Ultrastructural morphology and localisation of cisplatin-induced platinum-DNA adducts in a cisplatin-sensitive and -resistant human small cell lung cancer cell line using electron microscopy.

Ultrastructural morphology (transmission electron microscopy) and localisation of cisplatin-induced platinum (Pt)-DNA adducts (immunoelectron microscopy) were analysed in the human small cell lung cancer cell line GLC(4) and its 40-fold in vitro acquired cisplatin-resistant subline GLC(4)-CDDP, which is characterised by, among other things, a decreased DNA platination. Immunolabelling of Pt-DNA adducts was performed with the polyclonal antibody GPt, known to detect the main Pt-containing intrastrand and interstrand DNA adducts. Morphological analysis of GLC(4) and GLC(4)-CDDP at the ultrastructural level showed cells with a high nucleus/cytoplasm ratio with the majority of nuclei containing one or more nucleoli. GLC(4)-CDDP showed, in contrast to GLC(4), an extensive Golgi apparatus and an increased number of mitochondria. DNA platination was detectable in both GLC(4) and GLC(4)-CDDP. Immunoelectron microscopy showed Pt-DNA adducts primarily in the nucleus, preferentially at loci with high-density chromatin (e.g. heterochromatin, pars granulosa around nucleoli, condensed DNA in proliferating and apoptotic cells), and in mitochondria. The level of detectable Pt-DNA adducts was cell cycle status-dependent. In both cell lines, Pt-DNA adduct levels increased from non-dividing interphase cells to dividing cells and were highest in cells undergoing apoptosis. Overall localisation of Pt-DNA adducts was comparable in GLC(4) and GLC(4)-CDDP cells.

ATP- and glutathione-dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1.

The present study was performed to investigate the ability of the multidrug resistance protein (MRPI) to transport different cationic substrates in comparison with MDR1-P-glycoprotein (MDR1). Transport studies were performed with isolated membrane vesicles from in vitro selected multidrug resistant cell lines overexpressing MDR1 (A2780AD) or MRP1 (GLC4/Adr) and a MRP1-transfected cell line (S1(MRP)). As substrates we used 3H-labelled derivatives of the hydrophilic monoquaternary cation N-(4',4'-azo-in-pentyl)-21-deoxy-ajmalinium (APDA), the basic drug vincristine and the more hydrophobic basic drug daunorubicin. All three are known MDR1-substrates. MRP1 did not mediate transport of these substrates per se. In the presence of reduced glutathione (GSH), there was an ATP-dependent uptake of vincristine and daunorubicin, but not of APDA, into GLC4/Adr and S1(MRP) membrane vesicles which could be inhibited by the MRP1-inhibitor MK571. ATP- and GSH-dependent transport of daunorubicin and vincristine into GLC4/Adr membrane vesicles was inhibited by the MRP1-specific monoclonal antibody QCRL-3. MRP1-mediated daunorubicin transport rates were dependent on the concentration of GSH and were maximal at concentrations > or = 10 mM. The apparent KM value for GSH was 2.7 mM. Transport of daunorubicin in the presence of 10 mM GSH was inhibited by MK571 with an IC50 of 0.4 microM. In conclusion, these results demonstrate that MRP1 transports vincristine and daunorubicin in an ATP- and GSH-dependent manner. APDA is not a substrate for MRP1.

Transport of glutathione conjugates into secretory vesicles is mediated by the multidrug-resistance protein 1.

Intracellular glutathione-conjugate transport was evaluated in the human small cell lung carcinoma cell line GLC4 with low multidrug resistance protein (MRP1) expression and its 300x doxorubicin-resistant, MRP1-over-expressing, GLC4-Adr subline. Transport of non-toxic concentrations of monochlorobimane and 5-chloro-methyl fluorescein diacetate was evaluated using fluorescence microscopy. After exposure to these compounds, fluorescence was observed especially in intracellular vesicles in GLC4-Adr. Immunotransmission electron microscopy showed that MRP1 was present in the vesicle membranes and plasma membrane, while inside the vesicles the glutathione conjugate of 1-chloro-2,4-dinitrobenzene could be detected. Experiments with brefeldin A, which induces arrest in vesicle release from the Golgi complex, indicated that these vesicles may originate from the trans-Golgi network. In GLC4-Adr cells, doxorubicin also was transported in vesicles, with an arrest in vesicle release from the Golgi complex. Our results indicate that MRP1 functions as a glutathione-conjugate transporter not only at the plasma membrane but also in intracellular secretory vesicles.

Differential expression of DNA topoisomerase II alpha and -beta in P-gp and MRP-negative VM26, mAMSA and mitoxantrone-resistant sublines of the human SCLC cell line GLC4.

Sublines of the human small-cell lung carcinoma (SCLC) cell line GLC4 with acquired resistance to teniposide, amsacrine and mitoxantrone (GLC4/VM20x, GLC4/AM3x and GLC4/MIT60x, respectively) were derived to study the contribution of DNA topoisomerase II alpha and -beta (TopoII alpha and -beta) to resistance for TopoII-targeting drugs. The cell lines did not overexpress P-glycoprotein or the multidrug resistance-associated protein but were cross-resistant to other TopoII drugs. GLC4/VM20x showed a major decrease in TopoII alpha protein (54%; for all assays presented in this paper the GLC4 level was defined to be 100%) without reduction in TopoII beta protein; GLC4/AM3x showed only a major decrease in TopoII beta protein (to 18%) and not in TopoII alpha. In GLC4/MIT60x, the TopoII alpha and -beta protein levels were both decreased (TopoII alpha to 31%; TopoII beta protein was undetectable). The decrease in TopoII alpha protein in GLC4/VM20x and GLC4/MIT60x, was mediated by decreased TopoII alpha mRNA levels. Loss of TopoII alpha gene copies contributed to the mRNA decrease in these cell lines. Only in the GLC4/MIT60x cell line was an accumulation defect observed for the drug against which the cell line was made resistant. In conclusion, TopoII alpha and -beta levels were decreased differentially in the resistant cell lines, suggesting that resistance to these drugs may be mediated by a decrease in a specific isozyme.