Limits...
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.

Show MeSH

Related in: MedlinePlus

Serum TK1 activity and TK1 protein concentrations in different clinical samples. (A) Serum thymidine kinase 1 (sTK1) activity (pmol/min/mL) in sera from healthy dogs (•) and dogs with mammary tumors (■). The error bars represent means ± standard deviation (SD). (B) Serum TK1 protein levels in sera from healthy dogs (•) and dogs with canine mammary tumors (CMTs) (■). (C) Comparison of sTK1 activity in healthy dogs (•) and dogs with benign (■) and malignant (▲) CMTs. (D) Comparison of sTK1 protein levels in healthy dogs (•) and dogs with benign (■) and malignant (▲) mammary tumors. The error bars represent means ± standard deviation (SD).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Serum TK1 activity and TK1 protein concentrations in different clinical samples. (A) Serum thymidine kinase 1 (sTK1) activity (pmol/min/mL) in sera from healthy dogs (•) and dogs with mammary tumors (■). The error bars represent means ± standard deviation (SD). (B) Serum TK1 protein levels in sera from healthy dogs (•) and dogs with canine mammary tumors (CMTs) (■). (C) Comparison of sTK1 activity in healthy dogs (•) and dogs with benign (■) and malignant (▲) CMTs. (D) Comparison of sTK1 protein levels in healthy dogs (•) and dogs with benign (■) and malignant (▲) mammary tumors. The error bars represent means ± standard deviation (SD).

Mentions: Significant differences were found in mean sTK1 activity (P = 0.007; Figure 3A) and sTK1 protein levels (P < 0.0001; Figure 3B) between healthy and CMT dogs. Significant correlations were also found between the sTK1 activity and sTK1 protein levels in healthy dogs (r = 0.52, P = 0.01) and CMTs (r = 0.41, P = 0.03). Canine mammary tumors were further classified as benign or malignant and the sTK1 protein and activity levels were determined in these subgroups and compared to those in healthy dogs. There was no significant difference in sTK1 activity in sera from healthy, benign, and malignant dogs (Figure 3C). However, a significant difference was found in sTK1 protein levels between these subgroups (Figure 3D). To evaluate the performance of the two different assays, ROC curve analysis for CMTs vs. healthy dogs was done. The results showed that TK1 activity assay had an area under the curve (AUC) of 0.74, P = 0.0048 (95% confidence interval (CI) 0.59–0.88), with a cutoff value of 1.32 pmol/min/mL, sensitivity of 0.22, and specificity of 0.95 (Figure 4A). The TK1 protein assay, however, had an AUC of 0.96, P < 0.0001 (95% CI 0.92–1.01). The sensitivity in this case was 0.81 and the specificity 0.95, using a cutoff value of 17.5 ng/mL (Figure 4B). The specific activity of sTK1 (nmol dTMP/min/mg of sTK of 26 kDa), based on the immunoaffinity assay and 3[H]-dThd activity measurements in healthy dogs, was 49 ± 21 nmol/min/mg, which is significantly higher (P < 0.0001) compared to sera from the CMT group (20 ± 11 nmol/min/mg). The higher sTK1 specific activity in healthy dogs (about 2.5-fold), compared to dogs with CMTs, means that there is a larger fraction of inactive TK1 polypeptide in case of CMTs.Figure 3


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 TK1 activity and TK1 protein concentrations in different clinical samples. (A) Serum thymidine kinase 1 (sTK1) activity (pmol/min/mL) in sera from healthy dogs (•) and dogs with mammary tumors (■). The error bars represent means ± standard deviation (SD). (B) Serum TK1 protein levels in sera from healthy dogs (•) and dogs with canine mammary tumors (CMTs) (■). (C) Comparison of sTK1 activity in healthy dogs (•) and dogs with benign (■) and malignant (▲) CMTs. (D) Comparison of sTK1 protein levels in healthy dogs (•) and dogs with benign (■) and malignant (▲) mammary tumors. The error bars represent means ± standard deviation (SD).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Serum TK1 activity and TK1 protein concentrations in different clinical samples. (A) Serum thymidine kinase 1 (sTK1) activity (pmol/min/mL) in sera from healthy dogs (•) and dogs with mammary tumors (■). The error bars represent means ± standard deviation (SD). (B) Serum TK1 protein levels in sera from healthy dogs (•) and dogs with canine mammary tumors (CMTs) (■). (C) Comparison of sTK1 activity in healthy dogs (•) and dogs with benign (■) and malignant (▲) CMTs. (D) Comparison of sTK1 protein levels in healthy dogs (•) and dogs with benign (■) and malignant (▲) mammary tumors. The error bars represent means ± standard deviation (SD).
Mentions: Significant differences were found in mean sTK1 activity (P = 0.007; Figure 3A) and sTK1 protein levels (P < 0.0001; Figure 3B) between healthy and CMT dogs. Significant correlations were also found between the sTK1 activity and sTK1 protein levels in healthy dogs (r = 0.52, P = 0.01) and CMTs (r = 0.41, P = 0.03). Canine mammary tumors were further classified as benign or malignant and the sTK1 protein and activity levels were determined in these subgroups and compared to those in healthy dogs. There was no significant difference in sTK1 activity in sera from healthy, benign, and malignant dogs (Figure 3C). However, a significant difference was found in sTK1 protein levels between these subgroups (Figure 3D). To evaluate the performance of the two different assays, ROC curve analysis for CMTs vs. healthy dogs was done. The results showed that TK1 activity assay had an area under the curve (AUC) of 0.74, P = 0.0048 (95% confidence interval (CI) 0.59–0.88), with a cutoff value of 1.32 pmol/min/mL, sensitivity of 0.22, and specificity of 0.95 (Figure 4A). The TK1 protein assay, however, had an AUC of 0.96, P < 0.0001 (95% CI 0.92–1.01). The sensitivity in this case was 0.81 and the specificity 0.95, using a cutoff value of 17.5 ng/mL (Figure 4B). The specific activity of sTK1 (nmol dTMP/min/mg of sTK of 26 kDa), based on the immunoaffinity assay and 3[H]-dThd activity measurements in healthy dogs, was 49 ± 21 nmol/min/mg, which is significantly higher (P < 0.0001) compared to sera from the CMT group (20 ± 11 nmol/min/mg). The higher sTK1 specific activity in healthy dogs (about 2.5-fold), compared to dogs with CMTs, means that there is a larger fraction of inactive TK1 polypeptide in case of CMTs.Figure 3

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