We have studied the molecular basis of resistance of multiple human leukaemia CCRF-CEM sublines to the novel antifolates ZD9331, GW1843, AG2034, PT523 and edatrexate, which use the reduced folate carrier (RFC) as their main cellular uptake route and that target different folate-dependent enzymes. Antifolate-resistant sublines established by stepwise and singlestep selections displayed up to 2135-fold resistance to the selection drug, and up to 2323-fold cross-resistance to various hydrophilic antifolates. In contrast, these sublines were up to 17-and 20-fold hypersensitive to the lipophilic antifolates AG377 and trimetrexate, respectively. The total reduced folate pool of these antifolate-resistant sublines shrunk by 87-96 %, resulting in up to 42-fold increased folic acid growth requirement. These sublines lost 92-97 % of parental [$H]methotrexate influx rates. Genomic PCR single-strand conformational polymorphism analysis and sequencing revealed that most of these drug-resistant sublines harboured RFC mutations that surprisingly clustered in two

Smoking has a broad range of physiological effects, such as being a risk factor in osteoporosis, bone fracture incidence, and increased nonunion rates. Recent studies showed that nicotine has effects at the cellular level in human osteoblast cells. To identify possible mechanisms underlying nicotine-induced changes in osteogenic metabolism, we defined changes in proliferation and osteocalcin, type I collagen, and alkaline phosphatase gene expression after treating human osteosarcoma cells (MG63), with various concentration of nicotine. Nicotine affects cell proliferation in a biphasic manner, including toxic and antiproliferative effects at high levels of nicotine and stimulatory effects at low levels. Moreover, low levels of nicotine upregulated osteocalcin, type I collagen, and alkaline phosphatase gene expression. The increased cell proliferation and gene upregulation induced by nicotine were inhibited by addition of the nicotinic receptor antagonist D: -tubocurarine. High nicotine concentrations downregulated the investigated genes. Our results demonstrate, for the first time, that the addition of nicotine concentrations analogous to those acquired by a light to moderate smoker yields increased osteoblast proliferation and bone metabolism, whereas the addition of nicotine concentrations analogous to heavy smokers leads to the opposite effect. The inhibition of these effects by D: -tubocurarine suggests that nicotine acts via the nicotinic acetylcholine receptor (nAChR).

We have studied the molecular basis of drug resistance in human CCRF-CEM leukemia cells exposed to high dose intermittent pulses of novel polyglutamatable antifolates that target various folate-dependent enzymes. These include the dihydrofolate reductase (DHFR) inhibitors edatrexate, methotrexate and aminopterin, the thymidylate synthase (TS) inhibitors ZD1694 and GW1843, the glycinamide ribonucleotide formyltransferase (GARTF) inhibitor DDATHF as well as the multitargeted antifolate LY231514 inhibiting both TS, DHFR and GARTF. Fourteen antifolate-resistant sublines were isolated, 11 of which displayed a drug resistance phenotype that was based on impaired folylpoly-␥-glutamate synthetase ( Three-dimensional modeling of the human FPGS based on the crystal structure of Lactobacillus casei FPGS suggested that this mutation maps to the active site and interferes with the catalytic activity of the enzyme due to a putative bulky clash between the mutant Phe346 and a native Phe350 within ␣-helix A10 in a highly conserved C-terminal hydrophobic core. This was consistent with a 23-fold decreased affinity of the mutant Cys346Phe FPGS for L-glutamate. We conclude that decreased FPGS activity is a dominant mechanism of resistance to polyglutamylation-dependent novel antifolates upon a high-dose intermittent exposure schedule. The finding that cells may exhibit 5 orders of magnitude of resistance to polyglutamylation-dependent antifolates but in the same time retain parental sensitivity or hypersensitivity to polyglutamylation-independent antifolates or lipophilic antifolates offers a potentially promising treatment strategy in the overcoming of FPGS-based anticancer drug resistance.

Folate cofactors serve as one-carbon donors in the de novo biosynthesis of purines and thymidylate (1). As such, normal and neoplastic dividing cells have an absolute folate requirement for DNA replication (1). Disruption of folate biosynthesis with folic acid antagonists (i.e. antifolates) is the pharmacological basis for the antitumor activity of methotrexate (MTX) 1 and various antifolates (2). Because mammalian cells are devoid of folate biosynthesis, they rely on folate vitamin uptake from exogenous sources. Membrane transport of folates and MTX is mediated by several systems (3, 4): (a) the reduced folate carrier (RFC) is the major uptake route that functions as a bi-directional anion exchanger (5, 6) taking up folates through an antiport exchange mechanism with intracellular organic phosphates (7); (b) folate receptors mediate the unidirectional uptake of folate cofactors into mammalian cells via an endocytotic process (8); and (c) an apparently independent transport system with optimal folate uptake activity at low pH (9 -11).Apart from RFC, efflux of folates and MTX (12, 13) is mediated by multidrug resistance proteins (MRP) MRP1-4 (14 -19), which belong to the ATP-binding cassette superfamily (20,21). Members of the MRP family, currently comprising nine genes (i.e. MRP1-9), function as ATP-driven efflux transporters of various natural product anions and acidic charged drug conjugates (14, 15). Mammalian cells transfected with MRP1-4 accumulate decreased levels of MTX and consequently display resistance to this drug, particularly upon short term drug exposure (16 -19). Membrane vesicles isolated from MRP1-and MRP2-transfected cells exhibit ATP-dependent transport of MTX (16). Detailed kinetic analysis of folic acid, leucovorin (LCV; 5-formyl-tetrahydrofolate) and MTX transport into MRP1-and MRP3-rich membrane vesicles reveals K m values in the low millimolar range (22). Hence, the free intracellular level of folates and antifolates including MTX is determined by the net activities of these influx (i.e. RFC) and efflux (RFC and MRP) transport pathways.CEM-7A is a human leukemia CCRF-CEM subline previously established by gradual deprivation of LCV from the growth medium (23), resulting in RFC gene amplification (24) and carrier overexpression (23,24). Consequently, CEM-7A cells displayed a marked increase in the influx of MTX and LCV accompanied by a comparable increase in the steady-state transmembrane gradient of MTX (23). Surprisingly, however, there was no increase in the efflux rate constant for MTX (23). This is in contrast with previous studies (25) 1 The abbreviations used are: MTX, methotrexate; MRP, multidrug resistance protein; RFC, reduced folate carrier; LCV, Leucovorin (5-formyl-tetrahydrofolate); TMQ, trimetrexate; BCRP, breast cancer resistance protein; Pgp, P-glycoprotein; NHS-MTX, N-hydroxysuccinimide ester of MTX; AM, acetoxymethyl ester; HBS, Hepes-buffered saline.