Caspase-mediated apoptosis in AK-5 tumor cells: a cell-free study using peptide inhibitors and antisense strategy.
An in vitro system has been employed to study the apoptotic mechanisms in the AK-5 tumor which is a spontaneously regressing rat histiocytoma. Cytosolic extracts of tumor cells primed for apoptosis using dexamethasone and immune serum from tumor-regressing animals were able to induce apoptosis in intact nuclei and reproduce the classical morphological and biochemical features typical of apoptotic cells. The cleavage of lamin A and PARP to signature fragments by these extracts and the inhibition of the same using peptide inhibitors signify the pivotal role of ICE and ICE-related proteases in apoptosis. Lamin A cleavage was insensitive to YVAD but PARP cleavage was blocked by both YVAD and DEVD. Cell extracts derived from cells overexpressing the Bcl-2 gene and Nedd-2 antisense gene, respectively, failed to induce apoptosis in exogenously added nuclei, suggesting that Bcl-2 gene product is downregulating a key event in apoptotic cascade. The study also demonstrates the coherent action of different ICE-related proteases in apoptosis and their functional redundancy. This system may prove useful for analyzing complex molecular mechanisms underlying apoptosis in tumor cells.
Anjum
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R
Centre for Cellular and Molecular Biology, Hyderabad, India.
Khar
A
A
D000818:Animals; D016375:Antisense Elements (Genetics) / Q000494:pharmacology; D017209:Apoptosis; D053143:Caspase 2; D020169:Caspases; D002467:Cell Nucleus / Q000648:ultrastructure; D002474:Cell-Free System; D004249:DNA Damage; D018219:Histiocytoma, Benign Fibrous / Q000201:enzymology*; D034904:Lamin Type A; D034882:Lamins; D009365:Neoplasm Regression, Spontaneous; D009374:Neoplasms, Experimental; D009687:Nuclear Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D016375:Antisense Elements (Genetics); D034904:Lamin Type A; D034882:Lamins; D009687:Nuclear Proteins; D009842:Oligopeptides; D011480:Protease Inhibitors; D011065:Poly(ADP-ribose) Polymerases; D053143:Caspase 2; D020169:Caspases