Comparative antitumor efficacy of docetaxel and paclitaxel in nude mice bearing human tumor xenografts that overexpress the multidrug resistance protein (MRP)

BACKGROUND: Multidrug resistance has been associated with expression of the multidrug resistance protein (MRP). Recently, MRP-expression has been detected in human tumor samples of patients with breast cancer and non-small-cell lung cancer. Since taxoids are the most active drugs in the treatment of both tumor entities, the antitumor efficacies of paclitaxel and docetaxel were compared in nude mice bearing human tumor xenografts that express MRP. MATERIALS AND METHODS: Athymic nude mice (nu/nu) bearing tumor xenografts of parental human sarcoma HT1080 or MRP-expressing HT1080/DR4 cells (as confirmed by Northern blot analysis) were treated with the maximum tolerated doses (MTD) of doxorubicin ([Dx] 10 mg/kg i.v. push), paclitaxel ([PC] 50 mg/kg three-hour i.v. infusion), or docetaxel ([DC] 40 mg/kg three-hour i.v. infusion). In vitro, the activity of doxorubicin, paclitaxel and docetaxel was evaluated by the sulphorhodamine B (SRB) assay using the pyridine analogue PAK-104P (5 microM), a potent inhibitor of MRP-function. RESULTS: At their MTDs both taxoids showed significant activity against MRP-negative HT1080 xenografts with response rates of 80% (40% CR) for PC and 100% (60% CR) for DC. In contrast, DC was significantly more active than PC in nude mice bearing doxorubicin resistant MRP-expressing HT1080/DR4 tumor xenografts (overall response rates: 100% (60% CR) for DC; 10% (0% CR) for PC; 0% for Dx). Since treatment of mice with the MTD of PC or DC yielded similar overall toxicity (maximum weight loss for HT1080: PC 8.6 +/- 2.2%; DC 7.5 +/- 2.2% and for HT1080/DR4: PC 11.6 +/- 3.0%; DC 7.6 +/- 1.8%, respectively), these results demonstrate the increase in the therapeutic index for docetaxel against MRP-expressing tumors. In vitro, HT1080/DR4 cells were 270-fold, 6.4-fold and 2.8-fold more resistant than parental cells to doxorubicin, PC and DC, respectively. Pyridine analogue PAK-104P completely restored drug sensitivity to PC and DC, while no effect of PAK-104P on parental HT1080 cells was observed. CONCLUSIONS: Both taxoids, when given at their MTDs, showed significant efficacy against parental HT1080 tumor xenografts. However, docetaxel at its MTD was significantly more active against MRP-expressing tumor xenografts than paclitaxel. Furthermore, in vitro resistance of HT1080/DR4 cells was higher for PC (6.4-fold) than for DC (2.8-fold). Since PAK-104P completely restored sensitivity to both taxoids, the observed resistance appears to be related to MRP. These data suggest, that docetaxel is not as readily transported by MRP as paclitaxel leading to an increased therapeutic ratio in MRP-expressing tumors in vivo. Therefore, docetaxel may have therapeutic advantages in the clinical treatment of MRP-expressing tumors.     

Difloxacin reverses multidrug resistance in HL-60/AR cells that overexpress the multidrug resistance-related protein (MRP) gene

In this study, we have examined the in vitro chemosensitizing activity of difloxacin, a quinolone antimicrobial agent, in the multidrug-resistant human myeloid leukemia HL-60/AR cell line. HL-60/AR cells were found to overexpress multidrug resistance-associated protein (MRP) mRNA as compared to HL-60 cells. Difloxacin, in a concentration-dependent manner, increased the sensitivity of HL-60/AR cells to daunorubicin, adriamycin, and vincristine, and partially corrected the altered drug transport. In addition, difloxacin corrected subcellular distribution of adriamycin by inducing redistribution of the drug from the perinuclear region to the nucleus in HL-60/AR cells. The chemosensitizing effect of difloxacin was observed at clinically achievable concentrations. We conclude that difloxacin is an effective chemosensitizer of MRP-associated multidrug-resistant tumor cells and is a potential candidate for clinical use to reverse multidrug resistance.     

Expression of laminin in renal-cell carcinomas, renal-cell carcinoma cell lines and xenografts in nude mice

We studied the expression of laminin (Ln) chains (alpha1-alpha3, beta1-beta3, gamma1) in human renal-cell carcinomas (RCC), papillary renal neoplasms (PRN) and oncocytomas, in RCC cell lines and their xenografts. In RCCs the basement membranes (BM) showed immunoreactivity for chains of Ln-1 (alpha1-beta1-gamma1). Only in well-differentiated RCCs could vessel BMs be distinguished from those of carcinoma cell islets. RCCs and oncocytomas also exhibited an abundant immunoreactivity for Ln beta2 chain in both vessel and tumor cell BMs, while Ln alpha2 chain was not seen in any renal tumors. In distinction from RCCs, PRNs presented a strong BM immunoreactivity for Ln alpha3 and beta3 chains and for Ln-5, as well as lack of Ln beta2 chain. A more variable reactivity for Ln-5 was seen in oncocytomas. As PRNs and oncocytomas have been suggested to originate from collecting ducts, it is notable that in normal human kidney, we could detect immunoreactivity for Ln-5 and its chains only in BM of the tubules of the loop of Henle. In immunoprecipitation experiments, an abundant production of Ln-1, but not of Ln-5, was seen in cultured RCC cells, while in xenografts of the same cells BM-confined immunoreactivity for both Ln-1 and Ln-5 was seen. Ln beta2 chain was produced by 2 of the 4 RCC cell lines in culture but was found only in 1 of the xenografted tumors.     

Schedule-dependent interaction of doxorubicin, paclitaxel and gemcitabine in human breast cancer cell lines

We showed previously that a sequential treatment with doxorubicin (4 hr) followed by paclitaxel (24 hr) (Dox-->Pacl) induces a synergistic cytotoxic effect in the BRC-230 breast cancer cell line and in human primary breast cancer cultures. The validity of this experimental finding was confirmed in a clinical phase I/II study on advanced breast cancer patients. To improve the cytotoxic effect obtained by the Dox-->Pacl sequence, we analyzed the effect of adding gemcitabine (Gem) to the Dox-->Pacl sequence in a preclinical study. Our study was performed on BRC-230 and MCF-7 cell lines, and cytotoxic activity was evaluated by the sulforhodamine B assay and the type of drug interaction by Drewinko's test. When Gem (0.01 microg/ml for 24 hr) was given immediately or 24 hr after Dox-->Pacl, an antagonistic cytotoxic effect was observed. Conversely, a synergistic effect was found when Gem was given 48 hr after Dox-->Pacl. From results of flow cytometric analysis, the synergistic effect was attributed to cell cycle perturbation. Cells were arrested in G2-M (95% in treated vs. 21% in control samples) 24 hr after Dox-->Pacl treatment. The block progressively recovered thereafter, and after a further 24 hr, at the time of Gem treatment, the cells progressed into the G1-S phase boundary (the cell cycle phase susceptible to the cytocidal effect of the drug). Our findings suggest that the interactions of Dox, Pacl and Gem are highly schedule- and time-dependent and should be taken into consideration in the planning of clinical protocols.     

beta ig-h3: a transforming growth factor-beta-responsive gene encoding a secreted protein that inhibits cell attachment in vitro and suppresses the growth of CHO cells in nude mice

beta ig-h3 is a novel gene first discovered by differential screening of a cDNA library made from A549 human lung adenocarcinoma cells treated with transforming growth factor-beta 1 (TGF-beta 1). It encodes a 683-amino-acid protein containing a secretory signal sequence and four homologous internal domains. Here we show that treatment of several types of cells, including human melanoma cells, human mammary epithelial cells, human keratinocytes, and human fibroblasts, with TGF-beta resulted in a significant increase in beta ig-h3 RNA. A portion of the beta ig-h3 coding sequence was expressed in bacteria, and antisera against the bacterially produced protein was raised in rabbits. This antisera was used to demonstrate that several cell lines secreted a 68-kD beta IG-H3 protein after treatment with TGF-beta. Transfection of beta IG-H3 expression plasmids into Chinese hamster ovary (CHO) cells led to a marked decrease in the ability of these cells to form tumors in nude mice. The beta IG-H3 protein was purified from media conditioned by recombinant CHO cells, characterized by immunoblotting and protein sequencing and shown to function in an anti-adhesion assay in that it inhibited the attachment of A549, HeLa, and WI-38 cells to plastic in serum-free media. Sequencing of cDNA clones encoding murine beta ig-H3 indicated 90.6% conservation at the amino acid level between the murine and human proteins. Finally, the beta ig-h3 gene was localized to human chromosome 5q31, a region frequently deleted in preleukemic myelodysplasia and leukemia. The corresponding mouse beta ig-h3 gene was mapped to mouse chromosome 13 region B to C1, which confirms a region of conservation on human chromosome 5 and mouse chromosome 13. We suggest that this protein be named p68 beta ig-h3.     

In vivo (31)P magnetic resonance spectroscopy and morphometric analysis of the perfused vascular architecture of human glioma xenografts in nude mice

The relationship between the bioenergetic status of human glioma xenografts in nude mice and morphometric parameters of the perfused vascular architecture was studied using (31)P magnetic resonance spectroscopy (MRS), fluorescence microscopy and two-dimensional digital image analysis. Two tumour lines with a different vascular architecture were used for this study. Intervascular distances and non-perfused area fractions varied greatly between tumours of the same line and tumours of different lines. The inorganic phosphate-nucleoside triphosphate (P(i)/NTP) ratio increased rapidly as mean intervascular distances increased from 100 microm to 300 microm. Two morphometric parameters - the percentage of intervascular distances larger than 200 microm (ivd200) and the non-perfused area fraction at a distance larger than 100 microm from a nearest perfused vessel (area100), - were deduced from these experiments and related to the P(i)/NTP ratio of the whole tumour. It is assumed that an aerobic to anaerobic transition influences the bioenergetic status, i.e. the P(i)/NTP ratio increased linearly with the percentage of ivd200 and the area100.     

The efficacy of 2',2'-difluorodeoxycytidine (gemcitabine) and vinblastine combined with interferon in nude mice xenografts of human renal cell carcinoma

Recent in vitro experiments indicated strong activity of 2',2'-difluorodeoxycytidine (dFdC, gemcitabine) in human renal cell carcinoma (RCC) cell lines and an increase of efficacy by combined application of interferon (IFN). In the present study, nude mice with xenografts from ACHN- or SN12C cells were treated by dFdC, dFdC plus IFN-alpha or vinblastine (VBL) plus IFN-alpha. ACHN-xenografts were significantly more inhibited by dFdC+/-IFN-alpha than by VBL+IFN-alpha. Complete remissions (CR) were only seen by dFdC. An additional treatment with IFN-alpha shortened the time to commencement of tumor remission and increased CR of ACHN- and SN12C-tumors (40%; 7%) compared to a treatment with dFdC alone (20%; 0). dFdC+IFN-alpha reduced the number of pulmonary metastases compared to untreated animals. Survival was significantly prolonged by dFdC+/-IFN-alpha in ACHN-mice and dFdC+IFN-alpha or VBL+IFN-alpha in SN12C mice. In conclusion, experimental data confirm dFdC as a superior drug against human RCC compared to VBL. Combined therapy with IFN-alpha increased the efficacy of dFdC in terms of tumor response in immunodeficient nude mice, thus clinical studies are strongly recommended in patients with metastatic renal cell carcinoma.     

The correlation of membranous glycoprotein-gp-170, cytoplasmic glutathione and glucose-6-phosphate dehydrogenase levels with multidrug resistance in transitional cell carcinoma cell lines of the urinary tract

The expression of membranous glycoprotein gp-170, cytoplasmic glutathione (GSH) and energy-related glucose-6-phosphate dehydrogenase (G-6-PD) in cultured normal urothelial cells and transitional cell carcinoma (TCC) cell lines was analyzed by flow cytometric and enzymatic methods. The chemosensitivity of these tumor cells to four major types of anticancer drugs, including cisplatin, thiotepa, methotrexate, 5-fluorouracil, adriamycin and vinblastine, was correlated with biological activities in TCC cell lines. The TCC cell lines displayed a general sensitivity to anticancer drugs with a low incidence of highly resistant cell lines (23%). The expression of multidrug resistance was not related to cellular differentiation or invasiveness of cancer cells. Only 24% of TCC cell lines had an elevated expression of gp-170, but their expression was not related to drug resistance. Increased cytoplasmic GSH and G-6-PD was observed in over 90 per cent of TCC cell lines, but no correlation with drug resistance and cellular differentiation was observed. The biological activities of GSH and G-6-PD were not related to the drug resistance of TCC. The low expression rate of gp-170 in TCC cells indicates that other mechanisms should be involved in the development of MDR in TCC cells.     

Expression of human MRP6, a homologue of the multidrug resistance protein gene MRP1, in tissues and cancer cells

Arachidonic acid and eicosapentaenoic acid are important precursors for the production of prostaglandins and other hormone-like eicosanoid molecules. These fatty acids are synthesized by animals by elongating and desaturating precursor fatty acids such as linoleic acid (18:2Delta9,12) and alpha-linolenic acid (18:3Delta9, 12,15). We have identified a Delta5 fatty acid desaturase gene (fat-4) from the nematode Caenorhabditis elegans. We have expressed this gene product in Saccharomyces cerevisiae and demonstrate that it readily converts di-homo-gamma-linolenic acid (20:3Delta8,11,14) to arachidonic acid (20:4Delta5,8,11,14). The FAT-4 Delta5-desaturase also acts on a number of other substrates, including fatty acids that do not contain a double bond at the Delta8 position.     

Bacterial lipopolysaccharide confers resistance to G418, doxorubicin, and taxol in the murine macrophage cell line, RAW264

Many bacterial pathogens including Salmonella and Listeria replicate within macrophages. The susceptibility of these organisms to various antibiotics is dependent on the ability of macrophages to take up, retain, and deliver the antibiotic to the correct intracellular compartment. In this context, macrophages are known to express proteins that are involved in efflux of antibiotics and cytotoxic drugs, thereby reducing intracellular accumulation of such compounds. In our studies on the action of bacterial lipopolysaccharide (LPS) on the macrophage-like cell line, RAW264 we found that LPS treatment of these cells conferred resistance to the neomycin-related aminoglycoside G418 (geneticin). This phenotype was stable and was specific to LPS since colony-stimulating factor 1 and phorbol myristate acetate had no effect on G418 resistance. We have extended this observation to show that LPS induces transient resistance to the cytotoxic drugs taxol and doxorubicin. Macrophage resistance to cytotoxic drugs and antibiotics may have a number of important clinical consequences.     

Antitumor effect of DT-5461, a lipid A derivative, against human tumor xenografts is mediated by intratumoral production of tumor necrosis factor and affected by host immunosuppressive factors in nude mice

PURPOSE OF THE STUDY: The purpose of this report was to study the repercussions of rotator cuff condition on unconstrained shoulder arthroplasty results. METHODS: Between 1986 and 1993, 40 unconstrained shoulder prostheses were performed (24 total and 16 hemiarthroplasties). At the time of the operation, rotator cuff condition was reported to be normal in 15 shoulders, atrophic in 10, scarred in 3, torn in 12. Clinical and radiographical results were analyzed and supplemented with a rotator cuff echography. Postoperative follow-up averaged 42.25 months (12 to 97 months). RESULTS: When the rotator cuff was intact at the time of operation, clinical results were the best at the time of review. Constant score, overall mobility, forward elevation, external rotation with the elbow along the side were better for intact than for atrophic, scarred or torn rotator cuffs (p < 0.01). Ruptures repaired by local tissue transfer or trapezo deltoidal flap substitution (3 cases) gave satisfactory results, whereas two dacron cuff prostheses failed. In the non repaired ruptures (7 cases), arthroplasty resulted in 3/4 fair or poor results. At the time of review, the total incidence of postoperative rotator cuff tears was high (12 cases), functional repercussions were variable. Among the 15 rotator cuff tears observed at the time of the review, the long head of the biceps was present and in place in 9 cases and ruptured in 6 cases. In these latter cases, Constant score (p < 0.01), the forward elevation (p < 0.01), external rotation with elbow along side (p < 0.05) and overall mobility (p < 0.05) were worse; moreover, humeral head superior migration was greater (p < 0.01). DISCUSSION: Our study confirms the superiority of clinical results when the cuff was intact at the time of the operation. The repair of rotator cuff tears seems to be justified. When the rupture has not been repaired, clinical results were fair or poor in 5 cases out of 7, the initial rupture spread to the other tendons in 3 cases out of 7. Secondary ruptures were frequent (12 cases). No predictive factor was identified, but an overlapping of the greater tuberosity appears to be detrimental. Among rotator cuff tears present at the time of review, long head of the biceps rupture jeopardized clinical results and was associated with a proximal migration of the humeral head which was significantly more severe than with an intact non displaced long head of the biceps. CONCLUSION: During unconstrained shoulder prosthesis implantation, it is therefore recommend to systematically repair any associated rotator cuff rupture, in order to avoid any greater tuberosity overlap relative to the prosthetic head and to preserve the long head of the biceps tendon which limits upward migration of the humeral head and improves prosthetic kinetics.     

Expression and localization of the multidrug resistance protein 5 (MRP5/ABCC5), a cellular export pump for cyclic nucleotides, in human heart

The multidrug resistance protein 5 (MRP5/ABCC5) has been recently identified as cellular export pump for cyclic nucleotides with 3',5'-cyclic GMP (cGMP) as a high-affinity substrate. In view of the important role of cGMP for cardiovascular function, expression of this transport protein in human heart is of relevance. We analyzed the expression and localization of MRP5 in human heart [21 auricular (AS) and 15 left ventricular samples (LV) including 5 samples of dilated and ischemic cardiomyopathy]. Quantitative real-time polymerase chain reaction normalized to beta-actin revealed expression of the MRP5 gene in all samples (LV, 38.5 +/- 12.9; AS, 12.7 +/- 5.6; P < 0.001). An MRP5-specific polyclonal antibody detected a glycoprotein of approximately 190 kd in crude cell membrane fractions from these samples. Immunohistochemistry with the affinity-purified antibody revealed localization of MRP5 in cardiomyocytes as well as in cardiovascular endothelial and smooth muscle cells. Furthermore, we could detect MRP5 and ATP-dependent transport of [(3)H]cGMP in sarcolemma vesicles of human heart. Quantitative analysis of the immunoblots indicated an interindividual variability with a higher expression of MRP5 in the ischemic (104 +/- 38% of recombinant MRP5 standard) compared to normal ventricular samples (53 +/- 36%, P < 0.05). In addition, we screened genomic DNA from our samples for 20 single-nucleotide polymorphisms in the MRP5 gene. These results indicate that MRP5 is localized in cardiac and cardiovascular myocytes as well as endothelial cells with increased expression in ischemic cardiomyopathy. Therefore, MRP5-mediated cellular export may represent a novel, disease-dependent pathway for cGMP removal from cardiac cells.     

The small GTP-binding protein Rho1 is a multifunctional protein that regulates actin localization, cell polarity, and septum formation in the fission yeast Schizosaccharomyces pombe

BACKGROUND: The small GTP-binding protein Rho has been shown to regulate the formation of the actin cytoskeleton in animal cells. We have previously isolated two rho genes, rho1+ and rho2+, from the fission yeast Schizosaccharomyces pombe in order to investigate the function of Rho using genetic techniques. In this paper, we report the cellular function of Rho1. RESULTS: We found that Rho1 is essential for cell viability and cell polarity using gene disruption and by exogenous expression of botulinum C3 ADP-ribosyltransferase. In cells expressing either a constitutively active Rho1 or a dominant-negative Rho1, actin patches were delocalized. Both the cell wall and secondary septum were thick and stratified in cells expressing the constitutively active Rho1, while the cell wall of cells expressing the dominant-negative Rho1 seemed to be loosely organized. Furthermore, inactivation of Rho1 is apparently required for the separation of daughter cells. Cell fractionation studies suggested that Rho1 is predominantly membrane-bound. Moreover, we observed that Rho1 is localized to the cell periphery and to the septum. CONCLUSIONS: Rho1 is involved in actin patch localization, the control of cell polarity, the regulation of septation, and cell wall synthesis.     

Induction of Ad4BP/SF-1, steroidogenic acute regulatory protein, and cytochrome P450scc enzyme system expression in newly established human granulosa cell lines

We have established immortalized human granulosa cells by triple transfection of primary cells obtained from in vitro fertilization patients with SV40 DNA, Ha-ras oncogene, and a temperature sensitive (ts) mutant of the tumor suppressor gene p53 (p53val135). Forty-one clones were isolated, and their steroidogenic responses were analyzed. While all the cell lines proliferate rapidly and show only traces of progesterone production, upon stimulation with 50 microM of forskolin (FK), which elevates intracellular cAMP, they become steroidogenic as evidenced by progesterone production. The steroidogenic response of the cell lines was stable even after 20 generations and several cycles of freezing and thawing. A highly responsive cell line (HO-23) was further examined for characteristics of the steroidogenic response. Cells stimulated with FK and 8-Br-cAMP produced high levels of pregnenolone, progesterone, and 20alpha-hydroxy-4-pregnen-3-one (20alpha-OH-progesterone) comparable with amounts produced by highly differentiated primary human granulosa-luteal cells. Hydrocortisone and dexamethasone highly augment the cAMP-stimulated progesterone production, whereas testosterone and PRL enhanced cAMP-induced progesterone synthesis only moderately. Estradiol, insulin-like growth factor I, and insulin showed no significant effect on cAMP-induced steroidogenesis. The phorbol ester TPA, and basic fibroblast growth factor, dramatically suppress cAMP-induced production of progesterone, whereas bovine corneal endothelial cell ECM (BCE/ECM) enhanced cAMP-induced progesterone and antagonized basic fibroblast growth factor suppression of cAMP-induced steroidogenesis. Steroidogenic factor 1 (Ad4BP/SF-1) was expressed in control cells, and its expression was augmented by FK, whereas the steroidogenic acute regulatory protein showed low expression in the nonstimulated cells but was clearly elevated upon cAMP stimulation and was slightly decreased by TPA in cAMP-stimulated cells. Expression of the electron carrier adrenodoxin (ADX), which is a part of the cytochrome P450scc enzyme system, was very low in nonstimulated cells but was dramatically elevated in FK- and 8-Br-cAMP-stimulated cells, whereas no reduction of ADX was evident in cells costimulated with FK and TPA. Immunocytochemical studies revealed a weak staining of ADX in mitochondria of nonstimulated cells and intensive staining in highly clustered mitochondria of FK- or 8-Br-cAMP-stimulated cells. Only moderate reduction in ADX staining was evident in cells costimulated with FK and TPA. These unique cell lines can provide a useful model for the investigation of induced steroidogenesis in human granulosa cells.     

Combined expression of multidrug resistance protein (MRP) and glutathione S-transferase P1-1 (GSTP1-1) in MCF7 cells and high level resistance to the cytotoxicities of ethacrynic acid but not oxazaphosphorines or cisplatin

We tested the hypothesis that combined increased expression of human glutathione S-transferase P1-1 (GSTP1-1), an enzyme that catalyzes the conjugation with glutathione of several toxic electrophiles, and the glutathione-conjugate efflux pump, multidrug resistance protein (MRP), confers high level resistance to the cytotoxicities of anticancer and other drugs. To accomplish this, we developed MCF7 breast carcinoma cell derivatives that express high levels of GSTP1-1 and MRP, alone and in combination. Parental MCF7 cells, which express no GSTP1-1 and negligible MRP, served as control cells. We found that either MRP or GSTP1-1 alone conferred significant resistance to ethacrynic acid cytotoxicity. Moreover, combined expression of GSTP1-1 and MRP conferred a high level of resistance to ethacrynic acid that was greater than resistance conferred by either protein alone. Increased MRP was also associated with modest resistance to the oxazaphosphorine compounds mafosfamide, 4-hydroxycyclophosphamide, and 4-hydroperoxycyclophosphamide. However, coordinated expression of GSTP1-1 with MRP failed to augment this modest resistance. Similarly, GSTP1-1 had no effect on the sensitivities to cisplatin of MCF7 cells regardless of MRP expression. These results establish that coordinated expression of MRP and GSTP1-1 can confer high level resistance to the cytotoxicities of some drugs, including ethacrynic acid, but that such resistance is variable and does not apply to all toxic drugs that can potentially form glutathione conjugates in either spontaneous or GSTP1-1-catalyzed reactions.     

Epidermal growth factor (EGF) receptor blockade inhibits the action of EGF, insulin-like growth factor I, and a protein kinase A activator on the mitogen-activated protein kinase pathway in prostate cancer cell lines

For assays involving glycosyltransferases or transporters, several GDP-sugars are either commercially unavailable or expensive. We describe an enzymatic synthesis of GDP-d-[3H]arabinosep and GDP-l-[3H]fucose that yields 66-95% nucleotide-sugar from the appropriate radiolabeled sugar in less than 30 min. The coupled reaction requires Mg2+, ATP, and GTP along with the appropriate radioactive monosaccharide, sugar-1-kinase, and pyrophosphorylase. The latter two activities are present in a cytosolic fraction of Crithidia fasciculata, which is easily grown at room temperature in simple culture medium without serum or added CO2. Addition of commercial yeast inorganic pyrophosphatase shifts the equilibrium of the pyrophosphorylase reaction toward nucleotide-sugar formation. To verify that these nucleotide-sugars are biologically active, we tested their ability to serve as substrates for glycosyltransferases. GDP-l-[3H]fucose functions as the donor substrate for recombinant human fucosyltransferase V, and GDP-d-[3H]arabinosep serves as the donor substrate for the arabinosyltransferase activities present in Leishmania major microsomes.