Regulation of ongoing DNA synthesis in normal and neoplastic brain tissue

The treatment of human brain tumour is challenging in part due to the blood brain barrier and in part due to the specific biology of brain tumours that confer resistance to chemotherapy. For instance, the 5 years survival rate for patients carrying intracranial glioblastoma multiforme has remained at 4-5 % for the last 30 years. The knowledge of the brain tumour biology as well as the biology of the normal brain tissue would help to design new therapeutic strategies and to develop new and less toxic antineoplastic drugs for brain tumour treatment. Normal tissue must be studied in order to identify tumour-specific vulnerabilities and ways to inhibit toxicity in the host.

The present thesis describes a series of investigations of potential antineoplastic drugs performed in normal rat cerebral cortex, human brain tumour specimens and RG2 gliomas, performed "in vitro" in order to 1) better understand factors controlling the cell cycle and DNA replication in normal and neoplastic brain tissue, and thus, exploiting potential targets for new drugs 2) better apply the available antineoplastic drugs for the treatment of human brain tumours while producing no or low side effect on normal tissue.

A novel assay, which preserves the metabolic and proliferative properties of the tissue was developed and used to study ongoing DNA synthesis and its regulation by protein phosphorylation and proteolysis. The effect of low MW drugs (protein kinase and protease inhibitors) on these processes was evaluated, By analyzing the effects of different chemically unrelated inhibitors of protein kinases we found that many of these inhibitors might act through long term mechanism of action (e.g. inhibiting cell cycle transitions) rather than a direct effect on the DNA replication machinery, although some of these drugs are currently used as "DNA synthesis inhibitors". We suggest that, from the clinical point of view, it would be important to distinguish between these long and short-term mechanism of action. Our results also suggest that different sets of protein kinases and proteases yet not clearly identified regulate "ongoing DNA replication".

A more detailed study was carried out using roscovitine, a highly specific cyclin-dependent kinase inhibitor. The effect of roscovitine on DNA synthesis was evaluated in normal rat cerebral cortex, specimens obtained from human brain tumours and in a pilot experiment using a rat glioma model. We found that roscovitine is a potent inhibitor of ongoing DNA synthesis in the developing rat cerebral cortex as well as in human gliomas but showed little or no effect in adult normal tissue. Moreover, roscovitine inhibited preferentially DNA synthesis connected with replicative processes rather than DNA synthesis connected with DNA repair. In addition, some in vitro studies of redox regulation of topoisomerases and the effects of thiol reacting drugs on this enzyme are presented.

List of scientific papers

I. Yakisich JS, Siden A, Idoyaga Vargas V, Eneroth P, Cruz M (1998). Fast and sensitive method for simultaneous measurement of cell proliferation rate and drug sensitivity in rat cerebral cortex. Exp Neurol. 151(2): 194-202.
https://pubmed.ncbi.nlm.nih.gov/9628754

II. Yakisich JS, Siden A, Idoyaga Vargas V, Eneroth P, Cruz M (1998). Early inhibition of DNA synthesis in the developing rat cerebral cortex by the purine analogues olomoucine and roscovitine. Biochem Biophys Res Commun. 243(3): 674-7.
https://pubmed.ncbi.nlm.nih.gov/9500988

III. Yakisich JS, Siden A, Vargas VI, Eneroth P, Cruz M (1999). Early effects of protein kinase modulators on DNA synthesis in rat cerebral cortex. Exp Neurol. 159(1): 164-76.
https://pubmed.ncbi.nlm.nih.gov/10486185

IV. Yakisich JS, Boethius J, Lindblom IO, Wallstedt L, Vargas VI, Siden A, Cruz MH (1999). Inhibition of DNA synthesis in human gliomas by roscovitine. Neuroreport. 10(12): 2563-7.
https://pubmed.ncbi.nlm.nih.gov/10574370

V. Yakisich JS, Siden A, Eneroth P, Cruz M (2001). Disulfiram is a potent in vitro inhibitor of DNA topoisomerases. Biochem Biophys Res Commun. 289(2): 586-90.
https://pubmed.ncbi.nlm.nih.gov/11716515

VI. Yakisich JS, Siden A, Boethius J, Tasat DR, Hofer A, Idoyaga Vargas V, Eneroth P, Cruz M (2005). Preferential inhibition of replicative DNA synthesis in the developing rat cerebral cortex by the purine analogue Roscovitine. [Manuscript]