PubMed · 9775172
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M Auvinet, S Dahan, C Dingremont, S Ollier, H Juchet, P Arlet. 1998. [Yellow nails].. https://doi.org/10.1016/s0248-8663(98)80108-2
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7-Ethyl-10-hydroxycamptothecine (SN-38) is the active metabolite of the topoisomerase I inhibitor and antineoplastic agent, irinotecan (CPT-11). Here, we present a new and sensitive reversed-phase high-performance liquid chromatographic method for the determination of SN-38 in human plasma samples. Sample pretreatment involves a protein precipitation of 1-mL samples with 2 mL of acetonitrile, followed by a one-step solvent extraction with 5 mL of chloroform, with camptothecine used as internal standard. Chromatographic separation was achieved on an analytical column packed with Hypersil ODS material (100 x 4.6 mm i.d., 5 microm P.S.), and isocratic elution with a mixture of acetonitrile:0.1 M ammonium acetate containing 10 mM tetrabutylammonium sulfate (23:77, v/v), pH 5.3 (hydrochloric acid). The column effluent was monitored at excitation and emission wavelengths of 380 and 556 nm, respectively. The limit of quantitation of the method presented was at the low femtomole level ( approximately 8.4 fmol; equivalent to 5 pg/mL), with the standard curves being linear over nearly three orders of magnitude. Intraassay precision was <9%, while interassay variations were between 2 and 5%. The extraction efficiency was concentration independent and averaged 88.0 +/- 14.3% (mean +/- standard deviation; n = 59). The described method will be used in future studies to assess the extent of enterohepatic recirculation of SN-38 in cancer patients following intravenous CPT-11 treatment.
During apoptosis, the cell actively dismantles itself and reduces cell size by the formation and pinching off of portions of cytoplasm and nucleus as "apoptotic bodies." We have combined our previously established quantitative assay relating the amount of release of [3H]-membrane lipid to the degree of apoptosis with electron microscopy (EM) at a series of timepoints to study apoptosis of lymphoid cells exposed to vincristine or etoposide. We find that the [3H]-membrane lipid release assay correlates well with EM studies showing the formation and release of apoptotic bodies and cell death, and both processes are regulated in parallel by inducers or inhibitors of apoptosis. Overexpression of Bcl-2 or inhibition of caspases by DEVD inhibited equally well the activation of caspases as indicated by PARP cleavage. They also inhibited [3H]-membrane lipid release and release of apoptotic bodies. EM showed that cells overexpressing Bcl-2 displayed near-normal morphology and viability in response to vincristine or etoposide. In contrast, DEVD did not prevent cell death. Although DEVD inhibited the chromatin condensation, PARP cleavage, release of apoptotic bodies, and release of labeled lipid, DEVD-treated cells showed accumulation of heterogeneous vesicles trapped in the condensed cytoplasm. These results suggest that inhibition of caspases arrested the maturation and release of apoptotic bodies. Our results also imply that Bcl-2 regulates processes in addition to caspase activation.
It has been suggested that the ratio of Bcl-2 family proapoptotic proteins to antiapoptotic proteins determines the sensitivity of leukemic cells to apoptosis. However, it is believed that Bcl-2 family proteins exert their function on apoptosis only when they target to the mitochondrial outer membrane. The vinblastine-resistant T-lymphoblastic leukemic cell line CEM/VLB100 has increased sensitivity to tumor necrosis factor-alpha (TNF-alpha)-induced cytochrome c release, mitochondrial respiratory inhibition, and consequently apoptosis, compared with parental CEM cells. However, there was no difference between the two cell lines in the expression of Bcl-2 family proteins Bcl-2, Bcl-XL, Bcl-XS, Bad, and Bax at the whole cell level, as analyzed by Western blotting. Bcl-2 mainly located to mitochondria and light membrane as a membrane-bound protein, whereas Bcl-XL was located in both mitochondria and cytosol. Similar levels of both Bcl-2 and Bcl-XL were present in the resting mitochondria of the two cell lines. Although the proapoptotic proteins Bcl-XS, Bad, and Bax were mainly located in the cytosol, CEM/VLB100 mitochondria expressed higher levels of these proapoptotic proteins. Subcellular redistribution of the Bcl-2 family proteins was detected in a cell-free system by both Western blotting and flow cytometry after exposure to TNF-alpha. The levels of Bcl-2 family proteins were not altered at the whole cell level by TNF-alpha. However, after exposure to TNF-alpha, Bax, Bad, and Bcl-XS translocated from the cytosol to the mitochondria of both cell lines. An increase in Bcl-2 levels was observed in CEM mitochondria, which showed resistance to TNF-alpha-induced cytochrome c release. By contrast, decreased mitochondrial Bcl-2 was observed in CEM/VLB100 cells, which released cytochrome c from the mitochondria and underwent apoptosis as detected by fluorescence microscopy. We conclude that mitochondrial levels of Bcl-2 family proteins may determine the sensitivity of leukemic cells to apoptosis and that, furthermore, these levels may change rapidly after exposure of cells to toxic stimuli.