THE MAJOR CALPAIN ISOZYMES ARE LONG-LIVED PROTEINS - DESIGN OF AN ANTISENSE STRATEGY FOR CALPAIN DEPLETION IN CULTURED-CELLS

Calpains are intracellular Ca2+-dependent proteases that are thought t o participate in Ca2+-associated signal transduction pathways, It has been proposed that calpains are activated by an autoproteolytic mechan ism, If this is true one would expect a relatively short half-life for calpain protein in cells, To test this hypothesis, WI-38 human diploi d fibroblasts were pulse-labeled with [S-35]methionine, and calpain wa s immunoprecipitated at various times after chasing with nonradioactiv e methionine to determine residual radioactivity, The results demonstr ated that the two major calpain isozymes, m-calpain and mu-calpain, ha d metabolic half-lives of approximately 5 days, Calpains were long-liv ed proteins in several human cell lines, A-431, HeLa, VA-13, C-33A, an d TE2 cells, In addition, calpastatin, the calpain-specific inhibitor protein, also had a long metabolic half-life, These observations sugge st that the model for calpain activation by autoproteolysis requires r e-investigation. Based on a knowledge of calpain metabolic stability, a protocol was devised for chronic exposure of WI-38 cells and HeLa ce lls to a calpain small subunit antisense oligodeoxyribonucleotide. Dep letion of calpain small subunit after 5 or more days of treatment led to inhibition of cell proliferation that could be reversed by removal of antisense oligodeoxyribonucleotide from the culture medium, Togethe r with previous studies, these results indicate a requirement for calp ains in mammalian cell proliferation.