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High and low molecular weight DNA cleavage in ovarian granulosa cells: characterization and protease modulation in intact cells and in cell-free nuclear autodigestion assays.

Publication ,  Journal Article
Trbovich, AM; Hughes, FM; Perez, GI; Kugu, K; Tilly, KI; Cidlowski, JA; Tilly, JL
Published in: Cell Death Differ
January 1998

To continue elucidation of the biochemical and molecular pathways involved in the induction of apoptosis in granulosa cells (GC) of ovarian follicles destined for atresia, we characterized the occurrence and protease modulation of high and low molecular weight (MW) DNA fragmentation during rat GC death. Atresia of ovarian follicles, occurring either spontaneously in vivo or induced in vitro, was associated with both high MW and internucleosomal (low MW) DNA cleavage. Incubation of follicles in the presence of a putative irreversible and non-competitive inhibitor of caspase-1 (interleukin-1beta-converting enzyme or ICE), sodium aurothiomalate (SAM), completely prevented internucleosomal, but not high MW, DNA cleavage. As reported previously, morphological features of apoptosis (pyknosis, cellular condensation) and atresia (granulosa cell disorganization, oocyte pseudomaturation) remained detectable in SAM-treated follicles. The potential involvement of proteases in endonuclease activation was further analyzed in cell-free assays using nuclei from both GC (which autodigest their DNA) and HeLa cells (HC, which do not autodigest their DNA unless incubated with extracts prepared from other cell types). Crude cytoplasmic extracts prepared from GC induced both high MW and internucleosomal DNA cleavage in HC nuclei. The induction of low, but not high, MW DNA cleavage in HC nuclei by GC extracts was suppressed by pretreatment of the extracts with SAM or with any one of the serine protease inhibitors, dichloroisocoumarin (DCI), N-tosyl-L-leucylchloromethylketone (TLCK) or N-tosyl-L-phenylchloromethylketone (TPCK). Interestingly, SAM and DCI also prevented cation-induced low MW DNA fragmentation in GC nuclei; however, TLCK and TPCK were without effect. Our results support a role for cytoplasmic and nuclear serine proteases in the activation of the endonuclease(s) responsible for internucleosomal DNA cleavage during apoptosis.

Duke Scholars

Published In

Cell Death Differ

DOI

ISSN

1350-9047

Publication Date

January 1998

Volume

5

Issue

1

Start / End Page

38 / 49

Location

England

Related Subject Headings

  • Tosylphenylalanyl Chloromethyl Ketone
  • Tosyllysine Chloromethyl Ketone
  • Serine Proteinase Inhibitors
  • Rats, Sprague-Dawley
  • Rats
  • Ovarian Follicle
  • Oligopeptides
  • Molecular Weight
  • Humans
  • Hela Cells
 

Citation

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Trbovich, A. M., Hughes, F. M., Perez, G. I., Kugu, K., Tilly, K. I., Cidlowski, J. A., & Tilly, J. L. (1998). High and low molecular weight DNA cleavage in ovarian granulosa cells: characterization and protease modulation in intact cells and in cell-free nuclear autodigestion assays. Cell Death Differ, 5(1), 38–49. https://doi.org/10.1038/sj.cdd.4400299
Trbovich, A. M., F. M. Hughes, G. I. Perez, K. Kugu, K. I. Tilly, J. A. Cidlowski, and J. L. Tilly. “High and low molecular weight DNA cleavage in ovarian granulosa cells: characterization and protease modulation in intact cells and in cell-free nuclear autodigestion assays.Cell Death Differ 5, no. 1 (January 1998): 38–49. https://doi.org/10.1038/sj.cdd.4400299.
Trbovich, A. M., et al. “High and low molecular weight DNA cleavage in ovarian granulosa cells: characterization and protease modulation in intact cells and in cell-free nuclear autodigestion assays.Cell Death Differ, vol. 5, no. 1, Jan. 1998, pp. 38–49. Pubmed, doi:10.1038/sj.cdd.4400299.
Trbovich AM, Hughes FM, Perez GI, Kugu K, Tilly KI, Cidlowski JA, Tilly JL. High and low molecular weight DNA cleavage in ovarian granulosa cells: characterization and protease modulation in intact cells and in cell-free nuclear autodigestion assays. Cell Death Differ. 1998 Jan;5(1):38–49.

Published In

Cell Death Differ

DOI

ISSN

1350-9047

Publication Date

January 1998

Volume

5

Issue

1

Start / End Page

38 / 49

Location

England

Related Subject Headings

  • Tosylphenylalanyl Chloromethyl Ketone
  • Tosyllysine Chloromethyl Ketone
  • Serine Proteinase Inhibitors
  • Rats, Sprague-Dawley
  • Rats
  • Ovarian Follicle
  • Oligopeptides
  • Molecular Weight
  • Humans
  • Hela Cells