p53 plays a central role in determining how mammalian cells respond to stress. DNA damange, arrest of DNA or RNA sythesis, and inhibition of pyrimidine nucleotide synthesis all lead to both the accumulation and activation of p53. It is likely that modulation of the phosphorylation of serine residues plays a key role in each process, and acetylation of lysine residues may also be important. We have established a valuable experimental system, based on the human MDAH041 fibroblast cell line, derived from a Li-Fraumeni patient and lacking p53 protein. Re- expression of wild-type p53 at normal basal levels restores many normal p53-dependent properties to these cells. Using this system, we have discovered a novel p53-dependent cell cycle checkpoint which prevents entry into mitosis when DNA synthesis is blocked (by hydroxyurea). We will investigate in detail the mechanisms through which p53 mediates this important response and will also take advantage of this system to investigate how a wide range of normal p53 responses to stress are affected by mutation of key serine and lysine residues. The major underlying hypothesis is that different stresses cause distinct modifications of p53, and that differently modified p53 molecules effect distinct and different cellular responses.
Aim 1 : Determine the roles of specific amino acid residues in different p53-dependent responses. We will mutate multiple serine and lysine residues that are sites of covalent modification of p53 and study the effects in MDAH041 cells. Basal expression of p53 variant proteins from a tetracycline-inducible promoter will be normalized by adjusting the amount of tetracycline. The following important p53-dependent responses will be assayed: (1) The accumulation of p53 protein, (2) the ability of p53 to bind to known regulatory proteins, (3) the activation and repression of transcription, (4) the ability to block entry into mitosis when DNA synthesis is arrested, (5) the ability to block entry into S phase when DNA is damaged, (6) the ability to block entry into S phase when mitosis is arrested, (7) the ability to induce a fragile site on chromosome 17 and (8) the inability to give rise to N-(phosphonacetyl)-L-aspartate (PALA)-resistant colonies. The information obtained will help to define discrete pathways that modify p53 in response to different activating signals and discrete functions that depend on the patterns of modification.
Aim 2 : Investigate the mechanisms through which p53 mediates a novel biological response. We will investigate how p53 interacts with or affects known components that govern entry into mitosis, especially cyclin B1 and CDC2, and how these interactions are affected when specific covalent modifications of p53 are prevented by point mutations. Understanding the complexity of p53 activation and function is important in developing strategies to deal more effectively with the regulatory circuits involved in the origin and progression of tumors and will also be important in helping to understand the p53 dependence of the toxicity of chemotherapeutic agents now in use.
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