Cancer is a multi-step process typically involving the action of two or more deleterious oncogenes as well as the loss or mutation of tumor suppressor genes, such as p53. The long-term goal of the proposed research is to contribute to the understanding of the way in which wild- type p53 protein suppresses the growth of human cancers. This problem will be approached by studying the effects of a series of mutations on the structure and function of p53. The research will focus on the cysteine residues of murine p53 since cysteines are known to function in the DNA binding of a number of transcriptional regulatory proteins and p53 is thought to act as a transcriptional regulator. Eight of the twelve cysteine residues in murine p53 are located in the five highly conserved regions of the protein. In human malignancies, many of the equivalent cysteine residues in human p53 have undergone single-site mutation. The importance of each of the cysteine residues in normal p53 function will be explored by determining which cysteine mutations abolish the ability of normal murine p53 to suppress transformation of rat embryo cells by E1A + ras. Transformation assays will also be used to determine which cysteine mutations in p53 activate the protein such that is enhances transformation of rat embryo cells by E1A + ras. Other functional assays of the cysteine-mutant p53 proteins will be done to determine what specific functions of p53 account for the ability of the wild-type protein to suppress transformation and the mutant protein to enhance transformation. The mutant p53 proteins will be compared with the wild-type p53 protein with respect to the following functions: ability to regulate promoters involved in control of cell division, ability to bind zinc, ability to bind to specific DNA sequences, ability to interact with other transcriptional regulatory proteins, and ability to oligomerize into tetramers and multiples of tetramers. Knowing how alterations in the structure of p53 affect the function of the protein should provide further insight into the way in which loss or mutation of p53 plays a major role in the development of human cancer.
