug effects* / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology*; D005720:Gamma Rays; D006367:HeLa Cells; D006801:Humans; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013154:Spleen / Q000187:drug effects* / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology; D014227:Triazines / Q000494:pharmacology*
C495063:4-(4,6-dichloro-(1,3,5)-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D014227:Triazines; D004247:DNA; D004706:Endodeoxyribonucleases; C105124:deoxyribonuclease gamma; D019311:Staurosporine