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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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Acute lymphocytic leukemia (ALL) cell extract and serum analyzed by Superose 12 column chromatography. (A) The thymidine kinase 1 (TK1) activity in the fractions from the ALL cell extract (•). (B) Western blot analyses of the same fractions. (C) Serum TK1 (sTK1) activity in the fractions from the ALL dog (•). (D) Western blot analyses and sTK1 protein in the same fractions. Arrows indicate the elution position of the molecular weight (MW) markers.
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Fig5: Acute lymphocytic leukemia (ALL) cell extract and serum analyzed by Superose 12 column chromatography. (A) The thymidine kinase 1 (TK1) activity in the fractions from the ALL cell extract (•). (B) Western blot analyses of the same fractions. (C) Serum TK1 (sTK1) activity in the fractions from the ALL dog (•). (D) Western blot analyses and sTK1 protein in the same fractions. Arrows indicate the elution position of the molecular weight (MW) markers.

Mentions: A fresh blood sample collected from a dog with ALL and white blood cells were separated, as described in the Materials and Methods section. Cellular extract and sera from the same dog were applied to the Superose 12 column and the TK1 activity and protein levels in the fractions were determined. Native cellular TK1 activity eluted as major peak with molecular weights (MWs) of 40–66 kDa, but with a small peak and some enzyme activity in the fractions at the higher MW range (Figure 5A). Western blot analysis of cellular extract fractions showed a TK1 subunit (26 kDa) in the fractions eluted, corresponding to MWs of 40–66 kDa, with faint bands in the high MW region (Figure 5B). When analyzing sera from the same dog, about 90% of TK1 eluted as a peak in the high MW range corresponding to 200–720 kDa (Figure 5C). A TK1 polypeptide of 26 kDa was detected in fractions 1–12, but the band intensity did not correlate with the activity levels in these fractions (Figure 5D). These results indicate that active cellular TK1 isolated from ALL exists mainly as dimer, while active sTK1 from the same dog was found to occur as a high MW oligomer. A gel filtration experiment has been carried out with sera from another ALL dog and similar results were found (data not shown).Figure 5


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)

Acute lymphocytic leukemia (ALL) cell extract and serum analyzed by Superose 12 column chromatography. (A) The thymidine kinase 1 (TK1) activity in the fractions from the ALL cell extract (•). (B) Western blot analyses of the same fractions. (C) Serum TK1 (sTK1) activity in the fractions from the ALL dog (•). (D) Western blot analyses and sTK1 protein in the same fractions. Arrows indicate the elution position of the molecular weight (MW) markers.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Acute lymphocytic leukemia (ALL) cell extract and serum analyzed by Superose 12 column chromatography. (A) The thymidine kinase 1 (TK1) activity in the fractions from the ALL cell extract (•). (B) Western blot analyses of the same fractions. (C) Serum TK1 (sTK1) activity in the fractions from the ALL dog (•). (D) Western blot analyses and sTK1 protein in the same fractions. Arrows indicate the elution position of the molecular weight (MW) markers.
Mentions: A fresh blood sample collected from a dog with ALL and white blood cells were separated, as described in the Materials and Methods section. Cellular extract and sera from the same dog were applied to the Superose 12 column and the TK1 activity and protein levels in the fractions were determined. Native cellular TK1 activity eluted as major peak with molecular weights (MWs) of 40–66 kDa, but with a small peak and some enzyme activity in the fractions at the higher MW range (Figure 5A). Western blot analysis of cellular extract fractions showed a TK1 subunit (26 kDa) in the fractions eluted, corresponding to MWs of 40–66 kDa, with faint bands in the high MW region (Figure 5B). When analyzing sera from the same dog, about 90% of TK1 eluted as a peak in the high MW range corresponding to 200–720 kDa (Figure 5C). A TK1 polypeptide of 26 kDa was detected in fractions 1–12, but the band intensity did not correlate with the activity levels in these fractions (Figure 5D). These results indicate that active cellular TK1 isolated from ALL exists mainly as dimer, while active sTK1 from the same dog was found to occur as a high MW oligomer. A gel filtration experiment has been carried out with sera from another ALL dog and similar results were found (data not shown).Figure 5

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