Molecular characterization of equine thymidine kinase 1 and preliminary evaluation of its suitability as a serum biomarker for equine lymphoma.
Abstract: Thymidine kinase 1 (TK1) plays a key role in the synthesis of deoxythymidine triphosphate (dTTP) and is thus important for DNA replication and cell proliferation. The expression of TK1 is highest during S-phase, and it is rapidly degraded after mitosis. In cancer cells, TK1 is upregulated, resulting in leakage of excess TK1 into the blood. Consequently, serum TK1 has been used as a diagnostic and prognostic cancer biomarker, mainly in human medicine. The aims of this work were to characterize equine TK1 and to evaluate its suitability as a serum biomarker for equine lymphoma. Results: Equine TK1 was cloned, expressed in E. coli and affinity purified. The purified recombinant horse TK1 showed broad substrate specificity, phosphorylating pyrimidine deoxyribo- and ribonucleosides and, to some extent, purine deoxynucleosides, including anticancer and antiviral nucleoside analogues. ATP was the preferred phosphate donor. Serum TK1 activity was measured in samples collected from horses with confirmed or suspected lymphoma and control horses with and without concurrent diseases. Serum TK1 activity levels were significantly higher in horses with lymphoma (p < 0.0005) and suspected lymphoma (p < 0.02) and in tumour-free groups with diverse diseases (p < 0.03) than in controls without concurrent diseases. There was a significant difference between the lymphoma group and the tumour-free group with diverse diseases (p < 0.0006). Furthermore, receiver operating characteristic analysis revealed a sensitivity of 0.86, a specificity of 0.95 and an AUC (area under the curve) of 0.92 compared to the controls without concurrent diseases, with a sensitivity of 0.97, a specificity of 0.71 and an AUC of 0.88 when compared with the tumour-free group with diverse diseases. Conclusions: Equine TK1 showed high specific activity and broader substrate specificity than human TK1. Anticancer and antiviral thymidine analogues were efficiently phosphorylated by horse TK1, suggesting that these analogues might be good candidates for chemotherapy in horses. Serum TK1 activity was significantly higher in horses with lymphoma than in controls. ROC analysis indicated that serum TK1 could serve as a promising cancer biomarker in horses.
© 2021. The Author(s).
Publication Date: 2021-12-14 PubMed ID: 34906077PubMed Central: PMC8670147DOI: 10.1186/s12860-021-00399-xGoogle Scholar: Lookup
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Summary
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The research article presents the molecular study of Thymidine kinase 1 (TK1) in horses, and investigates its viability as a serum biomarker for equine lymphoma. The study concludes the potential benefits of using TK1 as a biomarker for cancer in horses due to its higher activity levels in horses with lymphoma.
Characterization of Equine TK1
- The research began with an examination of equine (horse) TK1 at the molecular level. TK1 plays a crucial role in DNA replication and cell growth by facilitating the synthesis of deoxythymidine triphosphate (dTTP). Its expression is notably high during the S-phase of the cell cycle and dramatically decreases after mitosis.
- In cancer cells, however, TK1 levels are abnormally high, leading to leakage into the bloodstream. This characteristic makes it a potential biomarker for cancer, and prior research mostly involves human medicine.
Cloning, Expression, and Purification
- The researchers cloned the equine TK1 gene and expressed it in the E. coli bacteria for practical study. They then purified the expressed equine TK1 using an affinity-based method.
Evaluation of TK1 Specificity
- The purified TK1 was tested for its substrate specificity. It showed a wide range of action, being able to phosphorylate not only pyrimidine deoxyribo- and ribonucleosides, but also to some extent, purine deoxynucleosides, including anticancer and antiviral nucleoside analogues. ATP was the preferred phosphate donor.
TK1 as a Biomarker
- Next, the researchers measured serum TK1 activity levels in horses with diagnosed or suspected lymphoma, as well as control horses with and without other diseases.
- They observed that horses with lymphoma and suspected lymphoma have significantly higher TK1 levels in their blood compared to healthy controls and horses with other diseases – more so than in humans.
- Furthermore, TK1 emerged as a potential efficient marker of lymphoma, with high sensitivity, specificity, and area under the Receiver Operating Characteristic (ROC) curve, making it a strong diagnostic tool.
Conclusions and Implications
- This study confirmed that equine TK1 is highly specific and has broader substrate specificity than human TK1. The nucleoside analogues that equine TK1 can phosphorylate include anticancer and antiviral compounds, indicating that these could be good chemotherapy candidates for treating cancer in horses.
- The research concludes the potential benefits of using TK1 as a biomarker for cancer in horses due to its higher activity levels in horses with lymphoma.
Cite This Article
APA
Wang L, Unger L, Sharif H, Eriksson S, Gerber V, Rönnberg H.
(2021).
Molecular characterization of equine thymidine kinase 1 and preliminary evaluation of its suitability as a serum biomarker for equine lymphoma.
BMC Mol Cell Biol, 22(1), 59.
https://doi.org/10.1186/s12860-021-00399-x Publication
Researcher Affiliations
- Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden. liya.wang@slu.se.
- Swiss Institute of Equine Medicine (ISME), Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, and Agroscope, Bern, Switzerland.
- Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
- Alertix Veterinary Diagnostic AB, SE-392 30, Kalmar, Sweden.
- Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
- Swiss Institute of Equine Medicine (ISME), Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, and Agroscope, Bern, Switzerland.
- Department of Clinical Science, Swedish University of Agricultural Sciences, Uppsala, Sweden.
MeSH Terms
- Animals
- Biomarkers, Tumor / genetics
- Horse Diseases / diagnosis
- Horses
- Lymphoma / diagnosis
- Lymphoma / veterinary
- Thymidine Kinase / genetics
Conflict of Interest Statement
HS is employed by Alertix Veterinary Diagnostic AB; SE is a consultant and cofounder, and HR is member of the scientific advisory board. SE, HR, HS, and LW own shares of the same company. The other authors declare no conflicts of interest.
References
This article includes 34 references
- Knowles E, Tremaine W, Pearson G, Mair T. A database survey of equine tumours in the United Kingdom.. Equine Vet J 2016;48(3):280–284.
- Taintor J, Schleis S. Equine lymphoma.. Equine Vet Educ 2011;23(4):205–213.
- Taylor S, Pusterla N, Vaughan B, Whitcomb M, Wilson W. Intestinal neoplasia in horses.. J Vet Intern Med 2006;20(6):1429–1436.
- Durham A, Pillitteri C, Myint M, Valli V. Two hundred three cases of equine lymphoma classified according to the world health organization (WHO) classification criteria.. Vet Pathol 2012;50:86–93.
- Miller C, Durham A, Schaeffer P, Ehrhart E, Powers B, Duncan C. Classification and clinical features in 88 cases of equine cutaneous lymphoma.. J Vet Diagn Invest 2015;27(1):86–91.
- Robertson J. Dealing with neoplasia in elderly horses.. Equine Vet Edu 2010;22(4):163–164.
- Gasparri F, Wang N, Skog S, Galvani A, Eriksson S. Thymidine kinase 1 expression defines an activated G1 state of the cell cycle as revealed with site-specific antibodies and ArrayScan assays.. Eur J Cell Biol 2009;88(12):779–785.
- Sun R, Eriksson S, Wang L. Mitochondrial thymidine kinase 2 but not deoxyguanosine kinase is up-regulated during stationary growth phase of the cultured cells.. Nucleosides Nucleotides Nucleic Acids 2014;33(4-6):282–286.
- Ke PY, Chang ZF. Mitotic degradation of human thymidine kinase 1 is dependent on the anaphase-promoting complex/cyclosome-CDH1-mediated pathway.. Mol Cell Biol 2004;24(2):514–526.
- Chen Z, Huang S, Wang Y, Yang A, Wen J, Xu X, Chen Y, Chen Q, Wang Y, He E, Zhou J, Skog S. Serological thymidine kinase 1 is a biomarker for early detection of tumours - a health screening study on 35, 365 people using a sensitive chemiluminescent dot blot assay.. Sensors (Basel) 2011;11(12):11064–11080.
- Zhou J, He E, Skog S. The proliferation marker thymidine kinase 1 in clinical use.. Mol Clin Oncol 2013;1(1):18–28.
- von Euler H, Einarsson R, Olsson U, Lagerstedt A-S, Eriksson S. Serum thymidine kinase activity in dogs with malignant lymphoma: a potent marker for prognosis and monitoring the disease.. J Vet Intern Med 2004;18(5):696–702.
- von Euler H, Eriksson S. Comparative aspects of the proliferation marker thymidine kinase 1 in human and canine tumour diseases.. Vet Comp Oncol 2011;9(1):1–15.
- Selting K, Sharp C, Ringhold R, Knouse J. Serum thymidine kinase 1 and C-reactive protein as biomarkers for screening clinically healthy dogs for occult disease.. Vet Comp Oncol 2015;13(4):373–384.
- Larsdotter S, Nostell K, von Euler H. Serum thymidine kinase activity in clinically healthy and diseased horses: a potential marker for lymphoma.. Vet J 2015;205(2):313–316.
- Théon A. Intralesional and topical chemotherapy and immunotherapy.. Vet Clin North Am Equine Pract 1998;14(3):659–671.
- Luethy D, Frimberger A, Bedenice D, Byrne B, Groover E, Gardner R, Lewis T, MacDonald V, Proctor-Brown L, Tomlinson J, Rassnick K, Johnson A. Retrospective evaluation of clinical outcome after chemotherapy for lymphoma in 15 equids (1991-2017). J Vet Intern Med 2018;33(2):953–960.
- Vissani M, Thiry E, Dal Pozzo F, Barrandeguy M. Antiviral agents against equid alphaherpesviruses: current status and perspectives.. Vet J 2016;207:38–44.
- Welin M, Kosinska U, Mikkelsen N, Carnrot C, Zhu C, Wang L, Eriksson S, Munch-Petersen B, Eklund H. Structures of thymidine kinase 1 of human and mycoplasmic origin.. Proc Natl Acad Sci U S A 2004;101(52):17970–17975.
- Sharif H, Jagarlamudi K, Wang L, He E, Eriksson S. Quaternary structures of recombinant, cellular, and serum forms of thymidine kinase 1 from dogs and humans.. BMC Biochem 2012;13:12.
- Munch-Petersen B, Cloos L, Tyrsted G, Eriksson S. Diverging substrate specificity of pure human thymidine kinases 1 and 2 against antiviral dideoxynucleosides.. J Biol Chem 1991;266(14):9032–9038.
- Munch-Petersen B, Tyrsted G, Cloos L. Reversible ATP-dependent transition between two forms of human cytosolic thymidine kinase with different enzymatic properties.. J Biol Chem 1993;268(21):15621–15625.
- Sharif H, von Euler H, Westberg S, He E, Wang L, Eriksson S. A sensitive and kinetically defined radiochemical assay for canine and human serum thymidine kinase 1 (TK1) to monitor canine malignant lymphoma.. Vet J 2012;194(1):40–47.
- Wang J, Choudhury D, Chattopadhyaya J, Eriksson S. Stereoisomeric selectivity of human deoxyribonucleoside kinases.. Biochemistry 1999;38(51):16993–16999.
- Vander Werf K, Davis E, Janardhan K, Bawa B, Bolin S, Almes K. Identification of equine herpesvirus 5 in horses with lymphoma.. J Equine Vet Sci 2014;34(6):738–742.
- Saulez M, Schlipf J, Jr, Cebra C, McDonough S, Bird K. Use of chemotherapy for treatment of a mixed-cell thoracic lymphoma in a horse.. J Am Vet Med Assoc 2004;224(5):733–738.
- Vander Werf K, Davis E. Disease remission in a horse with EHV-5-associated lymphoma.. J Vet Intern Med 2013;27(2):387–389.
- Schulte P. The use of biomarkers in surveillance, medical screening, and intervention.. Mutat Res 2011;592:155–163.
- Dang L, Ma H, Hei A, Xu S, Zhou J, He E, Skog S. A meta-analysis of serological thymidine kinase 1 as a marker for colorectal benign and malignant tumor risk assessment.. Mol Clin Oncol 2020;12(5):440–450.
- Kiran Kumar J, Sharif H, Westberg S, von Euler H, Eriksson S. High levels of inactive thymidine kinase 1 polypeptide detected in sera from dogs with solid tumours by immunoaffinity method: implications for in vitro diagnostics.. Vet J 2013;197(3):854–860.
- Wang L, Eriksson S. Cloning and characterization of full length mouse thymidine kinase 2: the N-terminal sequence directs import of the precursor protein into mitochondria.. Biochem J 2000;351(2):469–476.
- Schelling P, Folkers G, Scapozza L. A spectrophotometric assay for quantitative determination of Kcat of herpes simplex virus type 1 thymidine kinase substrates.. Anal Biochem 2001;295(1):82–87.
- Carnrot C, Wang L, Topalis D, Eriksson S. Mechanisms of substrate selectivity for bacillus anthracis thymidylate kinase.. Protein Sci 2008;17(9):1486–1493.
- Christen G, Precht C, van der Kolk J, Fouché N, Gerber V. Age over 25 years, but not plasma adrenocorticotropic hormone concentration above the seasonally adjusted reference range is predictive for eadiographically assessed changes of chronic laminitis in elderly horses.. Schweiz Arch Tierheilkd 2020;162(12):781–685.
Citations
This article has been cited 2 times.- Ostendarp C, Barton AK. Intraocular Tumors in Horses: Diagnosis, Tumor Classification, Oncologic Assessment and Therapy. Vet Sci 2025 Oct 17;12(10).
- Drozdzewska K, Gehlen H. Markers for internal neoplasia in the horse. Vet Med Sci 2023 Jan;9(1):132-143.
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