Fig. 7. Cellular distribution for p53 cancer mutants with and without suppressor amino acids. Expression plasmids for p53 proteins were transiently transfected into H1299 cells, nuclear and cytoplasmic extracts were prepared, and localization of p53 protein was determined by anti-p53 immunoblotting. The quality of nuclear and cytoplasmic extracts was assessed by immunoblotting for nuclear (DNA-topoisomerase IIa , b ) and cytoplasmic (a -tubulin) marker proteins. WT p53 was predominantly localized in the nucleus, indicating that nuclear import of p53 was efficient, despite significant overexpression of p53 in H1299 cells. In contrast with WT p53, the majority of p53 cancer mutants showed even distribution between nucleus and cytoplasm. These data suggest that p53 cancer mutations not only affect binding of p53 cancer mutants to p53 DBSs but also interfere with their proper localization to the nucleus. For eight p53 cancer mutants, no significant change in the distribution of p53 protein was observed in the presence of suppressor amino acids. However, for the other eight p53 cancer mutants, suppressor amino acids caused a substantial shift of protein from the cytoplasm to the nucleus, suggesting that suppressor amino acids may partly rescue p53 cancer mutants by improving import into the nucleus or preventing export from the nucleus (see C141Y, Y163C, Y173M, Y205C, G245C, R273H, R273L, and E286K). These results demonstrate the diverse behavior of p53 cancer mutants and indicate that suppressor amino acids are likely to affect p53 cancer mutants at several levels of p53 activity and regulation.