The interferons (IFNs) are a family of cytokines which function to limit cell proliferation by cytostasis or cell death and exhibit potent antitumor effects against certain human cancers. The molecular mechanisms by which IFNs elicit their growth inhibitory/cell death effects remain largely unknown. The 2-5A system is an RNA degradation pathway which functions through the activation of 2-5A-dependent RNASE-L is a critical mediator of cell growth inhibition by IF and is implicated in its antitumor activity; recent studies have identified a broader role for RNase-L in apoptosis. To determine how RNASE-L functions in the antitumor effects of IF, it is essential to understand the mechanisms underlying RNase-L mediated growth inhibition/cell death and how this activity is regulated in cells. To accomplish this broad objective, RNA substrates, downstream effectors and cell ular regulators of RNASE-L will be identified. Cell lines in which RNASE-L expression and activity can be modulated in both positive and negative directions will be used in complementary differential expression (aim 1) and function based (aim 2) methods.
In aim 1, mRNAs which are unregulated in RNase-L inhibited cells and downregulated following RNase-L induction represent candidate RNase-L substrates and downstream effectors and will be identified by differential display analysis. Expression of transfected RNASE-L renders NIH-3T3 cells sensitive to IF induced growth inhibition in conditions where control cells do not respond; thus, the specific modulation of RNase-L activity results in a selectable phenotypic change. To identify gene products which function in RNase-L-mediated growth inhibition (aim 2), a Genetic Suppressor Element (GSE) library, composed of short cDNA fragments encoding inhibition of gene products representing RNase-L regulators, substrates or downstream effectors will rescue RNase-L dependent growth arrest, providing a positive selection for cells expressing these GSEs. cDNAs identified in aims 1 and 2 will be characterized as upstream or downstream mediators of RNASE-L activity and sequenced to determine if they represent known or novel genes (aims 3ab). The regulation of endogenous RNase-L regulator/effector expression and the effects of transfected exogenous regulators/effectors on cell proliferation in normal and cancer cells lines will be examined to elucidate their role in growth inhibition (aims 3c,d). An in vitro RNA decay system will be developed to study how specific RNase-L substrates are targeted for degradation (aim 3e). The identification of RNASE-L regulators, substrates and effects will advance our understanding of several important biological issues: 1) the mechanism of IF induced growth inhibition; 2) the role of RNase-L in the antitumor effects of IF; and 3) the control of RNA stability in the regulation of gene expression.
| Li, X L; Blackford, J A; Judge, C S et al. (2000) RNase-L-dependent destabilization of interferon-induced mRNAs. A role for the 2-5A system in attenuation of the interferon response. J Biol Chem 275:8880-8 |
