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| 2. |
- Jagarlamudi, Kiran Kumar, et al.
(författare)
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High levels of inactive thymidine kinase 1 polypeptide detected in sera from dogs with solid tumours by immunoaffinity methods: Implications for in vitro diagnostics
- 2013
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Ingår i: Veterinary Journal. - : Elsevier BV. - 1090-0233 .- 1532-2971. ; 197, s. 854-860
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Tidskriftsartikel (refereegranskat)abstract
- STK1 activity in dogs with haematological tumours was significantly higher than in the solid tumour and healthy dog groups (mean +/- standard deviation [SD] = 65 +/- 79, 1.1 +/- 0.5, and 1.0 +/- 0.4 pmol/min/mL, respectively). Serum samples were analyzed after immunoaffinity isolation by western blot and the TK1 26 kDa band intensities quantified revealing that concentrations were significantly higher in dogs with haematological tumours and solid tumours compared to healthy dogs (mean +/- SD = 33 +/- 12, 30 +/- 13, and 10 +/- 5 ng/mL, respectively). Pre-incubation with the reducing agent dithioerythritol (DTE) showed a decrease in STK1 activity and protein levels in most samples, but an increase of about 20% in sera from healthy dogs and from those with haematological malignancies. Compared to animals with solid tumours, the specific STK1 activity (nmol [H-3]-deoxythymidine monophosphate (dTMP)/min/mg of TK1 protein of 26 kDa) was 30-fold higher in haematological malignancies and 2.5-fold higher in healthy dogs, respectively. The results demonstrate that there is a large fraction of inactive TK1 protein, particularly in sera from dogs with solid tumours. The findings are important in the use of STK1 as a biomarker. (C) 2013 Elsevier Ltd. All rights reserved.
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| 3. |
- Saellström, Sara, et al.
(författare)
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Serum TK1 protein and C-reactive protein correlate to treatment response and predict survival in dogs with hematologic malignancies
- 2022
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Ingår i: Research in Veterinary Science. - : Elsevier BV. - 0034-5288 .- 1532-2661. ; 145, s. 213-221
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Tidskriftsartikel (refereegranskat)abstract
- Thymidine kinase 1 (TK1), involved in DNA precursor synthesis, is used as a serum biomarker in cancer diagnostics in both human and veterinary medicine. We investigated the utility of serum TK1 protein (TK1p) and TK1 activity (TK1a) determinations for prognosis and monitoring of canine hematological malignancies. The combination of TK1p or TK1a with canine C-reactive protein (CRP) determinations was also investigated.Serum samples from 51 client-owned dogs with naive hematological malignancies and from 149 healthy subjects were included. Serum TK1p levels were determined using a prototype TK1-ELISA, TK1a using the [H-3]- dThd phosphorylation assay, and CRP using an immunoturbidimetric assay.Mean TK1p in sera from dogs with tumors was significantly higher than from healthy dogs (mean +/- SD = 3.9 +/- 5.9 vs. 0.45 +/- 0.15 ng/mL). Similarly, TK1a in hematological malignancies was significantly higher than in healthy dogs (mean + SD = 15.1 +/- 31.3 vs. 0.96 +/- 0.33 pmol/min/mL). The receiver-operating characteristic indicated that a combination of TK1p or TK1a with CRP gave higher sensitivity than either biomarker alone for the prognosis of hematological malignancies. Median pretreatment TK1p and TK1a levels were significantly higher than in dogs in remission and correlated with clinical outcome. Kaplan-Meier curve analysis showed that naive dogs with high TK1p, TK1a, and CRP had significantly shorter survival.This study present two new polyclonal antibodies used in an ELISA system to determine TK1p. The study also show that combining TK1p or TK1a with CRP gave higher sensitivity than either biomarker alone. Monitoring patients in the study while undergoing chemotherapy, suggests that the TK1 + CRP combination could be useful in a biomarker panel, possibly aiding the prognosis and therapy monitoring of hematological malignancies in dogs.
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| 4. |
- Sharif, Hanan, et al.
(författare)
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A monoclonal antibody-based sandwich ELISA for measuring canine Thymidine kinase 1 protein and its role as biomarker in canine lymphoma
- 2023
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Ingår i: Frontiers in Veterinary Science. - : Frontiers Media SA. - 2297-1769. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Dogs play an important role in society, which increased during the covid epidemics. This has led to a much higher workload for the veterinarians. Therefore, there is a need for efficient diagnostic tools to identify risk of malignant diseases. Here the development of a new test that can solve some of these problems is presented. It is based on serum Thymidine Kinase 1 (TK1), which is a biomarker for cell proliferation and cell lysis.Methods: Anti-TK1 monoclonal antibodies were produced against two different epitopes, the active site of the TK1 protein and the C-terminal region of canine TK1. The antibodies were developed with hybridoma technology and validated using dot blot, Quartz Crystal Microbalance (QCM) technology, western blots, immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA). Clinical evaluation of Canine TK1 ELISA was done by using sera from 131 healthy dogs and 93 dogs with lymphoma. The two selected Anti-TK1 monoclonal antibodies have Kd values in the range of 10(-9) M and further analysis with dot and western blots confirmed the high affinity binding of these antibodies. A sandwich Canine TK1 ELISA was developed using the anti-TK1 antibodies, and TK1 concentrations in serum samples were determined using dog recombinant TK1 as a standard.Results: Serum TK1 protein levels were significantly higher in dogs with lymphoma compared to those in healthy dogs (p < 0.0001). Receiver operating curve analysis showed that the canine TK1-ELISA obtain a sensitivity of 0.80, at a specificity of 0.95. Moreover, the Canine TK1 ELISA has a positive predictive value (PPV) of 97%, and the negative predictive value (NPV) of 83%, reflecting the proportion of test results that are truly positive and negative. Furthermore, Canine TK1 ELISA had significantly higher capacity to differentiate dogs with T-cell lymphoma from those with B-cell lymphoma compared to earlier used TK1 activity assays.Discussion: These results demonstrate that the Canine TK1 ELISA can serve as an efficient tool in the diagnosis and management of dogs with lymphomas.
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| 5. |
- Sharif, Hanan, et al.
(författare)
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A sensitive and kinetically defined radiochemical assay for canine and human serum thymidine kinase 1 (TK1) to monitor canine malignant lymphoma
- 2012
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Ingår i: Veterinary Journal. - : Elsevier BV. - 1090-0233 .- 1532-2971. ; 194, s. 40-47
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Tidskriftsartikel (refereegranskat)abstract
- Mean S-TK1 activities were 1.11 +/- 0.46 pmol/min/mL in healthy dogs and 1.15 +/- 0.32 pmol/min/mL in healthy humans. S-TK1 activities in dogs with hematological malignancies were 24.2 +/- 47.9 pmol/min/mL, and the receiver operating characteristic curve showed an area under the curve of 0.88. With a cut-off value of 1.9 pmol/min/mL (mean value +/- 2 SD), the sensitivity was 0.94 and the specificity was 0.68. Very similar results were obtained with human samples (healthy and lymphoma cases). S-TK1 activities measured during chemotherapy of six dogs with lymphoma were drastically reduced. In one case, S-TK1 activity increased prior to relapse. S-TK1 levels in dogs with solid tumors did not differ from the healthy group. S-TK1 activities correlated with those determined with the TK-REA and TK-Liaison assays (r = 0.92 and r = 0.96, respectively). In conclusion, this optimized [H-3]-dThd assay is fast, sensitive and economical for measuring S-TK1 activity and should increase its clinical use as biomarker. (C) 2012 Elsevier Ltd. All rights reserved.
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| 6. |
- Sharif, Hanan Mohamed, et al.
(författare)
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Elevation of serum thymidine kinase 1 in a bacterial infection : Canine pyometra
- 2013
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Ingår i: Theriogenology. - : Elsevier BV. - 0093-691X .- 1879-3231. ; 79:1, s. 17-23
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Tidskriftsartikel (refereegranskat)abstract
- Pyometra is a bacterial infection of the uterus that is common in dogs and is potentially life-threatening if delayed in diagnosis and/or treatment. Thymidine kinase 1 (TK1) is a cytosolic enzyme involved in DNA precursor synthesis, and it is also present in serum from patients with malignant diseases. TK1 has been used as a cell proliferation biomarker for many years in human medicine and recently in dogs. However, little is known regarding serum TK1 levels in individuals with bacterial infection. The objective of this study was to determine the activity of serum TK1 in dogs with pyometra and compare it with hematologic and biochemical parameters, e.g., acute phase proteins and inflammatory mediators such as C-reactive protein and Prostaglandin F-2 alpha. Serum and plasma TK1 activity of 40 healthy female dogs and 54 dogs with pyometra were analyzed using an optimized [H-3]-thymidine phosphorylation assay. TK1 activities in serum or plasma were significantly higher in dogs with pyometra as compared with healthy female dogs (mean +/- SD: 4.0 +/- 7.3 pmol/min/mL in the pyometra group and 1.07 +/- 0.34 pmol/min/mL in healthy control group). However, there was no difference in TK1 activity between systemic inflammatory response syndrome (SIRS) positive (n = 38) and SIRS negative (n = 16) pyometra cases. Furthermore, the plasma TK1 activity decreased in six and increased in one pyometra patients (n = 10), 24 h after ovariohysterectomy. No significant correlations (P > 0.05) were found between TK1 activity and hematological or other biochemical parameters. In conclusion, the TK1 activity was significantly elevated in dogs with pyometra. Further studies are needed to evaluate the mechanism and role of serum TK1 activity in bacterial infections and its possible diagnostic or prognostic value.
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| 7. |
- Sharif, Hanan
(författare)
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Molecular properties of serum thymidine kinase 1 and use of the canine enzyme in disease monitoring
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Thymidine kinase 1 (ATP: thymidine 5'-phosphotransferase, EC 2.7.1.21, TK1) catalyzes the transfer of the gamma phosphate group from a phosphate donor to the 5´- hydroxyl group of thymidine to form thymidine monophosphate, which is converted to thymidine triphosphate and then utilized in DNA synthesis. TK1 activity markedly increases in G1 and S phase of the cell cycle and is found mainly in dividing cells, but also in an extracellular form in the blood. Increased serum TK1 levels are considered as a sensitive and useful marker for cell proliferation and detection of malignancy in clinical medicine. The aim of this study was to investigate the molecular characteristics of canine thymidine kinase 1 and utilize several anti-TK1 antibodies to a better understanding its role in disease prognostics, risk assessment, and monitoring of cancer development. Study I was undertaken to clarify the structure of recombinant, cellular and serum TK1 in order to improve the usefulness of serum TK1 (STK1) as a proliferation biomarker in neoplastic diseases. Several forms of STK1 were observed with different enzymatic activities. The active form of STK1 was found mainly as an oligomer. Dimer and tetramer forms of STK1 were also seen in serum by immunoaffinity method, but they were enzymatically less active. Study II described a sensitive method using a [3H]-deoxythymidine phosphorylation assay to measure STK1 activity in dogs and humans with hematologic malignancies and dogs with solid tumors. The results revealed that the dThd phosphorylation assay was suitable for measurement of TK1 in hematologic malignancy (lymphoma and leukemia) and provided valuable information regarding the disease recurrence. Significant correlation was observed between the [3H]-dThd assay and the TK1 activity assays, TK-REA and TK Liaison. However, STK1 activity levels in dogs with solid tumors were very low. Therefore, another method to determine the levels of TK1 in serum based on immunoaffinity techniques was performed in study III. The concentration of STK1 protein in dogs with solid tumors was much higher than expected from the activity. Thus, methods to determine STK1 protein are more sensitive than activity tests in solid tumors. Study IV revealed that serum TK1 activity was elevated in 41% of bitches with pyometra, and thus TK1 determinations may be clinically valuable also in some non-neoplastic diseases.
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| 8. |
- Wang, Liya, et al.
(författare)
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Feline thymidine kinase 1: molecular characterization and evaluation of its serum form as a diagnostic biomarker
- 2021
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Ingår i: BMC Veterinary Research. - : Springer Science and Business Media LLC. - 1746-6148. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- Background Thymidine kinase 1 (TK1) catalyzes the initial phosphorylation of thymidine in the salvage pathway synthesis of dTTP, an essential building block of DNA. TK1 is a cytosolic enzyme with its highest level during the S-phase of the cell cycle. In cancer cells TK1 is upregulated and excess TK1 is leaked into the blood. Therefore, serum TK1 has been used as biomarker for cancer diagnosis and prognosis in human medicine. Feline TK1 shows high sequence similarity to TK1 from other species. The aim of this study was to characterize feline TK1 and evaluate if serum TK1 can be used as a diagnostic biomarker. Results Feline TK1 was cloned, expressed and affinity purified. The purified feline TK1 phosphorylated not only pyrimidine deoxyribonucleosides but also pyrimidine ribonucleosides and to some extent purine deoxynucleosides. A number of anticancer and antiviral nucleoside analogs also served as substrates with fairly high efficiency. ATP and dATP were the preferred phosphate donor. Serum TK1 activity in felines with malignant diseases was significantly higher than that in healthy individuals. ROC analysis revealed an area under the curve (AUC) of 0.98 with a sensitivity of 0.83 and a specificity of 0.95 for felines with lymphoma. Serum TK1 activity in felines with IBD or inflammatory disease was within the same range as healthy ones. Furthermore, in felines with lymphoma serum TK1 activity returned to normal levels in response to treatment. Conclusion Feline TK1 has high specific activity and a broader substrate specificity in comparison with TK1 from other species. Serum TK1 activity in felines with malignant diseases is significantly higher than that in normal felines and in felines with inflammatory diseases. These results suggest that serum TK1 may be a promising biomarker for the diagnosis and monitoring of malignant diseases and for the differential diagnosis of certain inflammatory disease.
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| 9. |
- Wang, Liya, et al.
(författare)
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Molecular characterization of equine thymidine kinase 1 and preliminary evaluation of its suitability as a serum biomarker for equine lymphoma
- 2021
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Ingår i: BMC molecular and cell biology. - : Springer Science and Business Media LLC. - 2661-8850. ; 22
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Tidskriftsartikel (refereegranskat)abstract
- Background 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. Conclusion 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.
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