A deficiency in nucleoside salvage impairs murine lymphocyte development, homeostasis, and survival.

The homeostasis of the immune system is tightly controlled by both cell-extrinsic and -intrinsic mechanisms. These regulators, not all known to date, drive cells in and out of quiescence when and where required to allow the immune system to function. In this article, we describe a deficiency in deoxycytidine kinase (DCK), one of the major enzymes of the nucleoside salvage pathway, which affects peripheral T cell homeostatic proliferation and survival. As a result of an N-ethyl-N-nitrosourea-induced mutation in the last α helix of DCK, a functionally null protein has been generated in the mouse and affects the composition of the hematopoietic system. Both B and T lymphocyte development is impaired, leading to a state of chronic lymphopenia and to a significant increase in the number of myeloid cells and erythrocytes. In the periphery, we found that mutant lymphocytes adopt a CD44(high)CD62L(low) memory phenotype, with high levels of proliferation and apoptosis. These phenotypes are notably the result of a cell-extrinsic-driven lymphopenia-induced proliferation as wild-type cells transferred into DCK-deficient recipients adopt the same profile. In addition, DCK also regulates lymphocyte quiescence in a cell-intrinsic manner. These data establish dCK as a new regulator of hematopoietic integrity and lymphocyte quiescence and survival.

Quantitative immunoassay of human deoxycytidine kinase in malignant cells.

Deoxycytidine kinase (dCK) is necessary for the activity of several nucleosides used for the chemotherapy of cancer and AIDS. However, the measurement of dCK catalytic activity in crude cell extracts may be imprecise, due to the presence of phosphatases and nucleotidases that degrade the enzyme products. We describe a simple immunoassay for dCK that can measure accurately as little as 5 ng enzyme protein in crude tissue extracts. The assay enabled us to show (i) that mutant cells deficient in dCK activity lack immunoreactive dCK protein, (ii) that dCK catalytic activity and immunoreactivity correlate closely in human tumors, and (iii) that immunoreactive dCK is particularly high in lymphocytes and lymphoid malignancies, although certain solid tumors may also contain the enzyme. The immunoassay of dCK could prove useful in the selection and monitoring of patients who are being treated with nucleosides that are activated by this enzyme.

Deoxynucleoside phosphorylating enzymes in monkey and human tissues show great similarities, while mouse deoxycytidine kinase has a different substrate specificity.

Three key enzymes in the anabolic phosphorylation of deoxyribonucleosides and deoxyribonucleoside analogs were purified i.e. cytoplasmic thymidine kinase (TK1), mitochondrial thymidine kinase (TK2) and cytoplasmic deoxycytidine kinase (dCK) from human, mouse and monkey liver and spleen. Their subunit structure and substrate specificities were compared. Extensive purification of TK1 and dCK from mouse spleen and TK2 from mouse and monkey livers revealed major polypeptide bands of 25, 30 and 28 kD, respectively, on sodium dodecyl sulphate-polyacrylamide gel electrophoresis which are very similar to the subunit molecular weights of the corresponding human enzymes. Affinity purified polyclonal antibodies against human dCK also cross-reacted with 30 kD bands in extracts from both mouse and monkey spleen. Thus, the molecular weights of the subunits of these three enzymes appeared to be very similar in all three species. TK1 and TK2 from these different sources appeared to have similar substrate specificities against several deoxyribonucleoside analogs. However, mouse dCK differed significantly from monkey and human dCK in its capacity to phosphorylate dAdo and 2',3'-dideoxycytidine (ddCyd) with a Vmax approximately 10-fold lower than that of the two latter enzymes. The Km and Vmax values for dCyd and arabinocytosine appeared to be very similar with the enzymes from all three species. The fact that mouse dCK shows low activity with dAdo and ddCyd explains differences reported previously in the metabolism of dAdo and ddCyd in mouse compared to that in human lymphocytes. These results argue against the use of mice as model systems for human deoxynucleoside metabolism.

Immunochemical characterization of pyrimidine kinase induced by varicella-zoster virus.

Thymidine kinase (TK) induced by varicella-zoster virus (VZV) was precipitated with ammonium sulphate and purified by Sephadex G-150, QAE-Sephadex and Blue Sepharose column chromatographies. The purified TK fraction also contained deoxycytidine kinase (dCK) activity and a 35000 mol. wt. (35K) polypeptide as a major component. The TK and dCK activities were both neutralized by anti-VZV serum. Antiserum to an extract of cells infected with a bromodeoxyuridine (BUdR)-resistant mutant virus contained no antibody to the viral TK and dCK activities or to the 35K polypeptide. Antiserum to the purified viral TK fraction was prepared and absorbed with a lysate of BUdR-resistant mutant virus-infected cells. The resulting absorbed antiserum (anti-vTK serum) neutralized the viral activities of both TK and dCK, and specifically immunoprecipitated a 35K polypeptide from the lysate of parental virus-infected cells, but did not immunoprecipitate any detectable polypeptide from cells infected with BUdR-resistant mutant virus. Anti-vTK serum stained mainly the nuclei of cells infected with the parental virus strain, but did not stain those infected with BUdR-resistant mutant virus by an indirect fluorescent antibody test. These results suggest that the 35K polypeptide produced in VZV-infected cells is responsible for the viral TK and dCK activities, and that the TK and dCK are mainly localized in the nuclei of infected cells.