P. Noordhuis

MRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method

Drug resistance of leukemic blasts is correlated to event-free survival and might be predicted by mRNA expression of drug resistance-related proteins. Methotrexate (MTX) is an important component in the treatment of childhood leukemia. Mechanisms of MTX resistance include (1) decreased transport via the reduced folate carrier (RFC), (2) altered levels of target enzymes, eg dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (3) decreased ratio of folylpolyglutamate synthetase (FPGS)/folylpolyglutamate hydrolase (FPGH). We designed competitive templates for each of these genes to measure mRNA expression by quantitative RT-PCR and normalized the expression to that of β-actin. T-lineage acute lymphoblastic leukemia (T-ALL), relatively MTX resistant compared to common/preB-ALL, displayed higher mRNA levels of DHFR and TS (three- and four-fold higher, respectively; P < 0.001), while FPGS expression was lower (three-fold, P = 0.006) compared to common/preB-ALL. The ratio of (DHFR × FPGH)/(RFC × FPGS) was more discriminating between T-ALL and c/preB-ALL (eight-fold higher; P < 0.001) than either target independently. Acute myeloid leukemia (AML) cells, considered MTX resistant, expressed two-fold lower levels of FPGS mRNA compared to c/preB-ALL (P = 0.04). The ratio of FPGH/FPGS was more discriminating between AML and c/preB-ALL (four-fold higher; P = 0.001) than either target independently. For the total group of 79 leukemic samples, mRNA expression of DHFR varied 549-fold and paralleled TS mRNA expression (r = 0.80; P < 0.001). Although variations in mRNA expression resembled variations in functional activity, no direct correlations were found for RFC (58-fold variation in mRNA expression), FPGS (95-fold) and FPGH (178-fold). In conclusion, differences in mRNA expression of MTX resistance parameters between leukemic subtypes as detected by competitive RT-PCR are in line with known differences in MTX resistance.

Prolonged retention of high concentrations of 5-fluorouracil in human and murine tumors as compared with plasma

SummaryConcentrations of 5-fluorouracil (5-FU) and its active metabolite 5-fluoro-2′-deoxy-5′-monophosphate (FdUMP) were measured in biopsy specimens of tumor tissue, normal mucosa, metastatic liver nodules, and normal liver tissue obtained from 39 patients and in two murine colon tumors (colon 26 and colon 38) after a single injection of 5FU at a therapeutic dose (500 mg/m2 and 100 mg/kg, respectively). These data were compared with plasma concentrations. Peak plasma concentrations (300–500 μm) of 5FU were comparable in human and murine plasma. The half-life of plasma elimination (during the period from 15 to 120 min) in both mouse and man ranged from 10 to 20 min, whereas at between 2 and 8 h, plasma concentrations varied from 0.1 to 1 μm, the half-life being about 100 min. In both species, 5FU could be measured in plasma at concentrations ranging from 0.01 to 1 μm for several days after 5FU treatment. 5FU concentrations in tissue samples obtained from 14 patients were measured during the time range of 1–6 h, those in samples taken from 7 patients, during the interval of 19–27 h; and those in samples obtained from 18 patients, within the interval of 40–48 h after injection. 5FU tumor concentrations varied between 0.78–21.6, 0.44–6.1, and 0.17–10.8 μmol/kg wet wt., respectively. Some of the 48-h samples were obtained from patients who had received leucovorin plus 5FU; coadministration of leucovorin did not alter 5FU tissue concentrations. At between 4 and 48 h, the tissue concentration/plasma concentration ratio was at least 10. 5FU concentrations in murine tumors were measured for up to 10 days after 5FU administration, with plateau 5FU tumor concentrations being about 50 μmol/kg wet wt. in colon 38 and about 200 μmol/kg wet wt. in colon 26 at 2 h after treatment; after 4 days, values of 0.5 and 4.8 μmol/kg, respectively, were obtained and after 10 days, respective concentrations of 0.1 and 0.07 μmol/kg were detected. The FdUMP concentrations measured in colon 26 and colon 38 tumors were 214 and 46 pmol/g, respectively, at 2 h after 5FU administration, and these values subsequently decreased to about 15 pmol/g in both tumors. In human tumors the initial FdUMP concentration ranged from 10 to 1000 pmol/g; at later time points the level of FdUMP was just above the detection limit of the assay. In liver metastases, high 5FU concentrations seemed to be related to high levels of FdUMP, which was likely of importance for the antitumor effect. The prolonged retention of 5FU should be taken into consideration in the design of biochemical modulation studies.

Antiproliferative activity, mechanism of action and oral antitumor activity of CP-4126, a fatty acid derivative of gemcitabine, in in vitro and in vivo tumor models

SummaryGemcitabine is a deoxycytidine (dCyd) analog with activity in leukemia and solid tumors, which requires phosphorylation by deoxycytidine kinase (dCK). Decreased membrane transport is a mechanism of resistance to gemcitabine. In order to facilitate gemcitabine uptake and prolong retention in the cell, a lipophilic pro-drug was synthesized (CP-4126), with an elaidic fatty acid esterified at the 5′position. CP-4126 was tested in cell lines resistant to cytarabine, another dCyd analog or gemcitabine. Activity of gemcitabine and the derivative was comparable in the parent cell lines, while in dCK deficient cells all compounds were inactive. However, inhibition of nucleoside transport increased the IC50 for gemcitabine up to 200-fold, but not for CP-4126, underlining the independence of a nucleoside transporter. For in vivo evaluation, nude mice bearing a human xenograft were treated intraperitoneally every third day for five doses at the maximal tolerated dose. In melanoma, sarcoma, lung, prostate, pancreatic and breast cancer xenografts, gemcitabine and CP-4126 were equally and highly effective; in four other xenografts moderately but equally active. In contrast to gemcitabine, CP-4126 could be administered orally, with a schedule and dose dependent toxicity and antitumor activity. In a colon cancer xenograft, antitumor activity of orally administered CP-4126 was equal to the intraperitoneally administered drug. In conclusion, CP-4126 is membrane transporter independent. Intraperitoneally administered CP-4126 was as effective as gemcitabine in several xenografts and CP-4126 is tolerated when orally administered. CP-4126 seems to be a promising new anticancer drug.

Oxaliplatin activity in selected and unselected human ovarian and colorectal cancer cell lines.

Oxaliplatin is used for treatment of colon cancer in combination with 5-fluorouracil or irinotecan. Oxaliplatin has similar, but also different resistant mechanisms as cisplatin. We studied the activity of oxaliplatin in ovarian and colon cancer cells with different resistance patterns to cisplatin. The 40-fold cisplatin-resistant cell line ADDP was only 7.5-fold resistant to oxaliplatin. The gemcitabine-resistant AG6000 cell line, 9-fold resistant to cisplatin, was not cross-resistant. LoVo-175X2, with mutant p53 showed no resistance compared to the empty vector control. However, LoVo-Li, with inactive p53, was 3.6-fold resistant corresponding to decreased accumulation and Pt adducts. Accumulation and DNA adducts formation showed no significant correlation with oxaliplatin sensitivity. Cell cycle distribution after exposure to oxaliplatin showed arrest in G2/M (A2780) or in S-phase (LoVo-92) for wt-p53 cells. ADDP and LoVo-Li showed G1 arrest followed by S-phase arrest and no changes in distribution, respectively. The cell cycle related proteins Cyclins A and B1 and (p)CDC25C were marginally affected by oxaliplatin. Expression of hCTR1 was decreased in ADDP, LoVo-Li and AG6000, OCT1 decreased in ADDP and AG6000 and OCT3 in LoVo-175X2, compared to the parental cell lines. In ADDP and LoVo-175X2 ATP7A and B were decreased but were increased in AG6000. From this study it can be concluded that changes in cell cycle distribution were cell line dependent and not related to changes in expression of Cyclin A or B1. Oxaliplatin accumulation was related to hCTR1 and, at low concentration, ATP7A to DNA adducts formation while the retention was related to hCTR1, OCT2 and ATP7B.

Antiproliferative Activity and Mechanism of Action of Fatty Acid Derivatives of Gemcitabine in Leukemia and Solid Tumor Cell Lines and in Human Xenografts

Gemcitabine is a deoxycytidine analog, which can be inactivated by deamination catalyzed by deoxycytidine deaminase (dCDA). Altered transport over the cell membrane is a mechanism of resistance to gemcitabine. To facilitate accumulation, the fatty acid derivative CP‐4125 was synthesized. Since, the fatty acid is acylated at the site of action of dCDA, a decreased deamination was expected. CP‐4125 was equally active as gemcitabine in a panel of rodent and human cell lines and in human melanoma xenografts bearing mice. In contrast to gemcitabine, CP‐4125 was not deaminated but inhibited deamination of deoxycytidine and gemcitabine. Pools of the active triphosphate of gemcitabine increased for over 20 hr after CP‐4125 exposure, while these pools decreased directly after removal of gemcitabine. In conclusion: CP‐4125 is an interesting new gemcitabine derivative.

Antitumour activity, toxicity and inhibition of thymidylate synthase of prolonged administration of 5-fluorouracil in mice

Continuous infusions of 5-fluorouracil (5-FU) are increasingly used in the treatment of cancer. Their optimal use, however, has still to be determined since the availability of suitable animal models is limited. We studied continuous infusions in mice using subcutaneously implanted pellets that release 5-FU over a period of 3 weeks. At the maximum tolerated dose (MTD) (based on the systemic toxicity in healthy animals) we assessed the antitumour activity, haematological toxicity, inhibition of thymidylate synthase (TS) in tumours and the concentration of 5-FU in plasma during the 3-week period. We also studied the addition of leucovorin in different schedules. The dose-limiting toxicity was weight loss, and at the MTD of 10 mg of 5-FU released in 21 days per mouse myelosuppression was tolerable (nadir for leucocytes and thrombocytes was approximately 40% of pretreatment levels). In several independent experiments using the 5-FU-resistant Colon 26 tumour, a good antitumour activity was observed during the first part of the infusion, but thereafter the growth of the tumours resumed; the overall effect of continuous infusions was thus comparable to that of bolus injections. Coadministration of leucovorin did not enhance the therapeutic results; depending on the schedule used, it proved ineffective or only increased toxicity. Similar results were obtained with head and neck squamous cell carcinomas and with the 5-FU-sensitive tumour Colon 38. In Colon 26 tumours the TS activity (FdUMP-binding assay) initially decreased to 20-30% of controls and returned to normal after 11 days. In the catalytic TS assay a slight inhibition was observed for the continuous infusion, followed after 11 days by a marked (4-fold) increase in activity. 5-FU plasma levels varied from 0.1 to 1 microM following a circadian rhythm (with a peak at 6 h after light onset), and were maintained during the entire period. Subcutaneously implanted pellets represent a suitable model to study prolonged administration of 5-FU in mice and to evaluate the effect of modulating agents in laboratory animals before transferring data obtained in vitro to the clinic.

No circadian variation of dihydropyrimidine dehydrogenase, uridine phosphorylase, beta-alanine, and 5-fluorouracil during continuous infusion of 5-fluorouracil.

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases and it catalyses the reduction of thymine and uracil to 5,6-dihydrothymine and 5,6-dihydrouracil, respectively. In mammals, the degradation of uracil by DPD is the only pathway leading to the biosynthesis of β-alanine. Furthermore, DPD is also responsible for the breakdown of the widely used antineoplastic agent 5-fluorouracil (5FU), thereby limiting the efficacy of the therapy. 5FU is one of the few drugs that shows some antitumour activity against various otherwise untreatable tumours including carcinomas of the gastrointestinal tract, breast, ovary and skin. Furthermore, 5FU is one of the few drugs for which a limited clinical effect has been shown when applied as a single agent during the treatment of advanced colorectal cancer. In order to exert its cyto-toxic effect against cancer, 5FU must first be anabolised to the nucleotide level. 5FU can be converted into FUMP by a sequential, two-step reaction consisting of the initial addition of a ribose by uridine phosphorylase (UP) to yield 5-fluorouridine (5FUrd), followed by phos-phorylation to FUMP by uridine kinase. The direct conversion of 5FU to FUMP is catalyzed by orotate phosphoribosyl transferase (OPRT) which transfers the ribose-phosphate moiety from phosphoribosyl pyrophosphate (PRPP) to 5FU. Although the cytotoxic effects of 5FU are probably directly mediated by the anabolic pathways, the catabolic route plays a significant role since more than 80% of the administered 5FU is catabolised by DPD. It has been reported that the bioavailability, efficacy as well as host-toxicity of 5FU follows a circadian rhythm in rodents1 and cancer patients2. In the present study, we investigated whether a circadian variation could be observed of the activity of DPD, UP and the plasma concentration of β-alanine and 5FU in patients treated with continuous infusion of 5FU.

Infants with acute lymphoblastic leukemia: no evidence for high methotrexate resistance.

The antifolate methotrexate (MTX) has contributed significantly to the great improvement in overall survival and central nervous system prophylaxis in patients with acute lymphoblastic leukemia (ALL) in the past 50 years. After transport into the cell, MTX is polyglutamylated with multiple glutamate residues to MTX-polyglutamates (MTXPGs), which have superior intracellular retention. Cellular resistance to MTX might contribute to treatment failure in childhood ALL. C/preB-lineage ALL ( 1 year) has a favorable prognosis and is in vitro more sensitive to MTX in the TSIA (short exposure) than T-lineage ALL.1 C/preB ALL also have more efficient accumulation of (long chain) MTX-PGs compared to T-ALL and acute myeloid leukemia.2–4 ALL diagnosed in infants less than 1 year of age is closely associated with a number of biological features, especiallyMLL gene (at chromosome 11q23) rearrangements and the proB (CD10-negative precursor B) immunophenotype, and still have a poor outcome.5 So far, little is known of the pharmacodynamics of MTX in infants with ALL, and the relationship thereof with the other biological characteristics frequent in infant ALL. The question then arises whether the poor prognosis of infants with ALL is associated with cellular resistance to MTX. Lymphoblasts isolated from bone marrow or peripheral blood of 47 infants 1 year with newly diagnosed, untreated ALL from the Dutch Childhood Leukemia Study Group (DCLSG), the German COALL study group, the Berlin–Frankfurt–Münster (BFM) Study Group and the Pediatric Oncology Group cell bank (POG) were used for this portion of the study. The distribution of important clinical parameters within the infants showed a high association with the proB immunophenotype and translocations involving the MLL gene (11q23, determined by karyotype, RT-PCR, and/or Southern blotting), and significantly higher white blood cell counts at presentation, features typical of infants with ALL. Since MTX cytotoxicity on primary ALL cells cannot be measured with the MTT assay, we used the thymidylate synthase (TS) inhibition assay (TSIA), which correlates strongly with IC50 values for MTX obtained for cell lines in the MTT assay.1 These data were expressed as the concentration of MTX necessary to inhibit 50% of the TS activity (TSI50), compared to the controls incubated without MTX. A large range of TSI50 values for MTX was observed for both the short (3 h, followed by 18 h drug-free) and continuous (21 h) exposure conditions. Infant ALL cells did not differ in MTX sensitivity from those of a reference group of 109 common(c)/preB ALL patients older than 1 year, with overlapping ranges and similar median TSI50 values (Table 1, Figure 1a and b). The total in vitro accumulation of MTX and the pharmacologically important long-chain polyglutamates (MTX-PG4–6, analyzed by HPLC) did not differ significantly between the infants and the older c/preB ALL group (Figure 1c and d). Hyperdiploid B-lineage ALL cells accumulate higher levels of MTXpolyglutamates.6 Because hyperdiploidy is found in one fifth of the older children but in no case of infant ALL, a comparison was also made of only those patients in both groups known to have a nonhyperdiploid DNA index, as determined by flow cytometry (hyperdiploidy was defined as a DNA index 1.16–1.35). Infants were as sensitive to MTX in the TSIA compared to non-hyperdiploid c/preB ALL patients 1 year (Table 1). In order to examine the influence of proB immunophenotype and MLL gene rearrangements on infant drug sensitivity, the impact of age was investigated within patients with proB immunophenotype and

In vitro antitumour activity of cis- and trans-5-fluoro-5,6-dihydro-6-alkoxy-uracils; effects on thymidylate synthesis.

A class of new 5-fluorouracil (FU) analogues, the 5-fluoro-5,6-dihydro-6- alkoxy-uracils was synthesised with a modification at the 6-position of the pyrimidine ring. At this position the analogues have a hydroxy or alkoxy group of different chain lengths either in the cis- or trans-configuration. The antiproliferative effect of these compounds was tested on five cell lines of different origin. Generally, the analogues with a cis-configuration had a higher activity than those with a trans-configuration. The growth inhibitory effect of the compounds decreased with increasing alkoxy chain length, but the compound with a hydroxy group had the lowest growth inhibitory effect. One analogue, cis-5-F-5,6-dihydro-6-methoxy-uracil had a higher antiproliferative effect than FU in one of the cell lines. Effects on thymidylate synthase (TS), the possible target of these analogues, were evaluated by thymidine rescue of growth inhibition and incorporation of tritiated deoxyuridine (3H-UdR) into DNA. In solid tumour cell lines addition of TdR reversed the antiproliferative effect. Inhibition of TS in intact cells was determined by measuring 3H-UdR incorporation in two cell lines. The effect of cis-5-F-5,6-dihydro-6-methoxy-uracil on incorporation of 3H-UdR was 2- to 5-fold stronger than that of FU in both cell lines. All other compounds produced a higher 3H-UdR incorporation than FU both at equimolar and equi-toxic concentration. Concluding from these results we regard cis-5-F-5,6-dihydro-6-methoxy-uracil as the most promising FU analogue of this series, because of its higher antiproliferative activity than FU and marked inhibition of TS in intact cells.

Selective Protection by Uridine of Growth Inhibition by 5-Fluorouracil (5FU) Mediated by 5FU Incorporation into RNA, But Not the Thymidylate Synthase Mediated Growth Inhibition by 5FU-Leucovorin

Fluorouracil (5FU) acts by RNA-incorporation and inhibition of thymidylate synthase; the first action is counteracted by uridine, and the second is enhanced by leucovorin (LV). Growth inhibition of C26-10 colon cancer cells by 5FU was enhanced by LV and rescued by uridine, but 5FU-LV was only partially rescued by uridine. In WiDr cells, 5FU sensitivity was not enhanced by LV, while both 5FU and 5FU-LV were rescued by uridine. Intermediate trends were found in SW948 and HT29 cells. Uridine rescue in mice allowed 1.5-fold increase in 5FU dose, leading to 2-fold increase in the antitumor effect and thymidylate synthase inhibition in resistant Colon-26 tumors. In the sensitive Colon-26-10 tumor, uridine rescue decreased 5FU-RNA incorporation > 10-fold, without affecting the antitumor activity. The use of LV and uridine can differentiate between two mechanisms of action of 5FU.