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

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 E100, E200 and E1000 sublines. ARA expression was not detected in the E16, 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 16p, 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.

Increased MRP expression is associated with resistance to radiation, anthracyclines and etoposide in cells treated with fractionated gamma-radiation.

The failure of chemotherapy is often associated with the failure of radiotherapy in the treatment of cancer. To investigate this relationship, the CCRF-CEM (CEM) human T-cell leukaemia cell line was treated with fractionated gamma-radiation totalling 75 Gy (10 cycles of 1.5 Gy daily for 5 days). This produced the CEMRR subline which was 1.5-fold resistant to radiation compared with the parental CEM cells. The CEMRR subline was also resistant to daunorbicin, idarubicin and etoposide but not to paclitaxel, cis-platinum or chlorambucil. Treatment with 50 microM buthionine sulphoximine, an inhibitor of glutathione synthesis, reversed the daunorubicin resistance in the CEMRR subline. Multidrug resistance-associated protein (MRP) mRNA was 6-fold higher in the CEMRR subline than in the CEM cells, and there was no detectable expression of P-glycoprotein in either the CEM cells or the CEMRR subline. Treatment of the CEM cells with 2 Gy of gamma-radiation caused an increase in MRP-mRNA within 4 hr which, by 24 hr, was greater than 5-fold that of the untreated CEM cells. No change in MRP mRNA was observed in the CEMRR subline with similar treatment. We conclude that MRP is involved in the immediate response to radiation and it may account for the drug resistance that often develops following radiation treatment.

The potential of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide to circumvent three multidrug-resistance phenotypes in vitro.

The effectiveness of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) relative to that of amsacrine, idarubicin, daunorubicin and paclitaxel against three different forms of multidrug resistance (MDR) was determined using two sublines of the CCRF-CEM human leukaemia cell line, the P-glyco-protein-expressing CEM/VLB100 subline and the MRP-expressing CEM/E1000 subline, and two extended-MDR sublines of the HL60 human leukaemia cell line, HL60/E8 and HL60/V8. DACA was effective against P-glycoprotein-mediated MDR and MRP-mediated MDR, whereas the extended-MDR phenotype showed only low levels of resistance (< 2-fold) to DACA. In comparison, idarubicin was ineffective against the MRP and extended-MDR phenotypes. Repeated exposure of the K562 human leukaemia cell line to DACA (55, 546 or 1092 nM for 3 days over 10 weeks) did not result in the development of any significant drug resistance. We conclude that DACA has the potential to treat refractory leukaemia.

The anthracycline resistance-associated (ara) gene, a novel gene associated with multidrug resistance in a human leukaemia cell line.

Multidrug resistance (MDR) in cancer cells is a major contributor to the failure of chemotherapy treatment. This paper describes a novel protein named the anthracycline resistance associated (ARA) protein. The ara gene is amplified in the MDR leukaemia line CCRF-CEM/E1000 and its mRNA is overexpressed. ARA belongs to the ATP binding cassette (ABC) family of proteins. Another ABC protein, the multidrug resistance-associated protein (MRP), has previously been reported to be overexpressed in the CEM/E1000 subline. The primary amino acid sequence of ARA indicates that it is 49.5 kDa without glycosylation, and that it has one potential glycosylation site. ARA has one ATP binding site and associated transmembrane regions. This is in contrast to MRP (190 kDa, 172 kDa deglycosylated) and most other higher eukaryote ABC proteins, which consist of two similar halves, each having one ATP binding site. In addition to ARA being coexpressed with MRP, comparison of amino acid sequences showed that, among known proteins, ARA is most similar to the C-terminal half of MRP.

Drug resistance mechanisms and MRP expression in response to epirubicin treatment in a human leukaemia cell line.

A drug resistant series of sublines were developed by treating the human leukaemia CCRF-CEM cell line with 16-1000 ng/ml of the anthracycline, epirubicin. The sublines developed resistance in two stages, neither involving detectable levels of P-glycoprotein. Treatment with up to 50 ng/ml epirubicin produced sublines with cross resistance limited to the anthracyclines and etoposide. Treatment with 100-1000 ng/ml epirubicin produced sublines with increased expression of the mrp gene, increased resistance to the anthracyclines and etoposide, additional cross resistance to vincristine and colchicine, decreased drug accumulation and reversal of resistance by verapamil and by buthionine sulphoximine (BSO; an inhibitor of glutathione synthesis). Our results indicate an interaction between MRP and glutathione metabolism as a mechanism for multidrug resistance.

Serum galactosyltransferase as a prognostic marker in patients with solid tumors.

The serum level of galactosyltransferase was measured in a group of 218 patients with a variety of solid tumors and most with advanced disease. The pretreatment enzyme level showed little potential as a diagnostic tumor marker, and its change with treatment did not reflect the initial response. There was, however, a significant correlation between the length of survival and the pretreatment enzyme level. Patients with normal levels survived over twice as long as those with elevated levels. When Cox's proportional hazards regression analysis was used to compare the prognostic potential of galactosyltransferase with a number of known clinical indicators of prognosis, the variable most related to survival was performance status (P less than 10(-4) followed by galactosyltransferase (P = 0.01) and then the extent of disease (P = 0.03). The other variables, such as previous therapy, the type, site, and size of primary tumor, did not contribute significantly to the relationship with survival. The pretreatment level of galactosyltransferase is therefore a relatively independent prognosticator of survival and, as such, could be potentially useful in patient management by increasing the accuracy of the initial assessment of prognosis.

Serum galactosyltransferase isoenzymes as markers for solid tumours in humans.

High resolution agarose isoelectric focusing was used to compare the galactosyltransferase isoenzyme patterns of serum from 9 healthy controls with those from 38 patients with either breast, lung, ovarian, stomach or colonic cancer. At least 12 peaks of enzyme activity were found in every sample, the healthy controls having major forms with isoelectric points of 4.74, 4.87, 4.96, 5.16 and 5.23. Thirty patients (79%) had elevated levels of at least one isoenzyme and 23 (61%) had at least 3 isoenzymes elevated compared to only 10 (26%) patients who had elevated total serum galactosyltransferase activity. The isoenzymes which were most often elevated in the cancer patient group had isoelectric points of 4.93, 5.16 and 4.61. One isoenzyme with an isoelectric point of 4.43 was preferentially elevated in patients with ovarian cancer. Those isoenzymes containing little or no sialic acid were rarely elevated in cancer patients. Although no cancer-associated isoenzyme was detected the quantitative differences observed in the cancer patient group were striking.