Roland Wolkowicz

Direct involvement of p53 in the base excision repair pathway of the DNA repair machinery

The p53 tumor suppressor that plays a central role in the cellular response to genotoxic stress was suggested to be associated with the DNA repair machinery which mostly involves nucleotide excision repair (NER). In the present study we show for the first time that p53 is also directly involved in base excision repair (BER). These experiments were performed with p53 temperature‐sensitive (ts) mutants that were previously studied in in vivo experimental models. We report here that p53 ts mutants can also acquire wild‐type activity under in vitro conditions. Using ts mutants of murine and human origin, it was observed that cell extracts overexpressing p53 exhibited an augmented BER activity measured in an in vitro assay. Depletion of p53 from the nuclear extracts abolished this enhanced activity. Together, this suggests that p53 is involved in more than one DNA repair pathway.

Mutant p53 protein expression interferes with p53-independent apoptotic pathways

Loss of normal p53 function was found frequently to interfere with response of cancer cells to conventional anticancer therapies. Since more than half of all human cancers possess p53 mutations, we decided to explore the involvement of mutant p53 in drug induced apoptosis. To further evaluate the relationship between the p53-dependent and p53-independent apoptotic pathways, and to elucidate the function of mutant p53 in modulating these processes, we investigated the role of a p53 temperature-sensitive (ts) mutant in a number of apoptotic pathways induced by chemotherapeutic drugs that are currently used in cancer therapy. To that end, we studied the M1/2, myeloid p53 non-producer cells, and M1/2-derived temperature-sensitive mutant p53 expressing clones. Apoptosis caused by DNA damage induced with γ-irradiation, doxorubicin or cisplatin, was enhanced in cells expressing wild type p53 as compared to that seen in parental p53 non-producer cells; mutant p53 expressing clones were found to be more resistant to apoptosis induced by these factors. Actinomycin D, a potent inhibitor of transcription, as well as a DNA damaging agent, abrogated the restraint apoptosis mediated by mutant p53. These observations suggest that while loss of wild type p53 function clearly reduces the rate of apoptosis, p53 mutations may result in a gain of function which significantly interferes with chemotherapy induced apoptosis. Therefore, to achieve a successful cancer therapy, it is critical to consider the specific relationship between a given mutation in p53 and the chemotherapy selected.

PACT: cloning and characterization of a cellular p53 binding protein that interacts with Rb

Cellular functions of tumor suppressor proteins can be mediated by protein-protein interactions. Using p53 as a probe to screen an expression library, a cDNA encoding a 250 kDa protein was isolated. Recombinant forms of this protein, designated PACT, bind to wild type p53 while two different mutations abolish this interaction. PACT protein can also interfere with p53 specific DNA binding. PACT contains a serine/arginine (SR) rich region and a C′ terminal lysine rich domain. The 250 kDa PACT protein can be precipitated from cell lysates by a method specific for SR proteins. snRNPs can be co-immunoprecipitated from cells with anti-PACT antibodies. These antibodies stain cell nuclei in a speckled pattern reminiscent of the distribution of known splicing factors. Recently, RBQ1, a truncated human homologue of PACT was identified by virtue of Rb binding. We show that RBQ1 is truncated as a result of a possible mutational event. PACT can interact with both cellular Rb and p53.

Systemic lupus erythematosus in mice, spontaneous and induced, is associated with autoimmunity to the C-terminal domain of p53 that recognizes damaged DNA.

The tumor suppressor molecule p53 features a regulatory domain at the C terminus that recognizes damaged DNA. Since damaged DNA might be involved in activating anti‐DNA autoantibodies, we tested whether autoimmunity to the C terminus of p53 might mark murine systemic lupus erythematosus (SLE). We now report that MRL / MpJ‐Faslpr mice, which spontaneously develop SLE, produce antibodies both to the C terminus of p53 and to a monoclonal antibody (PAb‐421) that binds the p53 C terminus. Anti‐idiotypic antibodies to PAb‐421 (sampled as monoclonal antibodies) could also bind DNA. Thus, the PAb‐421 antibody mimics DNA, and the anti‐idiotypic antibody to PAb‐421 mimics the p53 DNA‐binding site. This mimicry was functional; immunization of BALB / c mice to PAb‐421 induced anti‐DNA antibodies and antibodies to the C terminus of p53, and most of the mice developed an SLE‐like disease. Immunization of C57BL / 6 mice to PAb‐421 induced antibodies to p53, but not to its C‐terminal domain. The C57BL / 6 mice also did not develop anti‐DNA antibodies or the SLE‐like disease. Thus, network autoimmunity to the domain of p53 that recognizes damaged DNA can be a pathogenic feature in SLE in genetically susceptible strains of mice.

DNA binding and 3′–5′ exonuclease activity in the murine alternatively-spliced p53 protein

In this study we show that the naturally occurring C-terminally alternative spliced p53 (referred to as AS-p53) is active as a sequence-specific DNA binding protein as well as a 3′–5′-exonuclease in the presence of Mg2+ ions. The two activities are positively correlated as the sequence-specific DNA target is more efficiently degraded than a non-specific target. In contrast, a mutated AS-p53 protein that is deficient in DNA binding lacks exonuclease activity. The use of modified p53 binding sites, where the 3′-phosphate is replaced by a phosphorothioate group, enabled the inhibition of DNA degradation under the binding conditions. We demonstrate that AS-p53 interacts with its specific DNA target by two distinct binding modes: a high-affinity mode characterized by a low-mobility protein-DNA complex at the nanomolar range, and a low-affinity mode shown by a high-mobility complex at the micromolar range. Comparison of the data on the natural and the modified p53 binding sites suggests that the high-affinity mode is related to AS-p53 function as a transcription factor and that the low-affinity mode is associated with its exonuclease activity. The implications of these findings to a specific cellular role of AS-p53 are discussed.