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Fig. 5. Manipulation of intracellular redox state. Asynchronously growing HeLa cells were treated with 20 mM NAC (pH 7.0) and subjected to the RNA-protein gel mobility shift assay (A); Topo II mRNA levels were determined (B), as was NAC uptake (by HPLC) and glutathione content (by spectrophotometric recycling assay) (C). Changes in intracellular redox state were measured as the ratio of GSH (nmol/mg) to GSSG (2 x nmol/mg). Ethidium bromide-stained 28S ribosomal RNA levels were included for comparison of the Northern blot results.

of Anderson.20 Reduced (GSH) and oxidized (GSSG) glutathione are distinguished by addition of 2 of a 1:1 mixture of 2-vinylpyridine and ethanol per 30 ¡A of sample followed by incubation at room temperature for 1.5 hr before addition of SSA. NAC levels in cells are measured after derivatization with N-( I -pyrenyl)

20 M. E. Anderson, in "Handbook of Methods for Oxygen Radical Research" (R. A. Greenwald, ed.), p. 317. CRC Press, Boca Raton, FL, 1985.

maleimide, using a 15-cm C^ Reliasil column (Column Engineering, Ontario, CA) coupled with high-performance liquid chromatography with fluorescence detection.19 All biochemical determinations are normalized to the protein content of whole cell homogenates, using the method of Lowry et al.21

Results presented in Fig. 5C show that treatment of cells with NAC alters the intracellular redox state to a more reducing environment. Thus, NAC levels are approximately 5.6 nmol/mg protein after 6 hr of treatment. Consistent with these results, the ratio of GSH to GSSG increases approximately 1.5- to 2.0-fold during this time frame. Total cellular protein extracts are prepared in the absence of DTT and in vitro RNA-protein binding assays are performed according to the method described above. These results show that protein binding to the Topo II3' UTR increases 3- to 4-fold in NAC-treated cells compared with untreated controls (compare lanes 2 and 3 in Fig. 5A). These results provide in vivo evidence that a shift to a more reducing environment enhances protein binding to the 177-nt Topo II 3' UTR. Topo II mRNA levels are analyzed after a 6-hr treatment with NAC. An increase in intracellular reducing state induced by NAC treatment decreases Topo II mRNA levels by more than 90% (compare lanes 1 and 2 in Fig. 5B). Interestingly, the NAC-induced decrease in Topo II mRNA levels correlates with enhanced protein binding to the Topo II3' UTR (Fig. 5A). These results indicate that protein binding to the Topo II3' UTR is favored in a reducing environment, which appears to facilitate mRNA degradation. These results show the feasibility of manipulating the intracellular redox state and its subsequent effect on Topo II gene expression.

Conclusion

A growing body of literature suggests the importance of the intracellular redox state as the possible physiological regulator of cell proliferation. Although the molecular mechanisms are currently not fully understood, it is possible that at least some of the mechanisms could be at the level of cell cycle-coupled gene expression. The assays described here provide methods to study alterations in gene expression during the cell cycle and possible effects of alterations in the intracellular redox state on cell cycle-coupled variations in gene expression. Although the assays were optimized for the study of Topo II expression, similar approaches can be used to study the redox regulation of other cell cycle-coupled genes.

Acknowledgments

This work was supported by NIH Grants R29 CA-69593 (P.C.G.) and ROl HL-51469 (D.R.S.).

21 O. H. Lowry, N. J. Rosenbrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

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