Differential Expression of Multi Drug Resistance-Associated Protein (MRP 1) in phenotypically distinct neuroblastoma cell lines




Sonal Kashyap

Introduction

Neuroblastoma is the most common extracranial solid tumor of infancy which arises from embryonal neural crest cells. Neuroblastomas are categorized clinically by their location, age at diagnosis, spread/metastasis and degree of cellular maturation and heterogeneity. Long term studies undertaken in human cell lines have shown that there are three distinct neuroblastoma cell types—I-type stem cells, N-type neuroblastic/neuroendocrine precursors, S-type schwannian cell/melanoblastic precursors(1). The I-type stem cells are malignant neural crest cells and form tumors in the nude mice. The I-type cells show features common to both N and S type cells. The N-type cells adhere tightly to other cells forming structures like pseudoganglia and they form tumors only in athymic mice. The S type cells adhere tightly to the substrate and do not form tumors in nude mice. Studies have also shown that neuroblastomas contain multiple cell phenotypes and this feature is often used for the prognosis of the disease. I-cell types are present in tumors of all stages. Higher incidence of I type cells in most neuroblastoma is consistent with their high malignant potential in vitro (1, 6). The phenotypically distinct neuroblastoma cell lines have been reported to show varying levels and differential expression of MYCN gene. MYCN is the most widely used biomarker for neuroblastoma, higher the expression of MYCN, poorer is the disease prognosis. MYCN belongs to class of bHLH leucine zipper transcription factors which are involved in cell differentiation, apoptosis, cell growth and genomic instability (4). Some N and I type cell lines have higher levels of MYCN and some show overexpression of the MYCN gene. In the S-type cell line MYCN expression is reported to be down regulated (5, 6).
Some malignant neuroblastomas display chemoresistance. One of the factors causing chemoresistance in these tumors is the expression of Multidrug Resistance-associated Protein (MRP). MRP is overexpressed in most malignant and chemoresistant neuroblastomas. It is an ATP binding cassette protein which acts as an efflux pump. MRP is expressed at normally in many tissues and plays a significant role in efflux of unconjugated bilirubin in fetal hepatocytes and efflux of heavy metal ions from other tissues. Overexpression of MRP in different tumors results in pumping out of unrelated hydrophobic drugs from the cancer cells thereby resulting in chemotherapy failure. Overexpression of MRP have been reported in many cancers—Leukemia and Neuroblastoma. Alternative splicing of MRP gene can generate many variants.
It has been reported that MYCN may upregulate the expression of MRP gene, especially in the poor prognostic tumor types (2, 3, and 4). There is considerable literature demonstrating concomitant overexpression of MRP with MYCN gene in human neuroblastoma. It is possible that MYCN may act as transcriptional factor in regulating MRP gene expression but the exact mechanism still needs to be elucidated(2). Amplification of the MYCN gene is not always the primary factor involved in the neuroblastoma malignancy, presence of distinct cell phenotypes can also affect cancer prognosis. Differential cell lineages and states within a particular lineage have been reported to modulate the MYCN expression (5). The main objective of the present project was whether the MRP-1 expression correlates with a) overexpression of MYCN gene, or b) correlates with distinct neuroblastoma cell types, Resolution of this matter could help physicians select a more effective treatment of neuroblastoma.

Figures


Figure 1-Figure 1: Total RNA in RNAase free gel; Lane 1: SH-SY-5Y, Lane 2: SK-N-M17, Lane 3: SH-EP1, lane 4: LAI-5S, Lane 5: BE(2)C, Lane 6: SK-N-LP, Lane 7: BE(2)C ( isolated sample) and 100bp ladder on either sides


Figure 2-Figure 2 RT_PCR: Lanes 1-6 show MRP bands in different cell types; Total RNA.; Lane 1: SH-SY-5Y, Lane 2: SK-N-M17, Lane 3:SH-EP1, lane 4: LAI-5S, Lane 5: SK-N-LP, Lane 6: BE(2)C. Lanes 7-12 show GAPDH bands in different cell types; Lane 7: SH-SY-5Y, Lane 8: SK-N-M17, Lane 9: SH-EP1, lane 10: LAI-5S, Lane 11:SK-N-LP, Lane 12: BE(2)C. 100bp ladder on either sides.


Figure 3-Figure 3. Alignment of MRP-1 from SK-N-M17 with part of the MRP-1 mRNA (NCBI)


Multi drug resistance associated protein (MRP) belongs to an ATP binding cassette family of transmembrane proteins. It is implicated in non P-glycoprotein –mediated multi drug resistance in variety of neuroblastomas. Expression of MRP gene has been correlated with the MYCN gene which is overexpressed in the most poor prognostic neuroblastomas (2,3,4). Presence of distinct cell phenotypes—I type, S-type and N type determines the malignancy in neuroblastoma cell lines and tumors (1). Differential expression of MRP-1 was studied in selective neuroblastoma cell lines using RT-PCR and sequencing. Results show higher expression of MRP-1 in the cell lines which over express MYCN gene. MRP-1 expression seems to be higher in I-cell types as compare to the N cell types (with exception of SK-N-M17) and S cell types. The two I-cell types (SK-N-LP and BE(2)C) overexpress MYCN which explains the high expression of MRP-1 gene in these cell lines. Results ( graph 1 and table 1) supports that MYCN expression maybe modulating the expression of MRP-1 in neuroblastoma cell lines as previously cited by Haber et.al (2). The MYCN is a well known transcription factor which is over expressed in some neuroblastoma tumors and maybe involved in the upregulation of the MRP-1 expression but the exact mechanism is unknown (2, 3, and 4). MRP-1 expression was high in both the I-cell lines as compare to S-types, which makes sense since I-types are less differentiated (multi potent stem cell types) and show higher levels as well as over expression of MYCN gene. The S-cell types showed lower expression of MRP-1 as compare to the N-types and I-types because these are melanoblastic precursors and MYCN levels are down regulated in S-type cells (1). The LAI-5S ( S type cell line) has high levels of MYCN but the mRNA levels are low for this cell type which is why it show low levels of MRP-1. Though both the I-cell types showed high expression of MRP-1, no conclusion can be made regarding the correlation of MRP-1 expression and presence of I cell phenotype. To arrive at such a conclusion further research needs to be undertaken using larger sample size of I type cell lines including both over expressing MYCN and low expressing MYCN cell lines. Additionally, different isoforms of MRP can be studied in the neuroblastoma cell lines.

Full Paper

Acknowledgments

I like to thank Jinsong Qiu and Brian Fox for their continuous help, patience and encouragement. Additional thanks to Dr Robert A Ross and Jeanette Walton for the RNA samples and their helpful advice. I am grateful to Dr Sylvia Anderson for the primers and getting us started in the first place with the project. Sincere thanks to Dr Berish Rubin for his guidance and providing the opportunity to do the present project


For reference numbers mentioned above please see details in the enclosed full paper.


This document was last modified 01/31/2006.
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