Reversal of multidrug resistance-associated protein-mediated drug resistance in cultured human neuroblastoma cells by the quinolone antibiotic difloxacin.

BACKGROUND: We have recently shown that high-level expression of the multidrug resistance-associated protein (MRP) gene is a powerful independent predictor of poor outcome in neuroblastoma. The clinical implication of these findings is that MRP modulators may prove therapeutically useful. PROCEDURE: We therefore investigated the ability of difloxacin, a quinolone antimicrobial antibiotic, to increase drug cytotoxicity in unselected cultured human neuroblastoma cells. Drug cytotoxicity was determined using a microtiter assay in neuroblastoma cells expressing low (SH-EP), intermediate (NBL-S), or high [BE(2)-C] levels of MRP. RESULTS: Difloxacin (50 microg/ml) increased sensitivity to the MRP substrates, vincristine, doxorubicin, daunorubicin, and potassium antimony tartrate to an extent directly proportional to their level of MRP expression. No change in the response to cisplatin, which is not a substrate for MRP, was observed in any of the cell lines. CONCLUSIONS: The data demonstrate that difloxacin can reverse drug resistance in unselected human neuroblastoma cells and is therefore a potential candidate for future clinical trials.

MYCN expression is not prognostic of adverse outcome in advanced-stage neuroblastoma with nonamplified MYCN.

PURPOSE: The clinical significance of MYCN expression in children with neuroblastoma (NB) remains controversial. To determine the prognostic significance of MYCN expression in the absence of MYCN amplification, we analyzed MYCN mRNA and protein expression in tumors from 69 patients. PATIENTS AND METHODS: Sixty-nine NB tumor samples with nonamplified MYCN from patients with stage C or D disease were obtained from the Pediatric Oncology Group Neuroblastoma Tumor Bank. MYCN mRNA was analyzed using a real-time reverse transcriptase polymerase chain reaction assay, and MYCN protein was examined by Western blot analyses. RESULTS: The estimated 5-year event-free survival (EFS) and survival (S) rates plus SE for the cohort were 57% +/- 17% and 60% +/- 16%, respectively. Infants younger than 1 year had significantly higher rates of EFS and S than children >/= 1 year of age (P =.003 and P <.001, respectively); patients with stage C disease had better outcome than those with stage D NB (P <.001); and patients with hyperdiploid tumors had better outcome than those with diploid NB (P <.001). Surprisingly, outcome was slightly better for patients with high versus low levels of MYCN mRNA expression (4-year S, 70% +/- 13% v 50% +/- 16%; P =.290), and for patients with tumors that expressed MYCN protein (4-year S, 73% +/- 19% v 53% +/- 15%, respectively; P =.171). CONCLUSION: High levels of MYCN expression are not prognostic of adverse outcome in patients with advanced-stage NB with nonamplified MYCN. A trend associating high levels of MYCN expression with improved outcome was observed.

Altered expression of the MYCN oncogene modulates MRP gene expression and response to cytotoxic drugs in neuroblastoma cells.

We have recently shown a close correlation between expression of the Multidrug Resistance-associated Protein (MRP) gene and the MYCN oncogene and provided evidence that high MRP expression is a powerful independent predictor of poor outcome in neuroblastoma (Norris et al., New Engl. J. Med., 334, 231-238, 1996). The effect of MYCN down-regulation on MRP expression and response to cytotoxic drugs was investigated in NBL-S neuroblastoma cells transfected with MYCN antisense RNA constructs. Concomitant with MYCN down-regulation, the level of MRP expression was decreased in the NBAS-4 and NBAS-5 antisense transfectants. These cells demonstrated significantly increased sensitivity to the high affinity MRP substrates vincristine, doxorubicin, sodium arsenate and potassium antimony tartrate, but not to the poor MRP substrates, taxol or cisplatin. Similarly, transfection of full-length MYCN cDNA into SH-EP neuroblastoma cells resulted in increased MRP expression and significantly increased resistance specifically to MRP substrates. The results provide evidence for the MYCN oncogene influencing cytotoxic drug response via regulation of MRP gene expression. Our data also provide a link between the malignant and chemoresistant phenotypes of this childhood malignancy.

P-glycoprotein-mediated methotrexate resistance in CCRF-CEM sublines deficient in methotrexate accumulation due to a point mutation in the reduced folate carrier gene.

We have previously described a series of methotrexate (MTX)-selected CCRF-CEM sublines (CEM/MTX R1-3) displaying increased resistance to drugs associated with the multidrug resistance phenotype and have provided evidence that MDR1 P-glycoprotein contributes to multifactorial MTX resistance in these cells. We have also suggested that P-glycoprotein-mediated MTX transport arises in these cells due to a deficiency in the normal MTX transport route, the reduced folate carrier (RFC). We have now determined the nucleotide sequence of the RFC gene in CEM/MTX R1-3 cells and confirm that the carrier is defective in these cells as a result of a premature stop mutation at codon 99, which severely truncates the encoded protein. CEM/MTX R3 cells were removed from MTX, and a series of sublines with increasing MDR1 expression were derived, following selection with vincristine. These cells show increasing cross-resistance to vincristine as well as other drugs associated with the multidrug resistance phenotype. More importantly, the increased P-glycoprotein expression correlates with increased resistance to MTX, supporting the hypothesis that in cells with a defective carrier protein, MTX can become a substrate for P-glycoprotein. Our data have implications for the P-glycoprotein-mediated transport of other hydrophilic drugs in situations where the relevant carrier protein has been functionally inhibited.

Evidence that the MYCN oncogene regulates MRP gene expression in neuroblastoma.

We have recently shown that expression of the multidrug resistance-associated protein (MRP) gene is a powerful prognostic indicator in childhood neuroblastoma and have suggested that the MYCN oncogene may regulate MRP gene expression. To address this hypothesis, we have examined the relationship between MYCN and MRP gene expression in neuroblastoma tumours and cell lines. MYCN and MRP gene expression were highly correlated in 60 primary untreated tumours both with (P = 0.01) and without MYCN gene amplification (P < 0.0001). Like MRP, high MYCN gene expression was significantly associated with reduced survival, both in the overall study population and in older children without MYCN gene amplification (relative hazards = 13.33 and 19.61, respectively). Inhibition of MYCN, through the introduction of MYCN antisense RNA constructs into human neuroblastoma cells in vitro, resulted in decreased MRP gene expression, determined both by RNA-PCR and Western analysis. The data are consistent with MYCN influencing neuroblastoma outcome by regulating MRP gene expression.

Involvement of MDR1 P-glycoprotein in multifactorial resistance to methotrexate.

Cellular resistance to methotrexate (MTX) is believed to be unaffected by expression of MDR1 P-glycoprotein (Pgp), a pleiotropic efflux pump acting on different hydrophobic compounds that enter cells by passive diffusion. A series of human leukemic CCRF-CEM sublines, isolated by multi-step selection for very high resistance to MTX, exhibit multiple mechanisms of MTX resistance, including decreased carrier-mediated uptake of MTX and DHFR gene amplification. These sublines show cross-resistance to drugs of the multi-drug resistance (MDR) family, which is correlated with relative resistance to MTX. The MTX-selected sublines show increased expression and function of the MDR1 gene, based on the measurement of MDR1 mRNA, Pgp and rhodamine 123 accumulation. Sequence analysis of the MDR1 cDNA from MTX-selected CCRF-CEM cells revealed no mutations in the protein coding region. MTX resistance in these cell lines is partially reversible by a Pgp-specific monoclonal antibody (MAb) UIC2 and a monovalent FaB fragment of UIC2. Our results indicate that Pgp can contribute to multifactorial resistance to MTX.

Altered expression of M beta 2, the class II beta-tubulin isotype, in a murine J774.2 cell line with a high level of taxol resistance.

A series of taxol- and taxotere-resistant J774.2 cell lines has been characterized with respect to altered expression of beta-tubulin, the cellular target for these drugs. Vertebrates have six classes of beta-tubulin isotypes, each displaying a distinct pattern of expression. Although the functional significance of multiple beta-tubulins has not been fully defined, there is evidence that the individual isotypes contribute to differences in microtubule dynamics and drug binding. To determine if alterations in the expression of beta-tubulin isotypes play a role in taxol resistance, a PCR-based methodology was developed that permits highly specific amplification of each of the six known murine beta-tubulin isotypes. Two isotypes, M beta 5 and M beta 3, were expressed abundantly in the drug-sensitive parental J774.2 cells. Although expressed at an extremely low level in the parental cells, expression of the M beta 2 isotype was increased 21-fold (< 0.005) in the cell line most resistant to taxol. These findings suggest that a cell can alter its relative tubulin isotype composition in response to an external stress and specifically imply that altered expression of M beta 2, the class II beta-tubulin isotype, may contribute to the development of high resistance to taxol.

An assay for the determination of reduced methotrexate accumulation in cells displaying limited viability in vitro.

Amongst the mechanisms known to mediate resistance to methotrexate (MTX), a major component in the treatment of childhood leukemia, reduced drug accumulation appears to have direct clinical relevance. However, due to the poor viability of patient-derived acute lymphoblastic leukemia cells in vitro, determination of this parameter in clinical samples is associated with a number of difficulties. We have therefore developed an assay for reduced MTX accumulation, which controls for the metabolic state of the cell population under study by utilizing accumulation of the nucleoside thymidine as an independent indicator of this parameter. To establish this assay, we have utilized pediatric leukemic cell populations maintained as xenografts in nude mice, which, despite displaying sensitivity to MTX, demonstrated reduced accumulation of MTX when assayed using standard methodology. When accumulation of MTX by such cell populations was expressed, however, relative to their accumulation of thymidine, MTX accumulation was shown to be equal to that of drug-sensitive CCRF-CEM cells maintained in long-term culture. In contrast, significantly less MTX was accumulated, in this assay, by xenografted cell populations with demonstrated resistance to MTX. Identical results were obtained using either fresh or cryopreserved cells. The data thus indicate that by controlling for variable metabolic status of leukemic cells, it is possible to accurately assess MTX accumulation in leukemic samples displaying limited viability in culture.

Analysis of a novel cDNA encoding a C219-reactive peptide isolated from methotrexate-selected multidrug-resistant human leukemic cells.

We have isolated and sequenced a novel cDNA clone (D320), from methotrexate-selected multidrug-resistant (MDR) human leukemic CCRF-CEM cells, which encodes a peptide reactive with monoclonal antibody (mAb) C219. Despite having been cloned using this ostensibly P-glycoprotein-specific mAb, clone D320 has no significant homology with either the MDR-encoding gene or, at the amino acid (aa) level, with its product, P-glycoprotein. A putative C219-binding site has been identified in D320, which differs at two positions from the 6-aa C219 epitope previously described in P-glycoprotein.

Expression of the multidrug resistance-associated protein (MRP) gene correlates with amplification and overexpression of the N-myc oncogene in childhood neuroblastoma.

The MRP gene (Cole et al., Science (Washington DC), 258: 1650-1654, 1992) encodes a membrane-bound glycoprotein the expression of which correlates with non-P-glycoprotein-mediated multidrug resistance in a variety of cultured human cell lines. Using an RNA-polymerase chain reaction assay, expression of this gene was examined in the highly chemoresistant pediatric malignancy, neuroblastoma. MRP expression was observed in 5 human neuroblastoma cell lines and in all 25 primary neuroblastoma tumors of stage I through IVS. Tumors with amplification of the N-myc oncogene were found to have significantly higher MRP expression that those with no amplification (P = 0.0016). Expression of the MRP gene in the tumor specimens was highly correlated with expression of the N-myc gene (P = 0.0009), while expression of the MDR1 gene, encoding P-glycoprotein, was not related to expression of either the N-myc or MRP genes. Decreased expression of the N-myc oncogene in neuroblastoma cell lines SH-SY5Y and BE(2)-C, following treatment with retinoic acid, was paralleled by down-regulation of MRP gene expression, contrasting with increased expression of the MDR1 gene. Expression of the MRP gene is thus common in both primary neuroblastoma tumors and cultured cell lines, and correlates with amplification and overexpression of the N-myc oncogene, which is central to the malignant phenotype of this disease.

Ferritin production and desferrioxamine cytotoxicity in human neuroblastoma cell lines.

In patients with neuroblastoma, elevated serum ferritin is correlated with adverse outcome. In this investigation, three human neuroblastoma cell lines have been characterised in terms of their levels of both intracellular and secreted ferritin and their response to the iron-chelating agent, desferrioxamine (deferoxamine). The cell lines differed markedly in respect of ferritin production as determined by radioimmunoassay. Intracellular and secreted ferritin concentrations for SH-SY5Y and BE(2)-C cells were several fold higher than that determined for IMR-32 cells. IMR-32 cells were most sensitive to desferrioxamine, BE(2)-C intermediate and SH-SY5Y the most resistant to the drug in terms of the respective ID50 values. Combining the differentiating agent retinoic acid with desferrioxamine did not enhance cytotoxicity in the neuroblastoma cells. The present data suggest that neuroblastoma cells secreting a relatively low levels of ferritin may be most responsive to iron chelating agents.

Methotrexate cytotoxicity determination using the MTT assay following enzymatic depletion of thymidine and hypoxanthine.

Methotrexate, an important agent in the treatment of childhood acute lymphoblastic leukaemia, has generally failed to induce dose-dependent cytotoxicity of patient-derived leukaemic blasts when tested in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This effect is apparently due to salvage from the medium, by surviving leukaemic cells, of metabolites such as hypoxanthine and thymidine. In an attempt to address this problem, we have examined the effect, on leukaemic cell populations, of enzymatically depleting these metabolites from the culture medium employed during the MTT assay, using xanthine oxidase and thymidine phosphorylase. Specifically we have assessed methotrexate cytotoxicity in the paediatric acute lymphoblastic T cell leukaemia, GKTL, which is maintained as a xenograft, and like primary leukaemias, has poor viability in vitro. Although little cytotoxicity of GKTL cells was observed when the MTT assay was performed in supplemented RPMI-1640 medium, dose-dependent cytotoxicity of these cells was clearly apparent when the same medium was enzymatically depleted. In contrast, the ID50 for methotrexate of control CCRF-CEM cells was unaltered in enzymatically depleted medium. In the absence of methotrexate, enzymatic depletion of the medium did not affect leukaemic cell survival. We are currently investigating the general applicability of this approach for assaying the response to methotrexate of primary leukaemia samples.

Resistance to tetracycline, a hydrophilic antibiotic, is mediated by P-glycoprotein in human multidrug-resistant cells.

Two multidrug-resistant human leukemic CCRF-CEM sublines (CEM/VCR R and CEM/VLB100) were significantly more resistant to tetracycline, a hydrophilic antibiotic, than parental cells (P < 0.001). Verapamil and cyclosporin A completely reversed tetracycline resistance in CEM/VCR R cells, which also accumulated and retained significantly less [3H]tetracycline than CCRF-CEM cells. Like verapamil, addition of tetracycline to CEM/VCR R cells which had achieved steady-state vincristine levels resulted in augmented vincristine accumulation. [3H]Azidopine photoaffinity labelling of CEM/VCR R membrane proteins was inhibited by tetracycline in a dose-dependent manner. Although drugs associated with the multidrug-resistance phenotype are typically hydrophobic compounds, these data suggest that resistance to tetracycline, despite its hydrophilic nature, is mediated by P-glycoprotein in these cell lines.