Amplification and expression of the ABC transporters ARA and MRP in a series of multidrug-resistant leukaemia cell sublines

Abstract

Summary E1000, the most drug-resistant subline from the E-series (CCRF-CEM/E16 to E1000), has been previously shown to express high mRNA levels from two ABC transporter genes associated with multidrug resistance, ARA and MRP. The expression and amplification of both genes has now been characterized for each member of the E-series of drug-resistant sublines and is reported here. Both ARA [detected by reverse transcriptase polymerase chain reaction (RT-PCR)] and MRP (detected by Northern blot analysis) were expressed at low levels in the sensitive parental CEM cell line. An equivalent level of MRP mRNA expression was detected throughout the CEM, E16, E25 and E50 sublines, and there was increasing expression in the El 00, E200 and El 000 sublines. ARA expression was not detected in the El 6, E25, E50 and E100 sublines but was detected by both RT-PCR and Northern blot analysis in the E200 and E1000 sublines. Southern blot analysis indicated the increased levels of MRP and ARA expression resulted from gene amplification and that MRP was first amplified in the E100 subline and ARA in the E200 subline, suggesting that the two genes were not initially co-amplified. Cytogenetic analysis of E1000 cells demonstrated a large addition to chromosome 1 6p, around the region where the ARA and MRP genes are located. Increased expression of ARA is associated with increased colchicine resistance in the E-series of sublines and combined with MRP may account for their resistance phenotype.

MOAT-E (ARA) is a full-length MRP/cMOAT subfamily transporter expressed in kidney and liver

“…Since ARA, located at 16p13.1 (Kuss et al, 1998), is in close proximity to MRP (16p13.13), and both of these genes have been reported to be amplified in the drug resistant cell line from which ARA was isolated (Longhurst et al, 1996), it is possible that the ARA cDNA is the product of a rearrangement associated with the MRP amplicon. The detection of cytogenetic abnormalities at 16p in the cell line from which ARA was isolated is consistent with this possibility (O 'Neill et al, 1998). Together these observations suggest that ARA encodes a partial peptide of a larger transporter whose full-length cDNA has not yet been described.…”

Mammalian ABC Transporters in Health and Disease

“…Amplification of the 3Ј part of the MRP6 gene was found in leukemia cells selected for anthracycline (epirubicin) resistance (297)(298)(299). The anthracycline resistance was initially thought to be due to a new resistance determinant, called the anthracycline resistance gene, ARA.…”

Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

“…In addition, the amniotic membrane from term pregnancies expresses MRP5 on its apical and basolateral epithelial cells (Aye et al, 2007). MRP6 was first considered the anthracycline resistance-associated gene in resistant leukemia cell lines (Longhurst et al, 1996;O'Neill et al, 1998). Subsequent cloning demonstrated that the anthracycline resistanceassociated gene is a partial protein product of ABCC6 Kool et al, 1999a).…”