Alkylation of prohibitin by cyclohexylphenyl-chloroethyl urea on an aspartyl residue is associated with cell cycle G(1) arrest in B16 cells

Abstract

Background and purpose: Phenyl-chloroethyl ureas (CEUs) are a class of anticancer drugs that mainly react with proteins. Two molecules of this family, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU) induced G(1)/S and G(2)/M cell cycle blocks, respectively. We hypothesised that these observations were linked to a differential protein alkylation pattern.

Experimental approach: Proteins from B16 cells incubated with [(14)C-urea]-CCEU and [(125)I]-ICEU were compared by 2D-analyses followed by MALDI-TOF identification of modified proteins and characterisation of the CCEU binding. Protein expression was investigated by Western blot analyses and cell cycle data were obtained by flow cytometry.

Key results: Several proteins (PDIA1, PDIA3, PDIA6, TRX, VDAC2) were alkylated by both ICEU and CCEU but beta-tubulin and prohibitin (PHB) were specifically alkylated by either ICEU or CCEU respectively. Specific alkylation of these two proteins might explain the observed difference in B16 cell cycle arrest in G(2) and G(1) phases respectively. Mass spectrometry studies on the alkylated prohibitin localised the modified peptide and identified Asp-40 as the target for CCEU. This alkylation induced an increased cellular content of PHB that should contribute to the accumulation of cells in G(1) phase.

Conclusions and implications: This study reinforces our findings that CEUs alkylate proteins through an ester linkage with an acidic amino acid and shows that PHB alkylation contributes to G(1)/S arrest in CCEU treated B16 cells. Modification of PHB status and/or activity is an open route for new cancer therapeutics.

Figure 1

Chemical structure of CCEU and ICEU. The calculated monoisotopic masses for CCEU and ICEU are 280.13 and 323.95 Da, respectively, for the complete molecules, and 245.16 and 288.98 Da without the chlorine atom. ^*Shows the positions of the radiolabel. CCEU, cyclohexylphenyl-chloroethyl urea; ICEU, iodophenylchloroethyl urea.

Figure 2

CCEU and ICEU block B16 cell cycle at G₁/S and at G₂/M transition, respectively. (a) Typical flow cytometry analysis with propidium iodide showing a decrease of cells in S phase, whereas the number of cells in G₁ phase increased after CCEU treatment. In contrast, ICEU treatment leads to a major increase of cells in G₂ phase due to a block in the G₂/M transition, when compared to DMSO-treated cells (control). (b) Distribution of cells in the different cell-cycle phases expressed as percentages. Statistical differences are found between controls and treated cells by the χ² test (P<0.05). (c) Western blot analysis: ICEU, but not CCEU, modified the migration properties of β-tubulin in extracts of B16 cells. CCEU, cyclohexylphenyl-chloroethyl urea; ICEU, iodophenyl-chloroethyl urea.

Figure 3

CCEU and ICEU alkylate common and specific B16 cell proteins. 2D electrophoresis on 3–10 nonlinear pH range strips: proteins were extracted from B16 cells treated for 24 h with 100 μM CCEU (a) or 100 μM ICEU (b), unlabelled (left) or radiolabelled ([¹⁴C]-CCEU, 19 GBq/mmol and [¹²⁵I]-ICEU, 1.5 GBq/mmol) (right). They were stained with colloidal blue (left) or dried before autoradiography of ¹⁴C or ¹²⁵I (right, a and b, respectively). Spots equivalent to the radioactive spots (numbers) were recovered on Coomassie-stained gels and identified by MALDI-TOF-MS. From the six major labelled spots, spots 1–5 were alkylated by both CEUs whereas spots 6-a and 6-b were specifically identified after CCEU or ICEU treatment, respectively. ^* Corresponds to radiolabelled materials that could not be identified, as they did not correspond to defined spots on a coloured gel. CCEU, cyclohexylphenyl-chloroethyl urea; ICEU, iodophenyl-chloroethyl urea; MALDI-TOF-MS, matrix-assisted laser desorption/ionisation–time of flight–mass spectrometry.

Figure 4

CCEU covalently alkylates PHB on an Asp residue. (a) 2D gel analysis of proteins extracted from cells treated for 24 h with 100 μM CCEU (bottom) revealed one additional spot (P2) when compared to DMSO-treated cells (top). (b) Western blot analysis showed that two PHB spots were only present in CCEU-treated B16 cells, the basic one being labelled by [¹⁴C]-CCEU as demonstrated by autoradiography of the membrane. (c) MALDI-TOF-MS comparison of the two proteins identified as PHB. The ion at m/z 3125 corresponds to the peptide [Gly 44-Arg 70] containing the only Cys residue of PHB for the acidic PHB (top) or basic PHB (bottom). (d) MALDI-TOF-MS comparison of the PHB spots. An ion at m/z 720.41, corresponding to peptide [Ala 36-Arg 41] present in the original acidic PHB (top) was absent in the basic PHB, which in contrast contained an ion at m/z 964.56 (bottom). The mass difference (Δm=244.15) was in accordance with the presence of a CCEU residue on peptide [Ala 36-Arg 41]. ^* Corresponds to trypsin autolysis peptide (m/z=842.5100). CCEU, cyclohexylphenyl-chloroethyl urea; DMSO, dimethylsulphoxide; MALDI-TOF-MS, matrix-assisted laser desorption/ionisation–time of flight–mass spectrometry; PHB, prohibitin.

Figure 5

CCEU covalently alkylates PHB by an ester bond. (a) Fragmentation spectrum of the modified peptide [Ala 36-Arg 41]. Specific fragmentation ions are indicated in the figure. (b) Structure of the alkylated peptide, CEU# indicates a fragmented CEU. CEU, phenyl-chloroethyl urea; CCEU, cyclohexylphenyl-chloroethyl urea; PHB, prohibitin.

Figure 6

Time course of CCEU-induced cell-cycle arrest paralleled PHB accumulation in cellular extracts. (a) A G₁ increase, determined in kinetic studies of B16 cells treated with 100 μM CCEU occurred after 8 h and remained stable (75–80%) at 24 h, compared to DMSO-treated cells. (b) 2D electrophoreses of CCEU-treated B16 cell protein extracts showed that the additional spot appeared between 8 and 16 h incubation. (c) Analysis of PHB content in whole-cell extracts by Western blotting: an increased PHB amount was detected after 8–16 h of incubation, using anti-PHB and anti-β-tubulin antibodies for normalisation. Data presented above are representative of at least two independent experiments. CCEU, cyclohexylphenyl-chloroethyl urea; DMSO, dimethylsulphoxide; PHB, prohibitin.