Analysis of defective protein ubiquitylation associated to adriamycin resistant cells

DNA damage activated by Adriamycin (ADR) promotes ubiquitin–proteasome system-mediated proteolysis by stimulating both the activity of ubiquitylating enzymes and the proteasome. In ADR-resistant breast cancer MCF7 (MCF7ADR) cells, protein ubiquitylation is significantly reduced compared to the parental MCF7 cells. Here, we used tandem ubiquitin-binding entities (TUBEs) to analyze the ubiquitylation pattern observed in MCF7 or MCF7ADR cells. While in MCF7, the level of total ubiquitylation increased up to six-fold in response to ADR, in MCF7ADR cells only a two-fold response was found. To further explore these differences, we looked for cellular factors presenting ubiquitylation defects in MCF7ADR cells. Among them, we found the tumor suppressor p53 and its ubiquitin ligase, Mdm2. We also observed a drastic decrease of proteins known to integrate the TUBE-associated ubiquitin proteome after ADR treatment of MCF7 cells, like histone H2AX, HMGB1 or β-tubulin. Only the proteasome inhibitor MG132, but ...