NOVEL DEOXYRIBONUCLEOSIDE KINASES FOR GENE THERAPY  

    Introduction

    Novel deoxyribonucleoside kinases

    Project progress: Generating monoclonal kinase expressing cell lines

    References   

   Introduction  

Deoxyribonucleoside kinases catalyse the phosphorylation of deoxyribonucleosides (dN) to the corresponding deoxyribonucleoside monophosphates (dNMP). They are the key enzymes in the salvage of deoxyribonucleosides originating extracellularly or from intracellular breakdown of DNA. Subsequently dNMPs are phosphorylated into diphosphates (dNMP) and triphosphates (dNTPs), which are direct precursors of DNA. In humans, the salvage pathway is extremely important for activation of several anti-viral, and anti-cancer drugs (Van Rompay et al., 2000; Fig. 1).

Gene therapy based on deoxyribonucleoside kinases is a method of therapeutic intervention to treat various cancers and combat viral infections and it also has applications in transplantation technology. The basis of this therapy is that a heterologous kinase gene is introduced into target cells where the gene will be expressed.  Often, the target cells are cancer cells but can also be cells used for transplantation purposes.  The introduced kinase can then specifically multiply the activation of  otherwise harmless pro-drugs, which in the activated form are toxic, and will lead to cell death (Greco and Dachs, 2001). Herpes simplex virus 1 thymidine kinase (HSV1-TK) in combination with ganciclovir has already been used as a suicide gene in clinical trials (Greco and Dachs, 2001; Rainov, 2000).  However, it remains important to search for new and improved suicide enzyme/prodrug systems to maximize therapeutic efficacy.  Other kinases may be even better regarding kinetic properties and substrate specificity.

   Novel deoxyribonucleoside kinases

Recently a new enzyme, a multisubstrate ultra fast deoxyribonucleoside kinase (Dm-dNK EC 2.7.1.145) has been isolated from the fruit fly (Munch-Petersen et al., 1998);); and its 3D structure determined (Johanssen et al., 2001) (see the front page). This kinase has a turnover of thymidine, which is 70-fold higher than the turnover of HSV1-TK. Furthermore DmdNK is able to convert a range of medically interesting analogs. Therefore, it has a good potential to be used in gene therapy.

Several Dm-dNK mutants, with even more promising properties, have been developed, either by using random mutagenesis and screening (Knecht et al., 2000) or point directed mutagenesis based on the 3D structure (Knecht et al, 2002b). The Eukaryote Molecular Biology Group at BioCentrum-DTU is now exploring these mutants for use in gene-directed enzyme pro-drug therapy. In short, they are expected to (i) require even lower doses of the analogues to kill the cell, and (ii) convert analogues, which can otherwise not be converted by the human kinases at all.

In addition, to the Dm-dNK kinase and its mutant variants, we have also cloned several other deoxyribonucleoside kinases from a variety of organisms.  Among these are kinases from an amphibian  (Xenopus laevis) and  the silk worm Bombyx mori (Knecht et al, 2002a), and  other eukaryotes and prokaryotes.  In vitro characterisation of these enzymes, their substrate specificity and kinetics, is now in progress.

   Project progress: Generating monoclonal kinase expressing cell lines

The wild type Dm-dNK has previously been transfected into mammalian cell lines and shown to sensitize the cells towards different analogs (Zheng et al, 2000). On the average these cells exhibited app. 20 – 40 –fold elevated sensitivity on the tested drugs. In order to do such testing, it is necessary to generate stable, growing cell lines constitutively expressing kinase since the toxicity of most nucleoside analogs is growth dependent.  In our study, several different deoxyribonucleoside kinases have been sub-cloned into a mammalian expression vector carrying and transfected into two human cancer cell lines (Fig. 2).  From the many different polyclonal cell lines generated, the tedious task of isolating stabile, monoclonal cell lines is now in progress.  As controls, clones are also isolated from cells transfected with empty expression vector.  In spite of the selection pressure, only approximately 10-20 % of the cells express kinase. This means that a number of clones must be isolated and grown for each cell line to ensure the identification and isolation of an adequate number of clones expressing high amounts of kinase. So far, several of the monoclonal cell lines show 100-fold higher deoxyribonucleoside kinase activity than the control cell lines.

   References

Greco, O., Dachs, G. U. (2001) Gene directed enzyme/prodrug therapy of cancer: Historical appraisal and future prospectives.  J. Cell. Physiol. 187, 22-36.

Johansson, K., Ramaswamy, S., Ljungcrantz, C., Knecht, W., Piskur, J., Munch-Petersen, B., Eriksson, S., Eklund, H. (2001) Structural basis for substrate specificities of cellular deoxyribonucleoside kinases. Nature Struct. Biol. 8, 616-620.

Knecht, W., Ebert Petersen, G., Munch-Petersen, B., Piskur, J. (2002a) Deoxyribonucleoside kinases belonging to the thymidine kinase 2 (TK2)-like group vary significantly in substrate specificity, kinetics and feed-back regulation. J. Mol. Biol. 315, 529-540.

Knecht, W., Munch-Petersen, B., and Piskur, J. (2000) Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster. J. Mol. Bio. 301, 827-837.

Knecht, W. , Sandrini, M.P.B., Johansson, K., Eklund, H., Munch-Petersen, B., Piskur, J. (2002b) A few amino acid substitutions can convert deoxyribonucleoside kinase specificity from pyrimidines to purines. EMBO J.  21 (in press).

Munch-Petersen, B., Piskur, J., and Sondergaard, L. (1998) Four deoxynucleoside kinase activities from Drosophila melanogaster are contained within a single monomeric enzyme, a new multifunctional deoxynucleoside kinase. J. Biol. Chem. 273, 3926-3931.

Rainov, N. G. (2000) A phase III clinical evaluation of Herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme. Hum. Gene Ther. 11, 2389-2401.

Van Rompay, An R., Johansson, M., Karlsson, A. (2000) Phosphorylation of nucleosides and nucleoside analogs by mammalian nucleoside monophosphate kinases.  Pharmacol. Ther. 87, 189-198.

Zheng, X., Johansson, M., Karlsson, A. (2000) Retroviral transduction of cancer cell lines with the gene encoding Drosophila melanogaster multisubstrate deoxyribonucleoside kinase. J. Biol. Chem. 275, 35125-39129.  

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