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Properties of cellular and serum forms of thymidine kinase 1 (TK1) in dogs with acute lymphocytic leukemia (ALL) and canine mammary tumors (CMTs): implications for TK1 as a proliferation biomarker.

Jagarlamudi KK, Westberg S, Rönnberg H, Eriksson S - BMC Vet. Res. (2014)

Bottom Line: Our results showed that the sTK1 protein assay can differentiate benign tumors (early stage tumors) from healthy more efficiently than sTK1 activity assay.This preliminary data supports that sTK1 protein assay is clinically useful.Further studies are needed to evaluate the diagnostic or prognostic role of serum TK1 protein in CMTs.

View Article: PubMed Central - PubMed

Affiliation: Center of Clinical Comparative Oncology (C3O), Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, S-750 07, Sweden. henrik.von.euler@slu.se.

ABSTRACT

Background: Thymidine kinase 1 (TK1) is a deoxyribonucleic acid (DNA) precursor enzyme and a proliferation biomarker used for prognosis and treatment monitoring of breast cancer in humans. The aim was to determine if serum thymidine kinase 1 (sTK1) activity and sTK1 protein levels in dogs with mammary tumors could be useful in veterinary medicine.

Results: Serum samples from 20 healthy dogs and 27 dogs with mammary tumors were analyzed for sTK1 activity, using an [(3)H]-deoxythymidine (dThd) phosphorylation assay, and for sTK1 protein levels by immune affinity/Western blot assay. The molecular forms of sTK1 in acute lymphocytic leukemia (ALL), canine mammary tumor (CMT), and healthy sera were determined by size exclusion chromatography. Mean sTK1 activities in CMT were 1.0 ± 0.36 pmol/min/mL, differing significantly from healthy dogs (mean ± SD = 0.73 ± 0.26 pmol/min/mL). Serum TK1 protein (26 kDa polypeptide) levels were also significantly higher in CMTs compared to healthy dogs (mean ± SD = 28.5 ± 11.4, and 8.5 ± 4 ng/mL, respectively). Cellular TK1 isolated from ALL tumor cells was predominantly a dimer, while the serum TK1 activity eluted as a high molecular weight (MW) oligomer. In analyses of CMT tissue extracts, TK1 activity eluted in two peaks, a minor peak with a high MW oligomer and a major tetramer peak. Western blot analysis of chromatographic fractions showed that cellular TK1 protein in both ALL and CMT dogs, and to some extent serum TK1 from ALL dogs, correlated with activity profiles, but a large fraction of inactive TK1 protein was detected in CMT.

Conclusions: Serum TK1 protein and activity levels were significantly higher in CMT than in healthy dogs. Size exclusion chromatography demonstrated major differences in the molecular forms of sTK1 in ALL, healthy, and CMT dogs, with a large fraction of inactive TK1 protein in CMT. Our results showed that the sTK1 protein assay can differentiate benign tumors (early stage tumors) from healthy more efficiently than sTK1 activity assay. This preliminary data supports that sTK1 protein assay is clinically useful. Further studies are needed to evaluate the diagnostic or prognostic role of serum TK1 protein in CMTs.

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Related in: MedlinePlus

Serum from a healthy dog (No. 5) analyzed by Superose 12 column chromatography. (A) Serum thymidine kinase 1 (sTK1) activity in the fractions from the healthy dog (•). (B) Western blot analyses of sTK1 protein in the same fractions. Arrows indicate the elution position of the molecular weight (MW) markers. Numbers represent the fast protein liquid chromatography (FPLC) fractions.
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Fig7: Serum from a healthy dog (No. 5) analyzed by Superose 12 column chromatography. (A) Serum thymidine kinase 1 (sTK1) activity in the fractions from the healthy dog (•). (B) Western blot analyses of sTK1 protein in the same fractions. Arrows indicate the elution position of the molecular weight (MW) markers. Numbers represent the fast protein liquid chromatography (FPLC) fractions.

Mentions: A serum sample from a healthy dog (No. 5) was subjected to size exclusion chromatography. Thymidine kinase 1 activity was recovered as a single peak in fractions 1–5, corresponding to a MW range of 500–720 kDa (Figure 7A). Western blot analysis showed a TK1 polypeptide of 26 kDa in the same fractions (Figure 7B). These results demonstrate that sTK1 exists as an active high MW oligomer in sera of healthy dogs. When we analyzed another healthy dog serum on superose 12 column, similar results were found (Figure 8).Figure 7


Properties of cellular and serum forms of thymidine kinase 1 (TK1) in dogs with acute lymphocytic leukemia (ALL) and canine mammary tumors (CMTs): implications for TK1 as a proliferation biomarker.

Jagarlamudi KK, Westberg S, Rönnberg H, Eriksson S - BMC Vet. Res. (2014)

Serum from a healthy dog (No. 5) analyzed by Superose 12 column chromatography. (A) Serum thymidine kinase 1 (sTK1) activity in the fractions from the healthy dog (•). (B) Western blot analyses of sTK1 protein in the same fractions. Arrows indicate the elution position of the molecular weight (MW) markers. Numbers represent the fast protein liquid chromatography (FPLC) fractions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4195903&req=5

Fig7: Serum from a healthy dog (No. 5) analyzed by Superose 12 column chromatography. (A) Serum thymidine kinase 1 (sTK1) activity in the fractions from the healthy dog (•). (B) Western blot analyses of sTK1 protein in the same fractions. Arrows indicate the elution position of the molecular weight (MW) markers. Numbers represent the fast protein liquid chromatography (FPLC) fractions.
Mentions: A serum sample from a healthy dog (No. 5) was subjected to size exclusion chromatography. Thymidine kinase 1 activity was recovered as a single peak in fractions 1–5, corresponding to a MW range of 500–720 kDa (Figure 7A). Western blot analysis showed a TK1 polypeptide of 26 kDa in the same fractions (Figure 7B). These results demonstrate that sTK1 exists as an active high MW oligomer in sera of healthy dogs. When we analyzed another healthy dog serum on superose 12 column, similar results were found (Figure 8).Figure 7

Bottom Line: Our results showed that the sTK1 protein assay can differentiate benign tumors (early stage tumors) from healthy more efficiently than sTK1 activity assay.This preliminary data supports that sTK1 protein assay is clinically useful.Further studies are needed to evaluate the diagnostic or prognostic role of serum TK1 protein in CMTs.

View Article: PubMed Central - PubMed

Affiliation: Center of Clinical Comparative Oncology (C3O), Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, S-750 07, Sweden. henrik.von.euler@slu.se.

ABSTRACT

Background: Thymidine kinase 1 (TK1) is a deoxyribonucleic acid (DNA) precursor enzyme and a proliferation biomarker used for prognosis and treatment monitoring of breast cancer in humans. The aim was to determine if serum thymidine kinase 1 (sTK1) activity and sTK1 protein levels in dogs with mammary tumors could be useful in veterinary medicine.

Results: Serum samples from 20 healthy dogs and 27 dogs with mammary tumors were analyzed for sTK1 activity, using an [(3)H]-deoxythymidine (dThd) phosphorylation assay, and for sTK1 protein levels by immune affinity/Western blot assay. The molecular forms of sTK1 in acute lymphocytic leukemia (ALL), canine mammary tumor (CMT), and healthy sera were determined by size exclusion chromatography. Mean sTK1 activities in CMT were 1.0 ± 0.36 pmol/min/mL, differing significantly from healthy dogs (mean ± SD = 0.73 ± 0.26 pmol/min/mL). Serum TK1 protein (26 kDa polypeptide) levels were also significantly higher in CMTs compared to healthy dogs (mean ± SD = 28.5 ± 11.4, and 8.5 ± 4 ng/mL, respectively). Cellular TK1 isolated from ALL tumor cells was predominantly a dimer, while the serum TK1 activity eluted as a high molecular weight (MW) oligomer. In analyses of CMT tissue extracts, TK1 activity eluted in two peaks, a minor peak with a high MW oligomer and a major tetramer peak. Western blot analysis of chromatographic fractions showed that cellular TK1 protein in both ALL and CMT dogs, and to some extent serum TK1 from ALL dogs, correlated with activity profiles, but a large fraction of inactive TK1 protein was detected in CMT.

Conclusions: Serum TK1 protein and activity levels were significantly higher in CMT than in healthy dogs. Size exclusion chromatography demonstrated major differences in the molecular forms of sTK1 in ALL, healthy, and CMT dogs, with a large fraction of inactive TK1 protein in CMT. Our results showed that the sTK1 protein assay can differentiate benign tumors (early stage tumors) from healthy more efficiently than sTK1 activity assay. This preliminary data supports that sTK1 protein assay is clinically useful. Further studies are needed to evaluate the diagnostic or prognostic role of serum TK1 protein in CMTs.

Show MeSH
Related in: MedlinePlus