Figure 1

Expression, mitochondrial localization, and oligomerization of mutant p53 proteins. (A) HCT116 cells producing endogenous wt p53 were infected with retroviruses to express, from single gene copies per cell, either empty vector or C-terminally truncated 175H and 273H mutants (175ΔC, 273ΔC). p53 was detected with antibody DO-1 (1:2000) applied to 15 μg of total cell extract. The diagram shows the intensities of the signals produced by wt p53 and the truncated proteins, determined by densitometry and with the signal from wt p53 in vector-infected control cells arbitrarily set as one. (B) Western immunoblot analysis with anti-p53 antibody DO-1 (1:2000) on total and mitochondrial protein prepared at 24 h after the treatment of the three cell lines with 5FU (375 μM) or not. (C) Detection of mutant p53 oligomers in HCT116 p53-/- cells designed to express, again from single gene copies, one of the indicated mutants. The ΔO-mutants carry a deletion in the oligomerization domain. Exponentially growing cells were subjected to crosslinking by either bis-maleimidohexane (BMH) or glutaraldehyde (GLD), as specified in Materials and methods. Oligomers were detected by anti-p53 antibody DO-1 (1:2000) in 15 μg total protein samples that were run on standard SDS-PAGE. (D) Hetero-oligomerization between mutant p53 and a full-length p53 with mutated DO-1 epitope (asterisk). HCT116 p53-/- cells were transiently transfected with expression plasmids producing either construct 1 (p53-22,23) plus no protein (empty vector), construct 1 plus construct 2 (mutants 273H or 175H, with truncated C-terminus), or construct 1 plus construct 3 (mutants 273H or 175H, with deleted oligomerization domain). The deletion mutants were immunoprecipitated with antibody DO-1, and could co-immunoprecipitate p53-22,23 only through hetero-oligomerization. p53 was detected in immunoblots with the polyclonal anti-p53 antibody CM-1 (1:500).