The Double-Stranded RNA-Activated Protein Kinase Mediates Radiation Resistance in Mouse Embryo Fibroblasts through Nuclear Factor κB and Akt Activation

ABSTRACT Low oxygen gradients (hypoxia and anoxia) are important determinants of pathological conditions under which the tissue blood supply is deficient or defective, such as in solid tumors. We have been investigating the relationship between the activation of hypoxia-inducible factor 1 (HIF-1), the primary transcriptional regulator of the mammalian response to hypoxia, and 5′-AMP-activated protein kinase (AMPK), another regulatory system important for controlling cellular energy metabolism. In the present study, we used mouse embryo fibroblasts nullizygous for HIF-1α or AMPK expression to show that AMPK is rapidly activated in vitro by both physiological and pathophysiological low-oxygen conditions, independently of HIF-1 activity. These findings imply that HIF-1 and AMPK are components of a concerted cellular response to maintain energy homeostasis in low-oxygen or ischemic-tissue microenvironments. Finally, we used transformed derivatives of wild-type and HIF-1α- or AMPKα-null mouse embryo fibroblasts to determine whether AMPK is activated in vivo. We obtained evidence that AMPK is activated in authentic hypoxic tumor microenvironments and that this activity overlaps with regions of hypoxia detected by a chemical probe. We also showed that AMPK is important for the growth of this tumor model.

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Alphavirus Minus-Strand Synthesis and Persistence in Mouse Embryo Fibroblasts Derived from Mice Lacking RNase L and Protein Kinase R

Journal of Virology ◽

10.1128/jvi.77.3.1801-1811.2003 ◽

2003 ◽

Vol 77 (3) ◽

pp. 1801-1811 ◽

Cited By ~ 31

Author(s):

Dorothea L. Sawicki ◽

Robert H. Silverman ◽

Bryan R. Williams ◽

Stanley G. Sawicki

Keyword(s):

Protein Kinase ◽

Mouse Embryo ◽

Rnase L ◽

Minus Strand ◽

Protein Kinase R ◽

Persistent Infections ◽

Mouse Embryo Fibroblasts ◽

Mosquito Cells ◽

Embryo Fibroblasts ◽

Alphavirus Infection

ABSTRACT We report our studies to probe the possible role of the host response to double-stranded RNA in cessation of alphavirus minus-strand synthesis. Mouse embryo fibroblasts (MEF) from Mx1-deficient mice that also lack either the protein kinase R (PKR) or the latent RNase L or both PKR and RNase L were screened. In RNase L-deficient but not wild-type or PKR-deficient MEF, there was continuous synthesis of minus-strand templates and the formation of new replication complexes producing viral plus strands. Inhibiting translation caused minus-strand synthesis to stop and a loss of transcription activity of the mature replication complexes. This turnover of replication complexes that were stable in cells containing RNase L suggested that RNase L plays some role, albeit possibly indirect, in the formation of stable replication complexes during alphavirus infection. In addition, confluent monolayers of RNase L-deficient murine cells readily established persistent infections and were not killed. This phenotype is contrary to what has been observed for infection in vertebrate cells with a presumably functional RNase L gene and more resembled alphavirus replication in Aedes mosquito cells, in which the activity of replication complexes making plus stands was also found to decay with inhibition of translation.

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