Immunophenotypical markers, ultrastructure and chemosensitivity profile of metastatic melanoma cells.

The survival of patients affected by cutaneous melanoma has improved dramatically in the last 10 years, because of earlier diagnosis. Despite this, the therapeutic results obtained in metastatic melanoma (MM) are very disappointing due to its poor responsiveness to cytotoxic agents. In this type of solid tumor, tumor chemosensitivity assays have been suggested to be an important tool to predict clinical responsiveness to therapy. Metastatic melanoma cells (MMCs) were obtained from subcutaneous melanoma metastases of five patients and cultured for several consecutive passages. An immunofluorescence and an electron microscopic study were performed in order to establish the ultrastructural and physiopathological features of MMCs. A sulphorodamine-B test was used to measure in vitro sensitivity of MMCs to temozolomide, cisplatin, vindesine, taxol and interpheron alpha-2a. Following a 72 h exposure, maximum activity was obtained with vindesine (median inhibitory concentration, IC(50), 0.23 nM) and taxol (median IC(50) 0.31 nM). Cisplatin median IC(50) values were higher (4.6 microM) than taxol and vindesine, but still in the range of clinically achievable plasma concentrations. Temozolomide inhibited cell proliferation only at very high concentrations (median IC(50) 228 microM). No significant cell growth inhibitory effects (

Modulation of dihydrofolate reductase gene expression in methotrexate-resistant human leukemia CCRF-CEM/E cells by antisense oligonucleotides.

An increase in the cellular levels of dihydrofolate reductase (DHFR) is one of the most common mechanisms of tumor resistance to methotrexate (MTX), an antimetabolite that is widely used in the treatment of a variety of human malignancies. The MTX-resistant phenotype generally occurs as a consequence of DHFR gene amplification which in turn is responsible for DHFR gene overexpression. We have designed antisense oligodeoxynucleotides (aODNs) against the DHFR mRNA and tested their in vitro effect on human leukemia CCRF-CEM/E cells, overexpressing the DHFR gene about 20-fold in comparison with the CCRF-CEM/S parental cell line. An aODN complementary to a region encompassing the AUG translation start (DHFR1) of DHFR mRNA and a mixture of two aODNs complementary to the 5' untranslated region (DHFR2+DHFR3) have been used. A DHFR1 scrambled-sequence ODN and a fully degenerated ODN were the controls. All ODNs had a phosphodiester backbone. DHFR1 and the relevant scrambled ODN were also capped with two phosphorothioate derivatives at both the 5' and 3' ends in order to increase ODN stability against serum nucleases. ODNs were vehiculated with a cationic lipid, N-[1-(dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), known to enhance ODN cell uptake and biological activity. The effects of ODNs on DHFR gene expression were studied after a 4 day treatment by measuring both DHFR mRNA levels, using a semi-quantitative reverse transcription polymerase chain reaction method, and DHFR protein levels by flow cytometry. A marked reduction in DHFR mRNA levels (79.7 and 74.2%, respectively) was observed with both DHFR1 and DHFR2+DHFR3 aODNs, associated with a lower decrease in DHFR enzyme (44.8 and 61%, respectively). aODN effects on MTX cytotoxicity in CCRF-CEM/E cells were also assessed. No marked enhancement of in vitro MTX cytotoxicity was observed following co-exposure of cells with aODNs and the tested concentrations of the antifol (0.05 and 0.5 microM), indicating that no substantial reversal of the MTX-resistant phenotype was induced by the study aODNs.

Cytotoxicity, DNA damage, and cell cycle perturbations induced by two representative gold(III) complexes in human leukemic cells with different cisplatin sensitivity.

The gold(III) complexes [Au(phen)Cl2]Cl and [Au(dien)Cl]Cl2 were recently shown to exert important cytotoxic effects in vitro on human tumor cell lines. To elucidate the biochemical mechanisms leading to cell death, the effects produced by these gold(III) complexes on the leukemic CCRF-CEM cell line--either sensitive (CCRF-CEM) or resistant to cisplatin (CCRF-CEM/CDDP)--were analyzed in detail by various techniques. For comparison purposes the effects produced by equitoxic concentrations of cisplatin were also analyzed. First, the dependence of the IC50 values of either complex on the incubation time was investigated. Cytotoxicity experiments confirmed that both gold(III) compounds retain their efficacy against the cisplatin-resistant line: only minimal cross-resistance with cisplatin was detected. Notably, [Au(phen)Cl2]Cl is more cytotoxic than [Au(dien)Cl]Cl2, with IC50 values of 7.4 and 6.0 M at 24 and 72 h, respectively, on the resistant line. Results of the COMET assay point out that both gold(III) complexes directly damage nuclear DNA. Remarkably, DNA damage inferred by either gold(III) complex in the two cell lines is larger than that produced by equitoxic cisplatin concentrations. Finally, the effects that either gold(III) complex produces on the cell cycle were investigated by flow cytometry. It was found that both complexes cause only moderate and transient cell cycle perturbations. Larger cell cycle perturbations are induced by equitoxic concentrations of cisplatin. The implications of the present results for the mechanism of action of cytotoxic gold(III) complexes are discussed.

Inhibitory effect of luteinising hormone-releasing hormone analogues on human endometrial cancer in vitro.

We studied the effects of luteinising hormone-releasing hormone (LHRH) agonist leuproreline (1 microM for 96 h) and LHRH antagonist cetrorelix on the cell growth of primary cultures from nine human endometrial cancers using the sulphorhodamine colorimetric test. Histological examinations and reverse transcription and polymerase chain reaction amplification (RT-PCR) for LHRH receptors were also performed. The endometrial cancers examined had a medium to high degree of proliferative activity and a low degree of apoptotic power; furthermore, they expressed the LHRH receptor RNA variably, detectable in 71% of cases. The addition of leuproreline or cetrorelix to cell cultures inhibited growth in a statistically significant way compared to untreated control cells; nevertheless, the percentage of cell growth inhibition obtained was very variable. These data suggest that LHRH analogues can exert differential inhibitory effects on the growth of endometrial cancer, which seems to be independent of the expression of specific LHRH receptors.

Melatonin's growth-inhibitory effect on hepatoma AH 130 in the rat.

We tested the effects of daily melatonin treatment on the growth of the ascites hepatoma in rats, determining survival time, cell number and cell cycle phases at various stages of tumor development. Melatonin inhibited cellular proliferation, doubled mean life-time and increased survival. Thymidine incorporation in hepatoma cells from treated rats decreased significantly without changes in the apoptotic index. Flow cytometric analysis showed that melatonin slowed cell cycle progression by increasing the number of cells in phase G0G1. Thus, similar to in vitro models, melatonin's oncostatic action in vivo appears to be directed to specific cell cycle mechanisms, which remain to be elucidated.

Differential inhibitory effects on human endometrial carcinoma cell growth of luteinizing hormone-releasing hormone analogues.

In addition to its function as a key hormone in the regulation of the pituitary-gonadal axis, luteinizing hormone-releasing hormone (LHRH) probably also affects various extrapituitary tissues. LHRH binding sites and in vitro antiproliferative effects of LHRH analogues have been reported in human endometrial cancer. The effects of the LHRH agonist leuproreline and LHRH antagonist antide were studied on the cell growth, DNA synthesis, and cell cycle distribution of the human endometrial cancer cell lines HEC-1A and HEC-1B by the sulforhodamine B (SRB) method, [3H]thymidine assay incorporation, and propidium iodide DNA staining, respectively. In the presence of 1.0-100 microM leuproreline the proliferation of HEC-1A cells was significantly reduced as early as 3 days after drug exposure, with a minimum growth value of 69.9 +/- 3.6% (mean +/- SE) at the highest concentration tested (100 microM). Similar antiproliferative effects were obtained following a 6-day treatment with the LHRH antagonist antide. Also, inhibitory effects on [3H]thymidine incorporation into the DNA of the HEC-1A cell line were noted after a 6-day exposure to both LHRH analogues, in the above-mentioned concentration range. Cell cycle analysis of HEC-1A cells cultured in the presence of 10 microM leuproreline and antide showed a slight accumulation of cells in the G0/G1 phase, while the proportions of cells in the S and G2/M phases concomitantly decreased. No significant effects on proliferation, DNA synthesis, and cell cycle distribution were observed in HEC-1B cells with either leuproreline or antide (up to 100 and 10 microM, respectively) after a 6-day exposure. Both Northern blot analysis and reverse transcription polymerase chain reaction failed to detect expression of mRNA for the LHRH receptor in both HEC-1A and HEC-1B cell lines. In addition, the LHRH analogues did not affect the intracellular free calcium concentration, indicating that the classic signal transduction for LHRH is absent or impaired in HEC-1A cells. The observed direct inhibitory actions on HEC-1A cells support the concept that the two LHRH analogues may exert biological effects via cellular effectors distinct from the "classic" LHRH receptor. Although the mechanism by which these direct actions are produced is still obscure, these results might help to establish the basis for new approaches to the therapy of endometrial cancer.

A novel inward-rectifying K+ current with a cell-cycle dependence governs the resting potential of mammalian neuroblastoma cells.

1. Human and murine neuroblastoma cell lines were used to investigate, by the whole-cell patch-clamp technique, the properties of a novel inward-rectifying K+ current (IIR) in the adjustment of cell resting potential (Vrest), which was in the range -40 to -20 mV. 2. When elicited from a holding potential of 0 mV, IIR was completely inactivated with time constants ranging from 13 ms at -140 mV to 4.5 s at -50 mV. The steady-state inactivation curve (h(V)) was found to be independent of [Na+]o and [K+]o (2-80 mM) and could be fitted to a Boltzmann curve with a steep slope factor of 5-6, and a V1/2 around Vrest. Divalent ion-free extracellular solutions shifted h(V) to the left by about 28 mV. 3. Peak chord conductance, whose maximal value was approximately proportional to the square root of [K+]o, could be fitted to a Boltzmann curve independently of [K+]o, with a V1/2 value around -48 mV and a slope factor of 18. Extracellular Cs+ and Ba2+ blocked the IIR in a concentration- and voltage-dependent manner, but Ba2+ was less effective than it is on classical inward-rectifier channels. 4. Under control culture conditions the values of Vrest and V1/2 of h(V) varied widely among cells. The knowledge of V1/2 proved crucial or the theoretical prediction of Vrest. After cell synchronization in the G0-G1 phase of the cell cycle, or at the G1-S boundaries, the cells reduced their variability of h(V). The same occurred after cell synchronization in G1 by treatment with retinoic acid. 5. The experimental data could be fitted to a classical model of an inward rectifier, after removing the dependence of conductance activation on (V-EK), and incorporating an inactivation with an intrinsic voltage dependence. Moreover, the model predicts, for this novel inward rectifier and in contrast with the classical inward rectifier, the incapacity of maintaining, in physiological media, a Vrest more negative than -35 to -40 mV, which is an important feature of cancer cells.

Inhibition of MDR1 gene expression by antimessenger oligonucleotides lowers multiple drug resistance.

The multiple drug resistance of neoplastic cells is mediated by overexpression of the human MDR1 gene, which encodes the transmembrane efflux pump P-glycoprotein. In both cell lines and human tumors the MDR phenotype closely correlates with MDR1 mRNA and P-glycoprotein levels. Reversion of the MDR phenotype was attempted in human colorectal adenocarcinoma doxorubicin (Dx)-resistant cells (Lo Vo/Dx) by long-term administration of an equimolecular mixture of three unmodified ODNs (18mer) targeted to adjacent binding sites of the MDR1 mRNA and carried by a synthetic cationic lipid (DOTAP). Three different experimental parameters were used to evaluate the antimessenger agent's effectiveness in comparison with a random sequence ODN: the level of cell resistance to Dx; the level of P-glycoprotein (determined by flow cytometry); the level of MDR1 mRNA (determined by quantitative RT-PCR). Experimental data indicate that the level of both the MDR1 mRNA and the P-glycoprotein is reduced by approximately 50% by treatment of Lo Vo/Dx cells with a 10 microM total concentration of the aODN mixture every 24 h for 15 days. In agreement with these findings, sensitivity to Dx of the antimessenger agent-treated Lo Vo/Dx cells was almost doubled in comparison with random sequence ODN-treated controls.

In vitro effects of brodimoprim on human polymorphonuclear leukocyte functions. Preliminary results.

The in vitro effects of brodimoprim and trimethoprim on the functions of human polymorphonuclear (PMN) leukocytes have been studied comparatively evaluating chemotaxis, phagocytosis and production of superoxide anion. No significant effects of both diaminopyrimidines on chemotaxis and phagocytic activity of PMNs have been observed while both brodimoprim and trimethoprim enhanced the oxidative burst. A synergistic activity between the host immune system and the direct antimicrobial action of brodimoprim may occur while using this diaminopyrimidine in vivo.

Chromosomal characterization of methotrexate-resistant human T-lymphoblast leukemia cells (CCRF-CEM) with impaired polyglutamylation.

Two sublines of the human T-lymphoblast leukemia cell line CCRF-CEM, which were resistant to methotrexate (MTX) due to defective MTX polyglutamate synthesis, were karyologically characterized. No statistically significant differences in the modal number of chromosomes were noted in resistant cells (CCRF-CEM/P) as compared to parent cells (91, range, 86-123; and 93, range; 78-103, respectively). Fifteen marker chromosomes were identified and their origins at least partially established. An isochromosome 7q, (marker 13) was present in all MTX-resistant cells but was not found in any sensitive cell karyotype. This marker chromosome may be involved in the emergence of drug-resistant cells from the parental population of CCRF-CEM cells. In all cell lines, chromosomes 8, 9 and 14 appear to be highly unstable and are involved in the genesis of many marker chromosomes. These chromosomes are also implicated in the in vivo genesis of various leukemias and lymphomas, which suggests that both in vivo tumor progression and in vitro cellular adaptation are marked by chromosome mutations that may activate multiple oncogenes.

Biochemical modulation of fluoropyrimidines by antifolates and folates in an in vitro model of human leukemia.

Although 5-fluorouracil (FUra) is one of the most effective cytotoxic agents in the treatment of various solid tumors (carcinomas of the gastro-intestinal tract, breast, head and neck), remissions occur in only 20 to 30% of cases and usually are of short duration. Recently, preclinical studies have shown that the antitumor activity of FUra can be potentiated by modulating the metabolism of this drug by using other substances, in particular antifolates of folates. Pretreatment with antifolates may, by blocking de novo purine biosynthesis and consequently increasing phosphoribosyl pyrophosphate (PRPP) pools, enhance the conversion of FUra to active fluoronucleotide pools via orotate phosphoribosyltransferase. Methotrexate (MTX) pretreatment may also enhance binding of the fluoropyrimidine inhibitor, 5-fluodeoxyuridylate (FdUMP), to the target enzyme, thymidylate synthase (TS), indirectly by increasing dihydrofolate polyglutamates or directly, as MTX polyglutamates, by enhancing the formation of ternary complexes with FdUMP and TS. Exogenous folates, in particular 5-formyltetrahydrofolate (folinate, leucovorin, LV), can, by raising the intracellular levels of 5, 10-methylenetetrahydrofolate, lead to increased formation and stabilization of the ternary complex formed by TS, the folate coenzyme, and FdUMP. In vitro studies have also shown potentiation of FUra cytotoxicity by antifolates and folates against human lymphoblastic leukemia cell lines. Thus, while FUra may have little or no single agent activity in leukemias and lymphomas, it may be converted to an active drug in these neoplasms by appropriate modulation. Clinical studies of sequential MTX-FUra or combined LV-FUra based upon experimental tumor results reviewed herein, are warranted.

Induction of differentiation of HL-60 cells along the monocytic pathway by 5-methyltetrahydrofolate.

Many compounds of different chemical structure can induce the HL-60 cell line to differentiate along the monocytic or granulocytic pathway, but the mechanism(s) of differentiation by these agents is not known. Experimental evidence suggests that DNA and/or membrane phospholipid transmethylation reactions may be of importance. Based on this background, we have studied the effects of various concentrations of (dl)-5-methyltetrahydrofolate (mTHF), a folate coenzyme involved in transmethylation reactions, on differentiation of HL-60 cells. Differentiation along the monocytic pathway was evidenced in kinetic, functional, cytochemical and immunophenotypical studies when cells were treated with high-dose (dl)-mTHF (1 x 10(-3)M). Some hypotheses on the mechanism(s) of (dl)-mTHF induced HL-60 cell differentiation are discussed with particular regard to a possible enhancement of lipid or DNA methylation via methionine formation by the (l) form or to inhibition of folate-dependent metabolism by the unnatural (d) form, hence of purine and thymine nucleotide synthesis.

Impaired polyglutamylation of methotrexate as a cause of resistance in CCRF-CEM cells after short-term, high-dose treatment with this drug.

Two methotrexate-resistant sublines, CCRF-CEM R3/7 and CCRF-CEM R30/6, were selected from the human leukemia T-lymphoblast cell line, CCRF-CEM, after repeated exposures (7 and 6 times, respectively) for 24 h to constant concentrations (3 and 30 microM) of the drug. Analysis of the mechanism of resistance revealed no differences in levels of dihydrofolate reductase activity, its binding affinity for methotrexate, or in methotrexate transport between the CCRF-CEM parent and methotrexate-resistant cell lines. The development of resistance to methotrexate was associated with a marked decrease in the intracellular level of methotrexate polyglutamates. Although the resistant sublines were able to form substantial amounts of folate polyglutamates when measured with [3H]folic acid, the level of polyglutamates formed was decreased to about 50% of that formed by the parent cell line. No qualitative differences in folate polyglutamates formed were noted between the parental and resistant sublines. This is the first example of a cell line which displays resistance which is solely attributable to defective methotrexate polyglutamate synthesis.

Effects of 5-methyltetrahydrofolate on the activity of fluoropyrimidines against human leukemia (CCRF-CEM) cells.

The growth inhibitory effects of 5-fluorouracil (FUra) or 5-fluoro-2'-deoxyuridine (FdUrd) combined with 5-methyltetrahydrofolate (5-CH3-H4PteGlu) were determined, as a function of time, dose, and sequence of exposure, on human T-lymphoblast leukemia cells, CCRF-CEM. Synergistic inhibitory effects on cell growth were obtained when exponentially growing CCRF-CEM cells were exposed to 5-CH3-H4PteGlu (1-100 microM) for 4 hr and to FUra (250 microM) or FdUrd (0.5 microM) during the last 2 hr. Synergism was dependent on 5-CH3-H4PteGlu dose (100 greater than 10 greater than 1 microM) and did not occur at 0.1 microM. No clear dependence of synergism on sequence was observed with FUra and 5-CH3-H4PteGlu combinations (5-CH3-H4PteGlu----FUra,5-CH3-H4PteGlu + FUra, or FUra----5-CH3-H4PteGlu). With 5-CH3-H4PteGlu and FdUrd combinations, synergism was dependent on sequence of exposure (5-CH3-H4PteGlu + FdUrd, 5-CH3-H4PteGlu----FdUrd were synergistic, but FdUrd----5-CH3-H4PteGlu was not). Thymidine (0.1 microM), added after drug treatment, substantially rescued CCRF-CEM cells from 5-CH3-H4PteGlu----FUra cytotoxicity. L-methionine (1500 mg/l) completely protected CCRF-CEM cells from enhanced cytotoxicity of the combination, 5-CH3-H4PteGlu-FdUrd. The results are consistent with the hypothesis that the mechanism by which 5-CH3-H4PteGlu potentiates fluoropyrimidine cytotoxicity is the enhancement of complex formation between thymidylate synthase and 5-fluorodeoxyuridylate, as a consequence of an increase of intracellular levels of 5,10-methylenetetrahydrofolate generated from 5-CH3-H4PteGlu. Also, enhanced stability of the complex in the presence of high levels of this folate coenzyme may contribute to the synergism observed. These data provide a rationale basis for further trials of folate coenzymes and fluoropyrimidine combinations in the clinic.

Enhancement of fluoropyrimidine cytotoxicity by 5-methyltetrahydrofolate in a human leukemia cell line, CCRF-CEM.

The inhibitory effects of combined 5-methyltetrahydrofolate (5-CH3-THF), the physiological circulating folate species, and fluoropyrimidines, 5-fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd), on growth of human leukemia cells, CCRF-CEM, were determined as a function of time, dose, and sequence of exposure. Exposure of CCRF-CEM cells in exponential growth to 5-CH3-THF (1-100 microM) for 4 h and to FUra (250 microM) or FdUrd (0.5 microM) during the last 2 h resulted in having synergistic inhibitory effects on cell growth. Synergy was dependent on 5-CH3-THF dose (100 greater than 10 greater than 1 microM) and did not occur at 0.1 microM. No clear dependency of synergy on sequence was observed with FUra and 5-CH3-THF combinations (4 h exposure, 5-CH3-THF----FUra, 5-CH3-THF + FUra, or FUra----5-CH3-THF). With 5-CH3-THF and FdUrd combinations, synergy was dependent on sequence of exposure (5-CH3-THF----FdUrd and 5-CH3-THF + FdUrd were synergistic, but FdUrd----5-CH3-THF was not). Thymidine (0.1 microM), added after drug treatment, substantially rescued CCRF-CEM cells from 5-CH3-THF-FUra cytotoxicity. L-Methionine (1500 mg/l) completely protected CCRF-CEM cells from the toxicity of the combination 5-CH3-THF-FdUrd. The results are consistent with the hypothesis that the mechanism by which 5-CH3-THF potentiated fluoropyrimidine cytotoxicity is the enhancement of ternary complex formation between thymidylate synthase and 5-fluorodeoxyuridylate, the active metabolite of fluoropyrimidines, as a consequence of an increase of intracellular levels of 5-10-methylenetetrahydrofolate generated from 5-CH3-THF.

Alkaline elution of DNA as a method for assessing genotoxic effects of drugs: its application to a series of antiviral compounds.

Various techniques are available for the evaluation of the genotoxic potential of drugs and chemicals, some of which utilize as targets mammalian cells grown in vitro. Among these techniques alkaline elution of DNA has the advantage of rapidity and low cost, and can be performed on easily grown leukemia cell lines. To test the applicability of the method to pharmaceutical compounds, we used DNA-alkaline elution with a series of antiviral agents with variable DNA-damaging activity. 5-Iodo-2'-deoxyuridine (5-IdU), 5-bromo-2'-deoxyuridine (5-BrdU) and 5-trifluoromethyl-2'-deoxyuridine (F3dT) increased the DNA elution rate over controls in a dose-dependent fashion, 9-beta-D-arabinofuranosyladenine (ARA-A) had a marginal effect. 9-(2-Hydroxyethoxymethyl)guanine(acyclovir,ACV) and 5-(2-bromovinyl)-2-deoxyuridine (5-BrVdU), selective antiviral compounds, did not vary alkaline elution profiles. These results fully support the use of alkaline elution of DNA as a standard procedure in the evaluation of the genotoxic effect of drugs.

Modulation of fluoropyrimidine cytotoxicity by methotrexate or 5-methyltetrahydrofolate in human leukemia cells in vitro.

The effects of methotrexate, 5-fluorouracil, 5-fluoro-2'-deoxyuridine on the growth of human leukemic T-lymphoblasts, CCRF-CEM, were determined as a function of drug concentration and exposure time. Substantial inhibition of cell growth (greater than or equal to 90%) was obtained with short duration of exposure (4 h) for MTX (ED90 = 4.3 microM). 5-fluorouracil was a relatively ineffective cytotoxic agent for exposure of short duration (4 h). Only exposure of 24 and 72 h resulted in cell growth inhibition greater than or equal to 90% with this drug. In terms of a ED90, 5-fluoro-2'-deoxyuridine was about 190- and 1300-fold more active than 5-fluorouracil for 24 and 72 h exposures, respectively (0.4 vs 75 microM and 0.01 vs 26 microM). Sequential exposure to methotrexate (4 h) and 5-fluorouracil during the last 2 h of methotrexate exposure resulted in synergistic inhibitory effects on cell growth. Antagonistic inhibitory effects on cell growth of methotrexate and 5-fluoro-2'-deoxyuridine combinations were observed independently of drug concentrations. Pretreatment (4h) with 5-methyltetrahydrofolate, the reduced folate to which leucovorin is rapidly converted in vivo, potentiated cell growth inhibitory effects of subsequently administered 5-fluorouracil or 5-fluoro-2'-deoxyuridine. These results provide information on scheduling of methotrexate or reduced folates and fluoropyrimidines that might have potential importance in the development of clinical trials designed for patients with leukemia and lymphoma.

Clinical chemotherapeutic evaluation of fosfomycin plus amoxicillin (co-fosfolactamine): a prospective double-blind clinical trial.

Seventy-three hospitalized patients with urinary tract infections (UTI) were included in a double-blind study and received at random either fosfomycin or fosfomycin plus amoxicillin (CFL) in a pre-fixed combination, in order to evaluate the effectiveness of the CFL. CFL was administered orally to 39 patients at the daily dosage of 1 g every 8 h for 6 days (666 mg of fosfomycin and 334 mg of amoxicillin) while fosfomycin was given orally to 34 patients at the daily dosage of 666 mg every 8 h for 6 days. A bacteriological cure was observed in 97.4% of the patients treated with CFL, while in the fosfomycin group the bacterial eradication was obtained only in 82.3% (P less than 0.05). CFL showed a more rapid bactericidal activity than fosfomycin. In addition, CFL was more effective than fosfomycin against "difficult" Gram-negative bacteria such as Proteus spp. and Pseudomonas aeruginosa. Both antibiotics were well tolerated. Our limited experience revealed that CFL induces a negligible degree of resistant strains, and fewer than fosfomycin. Thus we conclude that CFL seems to be a useful antibiotic combination in the treatment of UTI.