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


METHODS
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.


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
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.
Bouchon
B
B
University Clermont 1, UFR Pharmacie, Clermont-Ferrand, France.
Papon
J
J
Communal
Y
Y
Madelmont
J-C
JC
Degoul
F
F
D000477:Alkylating Agents / Q000737:chemistry / Q000494:pharmacology; D000478:Alkylation / Q000187:drug effects; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001224:Aspartic Acid / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects; D015151:Immunoblotting; D007457:Iodine Radioisotopes; D015394:Molecular Structure; D000091402:Prohibitins; D040901:Proteomics / Q000379:methods; D012097:Repressor Proteins / Q000378:metabolism*; D016196:S Phase / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014404:Tubulin / Q000378:metabolism; D014508:Urea / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D050994:Voltage-Dependent Anion Channels / Q000378:metabolism
D000477:Alkylating Agents; D000970:Antineoplastic Agents; D002250:Carbon Radioisotopes; D007457:Iodine Radioisotopes; D000091402:Prohibitins; D012097:Repressor Proteins; D014404:Tubulin; D050994:Voltage-Dependent Anion Channels; C527418:cyclohexylphenyl-chloroethyl urea; C527419:iodophenyl-chloroethyl urea; D001224:Aspartic Acid; D014508:Urea
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