Induction of resistance to the lipophilic cytarabine prodrug elacytarabine (CP-4055) in CEM leukemic cells.

The deoxynucleoside analogs cytarabine (Ara-C) and gemcitabine (dFdC) are widely used in the treatment of cancer. Due to their hydrophilic nature they need the equilibrative (hENT) and concentrative (hCNT) nucleoside transporters to enter the cell. To bypass drug resistance due to decreased uptake, lipophilic 5'elaidic acid esters were synthesized, elacytarabine (CP-4055, from ara-C) and CP-4126 (from gemcitabine), which are currently in clinical development for solid and hematological tumors. We investigated whether resistance can be induced in vitro, and treated the CEM leukemic cell line with weekly increasing elacytarabine concentrations, up to 0.28 microM (10 times IC(50)). The IC(50) of the resistant CEM/CP-4055 was 35 microM, about 1,000 times that of the wildtype CEM, and comparable to that of CEM/dCK- (deoxycytidine kinase deficient) (22 microM). CEM/CP-4055 was also cross-resistant to Ara-C, gemcitabine and CP-4126 (28 and 33 microM, respectively). A low level of mRNA dCK was observed, and similar to CEM/dCK-, CEM/CP-4055 did not accumulate Ara-CTP after exposure to Ara-C or elacytarabine, which is consistent with a deficiency in dCK. In conclusion, elacytarabine induced resistance similar to Ara-C. This resistance was caused by downregulation of dCK.

Impact of cellular folate status and epidermal growth factor receptor expression on BCRP/ABCG2-mediated resistance to gefitinib and erlotinib.

The effect of folate status on breast cancer resistance protein (BCRP)-mediated drug resistance to epidermal growth factor receptor (EGFR)-targeted drugs, such as gefitinib and erlotinib, was investigated in two human colon cancer cell lines, WiDr and Caco-2, of which the latter displayed greater sensitivity to these drugs due to high EGFR expression. Caco-2 LF/LV cells, growing under low-folate (LF) conditions, showed increased BCRP protein expression compared with the high-folate (HF) counterpart, which was associated with 1.8-fold resistance to gefitinib. Of note, the BCRP-specific inhibitor Ko143 completely reverted this phenotype. WiDr LF cells also showed slightly increased BCRP expression compared with the HF cells, but no differences in gefitinib sensitivity were observed. Both Caco-2 LF/LV and WiDr LF cells showed 2.4- and 2.3-fold resistance to erlotinib, respectively, compared with their HF counterparts, which mechanistically seemed BCRP unrelated, as Ko143 had no effect on erlotinib activity. In conclusion, our data suggest that in EGFR-expressing Caco-2 cells, BCRP is one of the determinants of gefitinib resistance but not of erlotinib resistance. Beyond this, folate depletion can provoke an additional decrease in gefitinib and erlotinib activity by mechanisms dependent or independent of BCRP modulation.

Daily controlled physiotherapy increases survival time in dogs with suspected degenerative myelopathy.

The purposes of the study reported here were to evaluate the signalment and clinical presentation in 50 dogs with degenerative myelopathy, to evaluate whether mean survival time was significantly affected by various means of physiotherapy performed in 22 dogs, and to determine whether neurologic status, anatomic localization, or age at onset had an influence on survival time in dogs that received physiotherapy. We found a significant (P < .05) breed predisposition for the German Shepherd Dog, Kuvasz, Hovawart, and Bernese Mountain Dog. Mean age at diagnosis was 9.1 years, and both sexes were affected equally. The anatomic localization of the lesion was spinal cord segment T3-L3 in 56% (n = 28) and L3-S3 in 44% (n = 22) of the dogs. Animals that received intensive (n = 9) physiotherapy had longer (P < .05) survival time (mean 255 days), compared with that for animals with moderate (n = 6; mean 130 days) or no (n = 7; mean 55 days) physiotherapy. In addition, our results indicate that affected dogs which received physiotherapy remained ambulatory longer than did animals that did not receive physical treatment.

[Chronic inflammatory demyelinating polyradiculoneuropathy with hypertrophy of cervico-thoracal nerve roots in a dog]. / Chronische entzündliche demyelinisierende Polyradikuloneuropathie mit Hypertrophie zerviko-thorakaler Nervenwurzeln beim Hund.

A case of chronic inflammatory demyelinating polyradiculoneuropathy in a Magyar Vizsla dame, 7 months of age, is described. The neurological deficits such as movement disorders, hyporeflexia and muscle atrophy, were limited to the front legs. The hypertrophied cervico-thoracal nerve roots could be shown by magnetic resonance imaging. The diagnosis was additionally based on clinical findings, the relapsing course, the good response to therapy with prednisolone, the results of electrodiagnostic workup and muscle and nerve biopsy.

Neurodegenerative diseases in domestic animals: a comparative review.

Neurodegenerative diseases are characterised by selective damage to specific neurons in the nervous system. Interest in such diseases in humans has resulted in considerable progress in the molecular understanding of these disorders in recent decades. Numerous neurodegenerative diseases have also been described in domestic animals but relatively little molecular work has been reported. In the present review, we have classified neurodegenerative disease according to neuroanatomical criteria. We have established two large groups, based on whether the neuronal cell body or its axon was primarily affected. Conditions such as motor neuron diseases, cerebellar degenerations and neuroaxonal dystrophies are discussed in terms of their clinical and neuropathological features. In the most studied disorders, we also present what is known about underlying pathomechanisms, and compare them with their human counterparts. The purpose of this review is to re-kindle interest in this group of diseases and to encourage veterinary researchers to investigate molecular mechanisms by taking advantage of current diagnostic tools.

[Paraparesis in a dwarf goat: clarification by means of magnetic resonance imaging]. / Paraparese bei einer Zwergziege: Abklärung mittels Magnetresonanztomographie.

A 4-year old pygmy goat with chronic paraparesis of the hindlimbs was referred to the Ruminant Clinic of the University of Berne. The causative lesion was localized to the thoracolumbar spinal cord after a thorough clinical examination. Because a radiographic examination of the spine had not been diagnostic, magnetic resonance imaging (MRI) was performed. A mass compressing the spinal cord in the region of L2-L5 was detected. The goat was euthanized and autopsied, which allowed for the definitive diagnosis of lymphosarcoma. In addition to the changes in the lumbar area, further neoplastic masses were detected in the region of the thoracic vertebrae, near the thoracic aperture, on the lungs and on the pericardium. However, these processes had not yet caused clinical signs. MRI investigation allowed for the ante mortem diagnosis of an infiltrative mass in the spinal canal of this goat.

[Tetanus in cats: 3 case descriptions]. / Tetanus bei Katzen: 3 Fallbeschreibungen.

Three cats with spasticity on one leg or on all four limbs were presented between 1996 and 1998 at the Department of clinical veterinary medicine, Section of neurology, Vetsuisse-Faculty of Bern. The presumptive diagnosis was tetanus. A focal form was present in two cases and generalised tetanus in one cat. All cats had a history of injury at the affected legs respectively at the neck. The first clinical signs were seen between two days and three weeks after injury. The bacteriologic examination of serous fluid from the site of injury revealed an infection with Clostridium. EMG in one cat during anaesthesia showed motor united potentials (MUPs) on the spastic leg. All patients received antibiotics (Penicillin, respectively Amoxicillin/Clavulanic acid and Metronidazol). Supportive aid were initially sedation, wound revision and in one cat nutrition through oesophageal sonde. In a second phase physiotherapy was performed. All three animals were significantly better after a couple of weeks, two cats were without symptoms after eight and five weeks respectively.

Acquired resistance of human T cells to sulfasalazine: stability of the resistant phenotype and sensitivity to non-related DMARDs.

BACKGROUND: A recent study from our laboratory showed that induction of the multidrug resistance related drug efflux pump ABCG2 contributed to acquired resistance of human T cells to the disease modifying antirheumatic drug (DMARD) sulfasalazine (SSZ). OBJECTIVES: To investigate the duration of SSZ resistance and ABCG2 expression after withdrawal of SSZ and rechallenging with SSZ, and to assess the impact of SSZ resistance on responsiveness to other DMARDs. METHODS: Human CEM cells (T cell origin) with acquired resistance to SSZ (CEM/SSZ) were characterised for (a) SSZ sensitivity and ABCG2 expression during withdrawal and rechallenge of SSZ, and (b) antiproliferative efficacy of other DMARDs. RESULTS: ABCG2 protein expression was stable for at least 4 weeks when CEM/SSZ cells were grown in the absence of SSZ, but gradually declined, along with SSZ resistance levels, to non-detectable levels after withdrawal of SSZ for 6 months. Rechallenging with SSZ led to a rapid (<2.5 weeks) resumption of SSZ resistance and ABCG2 expression as in the original CEM/SSZ cells. CEM/SSZ cells displayed diminished sensitivity to the DMARDs leflunomide (5.1-fold) and methotrexate (1.8-fold), were moderately more sensitive (1.6-2.0 fold) to cyclosporin A and chloroquine, and markedly more sensitive (13-fold) to the glucocorticoid dexamethasone as compared with parental CEM cells. CONCLUSION: The drug efflux pump ABCG2 has a major role in conferring resistance to SSZ. The collateral sensitivity of SSZ resistant cells for some other (non-related) DMARDs may provide a further rationale for sequential mono- or combination therapies with distinct DMARDs upon decreased efficacy of SSZ.

Development of sulfasalazine resistance in human T cells induces expression of the multidrug resistance transporter ABCG2 (BCRP) and augmented production of TNFalpha.

OBJECTIVE: To determine whether overexpression of cell membrane associated drug efflux pumps belonging to the family of ATP binding cassette (ABC) proteins contributes to a diminished efficacy of sulfasalazine (SSZ) after prolonged cellular exposure to this disease modifying antirheumatic drug (DMARD). METHODS: A model system of human T cells (CEM) was used to expose cells in vitro to increasing concentrations of SSZ for a period of six months. Cells were then characterised for the expression of drug efflux pumps: P-glycoprotein (Pgp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2). RESULTS: Prolonged exposure of CEM cells to SSZ provoked resistance to SSZ as manifested by a 6.4-fold diminished antiproliferative effect of SSZ compared with parental CEM cells. CEM cells resistant to SSZ (CEM/SSZ) showed a marked induction of ABCG2/BCRP, Pgp expression was not detectable, while MRP1 expression was even down regulated. A functional role of ABCG2 in SSZ resistance was demonstrated by 60% reversal of SSZ resistance by the ABCG2 blocker Ko143. Release of the proinflammatory cytokine tumour necrosis factor alpha (TNFalpha) was threefold higher in CEM/SSZ cells than in CEM cells. Moreover, twofold higher concentrations of SSZ were required to inhibit TNFalpha release from CEM/SSZ cells compared with CEM cells. CONCLUSION: Collectively, ABCG2 induction, augmented TNFalpha release, and less efficient inhibition of TNFalpha production by SSZ may contribute to diminished efficacy after prolonged exposure to SSZ. These results warrant further clinical studies to verify whether drug efflux pumps, originally identified for their roles in cytostatic drug resistance, can also be induced by SSZ or other DMARDs.

[Cerebellar cortical abiotrophy in American Staffordshire terriers: clinical and pathological description of 3 cases]. / Zerebelläre kortikale Abiotrophie beim American Staffordshire Terrier: Klinische und pathologische Beschreibung von drei Fällen.

Three American Staffordshire Terriers were presented with gait abnormalities and loss of balance at the age of 4.5 (female) and 6 years (2 males). The onset varied between 3 and 5 years of age and the clinical signs were slowly progressive. The neurological examination revealed symmetrical generalized cerebellar ataxia with hypermetria, stiffness, and loss of balance with no evidence of paresis. The menace reflex was decreased in one dog and absent in another. A positional nystagmus was found in two dogs. The dogs were euthanized and a histopathological examination of each brain was performed. Pathological changes were confined to the cerebellum. The main finding was loss of Purkinje cells, as well as depletion of granular cell bodies and shrinkage of the granular and molecular cell layer. These findings are consistent with cerebellar cortical abiotrophy. A genetic basis is supposed, but the mode of inheritance is not determined yet. In contrast to some spinocerebellar ataxias in humans, the cause of Purkinje cell degeneration in cerebellar cortical abiotrophy of dogs is not known.

Analysis of the 5' region of the canine PAX3 gene and exclusion as a candidate for Dalmatian deafness.

The causative mutation in a gene related to hearing loss in Dalmatians has been elusive. Because of its role in melanocyte migration and differentiation as integral component of the inner ear, we hypothesized that the canine PAX3 (paired box homeotic gene 3) gene could be a candidate for Dalmatian deafness. Therefore, we isolated the canine PAX3 gene and searched for causative mutations within the coding region of important regulatory domains of PAX3. However, no mutations were identified when comparing the DNA sequences of healthy and affected dogs. These results were confirmed by a two-point linkage analysis in 203 Dalmatians transmitting deafness. Our data clearly show that the canine PAX3 gene can be excluded as candidate for Dalmatian deafness.

[Rehabilitation methods in small animal neurology]. / Rehabilitationsmassnahmen in der Kleintierneurologie.

Rehabilitation is an important part of the treatment of neurological diseases. The primary goal of these methods is an optimal functional restoring of the neuro-muscular system. Massages, thermo-, hydro- and electrotherapy, as well as therapy of movement are different treatment possibilities with their own indication, which are combined in a physiotherapy program. It follows an overview of the different physiotherapeutic methods and their application in some of the most common neurological diseases, as for example intervertebral disc problems or degenerative myelopathy.

Spontaneous lumbar intervertebral disc protrusion in cats: literature review and case presentations.

Reports on intervertebral disc disease in cats are rare in the veterinary literature. It has been postulated that intervertebral disc protrusion is a frequent finding during necropsy in cats, without having any clinical relevance (King and Smith 1958, King & Smith 1960a, King & Smith 1960b). However, a total of six cases with disc protrusions and clinically significant neurological deficits have been reported over the past decade. (Heavner 1971, Seim & Nafe 1981, Gilmore 1983, Littlewood et al 1984, Sparkes & Skerry 1990, Bagley et al 1995). As in dogs, there are also two types of intervertebral disc disease in cats: Hansen's type I (extrusion), and type II (herniation). Cervical spinal cord involvement was more commonly recognised in cats than the lumbar or the thoraco lumbar area. Cats over 15 years were mainly affected (King & Smith 1958, King & Smith 1960a, King & Smith 1960b). We describe two cats with lumbar intervertebral disc protrusions. Emphasis is placed on differential diagnoses, treatment and follow-up.

Clinical and genetic investigations of idiopathic epilepsy in the Bernese mountain dog.

A study was conducted to investigate the clinical aspects and to define the mode of inheritance of idiopathic epilepsy in the Bernese mountain dog. Pedigree analyses were carried out on an open, non-preselected population of 4005 dogs. Five different subpopulations with 50 epileptic dogs from 13 generations were included. Almost all epileptic patients showed generalised seizures of the grand-mal type with a well-defined prodromal and postictal phase. The majority (62 per cent) of the epileptic dogs had had their first seizures at between one and three years of age and it was found that the age at first seizure was significantly (P < 0.05) lower in dogs from affected parental animals than in dogs from healthy parental animals. A clear predisposition for males was also noted. Additionally, there was no correlation between inbreeding coefficient and age at first seizure or incidence rate of seizures. The increased occurrence of the disease in different subpopulations and different families of the same sires or dams showed that there was a genetic basis for the condition in the Bernese mountain dog. Furthermore, the results of the pedigree analyses and binomial test support the hypothesis that idiopathic epilepsy has a polygenic, recessive mode of inheritance in the breed. Additional objective test-mating programmes would however be necessary to define the exact mode of inheritance.

Multiple mechanisms of resistance to polyglutamatable and lipophilic antifolates in mammalian cells: role of increased folylpolyglutamylation, expanded folate pools, and intralysosomal drug sequestration.

Chinese hamster ovary PyrR100 cells display more than 1000-fold resistance to pyrimethamine (Pyr), a lipophilic antifolate inhibitor of dihydrofolate reductase. PyrR100 cells had wild-type DHFR activity, lost folate exporter activity, and had a 4-fold increased activity of a low pH folic acid transporter. Here we report on the marked alterations identified in PyrR100 cells compared with parental cells: 1) approximately 100-fold decreased folic acid growth requirement; 2) a 25-fold higher glucose growth requirement in Pyr-containing medium; 3) a 2.5- to 4.1-fold increase in folylpolyglutamate synthetase activity; 4) a 3-fold increase in the accumulation of [3H]folic acid and a 3-fold expansion of the intracellular folate pools; 5) a 4-fold increase in the activity of the lysosomal marker beta-hexoseaminidase, suggesting an increased lysosome number/PyrR100 cell; and 6) a small reduction in the steady-state accumulation of [3H]Pyr and no evidence of catabolism or modification of cellular [3H]Pyr. Consequently, PyrR100 cells were markedly resistant to the lipophilic antifolates trimetrexate (40-fold) and AG377 (30-fold) and to the polyglutamatable antifolates 5,10-Dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF) (26-fold) and AG2034 (14-fold). Resistance to these drugs was reversed in PyrR100 cells transferred into folate-depleted medium. In conclusion, these multiple resistance factors collectively result in a prominent increase in folate accumulation, an expansion of the intracellular folylpolyglutamate pool, and abolishment of the cytotoxic activity of polyglutamatable and lipophilic antifolates. The role of increased lysosome number per cell in sequestration of hydrophobic weak base drugs such as Pyr is also discussed as a novel mechanism of drug resistance.

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.

Lipoamino acid conjugates of methotrexate with antitumor activity.

The synthesis, characterization, and in vitro antitumor activity against a wild and a transport-resistant CCRF-CEM cell line is described for a series of alpha,gamma-bisamide lipoamino acid and oligomer conjugates of methotrexate. The influence of the lipophilicity of the conjugates on the cytotoxicity and the dihydrofolate reductase inhibition was investigated. All compounds were more active than their fatty acid conjugate analogues. Compound le with a 12-carbon atom aliphatic side chain showed the highest in vitro activity.

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.

Functional aspects of membrane folate receptors in human breast cancer cells with transport-related resistance to methotrexate.

Two methotrexate (MTX)-resistant human breast-cancer cell lines with impaired transport via the reduced folate carrier (RFC), one established in vitro (MTX(R)-ZR-75-1) and another inherently resistant (MDA-231), were adapted to grow in medium containing 2 nM folic acid. This induced the expression of previously undetectable membrane folate receptors (MFR) to levels of 8.2 and 2.3 pmol/10(7) cells, respectively. Polymerase chain reaction (PCR) quantitation revealed that MFR messenger-RNA levels of the isoform first described in human nasopharyngeal carcinoma KB cells (MFR-alpha) were increased in low-folate-adapted MTX(R)-ZR-75-1 cells, whereas placental transcripts (MFR-beta) coincided with MFR-alpha expression in low-folate (LF)-adapted MDA-231 cells. These cell lines were used to study the role of MFR in the uptake and growth-inhibitory effects of five different antifolates with varying affinities for MFR: N10-propargyl-5, 8-dideazafolic acid (CB3717) > 5,10-dideazatetra-hydrofolic acid (DDATHF) > N-5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-methyl) -N-methyl-amino]-2-theonyl}-glutamic acid (ZD1694) >> MTX > edatrexate (EDX). Expression of MFR only slightly decreased the resistant phenotype for MTX, EDX, and ZD1694, suggesting that these drugs are not transported intracellularly to cytotoxic concentrations at these levels of MFR expression. On the other hand, both cell lines became from at least 180- to 400-fold more sensitive to growth inhibition by CB3717 and DDATHF, which may be correlated with their high affinity for MFR. These sensitivity/resistance profiles were largely similar following cell culture in medium containing 1 nM L-leucovorin, a folate with an affinity for MFR 10-fold lower than that of folic acid, the one exception being the increased sensitivity for ZD1694 seen in the LF-adapted cells with the highest level of MFR expression (MTX(R)-ZR-75-1). These results illustrate that the efficacy of MFR in mediating antifolate transport and cytotoxicity depends on their affinity for the folate antagonist, their degree of expression, and the levels of competing folates.

Carrier- and receptor-mediated transport of folate antagonists targeting folate-dependent enzymes: correlates of molecular-structure and biological activity.

The transport properties and growth-inhibitory potential of 37 classic and novel antifolate compounds have been tested in vitro against human and murine cell lines expressing different levels of the reduced folate carrier (RFC), the membrane-associated folate binding protein (mFBP), or both. The intracellular targets of these drugs were dihydrofolate reductase (DHFR), glycinamide ribonucleotide transformylase (GARTF), folylpolyglutamate synthetase (FPGS), and thymidylate synthase (TS). Parameters that were investigated included the affinity of both folate-transport systems for the antifolate drugs, their growth-inhibitory potential as a function of cellular RFC/mFBP expression, and the protective effect of either FA or leucovorin against growth inhibition. Methotrexate, aminopterin, N10-propargyl-5,8-dideazafolic acid (CB3717), ZD1694, 5,8-dideazaisofolic acid (IAHQ), 5,10-dideazatetrahydrofolic acid (DDATHF), and 5-deazafolic acid (efficient substrate for FPGS) were used as the basic structures in the present study, from which modifications were introduced in the pteridine/quinazoline ring, the C9-N10 bridge, the benzoyl ring, and the glutamate side chain. It was observed that RFC exhibited an efficient substrate affinity for all analogues except CB3717, 2-NH2-ZD1694, and glutamate side-chain-modified FPGS inhibitors. Substitutions at the 2-position (e.g., 2-CH3) improved the RFC substrate affinity for methotrexate and aminopterin. Other good substrates included PT523 (N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine), 10-ethyl-10-deazaaminopterin, and DDATHF. With respect to mFBP, modifications at the N-3 and 4-oxo positions resulted in a substantial loss of binding affinity. Modifications at other sites of the molecule were well tolerated. Growth-inhibition studies identified a series of drugs that were preferentially transported via RFC (2,4-diamino structures) or mFBP (CB3717, 2-NH-ZD1694, or 5,8-dideazaisofolic acid), whereas other drugs were efficiently transported via both transport pathways (e.g., DDATHF, ZD1694, BW1843U89, or LY231514). Given the fact that for an increasing number of normal and neoplastic cells and tissue, different expression levels of RFC and mFBP are being recognized, this folate antagonist structure-activity relationship can be of value for predicting drug sensitivity and resistance of tumor cells or drug-related toxicity to normal cells and for the rational design and development of novel antifolates.