Decreased expression of hMYCN protein is achievable with the use of AS oligonucleotide treatment, even in the presence of hMYCN oncogene amplification. Antisense strategies targeting the hMYCN oncogene in vivo decrease mouse neuroblastoma tumorigenesis. Investigation of their clinical effect in children with neuroblastoma is warranted.

A series of taxol-and taxotere-resistant J774.2 cell lines has been characterized with respect to altered expression of ␤-tubulin, the cellular target for these drugs. Vertebrates have six classes of ␤-tubulin isotypes, each displaying a distinct pattern of expression. Although the functional significance of multiple ␤-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 ␤-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 ␤-tubulin isotypes. Two isotypes, M␤5 and M␤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␤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␤2, the class II ␤-tubulin isotype, may contribute to the development of high resistance to taxol.

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.

In the childhood cancer neuroblastoma (NB), the level of expression of the multidrug resistance-associated protein (MRP1) gene is strongly correlated with expression of the MYCN oncogene in primary NB tumors, suggesting that MRP1 may be a target for MYCN-mediated gene regulation. In this study, we show that MYCN induction in human NB cells results in increased MRP1 mRNA and protein levels, which in turn is accompanied by increased drug resistance and enhanced MRP1-mediated drug efflux. Furthermore, luciferase activity from MRP1 promoter/luciferase gene reporter constructs was significantly increased in NB cells with exogenous overexpression of MYCN, whereas activity was decreased in NB cells stably transfected with MYCN-antisense vectors. Decreased luciferase activity was observed with promoter constructs that lacked one or two E-box sequences or had E-box double point mutations, while a truncated MRP1 promoter lacking all three E-boxes exhibited only basal levels of activity. Specific electrophoretic mobility shifts of MRP1 E-box sequences were detected with nuclear extracts from NB cells with MYCN overexpression, and complex formation was inhibited with the addition of antibodies directed against MYCN or MYC. These findings indicate that by interacting with E-box elements within the promoter, MYCN can upregulate MRP1 expression and modulate drug resistance in NB.

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 e ect of MYCN down-regulation on MRP expression and response to cytotoxic drugs was investigated in NBL-S neuroblastoma cells transfected wtih 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 signi®cantly increased sensitivity to the high a nity 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 signi®cantly increased resistance speci®cally to MRP substrates. The results provide evidence for the MYCN oncogene in¯uencing 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.