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 M 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 type K 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 3 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 M 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.
5-FU is incorporated at detectable levels into RNA and DNA of human tumor tissue, but no relation between the efficacy of 5-FU treatment and incorporation was found, in contrast to TS.
Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of mixed lineage leukemia (MLL) gene rearrangements, a poor outcome, and resistance to chemotherapeutic drugs. One exception is cytosine arabinoside (Ara-C), to which infant ALL cells are highly sensitive. To investigate the mechanism underlying Ara-C sensitivity in infants with ALL, mRNA levels of Ara-C-metabolizing enzymes were measured in infants (n ؍ 18) and older children (noninfants) with ALL (n ؍ 24). In the present study, infant ALL cells were 3.3-fold more sensitive to Ara-C (P ؍ .007) and accumulated 2.3-fold more Ara-CTP (P ؍ .011) upon exposure to Ara-C, compared with older children with ALL. Real-time quantitative reverse trancriptase-polymerase chain reaction (RT-PCR) (TaqMan) revealed that infants express 2-fold less of the Ara-C phosphorylating enzyme deoxycytidine kinase (dCK) mRNA (P ؍ .026) but 2.5-fold more mRNA of the equilibrative nucleoside transporter 1 (hENT1), responsible for Ara-C membrane transport (P ؍ .001). The mRNA expression of pyrimidine nucleotidase I (PN-I), cytidine deaminase (CDA), and deoxycytidylate deaminase (dCMPD) did not differ significantly between both groups. hENT1 mRNA expression inversely correlated with in vitro resistance to Ara-C (r s ؍ ؊0.58, P ؍ .006). The same differences concerning dCK and hENT1 mRNA expression were observed between MLL gene-rearranged (n ؍ 14) and germ line MLL cases (n ؍ 25). An oligonucleotide microarray screen (Affymetrix) comparing patients with MLL gene-rearranged ALL with those with nonrearranged ALL also showed a 1.9-fold lower dCK (P ؍ .001) and a 2.7-fold higher hENT1 (P ؍ .046) mRNA expression in patients with MLL generearranged ALL. We conclude that an elevated expression of hENT1, which transports Ara-C across the cell membrane, contributes to Ara-C sensitivity in MLL generearranged infant ALL.
The recommended dose of S-1 in chemotherapy-naive or minimally chemotherapy-exposed patients is 40 mg/m(2) bid on 28 consecutive days, every 5 weeks. In heavily pretreated patients, the recommended dose is 35 mg/m(2) bid. Phase II trials are warranted in tumors known to be responsive to 5-FU treatment.
The incidence of thromboembolic events, possibly related to the particular regimen tested in this study, discourages further investigation of this regimen.
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