David G. Priest

The human ovarian cell line A2780 was exposed to either cisplatin (10 microM) or 5-fluorouracil (5FUra) (5 microM) for 1 hr. Cytotoxicity was less than 14% with either agent alone. Cisplatin (10 microM) and 5FUra (5 microM) in combination for 1 hr caused a 76% reduction in cell growth. Thymidine (dThd, 10 microM), if given concomitantly with the combination of cisplatin and 5FUra, completely protected the tumor cells. A 30-min exposure to cisplatin increased the intracellular pools of 5,10-methylenetetrahydrofolate and tetrahydrofolate 2.5-fold. The capacity of intact cells to form 5-fluorodeoxyuridylate (FdUMP)-thymidylate (dTMP) synthase complex when incubated with fluorodeoxyuridine (FdUrd) was enhanced 2.5-fold when the cells were pretreated with cisplatin. These experiments demonstrate that cisplatin can increase the availability of the reduced folate necessary for tight binding of FdUMP to dTMP synthase, thus enhancing the cytotoxicity of the cisplatin and 5FUra combination.

We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning DNA within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases the time and effort needed for integration of single or multiple DNA fragments. Additionally, clonetegration facilitates cloning and expression of genetic elements that are impossible to propagate within typical multicopy plasmids.

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 microM 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 Km value for MTX transport but a 31-fold and 9-fold lower Km 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 microM 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.

Studies have shown that conversion of leucovorin to the metabolite 5,10-methylenetetrahydrofolate (5,10-CH2FH4) is responsible for enhancement of the antitumor effects of fluorouracil given in combination with leucovorin, but the biochemical basis of this conversion in humans is not fully understood. To determine a possible sequence of metabolic steps, we studied the pharmacokinetics of leucovorin and its reduced folate metabolites in plasma in healthy volunteers. Groups of five subjects were given two equal doses of 10, 25, 125, 250, or 500 mg/m2 leucovorin, one orally and one intravenously at a 30-day interval. A sensitive radioenzymatic method that we developed previously was used to measure plasma concentrations of [S]5-formyltetrahydrofolate, 10-formyltetrahydrofolate (10-CHOFH4), 5-methyltetrahydrofolate (5-CH3FH4), and the combined 5,10-CH2FH4 plus tetrahydrofolate (FH4) pools. Intravenous administration of leucovorin resulted in dose-dependent accumulation of 5,10-CH2FH4 + FH4 exceeding 2 microM at peak levels. After oral and intravenous administration, 10-CHOFH4 and 5,10-CH2FH4 + FH4 exhibited peak levels earlier and were eliminated more rapidly than 5-CH3FH4. Accumulation of all metabolites after intravenous administration was linearly dose dependent, while oral administration appeared to result in saturation. We propose that the host activation of leucovorin suggested by these findings could be responsible for elevation of intratumor 5,10-CH2FH4 levels, thus enhancing the antitumor effects of fluorouracil. These results also suggest that 10-CHOFH4, 5,10-CH2FH4, and FH4 are intermediate metabolites and that 5-CH3FH4 is the terminal metabolite. In addition, our results indicate that attainment of high plasma levels of the metabolites active in modulation of the therapeutic effects of fluorouracil is best achieved through intravenous administration of high doses of leucovorin. Our future studies will address the proposed sequential conversion pathway and, thus, the mechanism by which pharmacologically relevant reduced folates accumulate in plasma after leucovorin administration.