Article

Thymidine Kinase 1 Upregulation Is an Early Event in Breast Tumor Formation

Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA.
Journal of Oncology 06/2012; 2012:575647. DOI: 10.1155/2012/575647
Source: PubMed

ABSTRACT Prognostic markers play an important role in our understanding of tumors and how to treat them. Thymidine kinase 1 (TK1), a proliferation marker involved in DNA repair, has been shown to have independent prognostic potential. This prognostic potential includes the novel concept that upregulation of serum TK1 levels is an early event in cancer development. This same effect may also be seen in tumor tissue. In order to demonstrate that TK1 upregulation is an early event in tumor tissue formation, tissue arrays were obtained and stained for TK1 by immunohistochemistry. Using a progressive breast tissue array, precancerous tissue including breast adenosis, simple hyperplasia, and atypical hyperplasia stained positive for TK1 expression. Different stages of breast carcinoma tissue also stained positive for TK1 including nonspecific infiltrating duct, infiltrating lobular, and infiltrating duct with lymph node metastasis carcinomas. This indicates that TK1 upregulation is an early event in breast carcinoma development, and may be useful in identifying precancerous tissue. Further work is needed to better understand the differences seen between TK1 positive and negative tissues.

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    ABSTRACT: Thymidine kinase 1 (ATP: thymidine 5’- phosphotransferase, EC 2.1.7. 21, TK1) is a cellular enzyme involved in salvage pathway for DNA precursor synthesis. TK1 activity fluctuates during cell cycle, it reaches peak in S phase and absent in M phase. Because of its tight regulation with cell cycle, TK1 has been used as proliferation marker for diagnosis and treatment monitoring of various malignancies in human and veterinary medicine. TK1 levels can be measured by activity based or antibody based assays. The main aim of the research described in this thesis was to develop TK1 antibody based assays for determining serum TK1 protein levels in comparison with TK1 activity from dogs with different malignancies. Further analysis revealed a significant difference in the molecular forms of TK1 in sera from canine leukemia and mammary tumours. In Study I, serum TK1 protein levels in dogs with different solid tumours were determined by using an antibody based assay i.e. immunoaffinity assay. TK1 protein levels were significantly higher in dogs with solid tumours than expected from the TK1 activity values. In contrast, the specific activity of TK1 in sera from healthy dogs was 2.5 fold higher than that of solid tumours. The molecular forms of recombinant, cellular and serum TK1 were investigated in study II. Dog recombinant and serum TK1 existed as oligomers with a molecular weight (MW) of 720-300 kDa. Cellular TK1 from both dogs and humans were mainly as tetramers. Human recombinant and serum TK1 activity eluted in two peaks: one at high and one at low MW, corresponding to 720-300 kDa and 200-50 kDa, respectively. In study III, TK1 protein levels in sera from dogs with mammary tumours were determined by immunoaffinity assay. The TK1 protein assay differentiated mammary adenomas efficiently from healthy dogs, and adenomas from carcinomas, but this was not possible with the TK1 activity assay. In mammary tumour sera, active TK1 eluted as high MW oligomer similar to leukemia however, TK1 protein was detected not only as high MW form but also in the fractions where no TK1 activity was found. This indicates that serum TK1 exits in multiple forms in mammary tumours with a large fraction of inactive TK1 protein. Study IV describes the development of TK1-ELISA, based on a polyclonal and a monoclonal anti TK1 peptide antibodies, that was used to measure TK1 protein levels in dog sera. TK1 protein levels were significantly higher in sera from dogs with haematological tumours as well as solid tumours in comparison with healthy dogs. Overall, the results demonstrate that TK1 protein assays provide valuable diagnostic information in a variety of canine malignancies.
    04/2015, Degree: PhD, Supervisor: Staffan Eriksson
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    [Show abstract] [Hide abstract]
    ABSTRACT: Thymidine kinase 1 (ATP: thymidine 5’- phosphotransferase, EC 2.1.7. 21, TK1) is a cellular enzyme involved in salvage pathway for DNA precursor synthesis. TK1 activity fluctuates during cell cycle, it reaches peak in S phase and absent in M phase. Because of its tight regulation with cell cycle, TK1 has been used as proliferation marker for diagnosis and treatment monitoring of various malignancies in human and veterinary medicine. TK1 levels can be measured by activity based or antibody based assays. The main aim of the research described in this thesis was to develop TK1 antibody based assays for determining serum TK1 protein levels in comparison with TK1 activity from dogs with different malignancies. Further analysis revealed a significant difference in the molecular forms of TK1 in sera from canine leukemia and mammary tumours. In Study I, serum TK1 protein levels in dogs with different solid tumours were determined by using an antibody based assay i.e. immunoaffinity assay. TK1 protein levels were significantly higher in dogs with solid tumours than expected from the TK1 activity values. In contrast, the specific activity of TK1 in sera from healthy dogs was 2.5 fold higher than that of solid tumours. The molecular forms of recombinant, cellular and serum TK1 were investigated in study II. Dog recombinant and serum TK1 existed as oligomers with a molecular weight (MW) of 720-300 kDa. Cellular TK1 from both dogs and humans were mainly as tetramers. Human recombinant and serum TK1 activity eluted in two peaks: one at high and one at low MW, corresponding to 720-300 kDa and 200-50 kDa, respectively. In study III, TK1 protein levels in sera from dogs with mammary tumours were determined by immunoaffinity assay. The TK1 protein assay differentiated mammary adenomas efficiently from healthy dogs, and adenomas from carcinomas, but this was not possible with the TK1 activity assay. In mammary tumour sera, active TK1 eluted as high MW oligomer similar to leukemia however, TK1 protein was detected not only as high MW form but also in the fractions where no TK1 activity was found. This indicates that serum TK1 exits in multiple forms in mammary tumours with a large fraction of inactive TK1 protein. Study IV describes the development of TK1-ELISA, based on a polyclonal and a monoclonal anti TK1 peptide antibodies, that was used to measure TK1 protein levels in dog sera. TK1 protein levels were significantly higher in sera from dogs with haematological tumours as well as solid tumours in comparison with healthy dogs. Overall, the results demonstrate that TK1 protein assays provide valuable diagnostic information in a variety of canine malignancies.
    04/2015, Degree: PhD, Supervisor: Staffan Eriksson
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    ABSTRACT: High levels of thymidine kinase 1 (TK1) and thymidine phosphorylase (TYMP) are key molecular targets by thymidine therapeutics in cancer treatment. The dual roles of TYMP as a tumor growth factor and a key activation enzyme of anticancer metabolites resulted in a mixed outcome in cancer patients. In this study, we investigated the roles of TK1 and TYMP on a thymidine quinoxaline conjugate to evaluate an alternative to circumvent the contradictive role of TYMP. TK1 and TYMP levels in multiple liver cell lines were assessed along with the cytotoxicity of the thymidine conjugate. Cellular accumulation of the thymidine conjugate was determined with organelle-specific dyes. The impacts of TK1 and TYMP were evaluated with siRNA/shRNA suppression and pseudoviral overexpression. Immunohistochemical analysis was performed on both normal and tumor tissues. In vivo study was carried out with a subcutaneous liver tumor model. We found that the thymidine conjugate had varied activities in liver cancer cells with different levels of TK1 and TYMP. The conjugate mainly accumulated at endothelial reticulum and was consistent with cytosolic pathways. TK1 was responsible for the cytotoxicity yet high levels of TYMP counteracted such activities. Levels of TYMP and TK1 in the liver tumor tissues were significantly higher than those of normal liver tissues. Induced TK1 overexpression decreased the selectivity of dT-QX due to the concurring cytotoxicity in normal cells. In contrast, shRNA suppression of TYMP significantly enhanced the selective of the conjugate in vitro and reduced the tumor growth in vivo. TK1 was responsible for anticancer activity of dT-QX while levels of TYMP counteracted such an activity. The counteraction by TYMP could be overcome with RNA silencing to significantly enhance the dT-QX selectivity in cancer cells.
    BMC Cancer 12/2015; 15(1):159. DOI:10.1186/s12885-015-1149-5 · 3.32 Impact Factor