Reverse transformation of multidrug-resistant human neuroblastoma stem cells is associated with up-regulation of CD133 and ABCG2, markers of a less differentiated, less malignant cell phenotype

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Reverse transformation –reduction or loss of malignant potential and resumption of contact inhibition –is one recurring concomitant of acquired high-level multidrug resistance in cultured tumor cell lines. Studies of multidrug-resistant rodent sublines linked reverse transformation with overexpression of P-glycoprotein, encoded in humans by the MDR1 (ABCB1) gene, but left open the possibility that differentiation state could also play a role. Using a series of well characterized multidrug-resistant sublines selected from the malignant human neuroblastoma BE(2)-C stem cell clone, we have analyzed expression of markers of differentiation as well as of other ATP-binding cassette (ABC) transporters. The seven resulting sublines could be divided into two groups based on tumorigenicity in nude mice. Group A sublines, selected with colchicine, form tumors in 100% of animals [as does BE(2)-C]. By contrast, Group B sublines, selected with actinomycin D or vincristine, exhibit a 23 to 80% reduction in tumor formation. Western analyses of two representative sublines from each group for levels of neuroblastic [chromogranin A (CGA), neurofilament 68 (NF68)] and non-neuronal [vimentin (VIM)] marker proteins revealed that Group B sublines contain 3- to 4-fold less CGA and NF68 and 20-fold less VIM than Group A sublines –expression patterns suggestive of a less differentiated, earlier stem cell type. Cell morphology also appears less differentiated and the Group B sublines no longer respond to common morphogens. Semi-quantitative RT-PCR analyses for mRNAs encoding proteins expressed in normal stem cells revealed expression patterns likewise consistent with an earlier differentiation stage: 4-fold higher levels of CD133 mRNA and activation of transcription of BCRP1 (ABCG2) in Group B cells compared to Group A. Expression levels of the developmentally earlier stem cell markers, the intermediate filaments cytokeratin 8 and 18, were similar between the groups. Of interest in neuroblastoma, MRP1 (ABCC1) increased in the tumorigenic, more neuroblastic Group A cells whereas MRP4 (ABCC4) decreased. Thus, reverse transformation related to high-level drug resistance correlates with –and may be the consequence of –expression of a less differentiated, intrinsically less malignant, stem cell phenotype.