Inhibitors of caspase homologues suppress an apoptotic phenotype in cultured rabbit corpora lutea.
Apoptosis is a morphologically defined type of cell death initiated by various stimuli that results in the activation of caspases (cysteine-containing aspartate-specific proteases). In the present study, it was determined that caspases are present during, and play a role in, corpus luteum (CL) apoptosis in vitro. Pseudopregnancy was induced in rabbits with 100 IU human chorionic gonadotrophin. On Day 11 of pseudopregnancy, CL were isolated and cultured for 0, 2, 4, 6, and 8 h in the absence of trophic support to induce spontaneous apoptosis. Total RNA was extracted and analysed for caspase-I expression by Northern blot analysis. The results demonstrated caspase-I expression from 4 h. In the second part of the study, CL were incubated without trophic support for 4 h with increasing concentrations of three general caspase inhibitors, sodium aurothiomalate (SAM), iodoacetic acid (IAA) and N-tosyl-L-phenylalanylchloromethylketone (TPCK), and two specific caspase inhibitors, N-acetyl (Ac)-Tyr-Val-Ala-Asp (YVAD)-chloromethylketone (CMK) (Ac-YVAD-CMK) and Ac-Asp-Glu-Val-Asp (DEVD)-aldehyde (CHO) (Ac-DEVD-CHO). At completion, DNA was isolated and integrity assessed. Treatment of CL with SAM, IAA or Ac-DEVD-CHO effectively suppressed apoptotic DNA fragmentation. The final component of the study was to examine caspase-3 protein expression. Western blot analysis revealed a significant increase in caspase-3 expression over the experimental time-course. The results of the present study clearly demonstrate a time-dependent link between the caspases, specifically caspase-3 and spontaneous apoptosis in the rabbit CL.
Abdo
M A
MA
Department of Anatomy and Human Biology, The University of Western Australia, Nedlands.
Richards
A
A
Atiya
N
N
Singh
B
B
Parkinson
S
S
Hisheh
S
S
Dharmarajan
A M
AM
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D020170:Caspase 1 / Q000235:genetics; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D003338:Corpus Luteum / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects; D005260:Female; D015870:Gene Expression; D006052:Gold Sodium Thiomalate / Q000494:pharmacology; D006801:Humans; D019807:Iodoacetic Acid / Q000494:pharmacology; D007700:Kinetics; D009842:Oligopeptides / Q000494:pharmacology; D009924:Organ Culture Techniques; D010641:Phenotype; D011555:Pseudopregnancy; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D006063:Chorionic Gonadotropin; D015853:Cysteine Proteinase Inhibitors; C098738:N-acetyl-tyrosyl-valyl-alanyl-aspartyl chloromethyl ketone; D009842:Oligopeptides; D012333:RNA, Messenger; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D006052:Gold Sodium Thiomalate; D014108:Tosylphenylalanyl Chloromethyl Ketone; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1; D019807:Iodoacetic Acid