Relationship between the induction of leukemia cell differentiation and the enhancement of reporter gene expression in 3T3 Swiss cells

Abstract

The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and hexamethylene bisacetamide (HMBA), which lacks HDAC inhibitory activity, both possess the capacity to induce leukemia cell differentiation and to enhance the expression of a wide range of transiently transfected reporter genes in 3T3 Swiss cells. In addition, known inducers of leukemia cell differentiation, including hypoxanthine, diazepam, 6-thioguanine and phorbol 12-myristate 13-acetate, also exhibited the ability to enhance reporter gene expression, while randomly chosen compounds that did not induce leukemia cell differentiation did not enhance reporter gene expression. The activity of TSA in the transfection system was modified by co-expression of histone acetyltransferase p300 and HDAC1; whereas, that of HMBA was enhanced by co-expression of the TATA-binding protein TBP. The stimulatory effects of diverse chemical inducers on transiently transfected genes suggest the existence of multiple exploitable targets for the selection of novel inducers of differentiation that function as modulators of gene activity.

Fig. 1

Stimulation of the expression of transiently transfected reporter genes in 3T3 Swiss cells by chemical inducers of differentiation. Cells were transfected with a reporter gene for 3 h and cultured in the absence or presence of chemical inducers for 20 h. Luciferase activities were normalized by cell number. Stimulation is expressed as the fold increase relative to the control value taken as 1.

Fig. 2

Characterization of PMA as a stimulator of reporter gene expression. (A) Cells were transfected with one of six independent reporter genes and stimulation of the expression of the reporter gene by PMA was determined as in Fig. 1. (B) Cells were transfected with pNF-κB for 3 h and exposed to 50 nM PMA, 60 nM TSA, or 15 mM HMBA for the indicated times. (C) Cells were transfected with pNF-κB for 3 h and cultured in the simultaneous presence of inducer (10 nM PMA, 30 nM TSA, or 7.5 mM HMBA) and bisindolylmaleimide II (BII) at a concentration of 0.2 or 0.8 μM for 20 h. (D) Gel shift assay for DIG-labeled NF-κB and AP-1 concensus oligonucleotides incubated with nuclear extracts from 3T3 Swiss cells treated with inducers for 4 h.

Fig. 3

Characterization of chemical inducers in histone modification and reporter gene expression. (A) Core histones were extracted from F-MEL cells treated with various inducers for 8 h, and the acetylation and phosphorylation status was determined by western analysis. Coomassie Brilliant Blue staining was used as a loading control. D₂ = diazepam. (B) 3T3 Swiss cells were transfected with the reporter HS2-421β^m and treated with graded concentrations of TSA plus a fixed concentration of 7.5 mM HMBA (left panel), or graded concentrations of HMBA plus a fixed concentration of 15 nM TSA (right panel). (C) 3T3 Swiss cells were co-transfected with 0.2 μg of both the reporter plasmid HS2-421β^m and the expression plasmid encoding the transcriptional modulator, and incubated with 20 nM TSA, 10 mM HMBA, or 50 nM PMA for 20 h.

Fig. 4

Up- and down-modulation of endogenous genes by chemical inducers. (A) Total cell RNA from F-MEL cells treated with inducers for 8 h was subjected to northern hybridization (left panel). Total cell RNA from F-MEL cells exposed to HMBA or TSA for the indicated times was subjected to northern hybridization (right panel). (B) The acetylation status of core histones from F-MEL cells exposed to TSA for the indicated times was analyzed by Western blotting. The methylation status of histones was unaltered and served as a loading control.