An Investigation of Enzyme/Prodrug Systems for Cancer Gene Therapy

Denning, Chris N (1997) An Investigation of Enzyme/Prodrug Systems for Cancer Gene Therapy. PhD thesis, University of Glasgow.

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Chemotherapy, radiotherapy and surgery, the current treatments for cancer, cure only fifty percent of patients. Gene therapy, where therapeutic genes are transferred directly to the tumour, offers a new form of cancer treatment. However, gene transfer techniques that are currently available can only achieve gene expression in a small proportion of the tumour cells, and this may hinder many gene therapy approaches. It may therefore be important to select genes for cancer therapy that offer the potential for a significant bystander effect, where cells not expressing the therapeutic gene are killed, as well as targeted cells. At present there appear to be two strategies for cancer gene therapy that achieve bystander killing, and hence require targeted expression in only a proportion of the cells. The first depends on stimulation of the immune system by, for example, expression of specific cytokines that increase proliferation of tumoricidal cells, while the second type of bystander effect is associated with enzyme / prodrug systems, which are the focus of the work described in this thesis. The mechanisms and efficacies of the Herpes Simplex virus thymidine kinase / ganciclovir (tk / GCV), human thymidine phosphorylase / 5'-deoxy-5-fluorouridine (tp / DFUR) and E.coli cytosine deaminase / 5-fluorocytosine (cd / FC) enzyme / prodrug systems have been examined. In culture, each enzyme / prodrug combination induced cell death in tumour cells expressing the suicide gene and, in mixed cultures of cells that do or do not express the gene, death occurred in both cell types, indicative of a bystander effect. With the tk / GCV system, in human and rodent cell lines, a correlation has been demonstrated between the bystander effect and gap junctional intercellular communication (GJIC). When cultures lack GJIC, no bystander effect was observed, but only a low level of communication was required to generate extensive bystander killing. Increased enzyme activity, on the other hand, improved the bystander effect, suggesting that high levels of tk expression will be important. Additionally, it has been shown that transfer of toxic metabolites from tk+ to tk- cells occurs within two hr of GCV application, before degenerative events were detected, indicating that apoptosis is the result, not the cause, of the tk / GCV bystander effect. Three of the cell lines used to investigate the tk / GCV system in culture were also used to examine the bystander effect in experimental tumours in nude mice. A correlation between the bystander effect in culture and in vivo was demonstrated in two of the cell lines but not the third, which suggests that other factors are important in vivo. In the tp / DFUR and cd / FC systems, bystander killing was due to transfer of toxic metabolites through the medium, and did not depend on GJIC or cell-cell contact. Bystander killing seen with these systems was weaker than that seen in the tk / GCV system. Combining the tk / GCV system with the tp / DFUR or cd / FC systems in culture was able to produce more extensive bystander killing than either system separately. The improvement was also observed using a cdtk fusion protein. The results in this thesis suggest that the choice of enzyme / prodrug therapy for cancer will be determined by the characteristics of each tumour. In addition they suggest the use of fusion proteins, to combine therapeutic proteins, warrants further investigation.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: John Pitts
Keywords: Oncology, Pharmacology
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-75855
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Dec 2019 09:15
Last Modified: 19 Dec 2019 09:15

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