Predictive value of thymidylate synthase and dihydropyrimidine dehydrogenase protein expression on survival in adjuvantly treated stage III colon cancer patients.

BACKGROUND: The predictive value of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) expression on long-term survival by influencing 5-fluorouracil (5-FU) effect were determined in primary tumours and node metastases of stage III colon cancer patients treated adjuvantly with 5-FU regimens (n=391). The effect of TP 53 mutation status, which is thought to be functionally linked to TS inhibition, was also examined. PATIENTS AND METHODS: TS and DPD protein expression was determined by immunohistochemical analysis using tissue microarrays of these colon tumours. Two hundred and twenty tumours had already been screened in a previous study for TP 53 mutations. RESULTS: Low TS protein levels in primary stage III colon tumours appeared to be associated with mucinous histology and low DPD protein levels with young age at time of randomisation. Concordance between TS and DPD expression in primary and metastatic tumours was low. No associations were found between disease-free survival (DFS) and TS or DPD protein levels. When stratified by TP 53 mutation status DFS did not differ with TS expression. CONCLUSIONS: Expression of TS and DPD proteins is not predictive for survival in patients with stage III colon cancer treated adjuvantly with 5-FU regimens. TS protein levels did not alter the effect of TP 53 mutation status.

Polymorphism of the thymidylate synthase gene and thymidylate synthase levels in colon cancer cell lines and different tissues of colorectal cancer patients.

In a panel of 18 colon cancer cell lines we found that the thymidylate synthase (TS) genotype was related to TS enzyme activity, but not to TS protein and mRNA levels. In addition, no relation with drug sensitivity was observed. TS genotyping of different tissues from 78 colorectal cancer patients revealed a high level of homology in polymorphic status between normal and malignant tissues and the heterozygous genotype to be the most frequent.

Comparison of mRNA expression levels determined with TaqMan and competitive template RT-PCR.

Two methods for measurement of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) mRNA expression were compared. Although the relative mRNA levels compared to beta-actin measured with competitive template RT-PCR were different from the data obtained with a TaqMan based PCR, a significant correlation between the two assays was found.

Exploring nature's plasticity with a flexible probing tool, and finding new ways for its electronic distribution.

Concepts and results are described for the use of a single, but extremely flexible, probing tool to address a wide variety of genomic questions. This is achieved by transforming genomic questions into a software file that is used as the design scheme for potentially any genomic assay in a microarray format. Microarray fabrication takes place in three-dimensional microchannel reaction carriers by in situ synthesis based on spatial light modulation. This set-up allows for maximum flexibility in design and realization of genomic assays. Flexibility is achieved at the molecular, genomic and assay levels. We have applied this technology to expression profiling and genotyping experiments.

Cross-resistance in the 2',2'-difluorodeoxycytidine (gemcitabine)-resistant human ovarian cancer cell line AG6000 to standard and investigational drugs.

Gemcitabine (2'-2'-difluorodeoxycytidine; dFdC) is a deoxycytidine analogue which is effective against solid tumours, including lung cancer and ovarian cancer. dFdC requires phosphorylation by deoxycytidine kinase (dCK) for activation. In the human ovarian cancer cell line A2780 and its 30,000-fold dFdC-resistant variant AG6000 (P<0.001), we investigated the cross-resistance profile to several drugs. AG6000, which has a complete dCK deficiency, was approximately 1000-10,000-fold resistant to other deoxynucleoside analogues such as 1-beta-D-arabinofuranosyl cytosine, 2-chloro-deoxyadenosine, aza-deoxycytidine and 2', 2'-difluorodeoxyguanosine (dFdG) (P<0.001). dFdG can be activated by dCK and deoxyguanosine kinase (dGK), but the latter enzyme was not altered in AG6000 cells. Thus dFdG resistance was only due to dCK deficiency. AG6000 was 1.6- and 46.7-fold resistant to 5-fluorouracil (5-FU) and ZD1694, respectively (the latter was significant; P<0.01), which may be due to the 1.7-fold higher thymidylate synthase (TS) activity, but AG6000 cells were also 2. 7-fold resistant to the lipophilic TS inhibitor AG337 (P<0.05). Remarkably, AG6000 cells were 2.5-fold more sensitive to methotrexate (MTX) (P<0.01) than A2780 cells, but 1.6-fold more resistant to trimetrexate (TMQ) (P<0.10). However, no differences in reduced folate carrier activity, folylpolyglutamate synthetase (FPGS) activity and polyglutamation of MTX were found between the cell lines. AG6000 cells were approximately 2 to 7.5-fold more resistant to doxorubicin (DOX), daunorubicin (DAU), epirubicin and vincristine (VCR) (the latter was significant; P<0.02) and approximately 4-fold more resistant to the microtubule inhibitors paclitaxel and docetaxel (P<0.001). Fluorescent activated cell sorter (FACS) analysis revealed no P-glycoprotein (Pgp) or multidrug resistance-associated protein (MRP) expression, but less fluorescence of intercalated DAU in AG6000 cells. An approximately 2-fold resistance to the topoisomerase I and II inhibitors etoposide, CPT-11 and SN38 was found in AG6000 cells. Topoisomerase I and IIalpha RNA expression was decreased in AG6000 cells. AG6000 was 2.4, 2.4, 2.3 and 3.7-fold more resistant to EO9 (P<0.02), mitomycin-C (MMC) (P<0.05), cisplatin (CDDP) (P<0.10) and maphosphamide (MAPH), respectively. DT-diaphorase (DTD), which activates EO9, was 2.2-fold lower in AG6000 cells. CDDP resistance might be related to a reduced retention of DNA adducts in AG6000. However, glutathione levels were equal in A2780 and AG6000 cells. A 24 h exposure to DOX, VCR and paclitaxel at equimolar and equitoxic concentrations, resulted in more double-strand breaks (1.5- to 2-fold) in A2780 than in AG6000 cells. MAPH at 1120 nM and 17 nM of EO9 did not cause DNA damage in either cell line. In conclusion, AG6000 is a cell line highly cross-resistant to a wide variety of drugs. This cross-resistance might be related to altered enzyme activities and/or increased DNA repair.

Clustering of mutations in the first transmembrane domain of the human reduced folate carrier in GW1843U89-resistant leukemia cells with impaired antifolate transport and augmented folate uptake.

We have studied the molecular basis for the resistance of human CEM leukemia cells to GW1843, a thymidylate synthase inhibitor. GW1843-resistant cells displayed a approximately 100-fold resistance to GW1843 and methotrexate but were collaterally sensitive to the lipophilic antifolates trimetrexate and AG337, which enter cells by diffusion. These cells exhibited a 12-fold decreased methotrexate influx but surprisingly had a 2-fold decreased folic acid growth requirement. This was associated with a 4-fold increased influx of folic acid, a 3.5-fold increased steady-state level of folic acid, and a 2.3-fold expansion of the cellular folate pool. Characterization of the transport kinetic properties revealed that GW1843-resistant cells had the following alterations: (a) 11-fold decreased transport K(m) for folic acid; (b) 6-fold increased transport K(m) for GW1843; and (c) a slightly increased transport V(max) for folic acid. Sequence analysis showed that GW1843-resistant cells contained the mutations Val-29 --> Leu, Glu-45 --> Lys, and Ser-46 --> Ile in the first transmembrane domain of the reduced folate carrier. Transfection of the mutant-reduced folate carrier cDNA into methotrexate transport null cells conferred resistance to GW1843. This is the first demonstration of multiple mutations in a confined region of the human reduced folate carrier in an antifolate-resistant mutant. We conclude that certain amino acid residues in the first transmembrane domain play a key role in (anti)folate binding and in the conferring of drug resistance.

Circumvention of methotrexate resistance in childhood leukemia subtypes by rationally designed antifolates.

Cellular methotrexate (MTX) resistance may cause treatment failure in childhood common/preB-acute lymphoblastic leukemia (c/preB-ALL), T-lineage ALL (T-ALL), and acute myeloid leukemia (AML). The ex vivo potency of several antifolates (MTX, trimetrexate [TMQ], GW1843U89, multitargeted antifolate [MTA], Raltitrexed, and ZD9331) was studied via in situ inhibition of thymidylate synthase (TS). After short-term exposure, relapsed c/preB-ALL (rALL, n = 21), T-ALL (n = 22), and AML (n = 22) were 3-fold, 10-fold, and 6-fold less sensitive to MTX (P

Differential methotrexate resistance in childhood T- versus common/preB-acute lymphoblastic leukemia can be measured by an in situ thymidylate synthase inhibition assay, but not by the MTT assay.

Methotrexate (MTX) is not cytotoxic to patient-derived acute lymphoblastic leukemia (ALL) cells in total-cell-kill assays, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, putatively due to the rescue effects of hypoxanthine and thymidine released from dying cells. This was mimicked by a diminished methotrexate (MTX) cytotoxicity for the cell lines HL60 and U937 in the presence of hypoxanthine, thymidine, or lysed ALL cells. However, enzymatic depletion or inhibition of nucleoside membrane transport did not result in MTX dose-dependent cytotoxicity in patient samples. Alternatively, a thymidylate synthase inhibition assay (TSIA), based on inhibition of the TS-catalyzed conversion of 3H-dUMP to dTMP and 3H2O, correlated with the MTT assay for antifolate sensitivity in four human leukemia cell lines with different modes of MTX resistance. For 86 ALL patient samples, TSI50 values after 21 hours exposure to MTX were not different between T- and c/preB-ALL (P =.46). After 3 hours incubation with MTX followed by an 18-hour drug-free period, T-ALL samples were 3.4-fold more resistant to MTX compared with c/preB-ALL samples (P =.001) reflecting the clinical differences in MTX sensitivity. TSI50 values correlated with MTX accumulation (r = -.58, P <.001). In conclusion, the TSIA, but not the MTT assay, can measure dose-response curves for MTX in patient-derived ALL cells and showed relative MTX resistance in T-ALL compared with c/preB-ALL.

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 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.

Ex vivo activity of methotrexate versus novel antifolate inhibitors of dihydrofolate reductase and thymidylate synthase against childhood leukemia cells.

Leukemic cells of 27 children [14 patients with initial acute lymphoblastic leukemia (iALL), 8 patients with relapsed ALL (rALL), and 5 patients with acute nonlymphoblastic leukemia (ANLL)] were evaluated for their sensitivity to methotrexate (MTX) and five novel antifolate drugs, which have the potential to circumvent MTX resistance. The novel antifolates include a polyglutamatable [edatrexate (EDX)] and a lipophilic (trimetrexate) inhibitor of dihydrofolate reductase and two polyglutamatable inhibitors (ZD1694 and GW1843U89) and one lipophilic inhibitor (AG337) of thymidylate synthase (TS). Drug activity was assessed via the determination of in situ inhibition of TS activity after exposing leukemic cells to antifolate drugs for: (a) 3 h, followed by a 15-h drug-free period; and (b) 18 h of continuous exposure. For human CEM leukemia cell lines with well-defined mechanisms of resistance to MTX, in situ TS inhibition correlated with the growth-inhibitory effects of MTX and the novel antifolates (r = 0.86-0.93; P < 0.01). Although a wide interpatient variability in MTX sensitivity was observed within the three leukemia groups, the median drug concentration required to inhibit TS activity to 50% of untreated controls (TSI50) for a 3-h exposure to MTX was similar for iALL and rALL cells but was up to 9-fold higher in ANLL cells. After a 3-h exposure, EDX, ZD1694, and GW1843U89 displayed a markedly (10-150-fold) increased potency over MTX in all leukemia groups with comparable TSI50 values for ANLL and iALL cells. Compared with a 3-h MTX exposure, continuous exposure resulted in lower TSI50 values for iALL (14-fold), rALL (14-fold), and ANLL cells (85-fold). In comparison to MTX, the TSI50 values in these groups were also lower for EDX (1.6-3.5-fold), ZD1694 (2.1-4.3-fold), and GW1843U89 (15-35-fold). On short-term exposure, the lipophilic drugs trimetrexate and AG337 displayed markedly less potency as compared with that of long-term exposure. In conclusion, the efficacy of novel antifolates against childhood leukemia cells can be tested with the in situ TS inhibition assay. These novel antifolates displayed a greater efficacy than MTX against childhood leukemia cells and may have potential for the circumvention of MTX resistance in ANLL cells.

Regulation of carrier-mediated transport of folates and antifolates in methotrexate-sensitive and-resistant leukemia cells.

Prolonged cell culture of human leukemia cells at folate concentrations in the (sub)physiological range (1-5 nM) rather than at 'standard' supraphysiological concentrations of 2-10 microM folic acid elicited a number of regulatory aspects of the reduced folate carrier (RFC), the membrane transport protein for natural reduced folate cofactors and folate-based chemotherapeutic drugs such as methotrexate (MTX). One subline of human CCRF-CEM leukemia cells grown under folate-restricted conditions (CEM-7A) exhibited a 95-fold increased Vmax for uptake of [3H]-MTX. The increased uptake of MTX in CEM-7A cells is based on at least two factors: (a) a constitutive 10-fold overexpression of the RFC1 gene and RFC1 message; and (b) a 7-9-fold up-regulation of RFC transport activity under low intracellular reduced folate concentrations. This second component appeared to be regulatable by changes in the cellular folate, purine and methylation status as judged from a 7-9 fold down-regulation of RFC transport activity after short term (1-2 hr) incubation of CEM-7A cells with reduced folate cofactors (25 nM LV), purines (100 microM adenosine) or S-adenosylmethionine (100 microM), respectively. Gradual folate restriction in the cell culture medium of CEM/MTX cells, a subline of CCRF-CEM resistant to MTX due to defective transport via the RFC, revealed the up-regulated expression of an altered RFC protein that is characterized by a 35-fold decreased Km for folic acid and a 10-fold decreased Km for the reduced folate cofactor LV compared to the RFC expressed in CCRF-CEM and CEM-7A cells. As a result of the markedly increased efficiency of folic acid uptake in CEM/MTX cells, intracellular folate pools were 7-fold higher than in CCRF-CEM cells when both cell lines were incubated in the presence of 2 microM folic acid. The high intracellular folate pools in CEM/MTX cells appeared to impair the polyglutamylation of antifolates and confer resistance to ZD1694, an antifolate drug that depends on polyglutamylation for its biological activity. Collectively, these studies provide a better insight into the basic regulation of RFC-mediated membrane transport of clinically active antifolates. In addition, these studies may also provide an opportunity to exploit the transport system as a target for biochemical modulation by which it may contribute to an improved efficacy of folate-based chemotherapy in a clinical setting.

A new aminoglycoside antibiotic complex--the seldomycins. III. The structures of seldomycin factors 1 and 2+.

The structures of seldomycin factors 1 and 2 have been determined by consideration of chemical degradation and spectral properties. Factor 1, also known as XK-88-1, is shown to be 6-O-(2-amino-2-deoxy-alpha-D-xylopyranosyl) paromamine (1) and factor 2, also known as XK-88-2, is shown to be 4'-deoxy-neamine (2). Mass spectral evidence has been obtained that suggests the most probable structure for seldomycin factor 3, also known as XK-88-3, is 6'-amino-6'-deoxyseldomycin factor 1 (12).