Decreased folylpolyglutamate synthetase activity as a mechanism of methotrexate resistance in CCRF-CEM human leukemia sublines.

D E Mccloskey,J J Mcguire,C A Russell,B G Rowan,J R Bertino,G Pizzorno,E Mini

doi:10.1016/s0021-9258(18)38101-8

D E Mccloskey, J J Mcguire + Show 5 more

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https://doi.org/10.1016/s0021-9258(18)38101-8

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Abstract

Determinants of methotrexate (MTX) resistance in cell lines resistant to short, but not continuous, MTX exposure were investigated since such lines may have relevance to clinical resistance. CCRF-CEM R30dm (R30dm), cloned from CCRF-CEM R30/6 (a MTX-resistant subline of the CCRF-CEM human leukemia cell line), had growth characteristics similar to CCRF-CEM. R30dm was resistant to a 24-h exposure to levels as high as 300 microM MTX but was as sensitive as CCRF-CEM to continuous MTX exposure. MTX resistance of R30dm was stable for greater than 68 weeks in the absence of selective pressure. Initial velocities of MTX transport were comparable for R30dm and CCRF-CEM, as were dihydrofolate reductase specific activity and MTX binding. A 2-fold thymidylate synthase activity decrease for R30dm from that of CCRF-CEM was not a significant factor in R30dm MTX resistance. Decreased MTX poly(gamma-glutamate) synthesis resulted in lower levels of drug accumulation by R30dm. Decreased polyglutamylation was attributable to folylpolyglutamate synthetase (FPGS) activity in R30dm extracts which was 1, 2, and less than or equal to 10% of CCRF-CEM extracts with the substrates MTX, aminopterin, and naturally occurring folates, respectively. Comparison of cell lines with varying levels of resistance to short term MTX exposure indicated that the extent of MTX resistance was proportional to the reduction of FPGS activity. The evidence supported decreased FPGS activity as the mechanism of resistance to short MTX exposure in the cell lines investigated.

Highlights

Determinants of methotrexate (MTX) resistance in The antifolate MTX’ is an anticancer agent used clinically cell lines resistant to short, but not continuous, MTX for treatmentof leukemia, breast cancearn, d choriocarcinoma exposure were investigated since such lines may haveas well as other cancers [2, 3]
CCRF-CEM R3Odm (R30dm), cloned from CCRF-CEM R30/6 (a MTX-resistant sublineof the CCRF-CEM human leukemiacell tiveness is resistance to the drug which is often developed by patientsduringtreatment[4].Tocircumventproblems of resistance it is desirable to understand the causative mechanisms
Characterization of RSOdm-The R30dm cell line was derived from CCRF-CEM R30/6 [6] by cloning under selective

Summary

RESULTS

Characterization of RSOdm-The R30dm cell line was derived from CCRF-CEM R30/6 [6] by cloning under selective. CCRF-CEM and R30dm cells, respectively, after 2 p~ MTX (1,180 f 140 pmol/h/mg of protein, n = 3) in crude extracts. The timeof recovery to 2N and 4lNevels of growth in R3Odm MTX resistance, growth inhibition by other thy- (Fig. 4) and theED50 values for a 14.5-h MTX exposure (not midylate synthase anddihydrofolate reductase inhibitors (un- shown) remained constant for R30dm cells over a 68-week der conditions of 120-h continuous exposure) was assessed. R30dm FPGS specific activity in Trimetrexate [8] was the dihydrofolatereductase inhibitor fresh cell extracts, with MTX and AMT as substrates, retested; the ED50 values were 2.3 and 0.6 nM for CCRF-CEM mained at low levels throughout a 71-week period (Fig. 4). 4-NH2-10-CHa-PteGlu,levels were determined by HPLC analysis as described under “Materials and Methods.”

Decreased FPGS Activity as a Mechanism of MTX Resistance

DISCUSSION

Maximum FPGS specific activiRtyatio

MTX AMT