Studies designed to better understand the involvement of cellular resistance to oxidative stress in mechanisms of cellular resistance to cisplatin were undertaken using H2O2-resistant variants of the HA1 Chinese hamster fibroblast cell line. H2O2-resistant cell lines were resistant to clonogenic inactivation mediated by cisplatin with dose modifying factors at 10% survival of 1.5-3.0, relative to HA1 cells. The most cisplatin resistant of these cell lines (OC5) also demonstrated fewer DNA-DNA crosslinks induced by cisplatin, relative to HA1. Since H2O2-resistant cells contained increased catalase activity as well as total glutathione (GSH) content, the involvement of these cellular antioxidants in the resistance to cisplatin toxicity was evaluated. Treatment of HA1 and H2O2-resistant cell lines (OC5, OC14) with 9 mM aminotriazole reduced catalase activity by 60-65% but had no effect on the cytotoxicity of cisplatin. In contrast, treatment with 5 mM buthionine sulfoximine reduced total GSH by 90% and sensitized the cells to cisplatin cytotoxicity. Furthermore, extracellular reaction of GSH with cisplatin prior to treating HA1 cells reduced the toxicity of the compound, indicating that this reaction is capable of participating in the detoxification of cisplatin. These results indicate that cellular adaptation to oxidative stress renders cells resistant to DNA damage as well as to cytotoxicity associated with cisplatin treatment. Furthermore, increases in total GSH content (but not catalase activity) appear to partially account for cisplatin resistance demonstrated by H2O2-resistant cells.

"MCM" was ranked as the DC-generating method yielding highest ("excellent") DCsubtype values .

No abstract

Dendritic cells (DC) and leukaemia derived DC (DCleu) are potent stimulators of anti-leukaemic activity in acute myeloid leukaemia (AML) and can be generated from mononuclear cells in vitro following standard DC/DCleu-generating protocols. With respect to future clinical applications though, DC/DCleu-generating protocols specifically designed for application in a whole-blood-(WB)-environment must be established. Therefore, we developed ten new DC/DCleu-generating protocols (kits; Kit-A/-C/-D/-E/-F/-G/-H/-I/-K/-M) for the generation of DC/DCleu from leukaemic WB, containing calcium-ionophore, granulocyte-macrophage-colony-stimulating-factor (GM-CSF), tumour-necrosis-factor-alpha, prostaglandin-E1 (PGE1), prostaglandin-E2 (PGE2) and/or picibanil (OK-432). All protocols were evaluated regarding their performance in generating DC/DCleu using refined classification and/or ranking systems; DC/DCleu were evaluated regarding their performance in stimulating anti-leukaemic activity using a cytotoxicity fluorolysis assay. Overall, we found the new kits capable to generate (mature) DC/DCleu from leukaemic WB. Through refined classification and ranking systems, we were able to select Kit-I (GM-CSF + OK-432), -K (GM-CSF + PGE2) and -M (GM-CSF + PGE1) as the most efficient kits in generating (mature) DC/DCleu, which are further competent to stimulate immunoreactive cells to show an improved anti-leukaemic cytotoxicity as well. This great performance of Kit-I, -K and -M in mediating DC/DCleu-based anti-leukaemic immunity in a WB-environment in vitro constitutes an important and directive step for translating DC/DCleu-based immunotherapy of AML into clinical application.

During studies to establish why pretreatment of 9L cells with a-difluoromethylornithine (DFMO) enhanced cell killing induced by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) (1) and reduced cell killing induced by cis-platinum (2), we reported, in 1982, an enhancement of BCNU-induced sister chromatid exchanges (SCE's) and a reduction in cis-platinum-induced SCE frequency with DFMO pretreatment (3 killing by DFMO pretreatment had proved certain, however, and the discrepancy was puzzling in view of repeated confirmation of the correlation between reduced cell killing and SCE frequency with cis-platinum (3).New experimental results reported by others (5) show that when DFMO remains in the cell culture medium during the period of BCNU treatment, the procedure enhances the number of BCNU-induced SCE's in 9L cells. When the DFMOcontaining medium is removed and the cells are rinsed before BCNU is added, however, the additional rinsing procedure greatly diminishes or eliminates the enhancement. We believe that our replicate experiments (4) deviated from our original protocol (3) by the introduction of an additional rinsing procedure, thereby accounting for the discrepant results. There is now evidence that slides containing DFMO-treated cells degrade over time (6). Such a degradation over the 5 years between the original scoring of the slides and the rescoring attempts could have contributed to our inability to confirm the original findings.