SUPPRESSION OF INTERLEUKIN-2 AND INTERLEUKIN-2 RECEPTOR BIOSYNTHESIS BY GOLD COMPOUNDS IN INVITRO ACTIVATED HUMAN PERIPHERAL-BLOOD MONONUCLEAR-CELLS

Objective. To further investigate the mechanism of action of gold compounds by studying their effects on interleukin-2 (IL-2) and IL-2 receptor (IL-2R) biosynthesis. Methods. We cultured phytohemagglutinin- or anti-CD3 antibody-activated normal peripheral blood mononuclear cells (PBMC), as well as the erythroleukemic K562 cell line, in the presence of gold sodium thiomalate or auranofin. Tritiated thymidine incorporation assays, cytotoxicity assays, immunofluorescence analysis, enzyme-linked immunosorbent assay, Northern blot, and RNA dot-blot hybridization were used. Results. Gold compounds, at concentrations attainable in vivo, inhibited the proliferation of normal PBMC, with no evidence of direct cytotoxicity. This inhibitory effect was associated with a dose-dependent suppression of both IL-2 and IL-2R messenger RNA accumulation. In contrast, the same concentrations of gold compounds failed to inhibit the spontaneous proliferation of the IL-2-independent K562 cells. Conclusion. Our findings suggest an IL-2/IL-2R-mediated mechanism for suppression of lymphocyte proliferation by gold compounds, which might account for the immunomodulatory effects of gold in patients with rheumatoid arthritis

INVIVO FORMATION AND REPAIR OF O-6-METHYLGUANINE IN HUMAN-LEUKOCYTE DNA AFTER INTRAVENOUS EXPOSURE TO DACARBAZINE

Blood leukocyte DNA obtained from 11 Hodgkin’s disease patients undergoing ABVD chemotherapy was analysed for the presence of the precarcinogenic adduct O6-methyl-guanine (O6-meG) at various times (1-2 h up to 49 h) after i.v. treatment with the methylating drug dacarbazine. Adduct formation was detected in all but one of the patients examined at levels ranging up to 0.45 fmol/mu-g DNA (7.2 x 10(-7) mol/mol guanine). The levels of the adduct decreased by approximately 30% over the 24 h following exposure and were usually not detectable 49 h after exposure. In five out of seven individuals examined after more than one treatment, consistent methylation responses were noted, while in the remaining two cases the responses were mixed. No correlation between the extent of adduct formation and lymphocyte levels of the repair enzyme O6-alkylguanine - DNA alkyltransferase was observed. The average extent of O6-meG formation 1 h after dacarbazine treatment was (4.3 +/- 3.1) x 10(-2) fmol/mu-g DNA per mg/kg dose [(1.2 +/- 0.8) x 10(-3 fmol/mu-g DNA per mg/m2 dose)]. Following exposure of rats to similar doses of dacarbazine, the corresponding levels of adduct in blood leukocyte DNA were 1.1 x 10(-2) fmol/mu-g DNA per mg/kg dose (2.6 x 10(-3) fmol/mu-g DNA per mg/m2 dose)

Intra- and intercellular variations in the repair efficiency of O-6-methylguanine, and their contribution to kinetic complexity

Following administration to rats of various doses of N-nitrosodimethylamine (NDMA), O-6-methylguanine (O-6-meG) was lost from the DNA of four tissues (liver, white blood cells, lymph nodes, bone marrow) over two, sharply demarcated phases with substantially differing repair rates. Repair during each phase followed approximately first-order kinetics in O-6-meG, even after a high dose of NDMA which caused substantial depletion of O-6-alkylguanine-DNA alkyltransferase (AGT), a suicide repair protein. This is compatible with rate-determining adduct repair being brought about by a distinct, minor pool of AGT molecules which is rapidly replenished by de novo AGT synthesis. Similar biphasic repair kinetics were also observed in HepG2 cells treated in vitro with NDMA. In this case, the first phase of repair was inhibited by alpha-amanitin, an inhibitor of RNA polymerase II-mediated transcription. However, no dependence on transcriptional activity was found when O-6-meG repair in specific gene sequences with different transcriptional status in rat liver was examined, suggesting that the effects of a-amanitin in HepG2 cells did not reflect inhibition of preferential repair of transcribed sequences. Repair was also examined in rat liver hepatocytes and non-parenchymal cells separately after administration of NDMA at non-AGT depleting doses. Within each cell-population, the repair followed single phase, first-order kinetics, with adduct loss from AGT-rich hepatocytes being significantly faster than from the relatively AGT-deficient non-parenchymal cells. In conclusion, differences in the AGT content of different cell subpopulations in the liver (and probably in other tissues), as well as additional cellular factors affecting repair efficiency, appear to determine the observed variation in the kinetics of repair of O-6-meG. The additional cellular factors involved appear not to be related to the transcriptional state of the sequences being repaired, but may reflect different states of chromatin condensation. (C) 2004 Elsevier B.V. All rights reserved