The p53 tumor suppressor gene is the most commonly altered gene in human cancer and its loss of function is considered to be a critical event in neoplastic progression for many cancers. As such, restoration of function of wild type (Wt) p53 in cancer cells that lack such function is considered an attractive approach to cancer therapy. One possible obstacle is that certain mutant p53s have been considered to be dominant-negative, i.e. they interfere with Wt p53 function. The data that have led to this conclusion are based primarily on rodent model systems. The few studies that have been performed with human cancer cells have led to conflicting conclusions. The intent of this proposal is to definitively evaluate the notion of loss-of-function in human cancer cells. Experimental approaches will involve the use of human cancer cells that lack expression of any p53 protein. To investigate dominant-negative function, p53 null cells will be co- transfected with wild type (Wt) and mutant p53 cDNA expression vectors. Using both constitutive and inducible (sheep metallothionein or tetracycline-responsive) promoter constructs, we will manipulate the relative levels of mutant:Wt p53 ranging from 1:1 and 10:1 ratios. Dominant-negative effects will be assayed using transactivation and transrepression transcriptional assays, in vitro growth inhibition assays, and in vivo tumor formation. In order to more rigorously obtain 1:1 mutant:Wt ratios, we will use bicistronic vectors that will facilitate equal levels of transcription of Wt and mutant p53. All of the above experiments (and those published by others) will result in overexpression of p53 - whether Wt and/or mutant - because of the use of strong heterologous promoters. In order to simulate physiological expression levels of p53, we will use bicistronic vectors that contain the homologous p53 promoter. At physiological levels of expression, Wt p53 should not suppress growth in vitro unless DNA damage occurs. Thus, we will be able to determine potential dominant-negative effects on the important parameters of G1/S block and growth suppression following exposure to gamma-irradiation and PALA - an inducer of genomic instability and amplification. To investigate possible gain-of-function mutants, p53 null cells will be stably transfected with various mutant p53 expression vectors. The stable transfectants will be examined for alteration of function in transcriptional assays, more aggressive growth in vitro - both as adherent populations and in soft agar - and for more aggressive growth in vivo during tumor formation. Particular attention will be placed on orthotopic implantation since this represents the optimal conditions for neoplastic growth, specifically in xenogenic systems that will be used in this study. We will measure the kinetics of primary tumor growth, local invasiveness, metastasis and relative angiogenesis. Resolution of the controversy regarding dominant-negative and gain-of- function mutations in p53 will have distinct significance for therapeutic strategies involving restoration of Wt p53 function as well as possible prognostic outcome of tumors that express certain mutant p53s.
