Selection for methotrexate resistance in mammalian cells bearing a <Emphasis Type="Italic">Drosophila</Emphasis> dihydrofolate reductase transgene

Antifolates, such as methotrexate (MTX), are the treatment of choice for numerous cancers. MTX inhibits dihydrofolate reductase (DHFR), which is essential for cell growth and proliferation. Mammalian cells can acquire resistance to antifolate treatment through a variety of mechanisms but decreased antifolate titers due to changes in drug efflux or influx, or alternatively, the amplification of the DHFR gene are the most commonly acquired resistance mechanisms. In Drosophila, however, a resistant phenotype has only been observed to occur by mutation resulting in a MTX-resistant DHFR. It is unclear if differences in gene structure and/or genome organization between Drosophila and mammals contribute to the observed differences in acquired drug resistance. To investigate if gene structure is involved, Drosophila Dhfr cDNA was transfected into a line of CHO cells that do not express endogenous DHFR. These transgenic cells, together with wild-type CHO cells, were selected for 19 months for resistance to increasing concentrations of MTX, from 50- to 200-fold over the initial concentration. Since Drosophila Dhfr appears to have been amplified several fold in the selected transgenic mammalian cells, a difference in genome organization may contribute to the mechanism of MTX resistance.     

Increased dihydrofolate reductase activity in methotrexate-resistant human promyelocytic-leukaemia (HL-60) cells. Lack of correlation between increased activity and overproduction.

Methotrexate(MTX)-resistant human promyelocytic-leukaemia cells (HL-60) derived from MTX-sensitive cells have a 20-fold increase in dihydrofolate reductase (DHFR) activity as compared with the sensitive cells. This increase is not associated with a concomitant increase in DHFR protein as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by immunological methods using mouse anti-DHFR antibody. The rate of DHFR synthesis is similar in both cell lines. Furthermore, both the sensitive and resistant cells have similar amounts of RNA hybridizing to a DHFR complementary-DNA probe, correlating well with the lack of increase in DHFR protein. DHFR-gene dosages were similar in both types of cells. We conclude that the 20-fold increase in DHFR activity present in these MTX-resistant cells is not due to the overproduction of DHFR but due to the expression of a more active form of the enzyme.     

Selective overproduction of human dihydrofolate reductase in a methotrexate-resistant human-mouse somatic cell hybrid

The development of methotrexate (MTX) resistance in cultured cells results in increased levels of the drug's target enzyme dihydrofolate reductase (DHFR). Stepwise-selected MTX-resistant sublines originating from an MTX-sensitive human-mouse hybrid expressed elevated DHFR levels and human-DHFR specific gene sequence amplification. By high resolution two-dimensional polyacrylamide gradient electrophoresis, human DHFR was shown to be selectively overproduced in VB2a-100 MTX-resistant cells whereas mouse DHFR protein "spots" present in MTX-sensitive parental hybrid were absent in these cells exhibiting 100 microM MTX resistance. These findings and those in a parallel study indicate that concurrent with overproduction of human DHFR and amplification DHFR sequences in VB2a-100, a loss of mouse-specific DHFR gene sequences occurred.     

Activity of dendrimer-methotrexate conjugates on methotrexate-sensitive and -resistant cell lines

Dendritic nanostructures can play a key role in drug delivery, due to the high density and variety of surface functional groups that can facilitate and modulate the delivery process. We have investigated the effect of dendrimer end-functionality on the activity of polyamido amine (PAMAM) dendrimer-methotrexate (MTX) conjugates in MTX-sensitive and MTX-resistant human acute lymphoblastoid leukemia (CCRF-CEM) and Chinese hamster ovary (CHO) cell lines. Two amide-bonded PAMAM dendrimer-MTX conjugates were prepared using a dicyclohexylcarbodiimide (DCC) coupling reaction: one between a carboxylic acid-terminated G2.5 dendrimer and the amine groups of the MTX (conjugate A) and another between an amine-terminated G3 dendrimer and the carboxylic acid group of the MTX (conjugate B). Our studies suggest that conjugate A showed an increased drug activity compared to an equimolar amount of free MTX toward both sensitive and resistant cell lines, whereas conjugate B did not show significant activity on any of the cell lines. Despite substantially impaired MTX transport by MTX-resistant CEM/MTX and RII cells, conjugate A showed sensitivity increases of approximately 8- and 24-fold (based on IC50 values), respectively, compared to free MTX. Co-incubation of the cells with adenosine and thymidine along with either conjugate A or MTX resulted in almost complete protection, suggesting that the conjugate achieves its effect on dihyrofolate reductase (DHFR) enzyme through the same mechanism as that of MTX. The differences in cytotoxicity of these amide-bonded conjugates may be indicative of differences in the intracellular drug release from the cationic dendrimer (conjugate B) versus the anionic dendrimer (conjugate A), perhaps due to the differences in lysosomal residence times dictated by the surface functionality. These findings demonstrate the feasibility of using dendrimers as drug delivery vehicles for achieving higher therapeutic effects in chemotherapy, especially in drug-resistant cells.     

Increasing methotrexate resistance by combination of active-site mutations in human dihydrofolate reductase

Methotrexate-resistant forms of human dihydrofolate reductase have the potential to protect healthy cells from the toxicity of methotrexate (MTX), to improve prognosis during cancer therapy. It has been shown that synergistic MTX-resistance can be obtained by combining two active-site mutations that independently confer weak MTX-resistance. In order to obtain more highly MTX-resistant human dihydrofolate reductase (hDHFR) variants for this application, we used a semi-rational approach to obtain combinatorial active-site mutants of hDHFR that are highly resistant towards MTX. We created a combinatorial mutant library encoding various amino acids at residues Phe31, Phe34 and Gln35. In vivo library selection was achieved in a bacterial system on medium containing high concentrations of MTX. We characterized ten novel MTX-resistant mutants with different amino acid combinations at residues 31, 34 and 35. Kinetic and inhibition parameters of the purified mutants revealed that higher MTX-resistance roughly correlated with a greater number of mutations, the most highly-resistant mutants containing three active site mutations (Ki(MTX)=59-180 nM; wild-type Ki(MTX)<0.03 nM). An inverse correlation was observed between resistance and catalytic efficiency, which decreased mostly as a result of increased KM toward the substrate dihydrofolate. We verified that the MTX-resistant hDHFRs can protect eukaryotic cells from MTX toxicity by transfecting the most resistant mutants into DHFR-knock-out CHO cells. The transfected variants conferred survival at concentrations of MTX between 100-fold and >4000-fold higher than the wild-type enzyme, the most resistant triple mutant offering protection beyond the maximal concentration of MTX that could be included in the medium. These highly resistant variants of hDHFR offer potential for myeloprotection during administration of MTX in cancer treatment.     

Variable content of double minute chromosomes is not correlated with degree of phenotype instability in methotrexate-resistant human cell lines.

Several variants resistant to 1.8 x 10(-4) M DL-methotrexate (MTX) have been isolated from the human cell lines HeLa BU25 and VA2-B by exposing them to progressively increasing concentrations of the drug. A striking variability of phenotype and chromosome constitution was observed among the different variants. All resistant cell lines exhibited a greatly increased dihydrofolic acid reductase (DHFR) activity and DHFR content; however, the DHFR activity levels varied considerably among the variants, ranging between about 35 and 275 times the parental level. In the absence of selective pressure, the increased DHFR activity was unstable, and in all cell lines but one was completely lost over a period ranging in different variants between 25 and 200 days. The MTX-resistant cells lines showed anomalies in their chromosome constitution, which involved the occurrence of a duplicated set of chromosomes in most cells of some of the variants and the presence of double minute chromosomes in all cell lines. An analysis of the correlation of loss of double minute chromosomes and loss of DHFR activity in the absence of MTX has given results consistent with the idea that the double-minute chromosomes contain amplified DHFR genes. However, the most significant finding is that, in contrast to what has been reported in the mouse system, the recognizable double-minute chromosomes varied greatly in number in different variants without any relationship to either the level of DHFR activity or the degree of instability of MTX resistance in the absence of selective pressure. These and other observations point to the occurrence in the human MTX-resistant variants of another set of DHFR genes, representing a varied proportion of the total, which is associated with the regular chromosomes, and which may be unstable in the absence of selective pressure.     

Increase in hepatic microsomal lipid peroxidation mediated by α-adrenergic system under cold stress and noradrenaline treatment

A membrane protein recognized by monoclonal antibody SQM1 was identified in human squamous carcinomas, including those originating in the head and neck (SqCHN), lung and cervix. Cell lines derived from SqCHN of previously untreated patients expressed high amounts of this protein. In contrast, many cell lines established from SqCHN of patients previously treated with chemotherapy and/or radiation showed diminished amounts of this SQM1 protein. The expression of SQM1 antigen was determined in several SgCHN cell lines made resistant by exposure to methotrexate (MTX) in vitro. The parent cell lines all exhibited strong binding to SQM1 antibody. The MTX-resistant sublines showed much lower membrane binding of SQM1. The lowest SQM1 reactivity was found in cell lines with high resistance to MTX and with diminished rate of MTX transport. Some highly MTX-resistant cell lines which had high levels of dihydrofolate reductase, but which retained a high rate of MTX transport, also retained high levels of SQM1 binding. Reduced SQM1 protein was also found in SgCHN cells which developed resistance to the alkylating drug cis-platinum (CDDP) and which showed reduced membrane transport of CDDP. Cell growth kinetics and non-specific antigenic shifts were not responsible for the differences in SQM1 binding between the parent cell lines and their drug-resistant sublines. The finding of a novel protein which is reduced in cells resistant to MTX and CDDP could contribute to our understanding of the basic mechanisms of drug resistance. By detecting SQM1 protein in clinical specimens, it may be possible to monitor the development of drug resistance in tumors.Abbreviations SqCHN Squamous Carcinoma of the Head and Neck - MTX Methotrexate - CDDP Cis-Platinum - DHFR Dihydrofolate Reductase     

Murine erythroleukemia cell variants: isolation of cells that have amplified the dihydrofolate reductase gene and retained the ability to be induced to differentiate

A series of murine erythroleukemia cell (MELC) variants was generated by selection for the ability to grow in increasing concentrations of the folate antagonist methotrexate (MTX). Growth of the parental MELC strain DS-19 was completely inhibited by 0.1 microM MTX. We isolated cells able to grow in 5, 40, 200, 400, and 800 microM MTX. Growth rates and yields were essentially the same in the presence or absence of the selective dose of MTX for all variants. MTX resistance was not the result of a transport defect. Dihydrofolate reductase (DHFR) from our variants and DS-19 was inhibited to the same extent by MTX. Variants had increased dihydrofolate reductase activities. The specific activity of DHFR was proportional to the selective concentration of MTX employed to isolate a given variant. DNA dot blotting established that the cloned variant (MR400-3) had a 160-fold increase in DHFR gene copy number relative to the parental strain (DS-19). Hybridization studies performed in situ established the presence of amplified DHFR genes on the chromosomes of the MTX-resistant but not the MTX-sensitive (parental) cells. Quantitation of DHFR mRNA by cytoplasmic dot blotting established that the amplified DHFR gene expression was proportional to gene copy number. Thus, MTX resistance was due to amplification of the DHFR gene. The variants retained the ability to be induced to differentiate in response to dimethyl sulfoxide and hexamethylenebis(acetamide) as evaluated by the criteria of globin mRNA accumulation, hemoglobin accumulation, cell volume decreases, and terminal cell division.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line

Properties of the methotrexate (MTX) transport carrier were examined in a stable single-step 16-fold MTX-resistant L1210 murine leukemia cell line with unchanged dihydrofolate reductase gene copy and thymidylate synthase and dihydrofolate reductase levels and activities. MTX influx was markedly depressed due to a decrease in Vmax without a change in Km. From this cell line a clonal variant with greater resistance to MTX was identified due solely to a further decrease in influx Vmax. Trans-stimulation of MTX influx by 5-formyltetrahydrofolate was induced in parental but not resistant cells. Analysis of specific MTX surface binding demonstrated a small increase in the number of carriers in the first- and second-step resistant lines. Affinity labeling of cells with an N-hydroxysuccinimide ester derivative of [3H]MTX demonstrated carriers with comparable molecular weights in the parent and second-step transport defective lines. In two partial revertants with increased MTX sensitivity isolated from the second-step resistant lines, MTX influx was increased but surface membrane-binding sites were unchanged suggesting that recovery of transport was due to normalization of carrier function rather than an increase in the number of carriers. These studies suggest that impaired MTX transport in these lines is not due to an alteration in the association of the transport carrier with its substrate at the cell surface. Rather, resistance may be due to an alteration in the mobility of the carrier possibly associated with a protein change in the carrier itself or the cell membrane that surrounds it.     

Elevated amounts of methotrexate-binding protein,different from normal dihydrofolate reductase,in a petunia MTXR-cell line

Summary A petunia cell line, 1ECB, was previously isolated by the stepwise selection procedure, for resistance to methotrexate (MTX), an antimetabolite for the enzyme dihydrofolate reductase (DHFR). Using ammonium sulfate precipitates of cell lysates of cell line 1ECB and its parental cell line (WT), it was found that the mutant has an increase of 400 fold in ³H-MTX binding capacity and a decrease in the affinity for MTX binding, at two orders of magnitude, in comparison with the WT. In addition, the DHFR specific activity in the mutant increased only moderately (5- to 10-fold), this activity is extremely sensitive to MTX inhibition as compared to the WT. It is evident that the MTX resistance of line 1ECB results mainly from overproduction of an MTX-binding protein which differs from the WT DHFR by four biochemical criteria. This protein may serve as a trap for the excess amounts of MTX to which the cells are exposed.     

Isolation of MTX-resistant Cell Line NP-19 of Nicotiana plumbaginifolia: Phenotypic, Genetic and Biochemical Study

Several cell lines of Nicotiana plumbaginifolia resistant tomethotrexate (MTX) were isolated upon gradual elevation of theMTX concentration in the growth medium. One of the MTX-resistantcell lines, NP-19, acquired resistance to 10 mmol m^–3MTX, that is, at least 50-fold higher than the lethal dose forthe wild-type cell suspension. Its resistance was stably maintainedupon prolonged withdrawal of the drug. The acquisition of resistancewas accompanied by severe deterioration of regeneration potential.Regenerated shoots still manifested resistance to 50 mmol m^–3.MTX-resistant colonies and tiny shoots were recovered followingasymmetric fusion between -irradiated NP-19 protoplasts andN. plumbaginifolia leaf mesophyll-derived protoplasts on a mediumcontaining 10 mmol m^–3 MTX. The development of the shootsderived both from NP-19 calli and following somatic fusion wasarrested at the stage of 6–8 leaves. No difference wasfound in uptake of ³H-MTX between cell suspension of NP-19 andthe parental cell line. A 30-fold increase in binding of ³H-MTXto protein extract was found in cell line NP-19, suggestingdifferential capacity of MTX binding as a mechanism involvedin the MTX resistance of this cell line. Since differentiatedorgans seem more sensitive to MTX than undifferentiated tissues,this cell line is a promising source for a gene(s) conferringenhanced MTX-tolerance both in non-differentiated and differentiatedtissues. Key words: Methotrexate resistance, MTX-binding capacity, MTX uptake, tobacco cell culture     

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

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.     

Construction of a dominant selectable marker using a novel dihydrofolate reductase.

Simian virus 40 promoter-enhancer-based mammalian expression plasmids using dihydrofolate reductase (DHFR)-encoding cDNA sequences originally isolated from two methotrexate (MTX)-resistant, DHFR-overproducing Chinese hamster lung cell lines were constructed. One, designated pSVA75, contains a DHFR cDNA that encodes leucine (Leu22) and corresponds to the wild type (wt), MTX-sensitive form of the enzyme [Melera et al., J. Biol. Chem. 263 (1988) 1978-1990]. The other plasmid, pSVA3, contains a cDNA that encodes a novel mutant form of the enzyme in which Leu22 has been changed to Phe [Melera et al., Mol. Cell Biol. 4 (1984) 38-48]. The resulting DHFR displays a 20-fold-enhanced resistance to inhibition by MTX, but maintains the catalytic activity of the wt enzyme [Albrecht et al., Cancer Res. 32 (1972) 1539-1546]. Transfection of DHFR- Chinese hamster ovary cells with either plasmid demonstrated that both were able to reconstitute the DHFR+ phenotype with equal efficiency (i.e., greater than 2.5 x 10(-3), indicating that both the wt and mutant enzymes were catalytically active in transfected cells. In addition, the mutant form of the enzyme was found to act as a dominant selectable marker when transfected into diploid DHFR+ cells, and to allow selection of resistant clones at low MTX concentrations (125 nM MTX) with a frequency of greater than 8 x 10(-4). Moreover, transfected clones were found to amplify their exogenous DHFR sequences to reasonably high levels (42-fold) at relatively low (888 nM) MTX concentrations, suggesting that substantial amplification of DHFR DNA and cotransfected sequences as well, can be achieved with this vector.     

Correlation of dihydrofolate reductase elevation with gene amplification in a homogeneously staining chromosomal region in L5178Y cells

A methotrexate (MTX)-resistant murine lymphoblastoid cell line has been obtained by serial passage in increasing concentrations of MTX which is greater than 100,000-fold resistant to MTX (L5178YR) and has dihydrofolate reductase (DHFR) levels 300-fold higher than the parental line. The L5178YR cell line synthesizes approximately 10-11% of its total soluble cell protein as DHFR regardless of growth phase, as measured by direct immunoprecipitation with a monospecific antiserum. Molecular hybridization of a purified [3H]DNA probe complimentary to DHFR specific mRNA with cellular DNA and RNA indicates that DHFR coding sequences are elevated several hundred fold in both nucleic acid species in the mutant cell line. Giemsa-banding studies of the diploid mutant line indicate the presence of a large homogeneously staining region on chromosome No. 2. In situ molecular hybridization studies indicate that the DHFR genes are localized in this homogeneously staining region. The homogeneously staining region probably consists of tandom repeats of a basic segment approximately 800 kilo base pairs long.     

Proteomic Analysis for the Identification of Proteins Related to Methotrexate Resistance

Methotrexate(MTX) is one of the most important and frequently used drugs in cancer therapy, but the efficacy of this drug is often compromised by the development of resistance in cancer cells. To seek and identify differentially expressed proteins related to MTX resistance and provide clues for the mechanism of MTX resistance, proteins from cell line MTX300 (resistant to 300 μmol/L MTX) and its control cell line 3T3R500 were separated by two-dimensional electrophoresis (2-DE). The colloidal Coomassie brilliant blue-stained 2-DE gels were subjected to image analysis, which revealed several spots with high levels of differential expression between MTX300 and 3T3R500. The protein spot with highest differential expression was submitted for tryptic peptide mass fingerprinting(PMF) for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MS analysis and database searches revealed it to be dihydrofolate reductase (DHFR), which was subsequently confirmed by Western blot. The result suggested that DHFR might play an important role in the MTX resistance.     

Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.