Selective killing of transformed cells by cyclin/cyclin-dependent kinase 2 antagonists

Abstract

Recent studies identified a short peptide motif that serves as a docking site for cyclin/cyclin-dependent kinase (cdk) 2 complexes. Peptides containing this motif block the phosphorylation of substrates by cyclin A/cdk2 or cyclin E/cdk2. Here we report that cell membrane-permeable forms of such peptides preferentially induced transformed cells to undergo apoptosis relative to nontransformed cells. Deregulation of E2F family transcription factors is a common event during transformation and was sufficient to sensitize cells to the cyclin/cdk2 inhibitory peptides. These results suggest that deregulation of E2F and inhibition of cdk2 are synthetically lethal and provide a rationale for the development of cdk2 antagonists as antineoplastic agents.

Figures

Figure 1

Peptide inhibition of cyclin/cdk2. (A) Phosphorylation of GST-RB in vitro by control (lane 1), anti-cyclin A (lanes 2–7), or anti-cyclin B (lanes 8–13) immunoprecipitates in the presence of γ-32P-ATP. Where indicated, TAT-LDL (final concentration 0.1, 1, and 10 μM as indicated by the triangles), TAT-Umt (10 μM), or TAT (10 μM) peptides were added before the addition of substrate. (B) Asynchronously growing U2OS osteosarcoma cells were treated with the indicated peptides (50 μM) for 4 hr, lysed, and immunoprecipitated with anti-cdk2 (lanes 1–5) or a control antibody (lane 6). In vitro kinase reactions were performed in the presence of γ-32P-ATP using GST-RB as substrate and resolved by SDS/PAGE. Phosphorylation of GST-pRB and recovery of cdk2 were monitored by autoradiography and anti-cdk2 immunoblot analysis, respectively.

Figure 2

Cell killing by peptidic cyclin/cdk2 antagonists. U2OS osteosarcoma (A and B) and MDA-MB-435 breast carcinoma cells (C and D) were treated with the indicated Tat (A and C) and penetratin (B and D) fusion peptides. Cell viability was measured by using an MTS assay.

Figure 3

Induction of apoptosis by peptidic cyclin/cdk2 antagonists. (A) Fluorescence microscopy of 4′,6-diamidino-2-phenylindole-stained U2OS cells treated with the indicated peptides. (B) DNA content, measured by fluorescence-activated cell sorting after staining with propidium iodide, of U2OS cells treated with indicated peptides for 6 hr at final concentration of 30 μM.

Figure 4

Preferential killing of transformed cells by peptidic cyclin/cdk2 antagonists. (A) The indicated cell lines were treated with the Tat-LDL peptide. Cell viability was measured by using an MTS assay. (B) WI38 and WI38/VA13 cells were treated with the indicated peptides, and cell viability was measured by MTS assay. (C). Phase-contrast (Left) and fluorescence microscopy (Right) of WI38/VA13 (Upper) and WI38 cells (Lower) treated with fluorescein-labeled Tat-LDL peptide.

Figure 5

Peptidic cyclin/cdk2 antagonists kill cells with deregulated E2F. Rat-1a fibroblasts stably transfected with a human E2F1 cDNA under the control of a zinc-inducible promoter (Rat-1a1093E2F1) were treated with the indicated peptides in the presence or absence of zinc. (A) Cell viability measured by MTS assay. (B) Cell-cycle distribution, measured by fluorescence-activated cell sorting after staining with propidium iodide (x axis) and FITC-conjugated anti-BrdUrd (y axis) of Rat-1a1093E2F1 cells in the presence (Upper) or absence (Lower) of zinc. Cells were treated with Tat-LDL (Left) or Tat-Umt (Right). Cells in G2/M are shown in blue and cells in S phase undergoing active DNA replication (as measured by BrdUrd incorporation) are shown in green.