Depudecin induces morphological reversion of transformed fibroblasts via the inhibition of histone deacetylase - PubMed (original) (raw)
Depudecin induces morphological reversion of transformed fibroblasts via the inhibition of histone deacetylase
H J Kwon et al. Proc Natl Acad Sci U S A. 1998.
Abstract
Depudecin is a fungal metabolite that reverts the rounded phenotype of NIH 3T3 fibroblasts transformed with v-ras and v-src oncogenes to the flattened phenotype of the nontransformed parental cells. The mechanism of detransformation induced by this agent had not been determined. Here, we demonstrate that depudecin inhibits histone deacetylase (HDAC) activity effectively both in vivo and in vitro. Depudecin induces similar morphological reversion in v-ras transformed NIH 3T3 cells as do other naturally occurring HDAC inhibitors such as trichostatin A or trapoxin. It competitively inhibits the binding of [3H]trapoxin in vitro and the nuclear binding of a trapoxin-coumarin fluorophore in vivo, suggesting that depudecin shares a nuclear binding protein and site on that protein with trapoxin. Furthermore, depudecin induces hyperacetylation of histones in a dose-dependent manner and at concentrations comparable with that required for detransformation. An in vitro histone deacetylase assay, using purified recombinant HDAC1, reveals that depudecin inhibits 50% of the enzyme activity at a concentration of 4.7 microM. These results demonstrate that depudecin is a novel HDAC inhibitor and suggest that its ability to induce morphological reversion of transformed cells is the result of its HDAC inhibitory activity.
Figures
Figure 1
The chemical structures of depudecin and other compounds discussed in text. (A) Depudecin and its inactive derivatives, mono-MTM-depudecin and depudecin-bisether. (B) HDAC inhibitors trapoxin, trichostatin, and butyric acid. (C) Radicicol. Trapoxin and other cyclic tetrapeptide inhibitors contain the functionally important amino acid (2_S_,9_S_)-2-amino-8-oxo-9,10-epoxydecanoic acid (Aoe). This side chain is approximately isosteric to that of ɛ-_N_-acetylated lysine residues in histone proteins and may form a bond to a nucleophilic residue in the enzyme active site.
Figure 2
The morphological change in v-_ras_-transformed NIH 3T3 cells by detransforming agents. Exponentially growing cells were treated for 24 h with test agents, and photographs were taken under a phase-contrast microscope. (A) Control v-ras transformed NIH 3T3 cells. (B) Cells treated with 4.7 μM synthetic depudecin. (C) Cells treated with 1.5 μM trichostatin A. (D) cells treated with 3.4 μM radicicol. The characteristic elongated cells having filamentous protrusions induced by depudecin and trichostatin A are indicated by the arrow. (Bar = 50 μm.)
Figure 3
The effect of depudecin and other detransforming agents on [3H]trapoxin binding with crude lysate of v-ras transformed NIH 3T3 cells. The assay was carried out as described in Material and Methods. A 100-fold excess amount of cold compounds was preincubated with crude lysate for 30 min at 25°C in the competition assay.
Figure 4
Visualization of HDACs and HDAC binding in the nucleus of v-ras NIH 3T3 cells using a competition binding assay involving K-trap–coumarin dye and various detransformation agents. The staining was carried out as described in Material and Methods. Upper and lower photos were taken using phase-contrast and fluorescence microscopy, respectively. Coumarin dye excites at 345 nm with blue light. (A and B) Coumarin dye control. (C and D) K-trap–coumarin dye control. (E and F) K-trap–coumarin dye in cells pretreated with 23.5 μM depudecin. (G and H) K-trap–coumarin dye in cells pretreated with 0.1 μM trapoxin. (I and J) K-trap–coumarin dye in cells pretreated with 3.4 μM radicicol.
Figure 5
The effect of depudecin and other detransforming agents on histone acetylation in v-ras transformed NIH 3T3 cells. Lane 1, control, treatment of v-ras transformed NIH 3T3 cells for 6 h with vehicle only; lane 2, 4.7 μM depudecin; lane 3, 23.5 μM depudecin; lane 4, 47 μM mono-MTM-depudecin; lane 5, 47 μM depudecin-bisether; lane 6, 3.4 μM radicicol; lane 7, 1.5 μM trichostatin A. Histones were extracted from the cells and visualized on AUT gel by Coomassie brilliant blue staining. Acetylated histone species have slower rates of migration relative to nonacetylated histones (H4-0, -1, -2, -3, -4).
Figure 6
The effect of depudecin and other detransforming agents on histone deacetylase in vitro. The assay was carried out as described in Materials and Methods. (A) HDAC assay using crude lysate as the source of enzyme. (B) HDAC assay using purified recombinant HDAC1-F.
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