Quantification by affinity perfusion chromatography of phosphorylated BRCAl and BRCA2 proteins from tumor cells after lycopene treatment.
A new procedure for the quantification of phosphorylated BRCA1 (P-BRCA1) and BRCA2 (P-BRCA2) proteins in breast cell lines after different treatments was carried out. Cells were cultivated with [35S]-methionine and extracts subjected to three perfusion chromatographies. First heparin affinity chromatography purified cellular DNA-binding proteins. Subsequent specific immunoprecipitation of BRCA1 and BRCA2 proteins was performed with antibodies raised against BRCA1 or BRCA2. The immune complexes were isolated by protein A affinity chromatography. Phosphorylated BRCA1 or BRCA2 proteins were then purified with a Poros 20 AL column where anti-phosphothreonine and anti-phosphoserine antibodies were previously bound. The percentage of phosphorylated BRCA1 or BRCA2 proteins was calculated as follows: 100 x dpm of P-BRCA1 or P-BRCA2 eluted from the POROS 20AL column/total dpm eluted from POROS 20AL column. Treatment with 10 microM lycopene increased P-BRCA1 and P-BRCA2 in the breast tumor cell line MCF7 but not in MDA-MB-231 or MCF-10a, breast tumor or fibrocystic cell lines, respectively.
Chalabi
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N
Département d'Oncogénétique, Centre Jean Perrin, INSERM UMR 484-UdA, Centre Biomédical de Recherche et Valorisation, 28 Place Henri Dunant, BP 38, 63001 Clermont-Ferrand, France.
Maurizis
J-C
JC
Le Corre
L
L
Delort
L
L
Bignon
Y-J
YJ
Bernard-Gallon
D J
DJ
D019313:BRCA1 Protein / Q000032:analysis*; D024682:BRCA2 Protein / Q000032:analysis*; D001943:Breast Neoplasms / Q000737:chemistry*; D002338:Carotenoids / Q000378:metabolism / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D000077276:Lycopene; D010766:Phosphorylation; D011960:Receptors, Estrogen / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D019313:BRCA1 Protein; D024682:BRCA2 Protein; D011960:Receptors, Estrogen; D002338:Carotenoids; D000077276:Lycopene