Thymidine utilization abnormality in proliferating lymphocytes and hepatocytes of the woodchuck

Effective incorporation of tritiated thymidine ([H-3]TdR) into proliferatin g lymphocytes is important because [H-3]TdR is a standard label to study pr oliferate T-cell responses. We analyzed the thymidine utilization of woodch uck peripheral blood lymphocytes (PBL) since the [H-3]TdR incorporation ass ay was not applicable to measure proliferative immune responses in the wood chuck, a current major virus/host model for human hepatitis B virus infecti on. Incorporation of [H-3]TdR into DNA as well as the activity of the salva ge pathway enzyme thymidine kinase (TK) of proliferating woodchuck PBL was low compared to human lymphocytes. Furthermore, [H-3]TdR incorporation of p roliferating woodchuck PBL remained residual regardless of the use of metho trexate, an inhibitor of the competitive deoxythymidine monophosphate de no vo synthesis pathway. Using a human probe, specific for the proliferation-a ssociated TK1, we proved the genomic presence and transcription of TK1 sequ ences in various species. TK1 sequences were detected in the genome of huma n, mouse, woodchuck, and chicken specimens. In contrast to proliferating hu man PBL and 3T3 mouse fibroblasts, no TK1 transcript was found in prolifera ting woodchuck PBL and hepatic cells. Transfection experiments with vectors containing the murine or human TK1 and selection assays demonstrated the a bility of woodchuck cells to transcribe TK1 and to express functional TK1 p roteins. Our study characterizes the unique failure of sufficient [H-3]TdR incorporation into proliferating woodchuck cells and demonstrates tritiated adenine and serine as alternative labels to monitor PBL proliferation in t he woodchuck. (C) 2001 Elsevier Science B.V. All rights reserved.