Differential Display Analysis of 2,3,7,8-Tetrachlorodibenzo-p-dioxin Identified Induction of Ras-related Nuclear Protein Binding Protein2 (RanBP2) Gene

A clonal cell line (56-42) that was stably and exclusively resistant to the toxic effects of the antileukemic agent 5-aza-2'-deoxycytidine (5-aza-CdR) was derived from C3H 10T1/2 C18 cells after multiple treatments with 5-aza-CdR. The 50% lethal dose of 5-aza-CdR for these cells was 1.3 microM, which was 15-fold greater than that for the parental cells. Cell line 56-42 was slightly cross-resistant to the ribo-analog 5-azacytidine, but was sensitive to the nucleoside analog 1-beta-D-arabinofuranosylcytosine and to colcemid. Both parental and resistant cell lines incorporated equimolar amounts of 5-aza-CdR into DNA. Resistance was therefore not due to decreased activation, increased detoxification, or reduced incorporation of the drug. The overall level of cytosine methylation in the resistant clone was 80% lower than the level in the sensitive cells. Therefore, the potential number of hemimethylated sites created by the incorporation of equivalent amounts of 5-aza-CdR into the DNA of the two cell types was much greater in the sensitive cells. Furthermore, 5-azacytosine-substituted DNA from the sensitive cells bound 100% more nuclear protein in the form of highly stable complexes. The incorporation of 5-aza-CdR opposite methylated cytosine residues in DNA of the sensitive cells thus resulted in increased nuclear protein binding at hemimethylated sites. This relative increase in tight-binding protein complexes was shown to occur in living cells and may well disrupt replication and transcription and instigate cell death. The differential binding of proteins to hypomethylated, azacytosine-containing DNA may thus mediate a novel mechanism of drug resistance.

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Exercise attenuates nuclear protein binding to gene regulatory sequences of hepatic fatty acid synthase

Journal of Applied Physiology ◽

10.1152/jappl.1999.87.3.1009 ◽

1999 ◽

Vol 87 (3) ◽

pp. 1009-1015 ◽

Cited By ~ 9

Author(s):

Russel Fiebig ◽

Mitchell T. Gore ◽

Li Li Ji

Keyword(s):

Fatty Acid ◽

Protein Binding ◽

Fatty Acid Synthase ◽

Nuclear Protein ◽

Exhaustive Exercise ◽

Regulatory Sequences ◽

Transcription Rate ◽

Response Element ◽

Hepatic Fatty Acid ◽

Fas Mrna

The effect of an acute bout of exhaustive exercise on hepatic fatty acid synthase (FAS) gene expression was examined in rats. Female Sprague-Dawley rats (age 8 wk) were fasted for 48 h (F, n = 6), or fasted, refed a high-fructose diet for 6 h, and killed at rest (R, n = 6) or killed after running on a treadmill at 27 m/min and 5% grade for 88 ± 7 min (E, n = 6). Gel mobility shift assay indicated that R rats had twofold higher liver nuclear protein binding to oligonucleotides corresponding to the insulin responsive sequence (−71/−50) and carbohydrate response element (+283/+303) on the FAS promoter, compared with F rats. Exercise severely attenuated this binding in liver nuclear extracts to the levels seen in F rats. Competition and supershift experiments revealed that the bound protein complexes contained the upstream stimulatory factors. Nuclear run-on experiment revealed a 49-fold increase in transcription rate of the FAS gene in R vs. F rats, whereas exercise suppressed the transcription rate. FAS mRNA abundance and FAS enzyme activity were dramatically increased with refeeding but were unaltered by exercise. The results reveal that dietary induction of hepatic FAS is stimulated by increased nuclear protein binding to insulin responsive sequence and carbohydrate response element, whereas exhaustive exercise attenuates the binding, which may precede downregulation of FAS mRNA and enzyme synthesis reported in our previous work (M. A. Griffiths, R. Fiebig, M. T. Gore, D. H. Baker, K. Esser, L. Oscai, and L. L. Ji. J. Nutr. 126, 1959–1971, 1996).

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