Therapeutic Approaches Related To p53 STATUS

As testified by the extensive bibliography, p53 is used as a tool for better diagnosis and prognosis, and also for development of new cancer therapies (Ref. [1]; for review, see Ref. [7]). There are several approaches for restoring normal p53 function, depending on the status of the gene. If the p53 gene is mutated, the most direct approach is gene therapy, in which normal p53 gene is introduced back into tumor cells using mostly replication-defective adenoviruses that deliver a human p53 cDNA sequence driven by strong viral promoters. 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 of function. However, several studies are exploring new vectors that have modified tissue and cellular tropism.

Another example of targeting cancer cells containing a high level of mutant p53 protein or without p53 uses an adenovirus hybrid, called ONYX-015, engineered to kill cells with mutant p53, but not wild-type p53. Briefly, the human adenoviruses infect quiescent cells and induce them into the S-phase of the cell cycle so that viral DNA replication can proceed. The E1A protein of human adenoviruses, which binds pRB (retinoblastoma oncosup-pressor gene) and other related proteins, is largely responsible for this entry into the S-phase. The E1B adenovirus gene encodes a 55-kDa protein that binds and inactivates the cellular p53 protein. In the ONYX-015 mutant adenovirus, the E1B gene product has been inactivated by mutation so that the virus cannot replicate in normal cells with a functional p53 protein. It has been reported that injection of this mutated virus into human p53-deficient solid tumors caused a significant regression of the tumor.

An alternative tumor targeting strategy exploits the loss of wild-type p53 function with regard to its dual role in the transcriptional regulation of gene expression. In fact, p53 can positively regulate the expression of target genes involved in cell growth inhibition or induction of apoptosis, but it is also able to suppress the transcription of other genes having a canonical promoter. Compelling evidence has indicated that such repressing activity is an important component of the tumor-suppressor function of p53. This gene therapy maximizes the expression of a potential therapeutic gene in tumors while simultaneously downregulates the same gene in normal cells. This system should be able to incorporate a number of different therapeutic genes, including prodrug-activating genes and immunomodulators. This approach makes use of two constructs. In the first construct, the potential therapeutic gene is placed under the control of the human heat shock protein 70 (Hsp70) gene promoter, which is upregulated in tumor cells in a more advanced stage and with poor prognosis. In addition, this promoter is activated by several p53 mutants and is repressed by wild-type p53. In this way, the therapeutic gene is selectively expressed only in tumoral cells with a mutated p53. In normal cells, residual expression of the therapeutic gene is repressed by using the second construct that drives a transcriptional repressor, or an antisense of the therapeutic gene under the control of a promoter activated by wild-type p53.

Other ambitious strategies aim to convert mutant p53 protein into its wild-type form by using small peptides, or by introducing drugs disrupting the interaction between MDM2 protein and p53.

The study of the allosteric and structural classes of mutant p53 protein has permitted researches to obtain the design of small peptides, which promote the stability not only of the wild-type p53, but also of mutant p53 to maintain an active conformation. Different classes of mutants require different rescue strategies, and before using activating drugs, it is crucial to define the type of p53 mutation.

Another attractive area of therapeutic development is the discovery of molecules that mimic the function of gene products whose synthesis is induced by p53. In particular, attention of researchers is focused on two principal genes: the cyclin-dependent kinase (CDK) inhibitor p21WAF1 and the proapototic gene bax.

If p53 is wild type in a tumor and p53 pathway is ablated, therapeutic strategies are addressed to the activation of endogenous p53 gene. Many therapeutic strategies have focused on MDM2, the principal negative regulator of the p53 pathway. By making use of MDM2 antisense, it is possible to inhibit MDM2 expression and to activate the p53 apoptotic response in tumor. Moreover, natural peptides have been found to bind MDM2 20 times more efficiently than p53, thus preventing the interaction between MDM2 and p53. In addition, several general inhibitors of transcription and the CDK inhibitor, roscovitine, are potent activators of p53-dependent transcription when delivered at moderate doses by selectively decreasing the expression of MDM2. Recently, it has been found that another small molecule, leptomycin

B, which inhibits the CMR1 nuclear exportin, is a potent activator of the p53 response. It can kill neuroblastoma cells in a p53-dependent manner while inducing only a reversible growth arrest in nontransformed cells.

This work has been supported by grants from the Italian Association for Cancer Research (AIRC), MIUR Italy (project Cluster C03-Ingegneria Molecolare, legge 488/92); MIUR Italy (project DD 9 ottobre 2002 prot. N. 1105).

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