Diagnostic implications of the molecular forms and levels of serum thymidine kinase 1 in different canine malignancies

摘要

Thymidine kinase 1 (ATP: thymidine 5’- phosphotransferase, EC 2.1.7. 21, TK1) is a cellular enzyme involved in salvage pathway for DNA precursor synthesis. TK1 activity fluctuates during cell cycle, it reaches peak in S phase and absent in M phase. Because of its tight regulation with cell cycle, TK1 has been used as proliferation marker for diagnosis and treatment monitoring of various malignancies in human and veterinary medicine. TK1 levels can be measured by activity based or antibody based assays.udud The main aim of the research described in this thesis was to develop TK1 antibody based assays for determining serum TK1 protein levels in comparison with TK1 activity from dogs with different malignancies. Further analysis revealed a significant difference in the molecular forms of TK1 in sera from canine leukemia and mammary tumours. udud In Study I, serum TK1 protein levels in dogs with uddifferent solid tumours were determined by using an antibody based assay i.e. immunoaffinity assay. TK1 protein levels were significantly higher in dogs with solid tumours than expected from the TK1 activity values. In contrast, the specific activity of TK1 in sera from healthy dogs was 2.5 fold higher than that of solid tumours. The molecular forms of recombinant, cellular and serum TK1 were investigated in study II. Dog recombinant and serum TK1 existed as oligomers with a molecular weight (MW) of 720-300 kDa. Cellular TK1 from both dogs and humans were mainly as tetramers. Human recombinant and serum TK1 activity eluted in two peaks: one at high and one at low MW, corresponding to 720-300 kDa and 200-50 kDa, respectively. udud In study III, TK1 protein levels in sera from dogs with mammary tumours were determined by immunoaffinity assay. The TK1 protein assay differentiated mammary adenomas efficiently from healthy dogs, and adenomas from carcinomas, but this was not possible with the TK1 activity assay. In mammary tumour sera, active TK1 eluted as high MW oligomer similar to leukemia however, TK1 protein was detected not only as high MW form but also in the fractions where no TK1 activity was found. This indicates that serum TK1 exits in multiple forms in mammary tumours with a large fraction of inactive TK1 protein. Study IV describes the development of TK1-ELISA, based on a polyclonal and a monoclonal anti TK1 peptide antibodies, that was used to measure TK1 protein levels in dog sera. TK1 protein levels were significantly higher in sera from dogs with haematological tumours as well as solid tumours in comparison with healthy dogs. Overall, the results demonstrate that TK1 protein assays provide valuable diagnostic information in a variety of canine malignancies.