Cellular and biochemical characterization of VX-710 as a chemosensitizer: reversal of P-glycoprotein-mediated multidrug resistance in vitro

VX-710 or (S)-N[2-Oxo-2-(3,4,5-trimethoxyphenyl)acetyl]-piperidine-2-carboxylic acid 1,7-bis(3-pyridyl)-4-heptyl ester, a novel non-macrocyclic ligand of the FK506-binding protein FKBP12, was evaluated for its ability to reverse P-glycoprotein-mediated multidrug resistance in vitro. VX-710 at 0.5-5 microM restored sensitivity of a variety of multidrug resistant cells to the cytotoxic action of doxorubicin, vincristine, etoposide or paclitaxel, including drug-selected human myeloma and epithelial carcinoma cells, and human MDR1 cDNA-transfected mouse leukemia and fibroblast cells. Uptake experiments showed that VX-710 at 0.5-2.5 microM fully restored intracellular accumulation of [14C]doxorubicin in multidrug resistant cells, suggesting that VX-710 inhibits the drug efflux activity of P-glycoprotein. VX-710 effectively inhibited photoaffinity labeling of P-glycoprotein by [3H]azidopine or [125I]iodoaryl azidoprazosin with EC50 values of 0.75 and 0.55 microM. Moreover, P-glycoprotein was specifically labeled by a tritiated photoaffinity analog of VX-710 and unlabeled VX-710 inhibited analog binding with an EC50 of 0.75 microM. VX-710 also stimulated the vanadate-inhibitable P-glycoprotein ATPase activity 2- to 3-fold in a concentration-dependent manner with an apparent k(a) of 0.1 microM. These data indicate that a direct, high-affinity interaction of VX-710 with P-glycoprotein prevents efflux of cytotoxic drugs by the MDR1 gene product in multidrug resistant tumor cells.     

Expression of the antisense cDNA for protein kinase C alpha attenuates resistance in doxorubicin-resistant MCF-7 breast carcinoma cells

Multidrug resistance is functionally associated with the expression of a plasma membrane energy-dependent drug efflux pump termed P-glycoprotein, the product of the mdr1 gene. Transfection of P-glycoprotein-expressing doxorubicin-resistant MCF-7 cells with an expression vector containing the cDNA for protein kinase C alpha in the antisense orientation reduces protein kinase C alpha levels and decreases total protein kinase C activity by 75%. This is accompanied by reduced phosphorylation of P-glycoprotein, a 2-fold increase in drug retention, and a 3-fold increase in doxorubicin cytotoxicity. These results provide further evidence that protein kinase C alpha can positively regulate multidrug resistance in MCF-7 cells through posttranslational phosphorylation of P-glycoprotein.     

Decreased drug accumulation without increased drug efflux in a novel MRP-overexpressing multidrug-resistant cell line

KB/7D cells represent a multidrug-resistant subclone of human nasopharyngeal carcinoma KB cells generated by continuous exposure to the topoisomerase II inhibitor VP-16 (etoposide). KB/7D cells also show cross-resistance to doxorubicin and vincristine. Phenotypic traits of the cell line include a 2-fold decrease in topoisomerase II levels and a decrease in the uptake of VP-16 without an increase in the rate of drug efflux or expression of P-glycoprotein, suggesting a novel mechanism associated with the uptake of anticancer drugs. This study demonstrated that the multidrug-resistance associated protein (MRP) is overexpressed in KB/7D cells, and that the loss of resistance in revertant cells correlates with the loss of MRP. The resistance to VP-16 and doxorubicin could be overcome, partially, and resistance to vincristine could be overcome completely, by the L-enantiomer of verapamil, but not by the D-enantiomer or by BIBW 22 (4-[N-(2-hydroxy-2-methyl-propyl)-ethanolamino]-2,7-bis[cis-2,6-++ +dimethylmorpholino)-6-phenylpteridin), an inhibitor of MDR-1. L-Verapamil was shown to be significantly more potent than D-verapamil in modulating the accumulation defect in KB/7D cells towards doxorubicin, as measured by flow cytometry and confocal microscopy, and towards VP-16, as measured by increases in protein-linked DNA strand breaks. This suggests that KB/7D cells are multidrug resistant due to decreases in topoisomerase II levels and the overexpression of MRP, that MRP leads to a decrease in drug accumulation, and that L-verapamil can modulate the MRP-associated accumulation defect and drug-resistance phenotype. This contrasts with previous studies that suggest that MRP causes multidrug resistance by exporting cytotoxic drugs out of the cell and that did not show modulation of MRP by verapamil.     

Cellular and karyotypic characterization of two doxorubicin resistant cell lines isolated from the same parental human leukemia cell line

Separate mechanisms underlying the multidrug resistant (MDR) phenotype were identified in 2 independent approaches to select tumour cells resistant to low concentrations of doxorubicin (Dox) from the sensitive T cell leukemia cell line CCRF-CEM. The CEM/A7 cell line was selected at an initial concentration of 0.005 microgram/ml of Dox and maintained at 0.07 microgram/ml. In contrast, the CEM/A5 line was selected using an initial concentration of 0.01 microgram/ml and maintained in Dox at a concentration of 0.05 microgram/ml. P-glycoprotein expression was demonstrated in the CEM/A7 line but not the CEM/A5 line. Amplification of the mdrI gene was not observed in the CEM/A7 cell line. Both cell lines showed cross-resistance to a number of structurally unrelated cytotoxic drugs including anthracyclines and etoposide (VP-16), although only the CEM/A7 line was cross resistant to Vinca alkaloids. Immunoblots of total cell lysates of the CEM/A5 line have revealed almost undetectable levels of topoisomerase II alpha and beta in this line. Cytogenetic analyses of both lines revealed numerous karyotypic abnormalities which were present in the parental cell line as well as both resistant cell lines. The CEM/A7 line also demonstrated a duplication of part of the long arm of chromosome 7 which included the region containing the mdrI gene, a finding not seen in the parental or CEM/A5 line. CEM/A5, however, demonstrated an abnormality of chromosome 7, outside the region of the mdrI gene, and it also contained a deletion of the short arm of chromosome 2. Abnormalities in this latter region of genome have been associated with non-P-glycoprotein-mediated MDR.     

Handling of doxorubicin by the LLC-PK1 kidney epithelial cell line

The characteristics of doxorubicin handling have been studied in the cultured kidney epithelial cell line LLC-PK1, which has structure and function similar to those of renal tubular cells and expresses P-glycoprotein. The uptake of doxorubicin by LLC-PK1 cells was time dependent, reaching a steady state at about 4 hr, and reduced at low temperature; the initial uptake was saturable. The efflux of doxorubicin from LLC-PK1 cells was also temperature dependent but, even at 37 degrees C, a significant percentage of the drug remained associated with the cells after 180 min, which suggests a strong cellular binding, and the fluorescence microscopy revealed that the drug was concentrated in intracellular organelles. Substances that are substrates for P-glycoprotein, such as verapamil, vinblastine, vincristine and quinidine, significantly increased doxorubicin concentrations in LLC-PK1 cells. Similar results were obtained with the metabolic inhibitors sodium metavanadate and 2,4-dinitrophenol. On the other hand, the uptake was not affected by the classic organic cation transport drugs cimetidine, decynium 22 or decynium 24, nor by the organic anion drug probenecid. These results indicate that, in LLC-PK1 cells, doxorubicin enters by passive diffusion, is trapped in intracellular organelles and then is extruded from cells by a mechanism that probably involves P-glycoprotein. On the contrary, substances that interfere with the renal organic cation or anion secretory system have no effect on doxorubicin net accumulation in these cells.     

Role of P-glycoprotein in colchicine and vinblastine cellular kinetics in an immortalized rat brain microvessel endothelial cell line

Uptake and efflux of colchicine and vinblastine, whose effects are related to their high-affinity binding to tubulin, were studied in the immortalized rat brain microvessel endothelial cell line RBE4. At 10 nM extracellular drug concentration, uptake equilibrium was approached at 45 hr for colchicine, but at only 3.5 hr for vinblastine. After 1 hr preincubation with 200 nM colchicine or vinblastine, drug efflux fitted biexponential kinetics with an initial fast phase (half-life = 2.2 min and 9.6 min, respectively) and a later slow phase (half-life = 3.6 hr and 1.8 hr, respectively). After 6 hr preincubation with 200 nM colchicine, only the slow phase (half-life = 3.6 hr) could be observed. The colchicine and vinblastine uptake rate was increased by cyclosporin A, an inhibitor of the drug efflux pump P-glycoprotein, which is expressed at the blood-brain barrier. Whereas cyclosporin A decreased vinblastine efflux, its effect on colchicine efflux was apparent after only 13 hr washout and was associated with the re-uptake by cells of colchicine molecules. Differences in uptake kinetics of colchicine and vinblastine could be related to differences in their lipid solubility, and mainly in their binding affinities to tubulin. Differences in efflux kinetics could in addition be explained by the involvement of P-glycoprotein in the efflux of vinblastine, whereas efflux of colchicine was not influenced by this pump. Indeed, binding of colchicine to tubulin would imply that most intracellular colchicine may be inaccessible to P-glycoprotein. In the case of a cytotoxic drug such as colchicine, which is tightly bound to intracellular receptors, the role of P-glycoprotein within the blood-brain barrier would be more to protect the brain against entry of this drug than to detoxify the brain by its extraction.     

Characterization of newly established human myeloid leukemia cell line (KF-19) and its drug resistant sublines

A new human myeloid leukemia cell line, designated KF-19, and its drug resistant sublines have been established. The KF-19 cell line was established from the pericardial effusion of a patient with acute myeloid leukemia clinically resistant to chemotherapy and KF-19 cells were characterized by expression of myeloid markers and differentiation into neutrophil- and macrophage-like cells upon optimal stimulations. KF-19AraC, KF-19ADR and KF-19VCR were established as sublines resistant to cytosine arabinoside (AraC), adriamycin (ADR) and vincristine (VCR), respectively. Efflux of the corresponding drugs was documented in each cell line. Expression of the MDR1 gene and the P-glycoprotein was found only in KF-19ADR, which showed a cross resistance to anthracyclines and vinca alkaloids; this resistance was reversed by verapamil or cyclosporin A. KF-19VCR lacking MDR1 gene and P-glycoprotein expression showed only resistance to vinca alkaloids, which was partially reversed by verapamil and cyclosporin A. Unexpectedly, KF-19ADR and KF-19VCR displayed cross resistance to AraC, despite lack of alterations of deoxycytidine kinase (dCK) and deaminase (dA) activities. KF-19AraC showed an efflux of AraC as well as a decreased level of dCK, but not of dA. In addition, KF-19AraC showed cross resistance to VCR in the efflux assay. The cell lines reported herein will provide new aspects on the mechanisms of drug resistance in leukemic cells.     

Correlations among Epworth Sleepiness Scale scores, multiple sleep latency tests and psychological symptoms

Mouse leukemic cell subline L1210/VCR exerts expressive multidrug resistance (MDR) that is mediated by P-glycoprotein. Cells originally adapted to vincristine are also extremely resistant to doxorubicin. Resistance to both vincristine and doxorubicin is connected with depression of drug uptake. While resistance of L1210 cells to vincristine could be reversed by verapamil as chemosensitizer, resistance of cells to doxorubicin was insensitive to verapamil. Action of verapamil (well-known inhibitor of PGP activity) on multidrug resistance was often used as evidence that MDR is mediated by PGP. From this point it may be possible that the resistance of L1210/VCR cells to vincristine is mediated by PGP and the resistance to doxorubicin is mediated by other PGP-independent system. Another and more probable explanation of different effect of verapamil on resistance of L1210/VCR cells to vincristine and doxorubicin may be deduced from the following fact: Using UV spectroscopy we found that doxorubicin dissolved in water buffered medium interacts effectively with verapamil. This interaction may be responsible for the decrease of concentration of both drugs in free effective form and consequently for higher survival of cells. In contrast to doxorubicin vincristine does not give any interaction with verapamil that is measurable by UV spectroscopy and resistance of L1210/VCR cells to vincristine may be fully reversed by verapamil.     

Doxorubicin-induced alterations of c-myc and c-jun gene expression in rat glioblastoma cells: role of c-jun in drug resistance and cell death

We studied the effect of doxorubicin on the expression of c-myc and c-jun in the rat glioblastoma cell line C6 and its doxorubicin-resistant variant C6 0.5, at equitoxic exposures. For quantitation, the mRNA levels of these oncogenes were related to those of two domestic genes, beta-actin and glyceraldehyde phosphate dehydrogenase. After a transient overexpression of the genes during the first hour of incubation, there was a selective, dose-dependent down-regulation of both genes by doxorubicin in the sensitive cells. In the resistant cell line, c-myc expression was also decreased in response to doxorubicin incubation, but the expression of c-jun remained unchanged over the whole range of concentrations. In contrast, vincristine had no effect on the amounts of c-myc and c-jun mRNAs in either line. The effect of doxorubicin on the mRNA levels of c-jun was also observed on the JUN proteins by immunoblotting, but the MYC protein levels remained unchanged upon doxorubicin treatment. There was a significant correlation between the levels of c-myc and c-jun gene expression and the degree of growth inhibition induced by doxorubicin. In addition, doxorubicin induced a fragmentation of DNA in sensitive cells, but not in resistant cells, thus revealing a resistance to apoptosis in this line. Doxorubicin-induced cell death did not appear to be mediated by p53 in either cell line.     

Characterization and chemosensitivity of a human epithelioid sarcoma cell line (SARCCR 2)

A new cell line was derived from the epithelioid sarcoma of a Caucasian woman who had previously received chemotherapy. The cells grew as an adherent monolayer, with a doubling time of 28 hr and had mainly epithelial morphology, but with areas of mesenchymal-like cytoplasmic extensions. The cells were tumorigenic in nude mice, with a short growth time, and a doubling time of 8 days. The cell line showed over-expression of P-glycoprotein by Western blot analysis, and its sensitivity to doxorubicin and vincristine was low. This sensitivity could be enhanced by reversants of multidrug resistance (MDR), such as cyclosporin or verapamil. This cell line constitutes an excellent model for studying compounds able to reverse MDR.     

Development and characterisation of novel human multidrug resistant mammary carcinoma lines in vitro and in vivo

Clinical chemotherapy of breast carcinomas must be considered insufficient, mainly due to the appearance of drug resistance. The multidrug resistance (MDR) phenotype, either intrinsically occurring or acquired, e.g., against a panel of different antineoplastic drugs, is discussed in relation to several MDR-associated genes such as the MDR-gene mdr1 encoding the P-glycoprotein (PGP), the MRP gene (multidrug resistance protein) encoding an MDR-related protein or the LRP gene encoding the lung resistance protein. Numerous experimental and clinical approaches aiming at reversing resistance require well-characterised in vitro and in vivo models. The aim of our work was to develop multidrug resistant sublines from human xenotransplanted breast carcinomas, in addition to the broadly used line MCF-7 and its multidrug resistant subline MCF-7/AdrR. MDR was induced in vitro with increasing concentrations of Adriablastin (ADR) for several weeks, resulting in a 3.5- to 35-fold increase in IC50 values using the MTT-test. Cell lines were cross-resistant toward another MDR-related drug, vincristine, but remained sensitive to non-MDR-related compounds such as cisplatin and methotrexate. The resistance toward Adriamycin and vincristine was confirmed in vivo by a lack of tumour growth inhibition in the nude mouse system. Gene expression data for the mdr1/PGP, MRP/MRP and LRP/LRP on both the mRNA (RT-PCR) and the protein levels (immunoflow cytometry) demonstrated that induction of mdr1 gene expression was responsible for the acquired MDR phenotype. Rhodamine efflux data, indicated by PGP overexpression, underlined the development of this MDR mechanism in the newly established breast carcinoma lines MT-1/ADR, MT-3/ADR and MaTu/ADR.     

Respiratory function tests in the newborn and the infant

AIM: To study the mechanisms of the resistance to harringtonine (Har) in the HL60 cells. METHODS: Growth inhibition, karyotype analysis, flow cytometry, Western blotting and polymerase chain reaction. RESULTS: The Har-resistant HL60 cell line, named HR20, showed cross resistance to homoharringtonine, doxorubicin, daunorubicin, vincristine, and colchicine. The growth doubling time and the cell numbers in G1 phase were increased. The accumulation of cellular daunorubicin in the resistant cells was obviously reduced, but distinctly increased by tetrandrine and verapamil. The numbers of telocentromeric chromosome increased and the chromosomal aberration more occured in the resistant cells. The resistant cells overexpressed multidrug resistant mdr-1 gene and P-glycoprotein 150 kDa. CONCLUSION: The Har-resistant HL60 cell strain belonged to a multidrug resistance strain, overexpressing mdr-1 gene and P-glycoprotein.     

Reversal of multidrug resistance by SDZ PSC 833, combined with VAD (vincristine, doxorubicin, dexamethasone) in refractory multiple myeloma. A phase I study

SDZ PSC 833, a non-immunosuppressive cyclosporin analogue reverses multidrug resistance (MDR) in vitro by inhibiting P-glycoprotein (P-gp) mediated drug efflux. We performed a dose escalation study of SDZ PSC 833 combined with VAD chemotherapy in refractory multiple myeloma (MM). Twenty-two MM patients who were refractory to doxorubicin/vincristine/dexamethasone (VADr, n=11) or had failed multiple regimens (n=6) or were melphalan-refractory (MELr, n=5), were treated with one to three cycles of VAD combined with oral SDZ PSC 833, which was administered at escalating dosages starting at 5 mg/kg/day to 15 mg/kg/day for 7 days. The median trough and peak blood levels of SDZ PSC 833 ranged from 461/1134 ng/ml at 5 mg/kg/day to 821/2663 ng/ml at 15 mg/kg, respectively. With addition of SDZ PSC 833 (5 mg/kg) the mean plasma AUC 0-->96 h of doxorubicin as compared with control patients treated with VAD increased from 779 to 1510 ng/ml/h (P=0.0071), while the doxorubicin clearance was reduced from 47.6 to 27.8 l/h/m2 (P=0.0002). The clearance of doxorubicinol was reduced accordingly. Because of the increased plasma AUC, the dose of doxorubicin and vincristine had to be reduced in 13 patients to 50% (n=1) or 75% (n=12). A further dose-escalation of SDZ PSC 833 did not lead to a proportional increase of doxorubicin AUC. Toxicity WHO CTC grade 2 or 3 included hypoplasia (18/22), constipation (10/22), hyponatremia (3/22) and infections (6/22). A partial response or stable disease was achieved in eight and six patients, respectively. In 17 evaluable patients the mean percentage of pretreatment bone marrow plasma cells which expressed P-glycoprotein was 40%. The pretreatment in vitro rhodamin retention in CD38++ myeloma cells was reversible by 2 microM SDZ PSC 833 with 15-98% in 7/9 tested patients. In 4/5 responding patients analyzed before and after treatment with VAD + SDZ PSC 833, a reduction of P-gp + plasma cells was observed. It is concluded, that the blood concentrations of SDZ PSC 833 attained in MM patients increase with dose after oral administration. It can be safely combined with VAD chemotherapy. SDZ PSC 833 diminishes the clearance of doxorubicin, leading to an increase of the plasma AUC of doxorubicin. In addition, it is an effective inhibitor of P-gp mediated efflux of doxorubicin in myeloma tumor cells in vitro. Therefore, a proportional dose-reduction of doxorubicin and vincristine is warranted. Phase II/III studies in refractory MM are in progress to evaluate the therapeutic efficacy of SDZ PSC 833 with VAD.     

Functional characterization of the rat mdr1b encoded P-glycoprotein: not all inducing agents are substrates

The multidrug resistance (mdr) genes encode P-glycoproteins, integral membrane proteins which function as drug efflux transporters. Exposure of animals in vivo and cells in vitro to a variety of xenobiotics leads to increased mdr1 gene expression and higher levels of P-glycoprotein. This response may protect cells from the cytotoxic effects of these compounds. In this investigation we functionally expressed the rat mdr1b gene in NIH 3T3 cells and assessed the ability of the encoded P-glycoprotein to protect these cells from the cytotoxicity of xenobiotics known to induce mdr1b expression. In long-term colony survival assays, stably expressed mdr1b conferred resistance to cytotoxic drugs such as colchicine, vinblastine and doxorubicin, but not to 5-fluorouracil nor to the carcinogens aflatoxin B1 and N-hydroxy-acetylaminofluorene. The mdr reversal agent verapamil restored cytotoxicity of colchicine, doxorubicin, actinomycin D, vinblastine and taxol, but had no effect on the sensitivity of these cells to 5-fluorouracil, aflatoxin B1 or N-hydroxy-acetylaminofluorene. In a competitive transport assay, verapamil and, to a lesser extent, colchicine blocked the increased efflux of the fluorescent dye rhodamine 123 from mdr1b-transfected cells, whereas aflatoxin B1 did not compete for this export. These data demonstrate that expression of the rat mdr1b encoded P-glycoprotein can protect cells from a diverse group of compounds previously identified to be mdr substrates, however, other effective inducers of mdr expression, such as aflatoxin B1 and N-hydroxy-acetylaminofluorene, remain potent cytotoxins despite high levels of P-glycoprotein. The fact that compounds which are not themselves substrates can induce P-glycoprotein expression may have implications for pharmacokinetic interactions and chemotherapy.     

Reversal of a novel multidrug resistance mechanism in human colon carcinoma cells by fumitremorgin C

We selected a human colon carcinoma cell line in increasing concentrations of mitoxantrone to obtain a resistant subline, S1-M1-3.2, with the following characteristics: profound resistance to mitoxantrone; significant cross-resistance to doxorubicin, bisantrene, and topotecan; and very low levels of resistance to Taxol, vinblastine, colchicine, and camptothecin. This multidrug resistance (MDR) phenotype, which was not reversed by verapamil or another potent P-glycoprotein (Pgp) inhibitor, CL 329,753, was dependent, in part, upon an energy-dependent drug efflux mechanism. Pgp and the multidrug resistance protein (MRP) were not elevated in the resistant cells relative to the drug-sensitive parent, suggesting that resistance was mediated by a novel pathway of drug transport. A cell-based screen with S1-M1-3.2 cells was used to identify agents capable of circumventing this non-Pgp, non-MRP MDR. One of the active agents identified was a mycotoxin, fumitremorgin C. This molecule was extremely effective in reversing resistance to mitoxantrone, doxorubicin, and topotecan in multidrug-selected cell lines showing this novel phenotype. Reversal of resistance was associated with an increase in drug accumulation. The compound did not reverse drug resistance in cells with elevated expression of Pgp or MRP. We suggest that fumitremorgin C is a highly selective chemosensitizing agent for the resistance pathway we have identified and can be used as a specific pharmacological probe to distinguish between the diverse resistance mechanisms that occur in the MDR cell.     

Newly synthesized dihydropyridine derivatives as modulators of P-glycoprotein-mediated multidrug resistance

Newly synthesized 1,4-dihydropyridine derivatives possessing alkyl chains at the 4-position screened whether they could overcome P-glycoprotein-mediated multidrug resistance in cultured cancer cells and also leukemia-bearing animals. Of these derivatives, some could overcome drug resistance to doxorubicin and vincristine in multidrug resistant human cancer cell lines. Combined administration of vincristine and some of the derivatives significantly increased the life span of P-glycoprotein overexpressing multidrug-resistant P388 leukemia-bearing mice. The calcium antagonistic activities, an undesirable effects, were weaker than that of verapamil. These results suggested that the introduction of alkyl groups at the 4-position were effective for both overcoming multidrug resistance and reducing the calcium antagonistic activity.     

P-glycoproteins: mediators of multidrug resistance

Multidrug resistance represents a major obstacle to successful chemotherapy of metastatic disease. Elevated levels in cancer cells of the product of the multidrug resistance gene, P-glycoprotein or the multidrug transporter, have been associated with the development of simultaneous resistance to a great variety of amphiphilic cytotoxic drugs. P-glycoprotein is an integral plasma membrane protein which contains 12 putative transmembrane regions and two ATP binding sites. It confers multidrug resistance by functioning as an energy-dependent drug efflux pump. Here we describe recent studies on the biosynthesis, structure, function, and mechanism of action of P-glycoprotein which have provided insights into the complexity of this multifunctional transport system and revealed an additional chloride channel activity. The physiological role of P-glycoprotein, however, still remains to be elucidated.     

Rapid recovery of a functional MDR phenotype caused by MRP after a transient exposure to MDR drugs in a revertant human lung cancer cell line

Prior studies have shown that, in some human tumour cells, increased expression of the multidrug resistance gene MDR1 can be induced in response to certain stress conditions such as a transient exposure to cytotoxic agents. Little is known about the possibility of increasing the expression of the recently cloned multidrug resistance-associated protein (MRP) in response to a transient exposure to cytotoxic drugs. In order to examine this possibility, we have used sensitive assays (RT-PCR, flow cytometry) and the sensitive large cell lung cancer cell line, COR-L23/P, and the revertant line (COR-L23/Rev), generated by growing the doxorubicin-selected, MRP-overexpressing resistant variant COR-L23/R without drug exposure for 24-28 weeks. COR-L23/Rev overexpresses MRP, but to a lesser extent than COR-L23/R. COR-L23/Rev rapidly recovered similar levels of MRP mRNA, protein expression, resistance and drug accumulation deficit as COR-L23/R after a 48-72 h exposure to cytotoxic concentrations of doxorubicin or vincristine but not cisplatin. The increase in MRP mRNA could only be detected 3 to 4 days after the transient exposure to drugs. However, when the parental line, COR-L23/P, was exposed to equitoxic doses of doxorubicin, vincristine or cisplatin, no increase in the levels of MRP mRNA could be observed at higher doses (5- to 10-fold the IC50) of doxorubicin or vincristine (but not of cisplatin), we detected a transient increase in the levels of MDR1 mRNA immediately after short-term exposure. In conclusion, we have shown that a human revertant lung cancer cell line (COR-L23/Rev) has the ability to recover quickly, similar levels of MRP expression and resistance as COR-L23/R after a transient exposure to the MDR-drugs doxorubicin and vincristine.     

Cell biological markers of drug resistance in ovarian carcinoma

The aim of the study is to review the mechanisms of resistance to four classes of drugs that are widely used in ovarian carcinoma: platinum (cisplatin/carboplatin) compounds, classical alkylating agents (cyclophosphamide/melphalan), natural drugs (doxorubicin), and "new drugs" (taxol and taxotere). Both platinum and classical alkylating agents mediate their cytotoxicity by the formation of drug-DNA adducts, resulting in DNA damage. Therefore, drug resistance mechanisms are (in part) comparable. In ovarian carcinoma cell lines increased repair of DNA damage and increased detoxification by binding of drugs to glutathione, possibly catalyzed by glutathione S-transferases, have been identified as the most prominent resistance mechanisms to these drugs. Studies on the role of DNA repair mechanisms and glutathione in human ovarian carcinoma are hampered by the complexity of enzyme systems involved in DNA repair and intratumor heterogeneity for glutathione. Resistance to doxorubicin appears to be mediated by enhanced efflux from the cell by increased expression of membrane glycoproteins acting as a drug efflux pump, such as P-glycoprotein. Resistance to doxorubicin can also be due to quantitative and/or qualitative changes in the nuclear target of doxorubicin, topisomerase (Topo) II. Finally, resistance to taxol may be mediated by enhanced expression of P-glycoprotein, while presumed other mechanisms such as alterations in tubulin structure, the cellular "target" of taxol, and changes in polymerization of tubulin are still largely unresolved. Several ways to modulate the reviewed resistance mechanisms are also described. In conclusion, this review shows that many cell biological factors may be involved in drug resistance. The relevance of the identification of most of these factors in ovarian carcinoma patients however remains to be established.