Thymidine Kinase-1 Activity of UV Induced B Lymphocytes

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Geoffrey G. Wood and Dr. Kim L. O’Neill, Microbiology and Molecular Biology

The normal life of a eukaryotic cell involves a dynamic struggle between survival and death. When a cell is no longer capable of living, certain pathways are initiated for natural cell death. These “programmed cell deaths” transpire in predictable patterns, and are regulated by many proteins and signals that either activate or inactivate the cell death pathways. Changes in the various homeostatic molecules initiate the apoptotic pathway. However, there are other agents within the cell that regulate these apoptotic triggers. Tumors can arise when these regulatory proteins are mutated, inactivated, or ignored, resulting in the aggressive replicating nature of tumor cells.

Tumor cells show increased activity of thymidine kinase-1 (TK-1) to facilitate their DNA replication needs. TK-1 is a cellular enzyme that that facilitates an alternate pathway for the incorporation of the nucleotide thymidine into DNA by converting thymidine into thymidine monophosphate in the presence of ATP, thereby circumventing the normal de novo synthesis of thymidine triphosphate. Cells that are rapidly replicating their DNA require an upregulation of TK-1 activity to assist in the incorporation of thymidine. Consequently, TK-1 has been shown to be a tumor marker by testing the serum in cancer patients. The scope of this research is to determine the changes in TK-1 activity in apoptotically dying cells compared cells dying of necrosis. We hypothesize that the apoptotic cell will have a lower level of TK-1 activity because the cell has already committed to death and is no longer carrying out DNA replication. In addition, we hypothesize that the media of the necrotic cell samples will have roughly the same TK-1 activity as the control cells because the cellular components of a necrotic cell are spilled out into the environment as the cell dies. Therefore, the necrotic core of some tumors might contribute to the rise of TK-1 activity in cancer patients.

The characteristic features of apoptosis are also useful in its detection. “Membrane blebbing” denotes the phenomenon of an apoptotic cell releasing membrane-bound packages of its intracellular. Another common feature of apoptosis is that the membrane bound phophatidylserine changes its orientation in the membrane, thus flipping out toward the extracellular matrix and exposing the serine residues. Furthermore, to ensure that an apoptotic cell’s genetic information is not incorporated and replicated by neighboring cells, an apoptotic cell will cut its DNA into small (50-300 kb) fragments.

We originally used heat shock as a physical damaging agent to induce apoptosis.2-5 Several cell lines were exposed to varying heat loads to determine the temperature windows for apoptosis and necrosis. We determined that the narrow apoptotic window for the Jurkat cell line ranged from temperatures of 41-45° C, peaking at 43° C, and necrosis occurred at temperatures greater than 45° C. The Jurkat cell line was used because of its high apoptotic and necrotic sensitivity. After heat shock exposure at the defined temperatures, cell extracts of 3×106 cells were prepared for further analysis using the TK-1 enzyme activity radioassay. The results of the TK-1 radioassay showed that the apoptotic cells had nearly one half of the activity of the control cells, while the necrotic cell activity was about 25% the activity of the control sample. We thought that the heat used to induced cell death also denatured the TK-1, and consequently skewed our results away from our original hypothesis.

It is not known if cellular heat shock proteins protect TK-1 denaturation in vivo. Nevertheless, we decided to use other physical damaging agents to induce cell death. Murahashi described the induction of apoptosis using UV radiation with peak emissions of 312 nm.6 The apoptotic window for Jurkat cells using UV radiation was found in similar manner as the heat shock method. Due to complexity, comet assays were replaced with flow cytometry analysis of cells stained with Annexin-V FITC and propidium iodide. Annexin-V FITC is a fluorescent tag that binds to the exposed phophatidylserine of apoptotic cells.

We determined that seven minutes of UV radiation exposure was the ideal damaging time length. Recovery periods after UV exposure that yielded the greatest percentage of apoptotic cells (41.0%) was four hours in a 37°C incubator, and 12 hours incubation for the greatest percentage of necrotic cells (20.3%). Cell extracts for the TK-1 radioassay were prepared as described above. Again, our TK-1 activity results showed a 50% reduction in activity for apoptotic cells compared to the control, and about a 75% reduction in TK-1 activity in necrotic cell extracts.

Apoptotic, necrotic, and control cell cultures containing 5×105 cells were also prepared for the thymidine incorporation assay. This assay was performed to determine the change in radioactive thymidine uptake in Jurkat cells as they went apoptotic or necrotic. As expected, our results showed that significantly less 3H-thymidine was incorporated in the apoptotic and necrotic cells compared to the control.

Our results demonstrated that induction of apoptosis and necrosis leads to a down-regulation of TK-1 activity. We expected the lower levels of 3H-thymidine incorporation in the apoptotic and necrotic cells due to the death of the cell; however, the TK-1 activity of the dying cells was not expected. Apoptotic cells consistently showed nearly one half of the activity of the control cells, while the necrotic cell activity was about 25% the activity of the control sample. We also did not expect the TK-1 activity of the medium of the control, apoptotic, and necrotic cells to show no significant difference in activity. From these results, we now hypothesize that TK-1 is either inactivated or degraded as the cell die; however, we are not sure what happens to the TK-1 in necrotic cells since it is not readily found in the media of necrotic cells. Understandingly, an apoptotic cell digests its DNA into small fragments and no longer has use for TK-1. In our continuing research of TK-1, we have decided to use Western blots with TK-1 monoclonal antibodies to determine if TK is inactivated or degraded. A better understanding of TK-1 regulation could lead to improved therapeutic and diagnostic possibilities.7

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  • Additional contributions from Levi Hilton, Marcos Lee-Hin, and Clifford Ellingson.