Background

As the number of those inflicted with cancer grows each day, the search for a more effective and less intrusive treatment becomes more intense. With the discovery of the correlation between the p53 gene and cancer, gene therapy might offer new alternatives. Successful gene therapy consists of two key factors: the identification of the critical cellular gene involved with the mutant or missing biological functions; and, an effective delivery method of the normal (wild- type) gene to the target genome of an individual carrying defective copies of the gene (i.e. transvection). If the gene therapy is successful, the transgene (i.e. transferred gene) will synthesize the missing gene product and restore normal phenotype (Stustad, 1997).

Research indicates that the loss of the molecular wild- type function of p53 might be involved in at least half of human cancers (Merke et al, 1998). Consequently, the recent focus of research is to identify an effective means of transvection and to explore its capabilities or consequences. The majority of p53 gene transfer experiments utilize an adenoviral vector. The reported advantages of using a recombinant adenoviral vectors include: they have broad host range, can be generated at high titer, are relatively harmless to humans as a pathogen, are efficient transferance into non-diving or dividing, and have high efficiency with relatively long-term expression (Kock 1996).

Adenoviruses were first identified in 1953 as the causative agents of the common cold. In 1977, Frank Graham developed a cell line which enabled the first production of recombinant adenoviruses as gene transfer agents that required no additional assistance. Adenoviral virions (i.e. a complete, infective viral particle consisting of genetic material surrounded by a protein shell or capsid) are icosahedral in shape, 70-90nm in diameter, and are not enveloped. The majority of the viral capsid consists of the three proteins: penton based (involved in cell internalization and receptor binding), fiber, and hexon. The viral genome consists of a single double stranded DNA molecule 36-38 kilobases long. All recombinant adenoviral vectors have deletion of the E1A region which is the first gene transcribed upon host cell nuclear entry and essential for viral replication. Termed "replication-deficient," these vectors can infect a cell only once and with no viral propagation. As a result, the infected cell does not die. The E3 region, which might be involved in viral pathogenesis, is also removed from the viral genome to allow for incorporation of larger DNA inserts (Fritz, 1996).

The primary goal of wild- type p53 gene transfer is to suppress cancerous tumor growth. Researches speculate that wild- type p53 gene transfer via a recombinant DNA adenovirus will induce cell apoptosis and/or cell arrest which have been disrupted by missing or mutant p53 gene. In normal cells undergoing apoptosis (programmed cell death) separation from neighboring cells occurs as cell membranes become crossed linked. Endonucleases cleave cellular DNA into 200 bp fragments giving rise to a characteristic "apoptotic DNA ladder." These cells are rapidly phagacytosed by adjacent cells or macrophages. The p53 gene affects other genes such as activation of proapoptotic genes and transcriptional repression of antiapoptotic genes. p53 is also involved in the regulation of cell cycle and proliferation affecting activities of genes that induce during growth arrest (prior to progression into S phase), and DNA repair (McKenna, 1999), (Nielsen,1998).

While the goal of p53 gene transfer is clear, current experimental data provides conflicting results that show both promising and inconclusive results. Researchers are conducting experiments using a variety of malignant cell lines (e.g. glioblastoma, soft tissue sarcoma, hepatocellular carcinoma, nasopharengeal carcinoma, breast and ovarian cancers, and others) as host cell for the recombinant adenoviral transfer. Both in vitro and in vivo experimental data have been collected. Experimental administration technique varies including intratumoral, intraperitoneal, and intrahepatic arterial. Researchers are also considering the relationship between enhanced treatment prognosis and the combination of gene transfer in conjunction with irradiation therapy. As the repertoire of p53 gene knowledge grows, it brings forth other points of interest such as using the amount of mutant p53 product accumulation or the level of mutant p53 antibodies in the blood as a prediction of prognosis and/or potential resistance to irradiation or chemotherapy. While results among experiments vary concerning the effectiveness of each potential treatment, all conclude that while much research remains to be done, p53 therapy may offer important new options.

A particularly promising investigation conducted by Katsuyuki Hamada (1996) suggests that wild-type p53 gene transfer is potentially novel approach in treating cervical cancer. Cervical cancer, the second most common malignancy in women in the US, account for 15% of all cancers diagnosed in women and have an overall 5 year survival rate of only 40%. The most important risk factor for cervical cancer is HPV (human papillomavirus) present in 90% of individuals inflicted. Continued expression of E6 and E7 protein products of HPV genome appears necessary for maintaining malignant phenotype. These protein products promote degradation of p53 via the ubiquitin-dependent protease system. This selective degradation of negative regulatory proteins then provides a mechanism for dominant acting oncoproteins.

The in vitro portion of the experiment (Hamada,1996) used eight cervical cancer cell lines. They used recombinant p53 adenoviral vector Ad5CMV-p53 containing a cytomegalovirus promotor, wild--type p53 cDNA, and an SV40 polyadenylation signal position into the E1-deleted region of a modified adenovirus Ad5. The control, Ad5CMV-poly5 did not contain the wild- type gene. Research obtained data using immunohistochemical analysis (p53 antibody count via staining technique), Tunnel assay (apoptotic cell detection via staining technique), Western Blot analysis (relative p53 protein density), cell count assay (cell viability), and [³H] thymidine incorporation assay (cell viability). The results are summarized below:

· All cell lines showed high transduction efficiencies.

· The most promising results were demonstrated in the SiHa cell line.

· Data showed increased levels of p53 antibodies, apoptotic activity, growth inhibition, and wild-type protein count that peaked at 3 days and continued for 15 days.

The first in vivo experiment (Hamada 1998) involved p53 null female mice with s.c. tumor modules induced by injection at Day 0 of the eight cancerous cell lines. Measures of treatment effectiveness included turmoriginicty assay (tumor volume) and inhibition of tumor growth. Those mice in the experimental group receiving the AdSCMV-p53 virus also on Day 0 never formed tumors while those in the control group, that received Ad5CMV-poly5, did. In the second part of the in vivo experiment, mice had pre-existing tumors, (i.e. induced tumors were allowed to grow for 20-25 days to diameter of 5-6mm.) Three trials were performed to examine for effects of treatment protocol: 1) AdSCMV-p53 administered at Day 0; 2) At Day 0,2,4; and 3)At Day 0,1,2,3,4,5. Results shown Appendix II display the dramatically increasing effectiveness of multiple administration. The researchers concluded that their results seem to provide a sound basis for future clinical trials of gene therapy to improve prognosis of cervical cancer.

Since current anticancer treatment can be rather toxic to those inflicted and have varying degrees of efficacy, gene therapy success is an attractive approach. Researchers are experimenting with combining p53 gene transfer with conventional approaches to explore the possible synergistic effect of combining p53 gene transfer with irradiation and/or chemotherapy. Synergy between two agent is an in vitro empirical phenomenon in which the observed effects of the combination is more than would be predicted from the effects of each agent working alone. Although synergy is not always quantitatively provable in clinical settings, in vitro experiments do suggest a favorable outcome of using two therapeutic in vivo (Nielson et, 1998).

Several experiments have demonstrated synergy between p53 gene transfer and chemotherapy (Ogawa 1997) (see Appendix III), however many had conflicting results. The majority of research agrees that there is usually a correlation between poor patient prognosis and p53 dysfunction (Gjerset 1995). Research also universally indicates that apoptosis caused by DNA damage induced with chemotherapy or irradiation is enhanced in cells expressing wild--type p53 and resisted in mutant p53 expressing cells as compared to p53 non-producing cells (Sutphin et al, 1998). A problem incurs concerning whether wild-type p53 transfer will produce continuous and dominant expression of protein product. Additionally, research demonstrates that mutant p53 accumulation resulting from an increased half-life of mutant p53 protein can actually act as a dominant negative effect or even a gain-of-function (i.e. preventing apoptosis and promoting entry into mitotic division), disrupting the ability of transferred wild--type gene to produce protein product. These affects are presumed to be a consequence of multiple protein-protein interactions (Weller, 1997).

Some researchers are utilizing this new experimental information to explore the correlation between the mutant p53 accumulation and possibilities for predicting degree of prognosis. By evaluating levels of p53 antibodies in the blood or protein count in the tumors, some experimental data indicates a correlation between tumor suppression and lowered concentrations. Monitoring these levels during remission may allow for treatment prior to clinical detection and relapse (Zalcman, 1998).