An inhibitor of nuclear scaffold protease blocks chemical transformation of fibroblasts.
A nuclear scaffold (NS) protease has previously been implicated in production of the M(r) 46,000 ATP-binding protein in NS (which may acquire nucleoside triphosphatase activity and participate in nucleocytoplasmic transport) by cleavage of a subset of lamins A/C. In a preceding paper (G. Clawson, L. Norbeck, C. Hatem, C. Rhodes, P. Amiri, J. McKerrow, S. Patierno, and G. Fiskum, Cell Growth & Differ., 3: 827-838), this NS protease was identified as a novel, Ca(2+)-regulated serine protease, which was found only in the NS and which appears to represent a unique multicatalytic protease complex. Based upon its predominantly chymotrypsin-like substrate preference, a peptide-chloromethylketone inhibitor (succinyl-AAPF-chloromethylketone, AAPFcmk) was identified. AAPFcmk showed a KI = 56 nM for the NS protease versus 1.4 microM for the endoplasmic reticulum activity. Treatment of C3H/10T1/2 mouse embryo fibroblast cells with 1 microM AAPFcmk produced effects which were confined to the nuclear (and to a lesser extent the endoplasmic reticulum) compartment. In this report, we examine the effects of the AAPFcmk inhibitor on cellular transformation and growth. Growth of C3H/10T1/2 cells was decreased by 34% and 56% at 25 microM and 50 microM AAPFcmk, respectively. Growth inhibition occurred without any major change in DNA content distribution, suggesting effects throughout the cell cycle. Growth inhibition was not observed at lower (< or = 10 microM) concentrations, which decreased transformation of C3H/10T1/2 fibroblasts in a dose-dependent manner by up to 90%, even at femtomolar concentrations of AAPFcmk (in the absence of growth inhibition). Inclusion of irrelevant inhibitors was without affect.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Pathology, Pennsylvania State University, Hershey 17033.
G A
GA
Clawson
L L
LL
Norbeck
J P
JP
Wise
S R
SR
Patierno
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects; D051379:Mice; D009842:Oligopeptides / Q000494:pharmacology*; D012697:Serine Endopeptidases / Q000187:drug effects*
D000590:Amino Acid Chloromethyl Ketones; D009842:Oligopeptides; C083541:succinyl-alanylalanyl-prolyl-phenylalanine chloromethylketone; D012697:Serine Endopeptidases; C078807:nuclear scaffold serine protease