Figure 5

p53 monomers and oligomers in the cell and at the mitochondria. (A) HCT116 cells treated with the apoptosis-inducing drug 5FU (375 μM) for 24 h show more oligomers of the apoptotic BH123 protein Bax in the mitochondrial fraction. These were detected in a standard SDS-PAGE after chemical crosslinking with BMH and subsequent immunoblotting with anti-Bax antibody sc-493 at 1:500 dilution (See Materials and methods for details). Total: total cell extract; mito: mitochondrial fraction. Oligo: oligomers; mono: monomers. (B) Analogous study of p53 oligomerization in the indicated cell lines and after treatment with 5FU. BMH: bis-maleimidohexane; GLD: glutaraldehyde. Again, oligomers were detected in a standard SDS-PAGE; the anti-p53 antibody was DO-1 (1:2000). wt p53: HCT116 cells infected with vector-only. 273H, 175H: HCT116 p53-/- cells infected with a retroviral vector producing either mutant 273H or 175H. (C) Wt p53 in MCF-7 cells translocates to the mitochondria upon treatment with α-amanitin (10 μM; A), 5FU (375 μM; F), or both combined (FA). Cultures were mock-treated or treated as indicated for 24 h, and were then fractionated as described in Materials and methods. 15 μg of total protein (t) or mitochondrial protein (mt) were subjected to Western immunoblot analysis with either anti-p53 antibody DO-1 (1:2000) or anti-cytochrome oxidase IV (OX IV) antibody (1:1000). (D) In analogy to (a) and (b), MCF-7 cells were treated with 5FU for 24 h, and p53 oligomerization in the cells and in the mitochondrial fraction was monitored by standard SDS-PAGE and immunoblotting after BMH or GLD crosslinking. p53 was detected with antibody DO-1 (1:2000). (E) Mutant p53DD does not translocate to the mitochondria. In HCT116 cells retrovirally infected to express p53DD, endogenous wt p53 is stabilized and mitochondria-associated upon 5FU treatment whereas p53DD is not.