Manfred Frey

Anti-p53 in Breast Cancer: Concordance of Different Assay Procedures and Association with p53 Antigen Expression

Objective: Anti-p53 levels detected by different methods were compared in a predefined group of patients with breast cancer and correlated with p53 antigen expression in the corresponding tumors. Methods: P53 autoantibodies were investigated in 165 patients with primary breast cancer using ELISAs with recombinant or native p53. Immunoblot and indirect immunofluorescence (Huh7) were used for confirmation, p53 antigen expression in the tumor was determined immunohistochemically. Results: Using ELISA, overall 18/165 positives (11%) were detected, with only partly concordant results between the assays. Five positive sera were confirmed by immunoblot, and three also by indirect immunofluorescence. Anti-p53-positive patients detected by more than two assays showed accumulated p53 in the tumor (6/6) and mostly suffered from recurrent tumors (4/6; p = 0.02). In these cases, a trend towards a shortened disease-free interval was found (26 vs. 49 months; n.s.). In patients with a positive or borderline result in only one of the serological methods, there was no increased rate of p53 accumulation compared to anti-p53-negative patients (4/19 versus 35/126). Conclusions: Lack of assay standardization may partly explain the divergence in reports on anti-p53 and its clinicopathological associations. We speculate that, in different groups of patients, anti-p53 might be induced by different mechanisms.

Functional Interactions of PARP-1 with p53

A close correlation between the frequency of specific mutations of oncogenes and/or tumor suppressor genes in mammals and cancer has been suspected for a long time. For instance, either spontaneous or forcefully inflicted mutations of a tumor suppressor gene coding for a protein known as p53 are usually associated with a variety of malignant tumors. Overwhelming experimental evidence indicates that more than 50% of human neoplasias1 contain one or multiple mutations in one or both alleles of p53. Therefore, the expression product of this pivotal gene, when mutated, appears to play a major role in carcinogenesis. Further significance of p53, as a tumor suppressor protein, is underscored by the fact that over 90% of all tumor-derived mutations associated with it, result in structural and/or functional alterations of its sequence-specific DNA-binding domain2 (Seq-Sp DBD, Fig. 1). Interestingly, the high mutation frequency observed with p53 in malignant tissues initially lead to the misidentification of a mutant of this chromosomal locus as an oncogene rather than its wild type version which functions as the opposite, a tumor suppressor product. An overwhelming amount of work has been done in the last few years to unveil the physiological, biochemical and molecular significance of p53, especially at the protein level. However, in this review the discussion centers on the relevance of the p53 structure and function relationships with poly(ADP-ribose) polymerase-1 (PARP-1), a prominent DNA-strand break sensor in higher eucaryotes, and the biochemical pathway of protein-poly(ADP-ribosyl)ation. A special emphasirity. Therefore, we will present the primary sequence and domain structure of both proteins first.

Up-Regulation of Two Distinct p53-DNA Binding Functions by Covalent Poly(ADP-ribosyl)ation: Transactivating and Single Strand Break Sensing

We used a [32P] p53 sequence-specific oligodeoxynucleotide and Electrophoretic-Mobility-Shift-Assays to monitor p53 DNA sequence-specific binding with p53-R267W, a nonbinding point mutant; and p53-Δ30, a deletion-mutant which lacks the carboxy-terminus that recognizes DNA-strand-breaks. Recombinant p53 and poly(ADP-ribose)polymerase-1 (PARP-1) were incubated with labeled βNAD+ with/without DNA. The poly(ADP-ribosyl)ation of each protein increased with incubation-time and βNAD+ and p53 concentration(s). Since p53-Δ30 was efficiently labeled, poly(ADP-ribosyl)ation target site(s) of wt-p53 must reside outside its carboxy-terminal-domain. The poly(ADP-ribosyl)ation of p53-Δ30 did not diminish its DNA binding; Instead, it enhanced DNA-sequence-specific-binding. Therefore, we conclude that DNA-sequence-specific-binding and DNA-nick-sensing of mutant-p53 are differentially regulated by poly(ADP-ribosyl)ation.