Independent regulation of ppp(A2'p)nA-dependent RNase in NIH 3T3, clone 1 cells by growth arrest and interferon treatment.

D Krause,A Panet,G Arad,C W Dieffenbach,R H Silverman

doi:10.1016/s0021-9258(17)39392-4

Abstract

The regulation of ppp(A2'p)nA-(2-5A)-dependent RNase (RNase L or RNase F) was investigated in NIH 3T3, clone 1 cells using 2-5A-binding and nuclease activity assays. Minimal levels of 2-5A-dependent RNase were detected in actively dividing clone 1 cells; these levels were independently induced by growth arrest or interferon treatment. Accordingly, levels of the RNase were enhanced during growth arrest by confluency regardless of the presence or absence of interferon or antibody to interferon in the media. Measurement of 2-5A-dependent RNase was unaffected by the addition of any of six different proteinase inhibitors to the cells prior to extraction. The expression of 2-5A-dependent RNase in growth-arrested, interferon-treated cells was still relatively low (about one-third to one-half of that found in similarly treated murine Ehrlich ascites tumor cells). Although this amount of 2-5A-dependent RNase could not be detected by 2-5A-mediated ribosomal RNA cleavage, the activity was identified using a more sensitive novel assay for 2-5A-dependent RNase. In addition, introduction of 2-5A or poly(I) X poly(C) into growth-arrested, interferon-treated cells resulted in some inhibition of protein synthesis. The results indicated that the expression of 2-5A-dependent RNase in NIH 3T3, clone 1 cells is regulated under different physiological conditions and that low levels of 2-5A-dependent RNase were insufficient to significantly inhibit encephalomyocarditis virus replication.

Highlights

The regulation of ppp(AZ’p),A-(2-5A)-dependent RNase (RNase L or RNase F) was investigated in NIH 3T3, clone 1 cellsusing2-5A-binding and nuclease activityassays
It was reported that some 2-5A-dependent RNasecould, be detectedfrom clone 1 cells but onlywhen the cells were extracted in the presenceof the proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF’) [16]
The enzyme was found to be independently regulated by either growth arrest or by interferon treatment.Regardless, 2-5A-dependent RNaselevels remained relatively low and encephalomyocarditis virus (EMCV) replication was not significantly inhibited in response to interferon

Summary

MATERIALS AND METHODS

Cell Culture tions and that lowlevels of 2-5A-dependent RNase NIH3T3, clone 1 cells Ascites tumor (EAT) cells were grown in spinner cultures in Eagle’s MEM (modifiedfor spinner conditions) containing 10% Nu-serum (Collaborative Research).Media were supplemented with glutamine, penicillin, streptomycin, and gentamicin. This article must be PBS, phosphate-bufferedsaline; EAT, Ehrlich ascites tumor; EMCV, hereby marked “aduertisement” in accordance with18U.S.C. Section encephalomyocarditis virus; MEM, minimal essential medium; TdR, 1734 solely to indicate thisfact. The cells were assay) were incubated a t 0 "Cin mM Tris-HCI, p H 7.6, 2 mM washed twice with Dulbecco's PBS and challenged with EMCV a t a magnesiumacetate, 0.4 mM ATP, 2% (v/v) glycerol, and between multiplicity of infection of 1. The cell pellet was resuspended in minetetraacetic acid, heated to 90 "C for 5 min, and centrifuged at 1.0 ml of ice-cold PBS-TdR to which was added 1.0 ml of ice-cold.

RESULTS

DISCUSSION

16. Nilsen