CANCERRESEARCH57. 4118—4122.SeptemberIS. I997J

ABSTRACT

To explore the feasibilityof targeting human tumor cells via their transport systems, dipeptide uptake was Studied in the human fibrosarcoma cellline HT1OSOand the human fibroblast cell line IMR-90 by the use of hydrolysis-resistant glycytsarcosine (Gly-Sar). The uptake of [‘4CJGly-Sar into

HT1OSOwas time dependent Kinetic analysis of the concentration dependence ofthe initial rate of['4C)Gly-Sar uptake showed that a carrier-mediatedtransport system with a Km01@11.4 ±33 IfiMlIfldVpj@of26.8 ±4.0 (nmolIl5mhx/ing protein) and a nonsaturable component (kd of 0.80 @df15mm/mgprotein) were @p@nsibkfor the dipeptide uptake by HT1OSOcells. Theoptimal pH for the maximal uptake was around 6.0. [‘4C]Gly-Saruptake wasinhibited by various di- and tripeptides and peptide-mimetic dnigs, such asbestatin and cefadroxil. [‘4CJGIy-Saruptake was not affected by the pr@enceof amino acids or tetra- or pentapeptides. The uptake of cefadroxil wasreduced significantly by unlabeled Gly-Sar. Moreover, Gly-Gly and GIy-Leuproduced an increase in the apparent Km0f the uptake of Gly-Sar withoutaltering V@. On the other hand, dipeptide uptake by IMR-90, which is anormal diploid cell line (not malignant), showed no saturable transportThese results suggest that HT1OSOcells take up dipepddes via a pH-dependent transporter. This is the first report showing that a dipeptide transportsystem, which is similar but not identical to the well-characterized oligopep

tide transporters PepTi and PepT2, exists in fibroblast-denved tumor cellsbut not in normal fibroblasts. The present finding could be the basis ofa novelstrategy for the specific delivery of oligopeptide-mimetic anticancer drugsinto tumor cells.

INTRODUCTION

It is well known that tumor cells possess several transport systems totake up nutrients, including amino acids (I), nucleosides (2), glucose (3),and folate (4). Various studies on the delivery of anticancer drugs totumor cells by using such transport systems have been reported. Melpharan, which is a cancer-therapeutic, phenylalanine-conjugated nitrogenmustard, was taken up by Ll2lO cells via the amino acid transport system(5, 6). Moreover, a sugar-linkednitrogen mustard derivative has beendeveloped as an anticancer drug (7). Methotrexate, an antitumor activeinhibitor ofdihydrofolate reductase, is taken up in a concentrative mannerby many tumor cells, such as leukemia cells of rodents and human, viathe folate transporter (4, 8, 9).

Interest in the transport of oligopeptides has been heightened by therecent cloning of a novel family of peptide-proton cotransporters. Thisfamily includes the rabbit small intestinal PepTl3 (10), the human andrat homologues of PepTl (1 1—13),and a second member of thefamily, PepT2, in the kidney (14, 15). However, there has been noreport on the presence of an oligopeptide transporter in tumor cells. If

Received 4/22/97; accepted 7/17/97.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance withI8 U.S.C. Section I734 solely to indicate this fact.

I This work was supported in part by a Grant-in-Aid for Scientific Research from the

Ministry of Education, Science and Culture, Japan, and by a grant from the Japan HealthSciences Foundation Drug Innovation Project.

2 To whom requests for reprints should be addressed, at Department of Pharmacobio

dynamics, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-I Takara-machi,Kanazawa 920, Japan. Phone: 8 1-76-234-4478; Fax: 81-76-234-4477.

3 The abbreviations used are: PepT. peptide transporter; Gly-Sar, glycylsarcosine;

MES, 2-(N-morpholino)ethanesulfonic acid; HPLC, high-performance liquid chromatography.

such a transporter does exist in tumor cells, it should be possible to useit for the selective delivery of drugs to tumor cells because expressionof PepTs is thought to be limited to epithelial cells in the smallintestine and kidney ( I 1, 14—I6).

We have examined oligopeptide transport activity in the humanfibrosarcoma cell line HT1O8O (17) and the human fibroblast cell lineIMR-90 (18) by using hydrolysis-resistant Gly-Sar. Here, we presentthe new finding that HT1O8O cells have an oligopeptide transportactivity exhibiting similar but not identical features to those of thewell-characterized intestinal and renal dipeptide transporters.

MATERIALS AND METHODS

Materials. IMR-90 cells were purchased from JCRB (Osaka, Japan).RPMI 1640 and MEM were obtained from Nissui (Tokyo, Japan). HT1O8Ocells were prepared in our laboratories. FCS and nonessential amino acids wereobtained from Life Technologies, Inc. (Grand Island, NY). L-Glutamine andGly-Gly were obtained from Wako Pure Industries Ltd. (Osaka, Japan). Rat tailcollagen (type I) was obtained from Collaborative Research Inc. (Bedford,MA). [Glycine-1-'4CJGly-Sar([‘4CJGly-Sar;2.22 GBq/mmol)and[‘4Cjinulin(210 MBq/g) were purchased from Amersham (Buckinghamshire, UnitedKingdom) and DuPont (Wilmington, DE), respectively. Cefadroxil was a giftfrom Bristol Banyu (Tokyo, Japan). Unlabeled Gly-Sar, Gly-Pro, Arg-Ala,bestatin, and amastatin were purchased from Sigma Chemical Co. (St. Louis,MO). The protein assay kit was purchased from Bio-Rad (Melville, NY). Allother chemicals were commercial products of reagent grade. Human PepTlcDNA was a gift from Dr. F. H. Leibach (Medical College of Georgia, GA).

Cell Cultures. HTIO8Oand IMR-90 cells were grown at 37°Cin a 5%C02/95% air atmosphere. HTIO8O and IMR-90 cells were cultivated in RPMI1640 culture medium and MEM containing I % nonessential amino acids and2 mt@iglutamine, each containing 10% FCS without addition of antibiotics,

respectively (19). For the uptake study, HT1O8O and IMR-90 cells were seededat a density of 5.0 x l0@cells/cm2 and 1.0 X l0@cells/cm2, respectively, onmultidishes (four wells) coated with collagen (20). HTIO8O and IMR-90 cells

were grown for 2 days and 7—8 days, and their passage numbers were in theranges of 20—50and 3—7,respectively.

Uptake Experiments. Uptake of [‘4C]Gly-Saror cefadroxil by the cultured cells was examined at 37°Cby the use of a reported method (21). Theflux was measured in HBSS (0.952 mM CaCl2, 5.36 mM KCI, 0.441 mMKH2PO4,0.812 mM MgSO4, 136.7 mM NaCl, 0.385 mM Na2HPO4,25 mMo-glucose, and 10 mt@MES or HEPES) adjusted to pH 6.0 with NaOH. Theosmolality of the HBSS was maintained at 310 mOsmlkg. To assess the effectof extracellular pH, the HBSS contained either 10 mMMES (pH 5.5—6.5)or10 mM HEPES (pH 7.0 and 7.5) and was titrated to a desired pH with 5 MNaOH. L‘4C]Inulinwas used as a marker for the extracellular fluid that adheredto the cells during the washing procedure and to estimate the zero time for thenonspecific adsorption that is involved in the apparent uptake of test com

pounds. To quantify the amounts of [‘4CIG1y-Sar and [‘4Clinulin in the cells,

the washed cells were solubilized by the addition of 5 N NaOH (0.25 ml),followed by shaking for 2 h. The resultant lysates were neutralized with HCIand mixed with 4 ml ofliquid scintillation cocktail, Cleasol-I (Nacalai Tesque,Kyoto, Japan). Radioactivity was determined using a liquid scintillation counter (LSC-l000, Aloka Co. Ltd., Tokyo, Japan). The uptake of cefadroxil wasdetermined by HPLC analysis. Fresh solutions of the test compounds were

prepared for each experiment.HPLC Analysis of [‘4C]Gly-Sarand Cefadroxil. [‘4C]Gly-Sarin the

uptake medium was analyzed by HPLC for the initial 15 mm. A 20-id samplewas separated on an analytical HPLC column, TSK gel ODS 80 Ts, 4.6 X 150mm (Tosoh, Tokyo), using 0.2% trichloroacetic acid solution as the mobile

4118

Carrier-mediated Transport of Oligopeptides in the Human FibrosarcomaCell Line HT1O8O'

Takeo Nakanishi, Ikumi Tamai, Yoshimichi Sai, Takuma Sasaki, and Akira Tsuji2

Department of Phannacobio-dvnaniics. Faculty of Pharmaceutical Sciences IT. N., 1. T., Y. S.. A. TI, and Cancer Research Institute IT. S.], Kanazawa University, Kanazawa 920,Japan

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PEPTIDE TRANSPORT IN TUMOR CELLS

IMR-90 cells. Fig. 1A (left) shows that the uptake of 20 p.M[‘4C]GlySar by HT1O8O cells was linear during the first 15 mm (Fig. 1A, right).Steady-state uptake of the dipeptide was achieved within 120 mm. Incontrast, when the cells were incubated at 4°C(open circles), theuptake was very low and reached a plateau within 30 mm, indicatingthat the binding to the cells was not responsible for the concentrativeuptake observed at 37°C.Moreover, the cells at 37°Caccumulatedapproximately three times more dipeptide than that observed at 4°C.These data suggest that [‘4C]Gly-Saruptake occurs by carrier-mediated transport and that the dipeptide is apparently accumulated intracellularly. On the basis of the results obtained in Fig. 1A, the uptakerates were determined at 15 mm in subsequent experiments.

To examine the dipeptide transport activity in normal diploid cells,dipeptide uptake was measured in the IMR-90 fibroblast cell line,which was established from human normal lung tissue. Fig. lB showsthe time courses of uptake of 20 p@ [‘4C]Gly-Sarat 37 and 4°Candof 1.0 mg/ml [‘4C]inulinas a marker of extracellular fluid thatadhered to the cells. Interestingly, there was no significant differencebetween dipeptide uptakes at 4 and 37°Cwithin 120 mm in IMR-90cells. In addition, the extent of dipeptide uptake was comparable tothat of inulin. These results suggest that no specialized transporter isinvolved in the [‘4C]Gly-Saruptake in IMR-90 cells.

phase at the flow rate of 1.0 mI/mm, with monitoring at 210 nm. The intact

Gly-Sar was eluted as a single peak at 5.6 mm. The eluate was collected for 12mm, and the radioactivity of aliquots was counted.

Uptake of unlabeled cefadroxil was determined by HPLC analysis. Cellswere lysed, and the proteins were precipitated with 60% acetonitrile. A 20-@1aliquot of the resultant supernatant was subjected to HPLC using an isocraticsolvent system as follows: analytical column, TSK gel ODS 80 Ts, 4.6 X 150

mm; mobile phase, 10miiiammonium acetate in acetonitrile-water (4:96); flowrate, 1.0 ml/min; and monitoring at 240 nm. The area of the peak at 5.8 mmwas used to calculate the amount of drug from a standard curve. Protein wasmeasured by the protein-dye binding method using BSA (fraction V; Sigma) asthe standard (22).

Data Analysis. The uptakewas usually expressed in termsof the uptakecoefficient Q.d/time/mg protein) obtained by dividing the uptake rate by thedrug concentration in the uptake medium. The percent of inhibition wascalculated on the basis of the control uptake measured in each experiment. The

kinetic parameters were calculated by the nonlinear least-squares regressionanalysis program, MULTI (23).

RESULTS

Time Course of [54C]Gly-Sar Uptake by HT1O8O and IMR-90Cells. As a first step in the characterization of dipeptide uptake, theaccumulation of [‘4C}Gly-Sarwas examined at pH 6.0 in HTIO8O and

A

IC.5

3

I

3

2

1'

30 60

Time (mm)

120 0 5 10 15

Time (mm)Fig. 1. Time courses of [‘4CJGIy-Saruptake by

HT1O8O and IMR-90 cells. Uptakes of I'4CJGlySar (20 @.tM)by HT1O8Ocells (A) and IMR-90 cells(B) were measured at 37 (•)or 4°C(0) by incubating cells in HBSS at pH 6.0. A, uptake of[‘4C)inulin(1.0 mg/mi) in IMR-90 cells. Datapoints, means of four experiments; bars, SE.

B

C.520.

30

I

60

Time (mm)

90 120

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0 10 20 30 40

Table1 inhibitoryeffectsof vanoaccompounds on (‘4CJGHTIO8O cellsly-Sar

uptake byculturedRelative

uptake―InhibitorConcentration(mM)(% ofcontrol)Gly1097.2

±7.3Pro10112.5±4.9Gly-Gly

Gly-LeuGly-SarGly-ProArg-AlaArg-@-AlaBeatatin10

10101010101056.0

±4.7―42.3 ±4.8―27.2±8.3―76.4 ±4.8―14.6 ±0.8―84.6 ±5.2―40.9 ±5.3―Cefadroxil1039.3

±4.9―Gly-Gly-Gly1059.0±7.2―Gly-Gly-Gly-Gly1093.9±2.1Gly-Gly-Gly-Gly-Gly1098.4±4.9

PEPTIDE TRANSPORT IN TUMOR CELLS

Concentration Dependence of Gly-Sar Uptake. To examine thekinetics of Gly-Sar uptake by HT1O8O cells, initial rates of Gly-Saruptake were measured at pH 6.0 over the concentration range of0.02—40 mM in the medium at 37 and 4°Cto estimate total andnonsaturable uptake, respectively. As shown in Fig. 2, at 4°Cthedipeptide uptake was linear over this concentration range. In contrast,when the cells were incubated at 37°C,the uptake rate tended tosaturate as the Gly-Sar concentration was increased. An EadieHofstee plot of the uptake rates after correction for nonsaturableuptake gave no indication of multiple transport systems (inset), so thedata were fitted to the following equation, which consists of singlesaturable and single nonsaturable linear terms, using the nonlinearleast-squares regression analysis program, MULTI (23):

V [S]v= max +kd[S].Km + [SI

The apparent kinetic constants obtained for Gly-Sar uptakewere a half-saturation constant (Km) of 11.4 ±3.3 mi@iand a maidmum uptake rate (Vm@)of 26.8 ±4.0 nmol/l5 rain/mg protein for thesaturable component and a first-order rate constant (kd) of 0.80 @d/l5mm/mg protein for the apparently nonsaturable component.

pH Dependence of Uptake. The effect of varying the extracellularpH from 5.5 to 7.5 on the rate of [14C]Gly-Sar uptake was measured.As shown in Fig. 3, Gly-Sar uptake was influenced by the pH of theuptake medium. The optimal pH was about 6.0. This result impliesthat protons may be involved as the driving force for the concentrativetransport of Gly-Sar in HT1O8O cells.

Substrate Specificity of [‘4CJG1y-Sar.To study the substratespecificity of the transport system in cultured HT1O8O cells, the

uptake of Gly-Sar was determined in the presence of various compounds. As shown in Table 1, the uptake of the [14C]Gly-Sar was notinfluenced by free amino acids but was significantly reduced byvarious dipeptides. HPLC analysis confirmed that [‘4CJGly-Sarwasstable in the medium for the initial 15 mm (data not shown). In thepresence of 10 p.M amastatin, an aminopeptidase inhibitor, uptake of

5:5@ 6:5 7 7.5

pHFig. 3. pH Dependency of [‘4CJGly-Saruptake by HT1O8O cells. Uptake of [‘4C]Gly

Sar (20 pM) was measured at 37°Cby incubating celia for 15 miss in HBSS of various pHvalues. HBSS contained 10 msi MES for a pH of 5.5, 6.0, or 6.5 or 10 inst HEPES for apH of 7.0 or 7.5. Data points, means of four experiments; bars, SE.

a The uptake of['4C]Gly-Sar(20 psi) was measured at 37°C for 15 mm by incubating

cells in HBSS buffer at pH 6.0 in presence of each inhibitor. Each value represents % ofcontroluptakeandis shownas the mean±SEof fourexperiment.

b Significantly different from the control value by Student's t test (P < 0.05).

Gly-Sar was not changed (data not shown). These observations mdicate that the dipeptide was taken up as the intact molecule.

Arg-13-Ala, which contains a f3-amino acid, was a less effectiveinhibitor than the peptide containing an a-amino acid (Arg-Ala).Furthermore, the inhibitory effect of Gly-Pro, which contains animino acid, on [14C]Gly-Sar uptake was also weak. Gly-Sar, containing a methylated N'@2 terminus, was as inhibitory as Gly-Gly andGly-Leu. The pseudodipeptide bestatin, an anticancer drug containingan NH2-terminal a-hydroxy-@-amino acid, was clearly inhibitory.Cefadroxil, a f3-lactam antibiotic drug having a tripeptide structure, isa substrate of the well-characterized intestinal and renal oligopeptidetransporters, PepTl and PepT2 (24—26).In the presence of 10 ma@cefadroxil, [14C]Gly-Sar uptake was reduced by 60%. When theuptake of cefadroxil was measured in cultured HT1O8O cells, theuptake was reduced significantly to 66% of the control in the presenceof 20 mM Gly-Sar (data not shown). This result indicates that thepseudopeptide drug cefadroxil uses the same transporter as does thenative dipeptide in HT1O8O cells. Moreover, to determine the effect ofbackbone chain length on the uptake of Gly-Sar, a tetrapeptide,Gly-Gly-Gly-Gly, and a pentapeptide, Gly-Gly-Gly-Gly-Gly, were

,0.

(SIt)

GIy-Sar (mM)

Fig. 2. Concentration dependencies of Gly-Sar uptake by HTIO8O cells. Uptake ofGly-Sar was measured at 37 (•)or 4°C(0) by incubating cells for 15 mm with variedconcentrations of Gly-Sar at pH 6.0. A, saturable component of the uptake rate calculatedfrom the kinetic parameters, obtained as described in “Results.―inset, Eadie-Hofatee plotof the uptake rates after correction for the nonsaturable component. The line was drawnby the method ofleast-squarea (correlation coefficient = 0.97). Data points, means of 3-8experiments; bars, SE.

4120

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PEPTIDE TRANSPORT IN TUMOR CELLS

that most likely the uptake of intact [‘4CJGly-Sar,not the degradativeproduct, [14C]glycine, was measured in the present study.

As shown in Fig. 1, the dipeptide uptake proceeded in a time-dependent manner. In contrast, when the incubation temperature was loweredto 4 from 37°C,the initial rate of uptake was reduced, and no significantincrease of uptake with time was observed. The uptake rate was concentration dependent and followed Michaelis-Menten kinetics (Fig. 2). Uptake at pH 6.0 was stimulated by an inward-directed proton gradient (Fig.

3). Gly-Sar uptake was reduced by di- or tripeptides and peptide analogues, such as bestatin and cefadroxil, whereas amino acids or tetra- orpentapeptides had little or no effect (Table 1). In addition, Gly-Gly andGly-Leu inhibited Gly-Sar uptake in a competitive manner (Fig. 4), andthe uptake of cefadroxil was reduced in the presence of Gly-Sar (data notshown), showing mutual inhibition between them. These results stronglyindicate that the HT1O8Ohuman fibrosarcoma cells take up oligopeptideand peptide-like drugs via a di- and tripeptide-specific, pH-dependent,cather-mediated transport mechanism.

The peptide uptake observed in HT1O8O cells exhibited pH dependency similar to that of the intestinal and renal PepTs, PepTI andPepT2, indicating that a proton gradient is the driving force (31, 32).The substrate specificity of the transporter on HT1O8O cells alsoexhibited close similarities with those of the well-characterized PepTland PepT2, which transport not only di- and tripeptides but alsoseveral peptide-mimetic drugs, such as j3-lactam antibiotics and bestatin (25, 26, 33—35).Like PepTl and PepT2, the transporter onHT1O8O cells seems to accept peptide-mimetic drugs and a wide rangeof native peptides. However, in terms of the kinetic constant Km forthe uptake of Gly-Sar, the PepT on HT1O8O cells is different fromPepTl and PepT2. The apparent Km value of rabbit PepTI obtainedfrom the Xenopus oocyte expression system is 1.9 nmi for Gly-Sar(10). The Km5 fiM@Gly-Sar uptake by human PepTl and PepT2cDNA-transfected HeLa cells are 290 @M(1 1) and 74 @M,respectively (36). PepT2 is a high-affinity proton/peptide cotransporter, incontrast to PepTl. We found that the Km value of the transporter onHT1O8Ofor Gly-Sar was 11.4 mr@i,indicating that the affinity is muchlower than those of PepTi and PepT2. Furthermore Northern blotanalysis using human PepTl cDNA as the probe did not show acorresponding signal in HT1O8O cells. Taken together, the resultsindicate that the oligopeptide transporter expressed in HTIO8O cell isa novel transporter, distinct from PepTl and PepT2.

Interestingly, the present study indicated that Gly-Sar was notactively transported in IMR-90 cells, which are normal diploid cellsfrom human lung connective tissue, suggesting that a dipeptide transporter exists in malignant cells, but not in normal diploid cells. Thisis important from the viewpoint of targeting anticancer drugs becausethe transporter becomes a good candidate for tumor cell-specificdelivery of oligopeptide-mimetic anticancer drugs.

In conclusion, we have demonstrated the existence of an oligopeptide transporter on HT1O8O cells, and we showed that Gly-Sar transport by this transporter is sensitive to pH and temperature and issaturable. Furthermore, the substrate specificity is very similar but notidentical to those of well-characterized PepTl and PepT2. Molecularidentification of the oligopeptide transporter on HT1O8O cells andconfirmation of its selective expression in various tumor cells but notnormal cells will be necessary to validate the feasibility of using thistransporter for tumor-specific drug delivery.

REFERENCES

I. Halvor, N. C., and Marie, L. An amino acid transport system of unasaigned functionin the Ehrlich ascites tumor cell. J. Biol. Chem., 240: 3601—3607. 1965.

2. Belt, J. A., Marina, N. M, Phelps, D. A., and Crawford, C. R. Nucleoaide transporterin normalandneoplasticcells.Adv.EnzymeRegul.,33: 235—252,1993.

3. Brown, R. S., and WahI. R. L Overexpressionof Glut-I glucose transporter in human

0.6

0.5

0.4

0.3

0.2

0.1

0EC

•I0C

,-@-

E

Fig. 4. Lineweaver-Burk plots for Gly-Sar uptake by cultured HTIO8O cells. Uptakerate was measured in the absence (0) or presence of 10 ms@Gly-Gly (@) and 10 ms@Gly-Leu (A). Incubation conditions were identical to those described in the Fig. I legend.Data points, means of 3—8experiments; bars, SE.

studied. These peptides did not have any effect on the uptake ofGly-Sar.

The inhibitory mechanism of Gly-Gly and Gly-Leu was kineticallyanalyzed. The uptake of Gly-Sar was determined in the presence of 10mM Gly-Gly or Gly-Leu over the Gly-Sar concentration range of 2—40

mM. Fig. 4 shows the Lineweaver-Burk plots of the uptake of Gly-Sar

by cultured HT1O8O cells in the absence and presence of 10 nmiGly-Gly and Gly-Leu. These peptides produced an increase of apparent Km for Gly-Sar without altering@ Therefore, the inhibition byboth dipeptides is clearly competitive. The inhibition constants (K1)calculated from the Lineweaver-Burk plots for Gly-Gly and Gly-Leuwere 20.1 and 7.2 m@t,respectively.

DISCUSSION

Because of the physiological and pharmacological implications,previous studies on mammalian oligopeptide transport have mainlyfocused on the characterization of the intestinal and renal PepTs(24—28).Because the oligopeptide transporter has not been demonstrated to be present in cultured mammalian malignant cells, exceptfor the human carcinoma cell lines Caco-2 (29) and HT-29 (30), bothof which originated from epithelial cells, the present study is the firstto demonstrate that oligopeptide is transported by a carrier-mediatedpathway in tumor cells derived from nondifferentiative cells. Established epitheial-type cell lines, such as Caco-2 and HT-29, differentiate to polarized cells possessing intestinal enterocyte-like properties;they exhibit oligopeptide transport activity and can be used as modelsto studyabsorptionprocessesin the small intestine(29, 30).

Here, we have examined the characteristics of the transport ofGly-Sar in HT1O8Ocells, a human fibrosarcoma cell line. Gly-Sar isa hydrolysis-resistant peptide because of the methylated nitrogen onthe peptide bond. Although we cannot completely exclude the possibility of hydrolysis of Gly-Sar, the uptake of [‘4C]Gly-Sarwas notreduced in the presence of 10 mi@iglycine (Table 1), and [14C]Gly-Sarwas confirmed to be stable during the initial 15 mm of incubation withthe cells by HPLC analysis (data not shown). These results indicate

0.1 0.2 03 0.4 0.5

1/Concentration (mM1)

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I I. Liang, R., Fei, Y. J., Prasad, P. D., Ramamoorthy, S., Han, H., Yang-Feng, T. L.,Hediger, M. A., Ganapathy, V., and Leibach, F. H. Human intestinal H@/peptidecotransporter. J. Biol. Chem., 270: 6456—6463, 1995.

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13. Miyamoto, K., Shiraga, T., Morita. K., Yamamoto, H., Haga, H., Taketani, Y., Tamai,I., Sal, Y., Tsuji, A., and Takeda, E. Sequence, tissue distribution and developmentalchange in rat intestinal oligopeptide transporter. Biochim. Biophys. Acta, 1305:34—38.1996.

14. Liu, W., Liang, R., Ramamoorthy, S., Fei, Y. J., Ganapathy, M. E., Hediger, M. A.,Ganapathy, V., and Leibach, F. H. Molecular cloning of PepT2, a new member of theH'ipeptide cotransporter family, from human kidney. Biochim. Biophys. Acta, 1235:461-466, 1995.

15. Saito, H., Terada, 1., Okuda, M., Sasaki, S., and Inui, K. Molecular cloning and tissuedistribution of rat peptide transporter. Biochim. Biophys. Acta, 1280: 173—177,1996.

16. Sal, Y., Tamai. I., Sumikawa, H., Hayashi, K., Nakanishi, T., Amano, 0., Numata,M., Iseki, S., and Tsuji, A. Immunolocalization and pharmacological relevance ofoligopeptide transporter PepTI in intestinal absorption of @-lactamantibiotics. FEBSLett., 392: 25—29,1996.

17. Suraiya, R., Walter, A., Nelson, R., Eva, M. T., Paul, A., and Murray, B. G.Characterization of a newly derived human sarcoma cell line (HT1O8O). Cancer(Phila.), 33: 1027—1033,1974.

18. Nichols, W. W., Murphy, D. G., Cristofald, V. J., Toji, L. H., Green, A. E., andDwight, S. A. Characterization of a new human diploid cell strain, IMR-90. Science(Washington DC), 196: 60—63, 1971.

19. Pinto. M., Robine, L. S., Appay, M. D., Kedinger, M., Triadou, N., Dussaulx, I.,Lacroix, B., Simon, A. P., Haffen, K., Fogh, J., and Zweibaum, A. Enterocyte-likedifferentiation and polarization of the human colon carcinoma cell line Caco-2 inculture. Biol. Cell, 47: 323-330, 1982.

20. Mullin, J. M., Weibel, J., Diamond, L., and Kleinzeller, A. Sugar transport in theLLC-PK1 renal epitheial cell line: similarity to mammalian kidney and the influenceof cell density. J. Cell. Physiol., 104: 375—389,1980.

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