Abstract
BackgroundThymidine kinase 1 (TK1) is a salvage enzyme involved in DNA precursor synthesis, and its expression is proliferation dependent. A serum form of TK1 has been used as a biomarker in human medicine for many years and more recently to monitor canine lymphoma. Canine TK1 has not been cloned and studied. Therefore, dog and human TK1 cDNA were cloned and expressed, and the recombinant enzymes characterized. The serum and cellular forms of canine and human TK1 were studied by size-exclusion chromatography and the level of TK1 protein was determined using polyclonal and monoclonal anti-TK1 antibodies.ResultsCanine TK1 phosphorylated the thymidine (dThd) analog 3'-azido-thymidine (AZT) as efficiently as it did dThd, whereas AZT phosphorylation by human TK1 was less efficient than that of dThd. Dog TK1 was also more thermostable and pH tolerant than the human enzyme. Oligomeric forms were observed with both enzymes in addition to the tetrameric and dimeric forms. Cellular TK1 was predominantly seen in dimeric and tetrameric forms, in the case of both dog TK1 from MDCK cells and human TK1 from CEM cells. Active serum TK1 was found mainly in a high molecular weight form, and treatment with a reducing agent shifted the high molecular weight complex to lower molecular weight forms with reduced total activity. Western blot analysis demonstrated a polypeptide of 26 kDa (dog) and 25 kDa (human) for cellular and serum TK1. There was no direct correlation between serum TK1 activity and protein level. It appears that a substantial fraction of serum TK1 is not enzymatically active.ConclusionsThese results suggest that the serum TK1 protein differs from cellular or recombinant forms, is more active in high molecular weight complexes, and is sensitive to reducing agents. The results presented here provide important information for the future development and use of serum TK1 as a diagnostic biomarker in human and veterinary medicine.
Highlights
Thymidine kinase 1 (TK1) is a salvage enzyme involved in DNA precursor synthesis, and its expression is proliferation dependent
A form of TK1 is found at high levels in the sera of humans and animals with malignant tumors; serum TK1 activity has been used as a prognostic marker for several different tumor types, but primarily in leukemia and lymphoma [10,11,12]
The presence of DTE reduced the amount of the high molecular weight (MW) forms of recombinant TK1, and increased the dimer and tetramer forms, suggesting that disulfide bonds are involved. These results demonstrate a significant difference between canine and human recombinant TK1 regarding their sensitivity to reducing agents
Summary
Thymidine kinase 1 (TK1) is a salvage enzyme involved in DNA precursor synthesis, and its expression is proliferation dependent. The serum and cellular forms of canine and human TK1 were studied by size-exclusion chromatography and the level of TK1 protein was determined using polyclonal and monoclonal anti-TK1 antibodies. A form of TK1 is found at high levels in the sera of humans and animals with malignant tumors; serum TK1 activity has been used as a prognostic marker for several different tumor types, but primarily in leukemia and lymphoma [10,11,12]. A non-radiometric TK1 activity assay (the TK Liaison assay, DiaSorin Inc.) is used Both assays provide clinically valuable information in humans and dogs with leukemia and lymphoma, for monitoring therapy and predicting relapse [15]. The recent development of antibodies against human TK1 has enabled the determination of serum TK1 protein levels in different hematologic and solid tumors such as bladder carcinoma [16], breast carcinomas [17], and non-small cell lung cancer [18]
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