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BMC molecular and cell biology2021; 22(1); 59; doi: 10.1186/s12860-021-00399-x

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).
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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

BMC molecular and cell biology
ISSN: 2661-8850
NlmUniqueID: 101741148
Country: England
Language: English
Volume: 22
Issue: 1
Pages: 59

Researcher Affiliations

Wang, Liya
  • Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden. liya.wang@slu.se.
Unger, Lucia
  • Swiss Institute of Equine Medicine (ISME), Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, and Agroscope, Bern, Switzerland.
Sharif, Hanan
  • Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
  • Alertix Veterinary Diagnostic AB, SE-392 30, Kalmar, Sweden.
Eriksson, Staffan
  • Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Gerber, Vinzenz
  • Swiss Institute of Equine Medicine (ISME), Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, and Agroscope, Bern, Switzerland.
Rönnberg, Henrik
  • 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.

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Citations

This article has been cited 2 times.
  1. Ostendarp C, Barton AK. Intraocular Tumors in Horses: Diagnosis, Tumor Classification, Oncologic Assessment and Therapy. Vet Sci 2025 Oct 17;12(10).
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  2. Drozdzewska K, Gehlen H. Markers for internal neoplasia in the horse. Vet Med Sci 2023 Jan;9(1):132-143.
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