David G. Priest

A Structurally Altered Human Reduced Folate Carrier with Increased Folic Acid Transport Mediates a Novel Mechanism of Antifolate Resistance

CEM/MTX is a subline of human CCRF-CEM leukemia cells which displays >200-fold resistance to methotrexate (MTX) due to defective transport via the reduced folate carrier (RFC). CEM/MTX-low folate (LF) cells, derived by a gradual deprivation of folic acid from 2.3 μm to 2 nm (LF) in the cell culture medium of CEM/MTX cells, resulted in a >20-fold overexpression of a structurally altered RFC featuring; 1) a wild typeK m value for MTX transport but a 31-fold and 9-fold lower K m values for folic acid and leucovorin, respectively, relative to wild type RFC; 2) a 10-fold RFC1 gene amplification along with a >20-fold increased expression of the main 3.1-kilobase RFC1 mRNA; 3) a marked stimulation of MTX transport by anions (i.e. chloride); and 4) a G → A mutation at nucleotide 227 of the RFC cDNA in both CEM/MTX-LF and CEM/MTX, resulting in a lysine for glutamate substitution at amino acid residue 45 predicted to reside within the first transmembrane domain of the human RFC. Upon transfer of CEM/MTX-LF cells to folate-replete medium (2.3 μm folic acid), the more efficient folic acid uptake in CEM/MTX-LF cells resulted in a 7- and 24-fold elevated total folate pool compared with CEM and CEM/MTX cells, respectively (500 versus 69 and 21 pmol/mg of protein, respectively). This markedly elevated intracellular folate pool conferred a novel mechanism of resistance to polyglutamatable (e.g. ZD1694, DDATHF, and AG2034) and lipophilic antifolates (e.g. trimetrexate and pyrimethamine) by abolishing their polyglutamylation and circumventing target enzyme inhibition.

Evidence for involvement of tyrosine phosphorylation in taxol-induced apoptosis in a human ovarian tumor cell line

Taxol is an antineoplastic agent with significant activity against ovarian as well as breast cancer. To investigate mechanisms by which taxol exerts its cytotoxic action, taxol-induced apoptosis, characterized by morphologic changes and internucleosomal DNA fragmentation, was examined in a human ovarian tumor cell line. Time-dependent morphologic changes, characteristic of apoptosis, were observed over the same time as the appearance of internucleosomal DNA fragmentation. The specific protein tyrosine kinase inhibitors genistein and herbimycin A, and the ATP depletion agent sodium azide, interfered with taxol-induced DNA fragmentation and clonal cell death. Based on a quantitative reverse transcription-polymerase chain reaction technique, bcl-2 alpha oncogene expression was decreased in conjunction with taxol-induced DNA fragmentation, and this decrease could be blocked by genistein. These results strongly implicate protein tyrosine phosphorylation as an event that mediates apoptosis and, thus, the antitumor activity of taxol in ovarian cancer.

Resistance to multiple novel antifolates is mediated via defective drug transport resulting from clustered mutations in the reduced folate carrier gene in human leukaemia cell lines.

We have studied the molecular basis of resistance of multiple human leukaemia CCRF-CEM sublines to the novel antifolates ZD9331, GW1843, AG2034, PT523 and edatrexate, which use the reduced folate carrier (RFC) as their main cellular uptake route and that target different folate-dependent enzymes. Antifolate-resistant sublines established by stepwise and single-step selections displayed up to 2135-fold resistance to the selection drug, and up to 2323-fold cross-resistance to various hydrophilic antifolates. In contrast, these sublines were up to 17- and 20-fold hypersensitive to the lipophilic antifolates AG377 and trimetrexate, respectively. The total reduced folate pool of these antifolate-resistant sublines shrunk by 87-96%, resulting in up to 42-fold increased folic acid growth requirement. These sublines lost 92-97% of parental [(3)H]methotrexate influx rates. Genomic PCR single-strand conformational polymorphism analysis and sequencing revealed that most of these drug-resistant sublines harboured RFC mutations that surprisingly clustered in two confined regions in exons 2 and 3. The majority of these mutations resulted in frame-shift and/or premature translation termination and lack of RFC protein expression. The remaining mutations involved single amino acid substitutions predominantly residing in the first transmembrane domain (TMD1). Some RFC-inactivating mutations emerged during the early stages of antifolate selection and were stably retained during further drug selection. Furthermore, some sublines displayed a markedly decreased or abolished RFC mRNA and/or protein expression. This constitutes the first demonstration of clustering of multiple human RFC mutations in TMD1, thereby suggesting that it plays a functional role in folate/antifolate binding and/or translocation. This is the first molecular characterization of human RFC-associated modalities of resistance to various novel antifolates in multiple leukaemia sublines.

The role of multidrug resistance proteins MRP1, MRP2 and MRP3 in cellular folate homeostasis

Previously, we reported that the multidrug resistance proteins MRP1, MRP2 and MRP3 confer resistance to therapeutic antifolates by mediating their cellular extrusion. We now determined whether MRPs also play a role in controlling cellular homeostasis of natural folates. In MRP1, MRP2 and MRP3-transfected 2008 human ovarian carcinoma cells total cellular folate content was 32-38% lower than in 2008 cells (105+/-14pmolfolate/mgprotein) when grown in medium containing 2.3 microM folic acid (FA). Under these conditions cellular growth rates were not compromised. However, when cells were challenged under folate-depleted conditions with a short exposure (4 hr) to FA or leucovorin, MRP1 and MRP3 overexpressing cells were impaired in their growth. In contrast to wild-type cells, MRP1 transfected cells retained only 60% of the maximum growth when exposed to 500 nM leucovorin or 500 microM FA. For 2008/MRP1 and 2008/MRP3 cells FA growth stimulation capacity was dramatically decreased when, during a 4 hr exposure, metabolism into rapidly polyglutamatable and retainable dihydrofolate was blocked by the dihydrofolate reductase inhibitor trimetrexate. To retain growth under such conditions MRP1 overexpressing cells required much higher concentrations of FA (EC(50) > 500 microM) compared to 2008 cells (EC(50): 12 microM). These results suggest that down- and up-regulation of MRP1 (and MRP3) expression can influence cellular folate homeostasis, in particular when cellular retention by polyglutamylation of folates is attenuated.

Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for folate homeostasis

We determined the mechanisms of resistance of human CCRF-CEM leukemia cells to methotrexate (MTX) vs. those to six novel antifolates: the polyglutamatable thymidylate synthase (TS) inhibitors ZD1694, multitargeted antifolate, pemetrexed, ALIMTA (MTA) and GW1843U89, the non-polyglutamatable inhibitors of TS, ZD9331, and dihydrofolate reductase, PT523, as well as DDATHF, a polyglutamatable glycinamide ribonucleotide transformylase inhibitor. CEM cells were made resistant to these drugs by clinically relevant intermittent 24 hr exposures to 5-10 microM of MTX, ZD1694, GW1843U89, MTA and DDATHF, by intermittent 72 hr exposures to 5 microM of ZD9331 and by continuous exposure to stepwise increasing concentrations of ZD9331, GW1843U89 and PT523. Development of resistance required only 3 cycles of intermittent drug exposure to ZD1694 and MTA, but 5 cycles for MTX, DDATHF and GW1843U89 and 8 cycles for ZD9331. The predominant mechanism of resistance to ZD1694, MTA, MTX and DDATHF was impaired polyglutamylation due to approximately 10-fold decreased folylpolyglutamate synthetase activity. Resistance to intermittent exposures to GW1843U89 and ZD9331 was associated with a 2-fold decreased transport via the reduced folate carrier (RFC). The CEM cell lines resistant to intermittent exposures to MTX, ZD1694, MTA, DDATHF, GW1843U89 and ZD9331 displayed a depletion (up to 4-fold) of total intracellular reduced folate pools. Resistance to continuous exposure to ZD9331 was caused by a 14-fold increase in TS activity. CEM/GW70, selected by continuous exposure to GW1843U89 was 50-fold resistant to GW1843U89, whereas continuous exposure to PT523 generated CEM/PT523 cells that were highly resistant (1550-fold) to PT523. Both CEM/GW70 and CEM/PT523 displayed cross-resistance to several antifolates that depend on the RFC for cellular uptake, including MTX (95- and 530-fold). CEM/GW70 cells were characterized by a 12-fold decreased transport of [3H]MTX. Interestingly, however, CEM/GW70 cells displayed an enhanced transport of folic acid, consistent with the expression of a structurally altered RFC resulting in a 2.6-fold increase of intracellular folate pools. CEM/PT523 cells displayed a markedly impaired (100-fold) transport of [3H]MTX along with 12-fold decreased total folate pools. In conclusion, multifunctional mechanisms of resistance in CEM cells have a differential impact on cellular folate homeostasis: decreased polyglutamylation and transport defects lead to folate depletion, whereas a structurally altered RFC protein can provoke expanded intracellular folate pools.

Impact of dietary folic acid on reduced folates in mouse plasma and tissues. Relationship to dideazatetrahydrofolate sensitivity.

To investigate the role of dietary folic acid in dideazatetrahydrofolate (DDATHF) sensitivity, reduced folates were estimated in plasma and tissue of mice following dietary depletion and repletion. Previous studies showed that DDATHF, a new folate antagonist targeted against glycinamide ribonucleotide transformylase, produced unexpectedly severe toxicity in humans compared with mice. However, toxicity in the animal model also became pronounced upon the removal of folic acid from the diet. Further, modest dietary restoration of folic acid in the drinking water showed that toxicity could be alleviated while antitumor activity was maintained. To investigate the role of dietary folic acid levels on tissue folates in this system, all the natural reduced folates were evaluated by a ternary complex based assay in mice placed on folic acid deplete and replete diets. After 2 weeks on a folic acid deplete diet, total plasma folate had decreased by 85%, whereas red blood cell, liver, and intestinal folate fell by only 50%. Repletion of folic acid in the drinking water at a low level (0.0003%) caused partial restoration of reduced folates, while a higher repletion level (0.003%) resulted in restoration to control levels or above. Administration of folic acid and leucovorin by oral gavage to DDATHF-treated mice resulted in elevation of tissue folates in mice maintained on folic acid deplete and replete diets. Relatively high levels of folic acid were present in plasma following oral gavage of folic acid, while essentially no [S]5-formyltetrahydrofolate was observed after leucovorin. Reduced folate pools in a subcutaneously implanted mouse mammary adenocarcinoma responded more extensively to dietary folic acid depletion than folate pools in liver. Likewise, these pools were more sensitive to restoration by folic acid or leucovorin. This greater reduced folate response of tumor versus normal tissue, if confirmed in other systems, suggests a possible basis for selective antitumor activity.

Comparison of folylpolyglutamate hydrolases of mouse liver, kidney, muscle and brain.

SummaryThe folylpolyglutamate hydrolase activities of mouse liver, kidney, muscle and brain were examined by incorporation of methylenetetrahydrofolate polyglutamate reaction products into a stable ternary complex with tritiated fluorodeoxyuridylate and L. casei thymidylate synthetase. Complexes were separated electrophoretically on the basis of charge associated with the polyglutamyl moieties to determine distribution of chain lengths throughout the time course of the reaction. Tissue folylpolyglutamate hydrolase activities were allowed to utilize endogenous folylpolyglutamate as substrates by incubating crude tissue extracts at pH 7.4 and pH 4.5. Kidney and muscle contained relatively reactive hydrolases which were capable of generating intermediates of essentially all chain lengths from folylpentaglutamate, the predominant endogenous species. The relatively low activity in brain also gave rise to all possible intermediates. Liver contained a high concentration of methylenetetrahydrofolate but little hydrolase activity. The activity present in liver gave rise to essentially no intermediates but yielded only the monoglutamate form of the cofactor. When purified lysosomal preparations from liver and kidney were allowed to react with synthetic folylpolyglutamates, the same specificity with regard to reaction products was observed as with endogenous substrates.

Increased thymidylate synthetase in 5-fluorodeoxyuridine resistant cultured hepatoma cells.

Abstract A FdUrd resistant line of cultured mouse hepatoma cells has been obtained. The resistant cell line had 6- to 10-fold higher levels of thymidylate synthetase, but dihydrofolate reductase and thymidine kinase were unchanged. No impairment of FdUrd incorporation by the resistant cell line could be detected. The increased thymidylate synthetase in resistant cells had the same turnover number and I 50 for FdUMP as the enzyme found in sensitive cells, making it unlikely that a new gene product had been obtained. Sensitive cells could be completely rescued by the addition of thymidine, suggesting that the primary mode of drug action is to diminish thymidine metabolites. Resistant cells, removed from FdUrd for several generations, did not proliferate immediately upon reintroduction of the drug; however, loss of sensitivity was much more rapid than upon initial exposure. These results are interpreted in terms of a mechanism for resistance.

Tissue folylpolyglutamate chain-length characterization by electrophoresis as thymidylate synthetase-fluorodeoxyuridylate ternary complexes

Abstract Tissue folate polyglutamate chain lengths have been estimated by incorporation of the 5,10-methylenetetrahydrofolate polyglutamate form of the cofactor into ternary complexes with Lactobacillus casei thymidylate synthetase and [3H]fluorodeoxyuridylate. Complexes were separated electrophoretically on the basis of the charge associated with the free carboxyl function of each glutamate residue. The method is specific for methylenetetrahydrofolate and assures evaluation of chain length with minimal error introduced by pool equilibration or autooxidation. Introduction of formaldehyde into tissue extracts was used to convert tetrahydrofolate to the methylene derivative and show that the two pools have the same polyglutamate chain lengths. By this technique, mammalian liver tissue was shown to contain both pteroylpentaglutamate and pteroylhexaglutamate. Monkey, pig, and rabbit liver contain predominately hexaglutamate, mouse liver predominately pentaglutamate, and rat liver nearly equal proportions of each. Chicken liver contains mostly pentaglutamate and fish liver, hexaglutamate. The presence of penta-and hexaglutamates in L. casei and heptaglutamates in yeast confirms earlier observations by other methods and attests to the validity of this one.

Cancer cells activate p53 in response to 10-formyltetrahydrofolate dehydrogenase expression

A folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase; EC 1.5.1.6), is not a typical tumour suppressor, but it has two basic characteristics of one, i.e. it is down-regulated in tumours and its expression is selectively cytotoxic to cancer cells. We have recently shown that ectopic expression of FDH in A549 lung cancer cells induces G1 arrest and apoptosis that was accompanied by elevation of p53 and its downstream target, p21. It was not known, however, whether FDH-induced apoptosis is p53-dependent or not. In the present study, we report that FDH-induced suppressor effects are strictly p53-dependent in A549 cells. Both knockdown of p53 using an RNAi (RNA interference) approach and disabling of p53 function by dominant-negative inhibition with R175H mutant p53 prevented FDH-induced cytotoxicity in these cells. Ablation of the FDH-suppressor effect is associated with an inability to activate apoptosis in the absence of functional p53. We have also shown that FDH elevation results in p53 phosphorylation at Ser-6 and Ser-20 in the p53 transactivation domain, and Ser-392 in the C-terminal domain, but only Ser-6 is strictly required to mediate FDH effects. Also, translocation of p53 to the nuclei and expression of the pro-apoptotic protein PUMA (Bcl2 binding component 3) was observed after induction of FDH expression. Elevation of FDH in p53 functional HCT116 cells induced strong growth inhibition, while growth of p53-deficient HCT116 cells was unaffected. This implies that activation of p53-dependent pathways is a general downstream mechanism in response to induction of FDH expression in p53 functional cancer cells.