Introduction: The activity of the pyrimidine salvage pathway enzyme thymidine kinase 1 (TK1) is tightly cell cycle regulated and has been investigated as a prognostic indicator of cancer in a variety of tissues. However, using the in vitro assay of TK1 to rank order a series of unique tumor samples by their TK1 activity can be problematic due to the complex nature of TK1 enzyme substrate kinetics. We present a refined TK1 in vitro assay and method of analysis which address these problems.
Methods: Extracts were prepared of the resected lung lesions from eight patients and assayed for TK1 activity using an in vitro assay modified to account for nonlinearities in extract protein concentration. A separate extract of exponentially growing A549 human lung carcinoma cells was used as a cross-assay control.
Results: In extracts prepared from eight frozen samples of resected human lung lesions, TK1 activity (mean=0.0070+/-0.0077 pmol [(3)H]-TMP/microg protein/minute) was 2 orders of magnitude below that of exponentially growing A549 human lung carcinoma cells (mean=0.1572+/-0.0218 pmol [(3)H]-TMP/microg protein/minute; n=9). TK1 activity was nonlinear with respect to extract protein concentration in both groups, with A549 cell extracts exhibiting evidence of positive cooperativity which could not be explained by the presence of detergents in the cell lysis buffer. Lung tumor extracts demonstrated evidence of negative cooperativity.
Conclusions: The modified TK1 assay takes into account these nonlinearities by averaging the results of several complete time-course curves measured over a range of extract protein concentrations. An extract prepared from exponentially growing A549 cells is included in each assay for use as a cross-assay control. We demonstrate that these modifications allow for the accurate rank ordering of TK1 activity in solid tumors.