Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts

Abstract

Several groups have attempted to develop gene therapy strategies to treat cancer via introduction of the wild-type (wt) p53 cDNA into cancer cells. Unfortunately, these approaches do not result in regulated expression of the p53 gene and do not reduce expression of the mutant p53 that is overexpressed in cancerous cells. These shortcomings may greatly limit the utility of this gene replacement approach. We describe an alternative strategy with trans-splicing ribozymes that can simultaneously reduce mutant p53 expression and restore wt p53 activity in various human cancers. The ribozyme accomplished such conversion by repairing defective p53 mRNAs with high fidelity and specificity. The corrected transcripts were translated to produce functional p53 that can transactivate p53-responsive promoters and down-modulate expression of the multidrug resistance (MDR1) gene promoter. The level of wt p53 activity generated was significant, resulting in a 23-fold induction of a p53-responsive promoter and a 3-fold reduction in MDR1 promoter expression in transfected cancer cells. Once efficient delivery systems are developed, this strategy should prove useful for making human cancers more responsive to p53 activity and more sensitive to chemotherapeutic agents. I n many cancers, the p53 tumor suppressor gene is mutated, and the most frequent changes are missense point mutations (1) that cluster in the central region of the gene that encodes the DNA-binding domain of the p53 protein (2). p53 is a transcription factor (3, 4) that binds as a tetramer to specific DNA recognition sequences (5, 6) adjacent to p53-responsive genes that engender cell-cycle arrest (7) and apoptosis (8). The mutant p53s lose sequence-specific DNA-binding properties (2, 5, 6) and cannot regulate expression of those genes (3,4,7,8). Moreover, by binding to and forming inactive tetramers with wild-type (wt) p53, certain mutant versions of the p53 protein can transform cells neoplastically, presumably by inhibiting endogenous wt p53 function in a dominant-negative fashion (9, 10). Finally, certain mutant p53 proteins have acquired a ''gain-of-function,'' defined as the ability to augment cell proliferation in the absence of endogenous wt p53 (11) and resulting in phenotypic changes such as the overexpression of the multidrug resistance (MDR1) gene (11,12), which can confer cross-resistance to cytotoxic drugs (13).Several clinical trials that attempt to introduce expression cassettes for the wt p53 cDNA into cancer cells are underway to evaluate the safety and potential utility of this strategy to combat cancer (14). One significant limitation of this gene transfer approach is the inability to properly regulate gene expression after gene transfer. A second potential complication is that most p53 mutations found in human cancers are not null mutations but rather encode mutant versions of the p53 protein that may have unwanted activities such as a gain-offunction or be dominant negative inhibitors of wt p53 activity (11,15). Therefore to treat these types of...

MDR1 Genotype-Related Pharmacokinetics and Pharmacodynamics.

“…[124][125][126][127] In contrast, gene therapy to introduce wildtype p53 results in a reduction of MDR1 promoter expression. 128) In addition, various types of transcription factors have been suggested to be involved in MDR1 expression, such as EGR1, 129) NF-IL6 130) and NF-R2. 131) In rodents, effects of C/EBPb 132) and NF-kB 133) were also proposed.…”

5′ Exon replacement and repair by spliceosome-mediated RNA trans-splicing

“…The consensus sequence that defines the 5Ј donor site, AG/GURAGU, is less extensive than the 3Ј splice elements (Moore et al 1999), and it has remained uncertain whether this site could be efficiently exploited to effect trans-splicing repair. Group I ribozymes have been used to perform trans-splicing reactions in vitro to repair globin mRNA in sickle cell anemia-derived erythroid precursor cells (Lan et al 1998), and trans-splicing ribozymes have also been used successfully to induce changes in p53 and chloride channels (Watanabe and Sullenger 2000;Rogers et al 2002) by a 3Ј replacement process; to our knowledge, there is no published report demonstrating that ribozymes are capable of repair in a 5Ј manner. Additionally, in trypanosomes (Van der Ploeg 1986) and Caenorhabditis elegans (Krause and Hirsh 1987), it has been demonstrated that RNAs are commonly modified by the addition of a splice leader on the 5Ј terminus by transsplicing.…”

Transcriptional Repression by p53 through Direct Binding to a Novel DNA Element

“…By stably introducing a trans-dominant negative p53 into wildtype p53-expressing cells, Shuetz and co-workers (24) showed that down-regulation of wild-type p53 led to up-regulation of the endogenous MDR1 RNA and protein, consistent with the role of p53 in MDR1 repression. More recently, it has been shown that induction of wild-type p53 activity in human cancer cells using ribozymes that repair mutant p53 proteins results in reduced activity of the MDR1 promoter (25).…”