Thymidine utilization abnormality in proliferating lymphocytes and hepatocytes of the woodchuck

Abstract

Effective incorporation of tritiated thymidine ([ 3H]TdR) into proliferating lymphocytes is important because [ 3H]TdR is a standard label to study proliferate T-cell responses. We analyzed the thymidine utilization of woodchuck peripheral blood lymphocytes (PBL) since the [ 3H]TdR incorporation assay was not applicable to measure proliferative immune responses in the woodchuck, a current major virus/host model for human hepatitis B virus infection. Incorporation of [ 3H]TdR into DNA as well as the activity of the salvage pathway enzyme thymidine kinase (TK) of proliferating woodchuck PBL was low compared to human lymphocytes. Furthermore, [ 3H]TdR incorporation of proliferating woodchuck PBL remained residual regardless of the use of methotrexate, an inhibitor of the competitive deoxythymidine monophosphate de novo synthesis pathway. Using a human probe, specific for the proliferation-associated TK1, we proved the genomic presence and transcription of TK1 sequences in various species. TK1 sequences were detected in the genome of human, mouse, woodchuck, and chicken specimens. In contrast to proliferating human PBL and 3T3 mouse fibroblasts, no TK1 transcript was found in proliferating woodchuck PBL and hepatic cells. Transfection experiments with vectors containing the murine or human TK1 and selection assays demonstrated the ability of woodchuck cells to transcribe TK1 and to express functional TK1 proteins. Our study characterizes the unique failure of sufficient [ 3H]TdR incorporation into proliferating woodchuck cells and demonstrates tritiated adenine and serine as alternative labels to monitor PBL proliferation in the woodchuck.