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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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Canine mammary tumor (CMT) (dog No. 23) tumor tissue extract and serum analyzed by Superose 12 column chromatography. (A) Thymidine kinase 1 activity in the fraction from the CMT tissue extract (•). (B) Western blot analyses of the same fractions. (C) Thymidine kinase 1 activity in the fractions from the CMT serum (•). (D) Western blot analyses of 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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Fig6: Canine mammary tumor (CMT) (dog No. 23) tumor tissue extract and serum analyzed by Superose 12 column chromatography. (A) Thymidine kinase 1 activity in the fraction from the CMT tissue extract (•). (B) Western blot analyses of the same fractions. (C) Thymidine kinase 1 activity in the fractions from the CMT serum (•). (D) Western blot analyses of the same fractions. Arrows indicate the elution position of the molecular weight (MW) markers. Numbers represent the fast protein liquid chromatography (FPLC) fractions.

Mentions: Tissue extract prepared from a CMT patient (No. 23) was applied on the Superose 12 column and around 70% of total TK1 activity eluted as major peak with MWs of 40–100 kDa, but with a minor peak and some enzyme activity in the fractions corresponding to higher MW (Figure 6A). Western blot analysis of CMT extract showed a TK1 subunit (26 kDa) in the fractions corresponding to the peak and faint bands in the high MW region (Figure 6B). A serum sample from the same CMT dog was also analyzed and more than 95% of the total TK1 activity eluted in fractions 1–11, corresponding to the MW range 200–720 kDa (Figure 6C). Thymidine kinase 1 polypeptide of 26 kDa was detected in all fractions (Figure 6D) and there was no correlation with TK1 activity. These results indicate that sTK1 protein exists in multimeric forms in CMT (oligomers, dimers, and tetramers), many of them apparently inactive.Figure 6


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)

Canine mammary tumor (CMT) (dog No. 23) tumor tissue extract and serum analyzed by Superose 12 column chromatography. (A) Thymidine kinase 1 activity in the fraction from the CMT tissue extract (•). (B) Western blot analyses of the same fractions. (C) Thymidine kinase 1 activity in the fractions from the CMT serum (•). (D) Western blot analyses of 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

Fig6: Canine mammary tumor (CMT) (dog No. 23) tumor tissue extract and serum analyzed by Superose 12 column chromatography. (A) Thymidine kinase 1 activity in the fraction from the CMT tissue extract (•). (B) Western blot analyses of the same fractions. (C) Thymidine kinase 1 activity in the fractions from the CMT serum (•). (D) Western blot analyses of the same fractions. Arrows indicate the elution position of the molecular weight (MW) markers. Numbers represent the fast protein liquid chromatography (FPLC) fractions.
Mentions: Tissue extract prepared from a CMT patient (No. 23) was applied on the Superose 12 column and around 70% of total TK1 activity eluted as major peak with MWs of 40–100 kDa, but with a minor peak and some enzyme activity in the fractions corresponding to higher MW (Figure 6A). Western blot analysis of CMT extract showed a TK1 subunit (26 kDa) in the fractions corresponding to the peak and faint bands in the high MW region (Figure 6B). A serum sample from the same CMT dog was also analyzed and more than 95% of the total TK1 activity eluted in fractions 1–11, corresponding to the MW range 200–720 kDa (Figure 6C). Thymidine kinase 1 polypeptide of 26 kDa was detected in all fractions (Figure 6D) and there was no correlation with TK1 activity. These results indicate that sTK1 protein exists in multimeric forms in CMT (oligomers, dimers, and tetramers), many of them apparently inactive.Figure 6

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