In vitro studies on the immunosuppressive effect of 2',2'-difluorodeoxycytidine (dFdC) and its metabolite 2',2'-difluorodeoxyuridine (dFdU).
The effect of 2',2'-difluorodeoxycytidine (dFdC) on in vitro human lymphocyte response was assessed in comparison with that of its major metabolite 2',2'-difluorodeoxyuridine (dFdU). Peripheral blood mononuclear cells (PBMNC) from healthy human volunteers were used for assay of mixed lymphocyte reaction (MLR), blastogenesis and colony forming by PHA. Both substances inhibited mitogen and alloantigen responses of PBMNC in a dose-dependent manner, but dFdU was up to 10,000-fold less potent than its parent compound dFdC. The data indicate that activation by alloantigen is more sensitive to the action of dFdU and dFdC than the response to PHA. Thus, dFdU inhibits MLR-induced response at significantly lower doses than PHA-induced proliferation (IC50 +/- SD, 23.55 +/- 8 microM versus 133.2 +/- 12 microM) (p = 0.0003). dFdC also proved to be about 12.3-fold more potent against alloantigen response compared to PHA-induced proliferation of PBMNC (IC50 +/- SD, 2.28 +/- 0.5 nM versus 28.1 +/- 0.5 nM) (p = 0.0001). To get an insight into the toxic profile of dFdU and dFdC, both substances were additionally tested on the in vitro clonal growth of CD34+ cells. Cells were cultured in methylcellulose in the continuous presence of dFdU and dFdC in doses up to 640 microM and 16 nM, respectively. The results show a marked inhibition of erythroid (BFU-E) and myeloid progenitors (CFU-GM) in a dose-dependent manner, but BFU-E was more sensitive to the action of dFdU and dFdC than CFU-GM (p=0.0001). Compared to T-lymphocytes, however, similar or even higher doses of dFdU and dFdC were required for complete inhibition of colony formation obtained from CD34+ cells. To test the role of deoxycytidine kinase (dCK) in the metabolism of dFdU in comparison to that of dFdC, reversal studies with deoxycytidine (dCyd), the natural substrate of dCK, were performed on dFdU- and dFdC-treated HL-60 cells. The data show that relatively low concentrations of dCyd (10 microM) were sufficient to protect HL-60 cells from cytotoxicity of lethal doses of dFdU (160 microM), whereas 100-fold higher concentrations of deoxycytidine (dCyd) (1 mM) were required for a complete reversal of dFdC-mediated toxicity. This suggests that activation of dFdU is due to its phosphorylation by dCK, but dFdU has low affinity to dCK. These effects of dFdU and dFdC in relation to T-lymphocytes and CD34+ cells suggest their possible use as immunosuppressive agents.
Tiefenthaler
Martin
M
Department of Internal Medicine, University of Innsbruck, Austria.
Hohla
Florian
F
Irschick
Eveline
E
Strasser-Wozak
Elisabeth
E
Bacher
Nicole
N
Mühlmann
Oliver
O
Wein
Wolfgang
W
Konwalinka
Günther
G
D000305:Adrenal Cortex Hormones / Q000494:pharmacology; D018952:Antigens, CD34 / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D003842:Deoxycytidine Kinase / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004359:Drug Therapy, Combination; D005467:Floxuridine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000276:immunology; D008213:Lymphocyte Activation / Q000187:drug effects; D007959:Lymphocyte Culture Test, Mixed; D010835:Phytohemagglutinins / Q000494:pharmacology; D013234:Stem Cells / Q000187:drug effects; D000093542:Gemcitabine
D000305:Adrenal Cortex Hormones; D018952:Antigens, CD34; D007166:Immunosuppressive Agents; D010835:Phytohemagglutinins; D005467:Floxuridine; D003841:Deoxycytidine; D003842:Deoxycytidine Kinase; C084565:2',2'-difluoro-2'-deoxyuridine; D000093542:Gemcitabine