Novel method for studying the in-vitro effects of anti-cancer agents: the assay of thymidine kinase 1 (TK1) in cell culture utilising AroCell TK 210 ELISA


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Kiran Kumar Jagarlamudi1,Liya Wang2,Staffan Eriksson3
1AroCell AB www.arocell.com,2Swedish Agricultural University,3Swedish Agricultural University, AroCell AB

Abstract

Background

Thymidine kinase 1 (TK1) is a salvage pathway enzyme involved in DNA precursor synthesis and its expression is cell cycle regulated. TK1 is up-regulated as a result of DNA damage and released from dying cells during un-regulated cell proliferation. TK1 enzyme activity has been widely used to study haematological malignancies but little has been published on its use in in-vitro models of malignancy.

The AroCell TK 210 ELISA kit is a novel method for determining TK1 mass. The purpose of this study was to evaluate the changes in TK1 levels in an in-vitro culture of leukaemic cells

Here we report on studies using CEM lymphoblastic cells and Doxorubicin (DOX), an anthracycline drug that is an effective anti-neoplastic drug for both haematological and solid malignancies

Method

The effect of DOX on cellular TK1 levels in a lymphoblastic leukaemia-derived cell line (CCRF-CEM) was investigated. Cells were treated with 0 to 100 µM of DOX for 24 and 48 hours. After DOX exposure, TK1 protein levels were determined in culture media supernatant and cellular extracts, using the AroCell TK 210 ELISA kit.

Results

There was a linear relation between intra cellular TK1 concentration and number of cells (400 to 8000 cells per well in a 96 well microtiter plate). Intra-cellular TK1 increased about 2-fold with increasing concentrations of DOX up to 1 µM, while the levels in the media increased  significantly above 5 µM. These results demonstrate that changes in the intracellular and extracellular TK1 protein levels at different concentrations of DOX may provide information about the induction of DNA damage and release of TK1 protein during cell lysis.

Conclusion

The use of AroCell TK 210 ELISA provides opportunities for in-vitro studies of old and new anti-cancer drugs, targeting cell-proliferation, offering a new method for evaluating the effects of anti-tumour agents.