Digestion 1990;46(suppl 2):352—359

CD1990 S. Karger AG, Basel 0012-2823/90/0468-0352S2.75/0

Polyamine Uptake by Human Colon Carcinoma Cell Line CaCo-2 L. D'Agostinoa, S. Pignataa, B. Danielea, G. D ’A damo3, C. Ferraroa, G. Silvestrob, P. Tagliaferrib, A. Contegiacomob. R. Gentilec, G. Tritioa, /!./?. Biancob, G. Mazzacca3 aCattedra di Gastroenterologia, bCattedra di Oncología M edica,c Dipartimento di Biología e Patología Cellulare e Molecolare, 2a Facoltá di Medicina, Universitá di Napoli, Napoli, Italia

Key Words. Polyamines • Membrane uptake • Ornithine decarboxylase • a-Difluoromethylornithine • Small intestine

The polyamines putrescine, spermidine and spermine are ubiquitous highly charged cations [1]. Putrescine is synthesized from the aminoacid ornithine through its decar­ boxylation by ornithine decarboxylase

(ODC) and can be converted into spermi­ dine and spermine [1], The intracellular concentration of poly­ amines is highly regulated and high poly­ amine concentrations are associated with

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Abstract. The intracellular concentrations of the polyamines are highly regulated and high polyamine concentrations are associated with rapidly proliferating cells. Hormones, nu­ trients and growth factors that stimulate the proliferation of the intestinal epithelium, increase the intracellular polyamine concentration mainly by activating ODC expression. Other cell types stimulated to proliferate satisfy their requirement for polyamines by increas­ ing polyamine uptake. In the present study, we investigated polyamine uptake by a human colon carcinoma cell line, CaCo-2. Uptake of putrescine, spermidine and spermine by CaCo2 cells was saturable and temperature dependent and all polyamines appear to share a com­ mon carrier. The carrier of differentiated cells had an apparently higher affinity and lower activity than the carrier of replicating cells. Culture of CaCo-2 cells on porous filters showed that polyamine accumulation occurred mainly through the basolateral membrane in repli­ cating cells, while an increase in the rate of apical uptake was observed after differentiation. A significant increase in polyamine uptake and in ODC expression resulted from fresh medium replacement, a well-known stimulus to proliferation; no change in uptake occurred after ODC inhibition by DFMO. We conclude that CaCo-2 cells are able to increase their polyamine concentration by both enhanced synthesis and increased polyamine uptake.

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Polyaminc Uptake in CaCo-2 Cells

cells. Since in some epithelial cells, such as those involved in absorbing nutrients from the gut, carrier proteins are distributed asymmetrically in the plasma membrane, polyamine uptake was also studied in CaCo2 cells cultured on porous filters that allowed separate access to the basolateral and the apical sides of the cells.

Materials and Methods Chemicals The radioisotopes u C-putrescine, u C-spermine. MC-spermidine and l4C-ornithine were from Amersham, UK. Unlabeled putrescine, spermine and sper­ midine were from Sigma. All reagents for cell cultures were from Row Laboratories, UK. DFMO was a generous gift of Dr. P.S. Mamont, Merrell-Dow Research Institute (Strasbourg, France). Experimental Design Putrescine, spermine and spermidine uptake by actively replicating and differentiated CaCo-2 cells was assayed at the 6th and 14th day of culture, respec­ tively. The influence of temperature on polyamine uptake was evaluated by measuring the uptake at 0, 4, 20 and 37 °C. Polyamine uptake and ODC activity were also measured 3, 6, 8, 10 and 18 h after fresh medium replacement (MR) in cells at day 6 of culture; in these cells MR induced the maximal ODC activation [14], To assess if there is any correlation between ODC expression and polyamine uptake, some experiments were performed with medium containing 1 mM DFMO, a selective, irreversible ODC inhibitor. To verify if the accumulation of polyamines occurred through the basolateral or the apical membrane, we assessed the uptake in CaCo-2 cells maintained on porous filters at the 5th and 10th day of culture. Radiolabeled compounds were added to the medium facing either the basolateral or the apical membrane: the cells were then incubated for 120 min, and ra­ dioactivity was measured in cells and medium. The radioactivity found in the opposite side media was considered to be an expression of the amount of poly­ amines released from the cells.

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rapidly proliferating cells [2], The increase in intracellular concentration of polyamines depends on accelerated synthesis, regulated by ODC activity, or on polyamine uptake from the extracellular space [1, 3, 4], Al­ though the former appears to be the major regulatory mechanism of cellular polyamine concentrations, several cell types are able to accumulate polyamines through a diffusion process [3]. Hormones, nutrients and growth factors, that stimulate cells to proliferate, can induce both ODC activation and poly­ amine uptake [1, 4-7). The maintenance of the structure and function of the intestinal mucosa depends on a rapid cell turnover regulated by the com­ bined effects of luminal nutrients, hor­ mones, gastrointestinal secretions and growth factors [8], All these factors seem to act by regulating ODC expression and, ac­ cordingly, polyamine concentrations [8, 9], So far, there have been few investigations of polyamine uptake by intestinal cells: iso­ lated small bowel enterocytes [10, 11] and a colon cancer cell line [ 12] are able to accu­ mulate putrescine and spermidine by a car­ rier-mediated process. Polyamine uptake could be a mechanism of regulation of enterocytic proliferation and differentiation whose importance has been underesti­ mated. In the present study we investigated poly­ amine uptake by a human colon carcinoma cell line, CaCo-2, and the modifications in­ duced by inhibition of ODC activity by difluoromethylornithine (DFMO). Confluent CaCo-2 cells progressively differentiate in small intestinal enterocyte-like cells, express­ ing well structured microvilli and brush bor­ der hydrolases [13, 14], This experimental model allowed us to investigate polyamine uptake by proliferating and differentiated

354

Polyamine Uptake Assay Polyamine uptake was started by the addition of l4C-radiolabeled substrate to the culture medium at a 0.8-pA/ (0.1 pCi/ml) concentration. Initially uptake was measured over a time of 2 h (0, 15, 30, 45. 60, 90 and 120 min); high diffusion rates were reached after 60 min; thus, we decided to perform the experiments by assessing the uptake after 60 min. Unspecific uptake was measured in a parallel set of culture plates that were immediately placed on ice and kept there for 60 min. When porous filters were used, l4C-polyamines (0.8 pM; 0.1 pCi/ml me­ dium) were added separately to each side of the cell layer and radioactivity was measured in both cells and medium after 120 min. The longer incubation was necessary to observe if the absorbed polyamines were subsequently released. Transport was always terminated by the addition of cold compounds at a 5 mA/ concentration. After removal of the medium, cells were washed twice with ice-cold saline containing 7.5 IU/ml heparin in order to remove the compounds electrostatically linked to cell membranes and to the filters. The cells were then scraped by a rubber policeman and homogenised with an Ystral homogenizer in 0.1 M sodium-phosphate buffer, pH 7.2. Aliquots of the cell homogenates and medium were added to Insta-gel-containing vials, and radioac­ tivity was measured using a Packard Tricarb 4530

scintillation counter. Protein content was determined by the Lowry method [ 15], ODC was assayed as pre­ viously described [14]. Polyamine uptake was expressed as pmol/h/mg protein; ODC activity was expressed as nmol of putrescine released in 1 h at 37 ®C, pH 7.2.

Results Characteristics o f Polyamine Uptake After 6 days of culture, CaCo-2 cells growing in Petri dishes took up putrescine, spermine and spermidine added to the me­ dium at a concentration of 0.8 pM Figure 1 shows the relationship between incubation time and polyamine uptake. The diffusion was temperature dependent: at 20 °C the up­ take was about half as much as at 37 °C. At 4°C there was a further reduction of poly­ amine uptake, and only 8% of the overall diffusion was observed at 0 °C (fig. 2). The diffusion kinetics is shown in fig­ ure 3; the rate of diffusion of each polyamine reached a plateau at a 4-pM concentration. The Vmax and Km of uptake for each poly­ amine is shown in table 1. Differentiated cells express a higher affinity, lower activity carrier than proliferating cells. Polyamines inhibited each other’s up­ take. thus suggesting a common transport system; inhibition was complete when cold substrate was added to the culture medium at a 5 mM concentration. The 0.8 \iM con­ centration of the unlabeled substrate inhib­ ited about half the uptake (data not shown). To assess the importance of protein syn­ thesis on the expression of the carrier, we preincubated the cells for 2 h in culture me­ dium containing 50 pg/ml cycloheximide: no modification in polyamine uptake was recorded (data not reported in the figure).

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Cell Culture CaCo-2 cells were cultured in a humidified incu­ bator at 37 °C in 5% CCb-95% air and routinely grown in Eagle’s Minimum Essential Medium supple­ mented with 20% fetal calf serum, glutamine (20 mA/), non-essential amino acids (20 mA/), peni­ cillin (100 U/ml), and streptomycin (100 pg/ml), and buffered with N-2-hydroxy-ethylpiperazine-N'-2ethane sulfonic acid (20 mA/). The culture medium was changed every 3 days. CaCo-2 cells were seeded at 2 -105 cells/ml. Confluence was reached 6 days after the inoculum and the growth curve reached a plateau at the 12th day. Cells were also cultured on Millipore filters: cell suspensions were plated at 2 - 106 cells per 3 ml me­ dium inside presoaked 30-mm Millicell chambers HA 0.45 pm (Millipore Corporation, Bedford, Mass.) and placed in 6-well dishes. After 24 h the medium was removed, the cells were washed, and 1.5 ml of fresh culture medium was added to each side.

D'Agostino et al.

Polyamine Uptake in CaCo-2 Cells

355

Fig. 1. Effect of time on putrescine (PUT), spermidine (SPD) and spermine (SP) uptake by CaCo-2 cells. CaCo-2 cells at day 6 of cul­ ture were incubated with 0.8 \iM l4C-polyamines. Each point repre­ sents the mean of 3 observations.

Polyamine Uptake after Medium Replacement Figure 4 shows that MR increased poly­ amine transport by a factor of about four: maximal values were reached 8 h after MR. ODC activity increased after MR. reaching the peak value after 6 h (2.1 U/mg protein;

basal value = 0.2 U/mg protein). The com­ plete inhibition of ODC activity by 1 mM DFMO did not modify the rate of diffusion of the three polyamines. Data concerning ODC activity and polyamine uptake in DFMO-treated cells are not reported in the figure.

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Fig. 2. Temperature depen­ dence of putrescine (•), spermidine (o) and spermine (■) uptake. CaCo-2 cells at day 6 of culture were incubated in presence of 0.8 \iM substrate concentration. Each point represents the mean of 3 observations.

D'Agostino et al.

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Fig. 3. Effect of substrate con­ centration on putrescine (PUT), spermidine (SPD) and spermine (SP) uptake. CaCo-2 cells at day 6 (replicating cells) and 14 (differen­ tiated cells) were incubated with substrate concentrations ranged from 0.016 to 8 \iM. Each point represents the mean ± SD of 3 ob­ servations.

Putrescine Spermidine Spermine

Table 2. Basolateral (BL) vs. apical (A) uptake of putrescine, spermidine and spermine in replicating (day 5) and differentiated (day 10) CaCo-2 cells; results are expressed as percentages and refers to total cell uptake

Replicating cells

Differentiated cells

Vmax

Km

Vmax

Km

Putrescine

Spermidine

Spermine

0.70 0.45 0.11

0.75 0.91 0.45

0.15 0.07 0.03

0.41 0.30 0.12

BL

BL

BL

Km is expressed as \iM; Vmax is expressed as pmol/h/mg protein. Each point represents mean of 3 observations.

A

A

A

Day 5

87.3 12.7

77.8 22.2

66.6 33.4

Day 10

48.3 51.7

56.7 43.3

61.0 39.0

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Table 1. Vnlax and Km values for putrescine. sper­ midine and spermine uptake by CaCo-2 cells

Polyamine Uptake in CaCo-2 Cells

357

about 5% of spermidine and spermine was released into the basolateral medium (data not shown). Only negligible amounts of poly­ amines. diffused through the basolateral membrane, were released into the apical me­ dium by both replicating and differentiated cells.

Fig. 4. Effect of medium replacement on putrescine (PUT), spermidine (SPD) and spermine (SP) uptake by CaCo-2 cells. Polyamine uptake was calcu­ lated in cells at day 6 of culture, 3, 6, 8, 10 and 18 h after medium replacement. Each point represents the mean of 3 observations.

Polyamine Uptake in Cells Cultured on Porous Filters Table 2 shows the apical vs. basolateral uptake of 0.8-pAf polyamines in replicating (day 5) and differentiated (day 10) cells. Re­ plicating cells mainly accumulated poly­ amines from basolateral side, while in differ­ entiated cells an increase in the apical uptake was observed. Uptake of differentiated cells was about three-fold less than that of repli­ cating cells. Cells released some of the poly­ amines taken up through the apical side into the medium facing the basolateral mem­ brane: in replicating cells, 36% of the putrescine, 10.5% of the spermidine, and 5.8% of the spermine accumulated through the api­ cal membrane after 120 min was released into the basolateral medium, while in differ­ entiated cells 20% of the putrescine and

The results of this study demonstrate that there is a carrier-mediated diffusion of pu­ trescine, spermidine, and spermine into CaCo-2 cells. Uptake is saturable, is timeand temperature-dependent and is strongly enhanced by medium replacement. The in­ creased polyamine transport induced by me­ dium replacement did not depend on the lev­ els of intracellular ODC activity, since DFMO did not modify the rate of diffusion. CaCo-2 cells accumulated polyamines through both the apical and basolateral membranes. Polyamines are important factors in the regulation of normal and neoplastic growth [2], Intracellular polyamines are required for DNA synthesis, and polyamine depletion in­ duced by DFMO blocks DNA synthesis and cell division [1,3]. Hormones, nutrients and growth factors that stimulate the prolifera­ tion of intestinal epithelium increase the in­ tracellular polyamine concentration mainly by activating ODC expression [5-7, 16]. Other cell types stimulated to proliferate [3, 4, 17] satisfy their requirement for poly­ amines by increasing polyamine uptake. Re­ cent reports indicated that in isolated small intestinal cells a carrier-mediated diffusion is responsible for the uptake of putrescine and spermidine [10, 11], Similarly. LoVo colon cancer cells were able to accumulate

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Discussion

putrescine by a carrier-mediated process [12]. As experimental model we used the human colon carcinoma cell line CaCo-2; such cells, once confluent, spontaneously ex­ hibit features of small-bowel enterocytes [ 13, 14], CaCo-2 cells are also polarized cells con­ nected by tight junctions, forming domes and apical microvilli [13, 18, 19]. We found that CaCo-2 cells accumulated putrescine, spermine, and spermidine; up­ take showed saturation kinetics and was temperature dependent; all polyamines ap­ pear to share a common carrier since poly­ amines inhibited each other’s uptake. These findings suggest a carrier-mediated diffusion and are consistent with the results of other studies [10, 11], The carrier of differentiated cells had an apparent higher affinity and lower activity than the carrier of replicating cells. However, the presence of tight junc­ tions between differentiated cells may pre­ vent polyamines to reach the carriers located on the basolateral membrane. Therefore, re­ sults could not necessarily indicate a higher rate of uptake in rapidly growing cells. To probe this hypothesis, polyamine uptake was also investigated in both replicating and dif­ ferentiated cells cultured on porous filters. This system allowed us separate access to the basolateral and apical membrane. Moreover, in these experimental conditions polyamine uptake was about three times higher in repli­ cating cells than in differentiated cells. Replicating CaCo-2 cells accumulated polyamines mainly from the basolateral membrane. In differentiated cells, the in­ crease in polyamine uptake from the apical side is probably due to their increased func­ tional and structural maturity. In replicating cells, medium replacement increased both polyamine uptake and ODC expression; peak values were reached after

D’Agostino et al.

6-8 h, when diffusion was about four times higher than basal values. This finding indi­ cates that fresh medium replacement, which stimulates cells to proliferate, can increase the intracellular polyamine concentration by both enhancing the synthesis (increasing ODC expression) and increasing the up­ take. The observation that the maximal in­ creases in polyamine transport and in ODC activity, induced by MR, overlapped, en­ couraged us to inhibit ODC activity by add­ ing DFMO to the medium. In doing so, our aim was to investigate the possible relation­ ship between endogenous synthesis of poly­ amines and external uptake: polyamine up­ take after MR did not depend on cellular ODC activity. However, our results do not exclude the possibility that in cells cultured continuously in presence of DFMO poly­ amine depletion can induce an increase in polyamine uptake [17], In fact, the experi­ ment was exclusively designed to identify the mechanisms involved in the short-term regulation of polyamine uptake after MR. It is also possible that, in a condition of nonmaximal rate of polyamine uptake (using lower concentrations of fetal calf serum in the medium), DFMO can incresae poly­ amine uptake. In conclusion, these data indicate that CaCo-2 cells take up putrescine, spermine, and spermidine by carrier-mediated diffu­ sion. Polyamine accumulation occurs main­ ly through the basolateral membrane in re­ plicating cells, while an increase in the rate of apical uptake is observed after differentia­ tion. A significant increase in polyamine up­ take results from fresh medium replacement, a well known proliferative stimulus; no mod­ ification occurs after ODC inhibition by DFMO. We believe that CaCo-2 cells, due to

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Polyamine Uptake in CaCo-2 Cells

Acknowledgements The authors wish to thank Mr. Pasquale Esposito for technical assistance. This work has been partially supported by AIRC (Associazione Italiana per la Ricerca sul Cancro). Dr. Sandro Pignata is supported by a fellowship from AIRC. Dr. Bruno Daniele is sup­ ported by a fellowship from Dottorato di Ricerca in Scienze Gastroenterologiche, Université di Roma ‘La Sapienza’.

References 1 Tabor CW, Tabor H: Polyamines. Ann Rev Biochem 1984;53:749-790. 2 Janne J, Poso H, Raina A: Polyamines in rapid growth and cancer. Biochim Biophys Acta 1978; 473:241-293. 3 Pegg AE: Polyamine metabolism and its impor­ tance in neoplastic growth and as a target of che­ motherapy. Cancer Res 1988:48:759-774. 4 Pohyanpelto P: Putrescine transport is greatly in­ creased in human fibroblasts initiated to prolifer­ ate. J Cell Biol 1976;68:512-520. 5 Tabata K, Johnson LR: Ornithine decarboxylase and mucosal growth in response to feeding. Am J Physiol 1986;251:G270-G274. 6 Tabata K, Johnson LR: Mechanism of induction of mucosal ornithine decarboxylase by food. Am J Physiol 1986;251:G370-G374. 7 Feldman EJ, Aures D. Grossman Ml: Epidermal growth factor stimulates ornithine decarboxylase activity in the digestive tract of the mouse. Proc Soc Exp Biol Med 1978;159:400-402. 8 Dowling RH, Hosomi M. Stace NH, et al: Hor­ mones and polyamines in intestinal and pan­ creatic adaptation. Scand J Gastroenterol 1985; 20(suppl 112): 84-9 5.

9 Luk GD, Yang P: Distribution of polyamines and their biosynthetic enzymes in intestinal adapta­ tion. Am J Physiol 1988;254:G194-G200. 10 Kumagai J, Johnson LR: Characteristics of putrescire uptake in isolated rat enterocytes. Am J Physiol 1988;17:G81-G86. 11 Kumagai J, Rajeev J, Johnson LR: Characteristics of spermidine uptake by isolated rat enterocytes. Am J Physiol 1989:19:G905-G910. 12 Me Cormack S, Johnson LR: Putrescine uptake and release by colon cancer cells. Am J Physiol 1989;19:G868-G877. 13 Pinto M. Robine-Leon S, Appay MD, et al: Enterocyte-like differentiation and polarization of the human colon carcinoma cell line CaCo-2 in culture. Biol Cell 1983;47:323-330. 14 D'Agostino L, Daniele B, Pignata S, et al: Orni­ thine decarboxylase and diamine oxidase in hu­ man colon carcinoma cell line CaCo-2 in culture. Gastroenterology 1989:97:888-894. 15 Lowry OH, Rosebrough NJ, Farr AL. Randall RJ: Protein measurement with the Folin phenol re­ agent. J Biol Chem 1951;193:265-275. 16 D’Agostino L, Daniele B, Pignata S, Barone MV, D’Argenio G, Mazzacca G: Modifications in orni­ thine decarboxylase and diamine oxidase in small bowel mucosa of starved and refed rats. Gut 1987; 28(suppl 1): 135-138. 17 Kaninuma Y, Hoshino K, Igarashi K: Character­ ization of the inducible polyamine transporter in bovine lymphocytes. Eur J Biochem 1988; 176: 409-414. 18 Grasset E, Pinto M. Dussaulx E, Zweibaum A. Desjeux JF: Epithelial properties of human co­ lonic carcinoma cell line CaCo-2: electrical pa­ rameters. Am J Physiol 1984;247:C260-C267. 19 Hidalgo IJ, Raub TJ, Borchardt RT: Characteriza­ tion of the human colon carcinoma cell line (CaCo-2) as a model system for intestinal epithe­ lial permeability. Gastroenterology 1989;96:736— 749.

Dr. Luciano D’Agostino Cattedra di Gastroenterologia 2a Facoltà di Medicina Université di Napoli Via Sergio Pansini 5 1-80131 Napoli (Italy)

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their structural and functional analogies with small bowel enterocytes, represent a valuable experimental model to study the complex mechanisms regulated by nutrients and trophic factors and understand poly­ amine function in small bowel epithelial cells.

359

Polyamine uptake by human colon carcinoma cell line CaCo-2.

The intracellular concentrations of the polyamines are highly regulated and high polyamine concentrations are associated with rapidly proliferating ce...
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