Selecting agent hygromycin B alters expression of glucose-regulated genes in transfected Caco-2 cells

Annie Rodolosse1, Alain Barbat1, Isabelle Chantret1, Michel Lacasa1,2, Edith Brot-Laroche1, Alain Zweibaum1, and Monique Rousset1

1 Unité de Recherches sur la Différenciation Cellulaire Intestinale, Institut National de la Santé et de la Recherche Médicale, Unité 178, 94807 Villejuif Cedex; and 2 Université Pierre et Marie Curie, 75251 Paris Cedex 05, France

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Incorporation into plasmids of genes conferring resistance to aminoglycoside antibiotics such as hygromycin B is currently utilized for selection in experiments involving gene transfer in eukaryotic cells. Using a subclone of Caco-2 cells stably transfected with an episomal plasmid containing the hygromycin resistance gene, we observed that transformed cells subcultured in the presence of hygromycin B exhibit, compared with the same cells subcultured in antibiotic-free medium, a sixfold increase in the rates of glucose consumption and lactic acid production and dramatic changes, at mRNA and protein level, of the expressions of sucrase-isomaltase and hexose transporter GLUT-2, which are downregulated, contrasting with an upregulation of hexose transporter GLUT-1. This occurs without significant modifications of the differentiation status of the cells, as demonstrated by the normal expression of villin, ZO-1, dipeptidyl peptidase IV, or Na+-K+-ATPase. The plasmid copy number is, however, the same, whether or not the cells are cultured in the presence of hygromycin B. These results draw attention to the need to consider antibiotic-dependent alterations of metabolism and gene expression in transfection experiments.

glucose consumption; sucrase-isomaltase; GLUT-1; GLUT-2

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

AMINOGLYCOSIDE ANTIBIOTICS (10) are highly cytotoxic to eukaryotic cells. For this reason, incorporation into plasmids of genes that confer resistance to these antibiotics is extensively utilized for selection in experiments involving gene transfer in eukaryotic cells. Among these antibiotics, hygromycin B (1, 3) has been utilized for studying the regulation of a variety of transferred genes in several transfected cell types, including in the enterocyte-like cell line Caco-2. This cell line has been extensively utilized in the last decade as an in vitro model for human intestinal enterocytes since the first demonstration of its differentiation properties (21).

The purpose of the present report is to draw attention to the fact that the use of hygromycin B is not neutral, since, as in Caco-2 cells, it interferes per se with glucose utilization and expression of glucose-regulated genes.

Previous results obtained with Caco-2 cells have shown that all conditions of increased glucose consumption, whether obtained through pharmacological modulation as with forskolin (5, 14, 26), monensin (5), or inducers of cytochrome P-4501A1 (4) or by comparing clonal populations of cells selected on the basis of different rates of glucose consumption (6), result in marked modifications of the level of expression of a number of proteins involved in the uptake, transport or metabolism of glucose. High-glucose-consumption-associated modifications include, for example, a decreased expression of sucrase-isomaltase (SI) (4-6, 14, 26), hexose transporter GLUT-2 (4, 16), fructose 1,6- diphosphatase, and pyruvate kinase (24) and an increased expression of hexose transporters GLUT-1 and GLUT-3 (4, 16) and phosphoenolpyruvate carboxykinase (24), occurring without modifications of the morphological differentiation of the cells or of the expression of other differentiation-associated proteins such as villin or dipeptidyl peptidase IV (DPP-IV).

The results reported here were obtained during experiments conducted to define the elements of the promoter region of the SI gene through which glucose exerts its repressive transcriptional effect (25). For such studies, we have used clonal populations of the enterocyte-like Caco-2 cell line, which greatly differ in their glucose utilization and their level of SI expression (6). Among them, the TC7 clone exhibits, after confluency, low levels of glucose consumption and GLUT-1 mRNA (4, 16) and inversely expresses high levels of SI and GLUT-2 mRNA (4, 6, 16). We report here that, after transfection of TC7 cells with a plasmid containing the gene that confers resistance to hygromycin B and after selection of the stably transformed TC7 cells, the selecting agent hygromycin B induces by itself a marked increase in the rates of glucose consumption and lactic acid production and changes in the level of expression of SI, GLUT-1, and GLUT-2.

    MATERIALS AND METHODS
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Materials & Methods
Results
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References

Plasmid. The plasmid used in this study (Fig. 1) has been previously described (25). It contains the promoter-less luciferase gene inserted in the Nar I site of a derivative of plasmid p205-GTI (28). This plasmid contains the Epstein-Barr virus sequences for Ori-P and EBNA-1 that allow a stable and episomal maintenance of the vector in human cells (32) and the hygromycin resistance gene under control of the herpes simplex virus 1 thymidine kinase promoter.


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Fig. 1.   Structure of plasmid used for transfection experiments. EBNA-1 and Ori-P are Epstein-Barr virus sequences that allow a stable and episomal maintenance of vector in human cells.

Cell cultures, transfections, and selection of transformed cells. TC7 cells, a clonal population of the Caco-2 cell line (6), were routinely cultured in Dulbecco's modified Eagle's medium containing 25 mM glucose (Eurobio, Paris, France), 20% heat-inactivated (56°C, 30 min) fetal bovine serum (Boehringer, Mannheim, Germany), and 1% nonessential aminoacids (GIBCO-BRL, Glasgow, UK). Cultures were maintained in a 10% CO2-90% air atmosphere. For transfection experiments, 0.3 × 106 cells were seeded in 5 ml medium in 25-cm2 petri dishes (Corning Glassworks, Corning, NY). The medium was changed 2 days later, and transfections were performed 4-5 h later by using the CaPO4 procedure (11) and 20 µg of plasmid DNA. The medium was changed 15 h later and then daily. For the selection of transformed cells, hygromycin B (Sigma, St. Louis, MO) was added at confluency at a final concentration of 210 µg/ml, which was determined to be the minimal lethal concentration for the untransformed cells. Hygromycin-resistant cells were allowed to grow until subconfluent and were then subcultured for several passages in the presence of hygromycin. After several passages, the cells were either maintained in the presence of the drug or reversed to drug-free medium. When maintained in the presence of the drug, the cells were allowed to attach to the flask for 48 h without the drug, and then hygromycin B was added daily at a concentration increasing progressively from 40 to 210 µg/ml, depending on cell density. In all cases, the medium was changed 48 h after seeding and daily thereafter.

Plasmid extraction and DNA analysis of transformed cells. Extrachromosomal and genomic DNA were purified by a small-scale SDS lysis procedure, adapted from the method of Birnboim and Doly (2). DNA extracts were analyzed by slot blot and hybridization with a luciferase probe (a 759-bp EcoR I-Kpn I fragment from p205LUC) for quantification of the plasmid. Probe was 32P labeled, using a megaprime DNA labeling kit (Amersham). After 18 h of hybridization in the presence of 10% dextran sulfate (Pharmacia), the blots were washed twice in 2× standard sodium citrate (SSC)/0.1% SDS at 65°C for 30 min and then for 15 min in 0.1× SSC at 65°C.

RNA extraction and analysis. Total RNA was extracted by the guanidium isothiocyanate method (7). Samples of total RNA, denatured in 1 M glyoxal, were fractionated by electrophoresis through 1% agarose gels and transfered onto Hybond N (Amersham) (29). Prehybridization was performed at 42°C in the presence of 50% formamide, and hybridization was performed at 42°C in the presence of 40% formamide and 10% dextran sulfate. The blots were washed twice in 2× SSC/0.1% SDS for 10 min at room temperature, once in 0.1× SSC/0.1% SDS for 15 min at 50°C, and once in 0.1× SSC/0.1% SDS for 15 min at 65°C. The probes used were SI2 (12) for SI mRNA, DPI-101 (9) for DPP-IV mRNA, and cDNA V19 (22), which was obtained from D. Louvard (Institut Curie, Paris, France), for villin mRNA. The alpha 1-Na+-K+-ATPase mRNA was detected with a 3.6-kb cDNA, which was a generous gift from Dr. J. B. Lingrel (University of Cincinnati, Cincinnati, OH). The cDNA probes pGEM4Z-HepG2GT GLUT-1 (1.75-kb insert) and pBS-HTL210/hGLUT-2 (2.4-kb insert) were obtained from Dr. G. I. Bell (Howard Hughes Medical Institute, University of Chicago, Chicago, IL).

Electron microscopy. Transmission electron microscopy was performed on late postconfluent (day 20) cells grown in 25-cm2 plastic flasks. Samples embedded in Epon were reembedded to make sections perpendicular to the bottom of the flasks.

Immunofluorescence. Indirect immunofluorescence was performed on frozen cryostat sections of cell layer rolls, as already reported (6), after fixation in 3.5% paraformaldehyde in Ca2+/Mg2+-free PBS (15 min at room temperature). Mouse monoclonal antibodies HBB 2/614/88, 3/775/42 (15), and N1/123/33, specific for human SI, DPP-IV, and the alpha 1-subunit of the Na+-K+-ATPase, respectively, were obtained from Dr. H. P. Hauri (Biocenter of the University of Basel, Basel, Switzerland). Rabbit polyclonal antibodies against porcine villin (23) and the tight-junction protein ZO-1 (31) were obtained from Dr. D. Louvard (Institut Curie) and Dr. J. M. Anderson (Yale University, New Haven, CT) respectively. Rabbit polyclonal antibodies (L459) against SI from Caco-2 cells were produced in our laboratory (30). Rabbit polyclonal antibodies against GLUT-1 were from East Acres Biologicals (Southbridge, MA). Double immunofluorescence was performed, using as second antibodies anti-mouse and anti-rabbit fluorescein-coupled sheep antiglobulins (Institut Pasteur Productions, Marnes-la-Coquette, France) or rhodamine-coupled sheep antiglobulins (Boehringer).

Glycogen, glucose consumption, and lactic acid production assays. For glycogen assays, the cells were harvested 18 h after the medium changes for extraction and measurement with anthrone, as previously reported (6). Results are expressed as micrograms per 106 cells. Glucose consumption was determined every day, 16-18 h after the medium changes, using a Beckman glucose analyzer 2, and lactic acid production was determined, using a L-lactic acid measurement kit (Boehringer), as previously reported (4). Results are expressed as nanomoles per hour per 106 cells.

Sucrase and DDP-IV activity assays. Sucrase (EC 3.2.1.48) activity was measured according to Messer and Dahlqvist (17). DPP-IV (EC 3.4.14.5) activity was measured according to Nagatsu et al. (20), using 1.5 mM glycyl-L-proline-4-nitroanilide as substrate. Results are expressed as milliunits per milligram of protein. One unit is defined as the activity that hydrolyzes 1 µmol substrate/min at 37°C. Proteins were assayed with the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of hygromycin on rate of glucose consumption. The presence of hygromycin has no effect on the rate of cell division and growth curves of transformed cells (not shown), but it deeply affects the growth-related pattern of glucose consumption. As shown in Fig. 2, the pattern of evolution of the rate of glucose consumption from transformed TC7 cells grown in the absence of hygromycin is characterized, as already reported in untransformed TC7 cells (4, 6), by a rapid decrease starting at confluence (day 5) and reaching a plateau of low value at late confluence. By contrast, although decreasing after confluence, the rate of glucose consumption of transformed cells cultured in the presence of hygromycin is maintained, at late confluency, at a level that is sixfold higher than that observed in the cells cultured without the drug. The hygromycin-dependent elevated rate of glucose consumption is associated with an increased rate of lactic acid production and a lower glycogen content (see Table 1).


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Fig. 2.   Growth-related variations of glucose consumption in transformed TC7 cells cultured in the absence (first passage) (square ) or presence (black-square) of hygromycin. Results are means ± SD of 3 different cultures. Transformed cells were maintained for 3 passages in the presence of the drug before analysis. Analysis was done with cells from the same corresponding passages, either subcultured in the presence of the antibiotic or reversed for each single analyzed passage in hygromycin-free medium. Curves of glucose consumption from untransformed TC7 cells were undistinguishable from those of transformed cells cultured in the absence of hygromycin (not shown).

                              
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Table 1.   Reversibility of the effect of hygromycin on glucose utilization parameters

Effect of hygromycin on cell morphology. Morphology of late postconfluent (day 20) transformed TC7 cells grown with or without hygromycin is shown in Fig. 3. As shown on thin sections, the cells form a monolayer in both culture conditions. However, the cells cultured in the presence of hygromycin (Fig. 3b) appear enlarged, compared with the transformed cells cultured without the drug (Fig. 3a). At the ultrastructural level, the brush border, which is well organized in cells cultured without the drug (Fig. 3, c and e), appears disorganized in the cells cultured in the presence of hygromycin (Fig. 3, d and f).


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Fig. 3.   Effect of hygromycin on the morphology of postconfluent transformed TC7 cells. Cells from a same passage were analyzed after 20 days of culture in the absence (a, c, and e) or presence (b, d, and f) of the antibiotic. a and b: Thin section of the cell layer perpendicular to the bottom of the flask (bar, 17 µm); c and d: transmission electron microscopy of the same cell layers (bar, 2 µm); e and f: higher magnification of the apical brush border (bar, 1 µm).

Effect of hygromycin on expression of SI, GLUT-1, GLUT-2, alpha 1-Na+-K+-ATPase, DPP-IV, and villin. Because SI, GLUT-1, and GLUT-2 have been reported to show the highest variations in relation to the rate of glucose consumption (4, 6, 16), we chose to analyze the mRNA level of these three markers in differentiated postconfluent transformed cells (day 20). In hygromycin-treated cells, SI and GLUT-2 mRNA levels are considerably reduced, whereas GLUT-1 levels are considerably increased (Fig. 4). In contrast, no significant differences were observed between untreated and treated cells with regard to the levels of alpha 1-Na+-K+-ATPase, DPP-IV, and villin mRNAs (Fig. 4).


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Fig. 4.   Northern blot analysis of sucrase-isomaltase (SI), GLUT-1, GLUT-2, alpha 1-subunit of Na+-K+-ATPase, dipeptidyl peptidase IV (DPP-IV), and villin in transformed TC7 cells analyzed after 20 days of culture in the absence (-) or presence (+) of hygromycin. Same quantified amount of total RNA (20 µg) was loaded in each lane. Membrane was hybridized simultaneously with probes for SI and GLUT-1. After dehybridization, the same membrane was sequentially hybridized with the other probes. Similar results were obtained in cultures from 2 different passages.

Consistent with the results of mRNA, sucrase and DPP-IV activities measured at late postconfluence (day 20) are elevated in transformed cells cultured in the absence of hygromycin and similar to the values observed in untransformed cells (Fig. 5). In contrast, in transformed cells cultured in the presence of hygromycin, the activity of sucrase is decreased by ninefold, whereas DPP-IV activity is normal (Fig. 5).


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Fig. 5.   Activity of sucrase and DPP-IV in cell homogenates from postconfluent cultures (day 20) of control (open bars) and transformed TC7 cells cultured without (hatched bars) or under permanent exposure to (solid bars) hygromycin B. Results are the means of 3 different cultures.

Also consistent with the low levels of SI mRNA and sucrase activity in transformed cells cultured in the presence of hygromycin is the poor expression of the protein as detected by immunofluorescence (Fig. 6). Only a few cells show a discrete apical expression of SI (Fig. 6d), contrasting with the strong positivity of the entire apical domain of the cells cultured without hygromycin (Fig. 6b). Consistent with the high level of GLUT-1 mRNA, GLUT-1 is expressed in all hygromycin-treated cells (Fig. 6c), whereas, in transformed cells cultured in drug-free medium, only a few cells, grouped into clusters, express GLUT-1 (Fig. 6a). As already observed at the mRNA level, villin (Fig. 6, e and g), DPP-IV (Fig. 6, f and h), and alpha 1-Na+-K+-ATPase (Fig. 6, j and l) appear similarly expressed in both conditions, with this also being true for ZO-1 (Fig. 6, i and k).


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Fig. 6.   Double immunofluorescence staining of cryostat sections of the cell layers from postconfluent cultures (day 20) of transformed TC7 cells cultured without (a, b, e, f, i, and j) or under permanent exposure to hygromycin B (c, d, g, h, k, and l); a and c: GLUT-1; b and d: SI; e and g: villin; f and h: DPP-IV; i and k: ZO-1, j and l: Na+-K+-ATPase. Bar (l) = 40 µm.

Effect of hygromycin on plasmid copy number. The plasmid copy number was assessed during the culture of transformed TC7 cells grown in the presence or absence of hygromycin. During the course of these experiments, we found that the presence of the antibiotic was not necessary to maintain a similar amount of plasmid, as the same results were obtained with or without the drug in both dividing (day 4) and late postconfluent (day 20) cells (Fig. 7). It must be noted that, even in the absence of hygromycin, the amount of plasmid was found to be higher in postconfluent than in dividing cells.


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Fig. 7.   Slot-blot analysis of extrachromosomal DNA, as measured with the luciferase probe (see MATERIALS AND METHODS), in exponentially growing (day 4) and late postconfluent (day 20) transformed TC7 cells cultured in the absence (-) or presence (+) of hygromycin B.

Reversibility of effects of hygromycin B. Hygromycin was removed after 7 days of treatment, i.e., at confluency, and the cells were analyzed 7 days later for the parameters of glucose utilization. As shown in Table 1, the rates of glucose consumption and lactic acid production are lower than in cells maintained in hygromycin, although these rates remain still higher than in untreated cells. Also, glycogen content is increased but is still lower than in untreated cells. This is paralleled by an increased activity of sucrase that even after 7 days, remains, however, much lower than in cells cultured in the absence of drug from the beginning of the culture (Fig. 8).


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Fig. 8.   Reversibility of the effect of hygromycin B on the activity of sucrase in transformed TC7 cells cultured from the beginning of the culture in the absence (hatched bars) or presence of hygromycin B (solid bars) or switched from day 7 on from hygromycin B treatment to drug-free medium (open bars). Activity of sucrase was measured in the cell homogenates at the indicated days. Results are the means ± SD of 3 different cultures.

    DISCUSSION
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Materials & Methods
Results
Discussion
References

The present results show that permanent exposure to hygromycin B of stably transformed Caco-2 cells, selected for their resistance to this antibiotic, results, at late postconfluence (i.e., when the cells are fully differentiated), in a dramatic increase in the rate of glucose consumption and lactic acid production, a lower glycogen content, and marked changes, at both mRNA and protein levels, of the expression of SI and GLUT-2, which are downregulated, contrasting with an upregulation of GLUT-1. This occurs without apparent modifications of the expression of other differentiation-associated gene products, such as villin, DPP-IV, or the alpha 1-subunit of Na+-K+-ATPase. Although hygromycin B induces some degree of morphological changes, such as a partial disorganization of the brush border, it has no effect on the polarization of the cells, as substantiated by the apical expression of villin, DPP-IV, and the tight junction-associated protein ZO-1 or the basolateral expression of the Na+-K+-ATPase.

In view of our experience with Caco-2 cells, it is likely that the changes in the expression of SI, GLUT-2, and GLUT-1 are a consequence, at the transcriptional level, of the effect of the drug on glucose consumption, through a mechanism that remains to be elucidated.

Hygromycin B is a member of the aminoglycosides antibiotics, which have been extensively studied for the mechanisms leading to their bactericidal effect (for review, see Ref. 10). These antibiotics have been reported to induce membrane damage in bacteria, which results in a nonspecific increased permeability to small molecules such as anions, cations, and small uncharged molecules (10). To date, such an effect has not been documented in resistant transfected eukaryotic cells, selected and maintained in the presence of the selecting agent. It has been shown, however, that in normal kidney cells, i.e., not resistant transfected cells, the nephrotoxic effect of aminoglycoside antibiotics is associated with an impairment of the catabolism of phospholipids and a change in membrane permeability and membrane aggregation (18). More recently, it has been reported that aminoglycoside antibiotics could exert their cytotoxic effect through a persistent activation of phospholipase C (19). Whether alterations of the same metabolic pathways account for the changes in glucose utilization and glucose-related modifications of gene expression observed in our study remains to be investigated. Also questionable is whether other metabolic pathways or the expression of genes not analyzed here are also modified as an effect of the drug.

Whatever the answers to these questions, it is clear that the occurrence of changes in the metabolism of the cells induced by aminoglycoside-selecting agents during transfection experiments must be considered. This is particularly true for Caco-2 cells. Indeed, since the first observation of the differentiation properties of Caco-2 cells (21), a considerable number of studies have been conducted in these cells to analyze the transcriptional regulation of genes involved in the process of intestinal cell differentiation. Because the differentiation process occurs late in the culture, stably transformed cells obtained after selection and cloning have been utilized in a number of studies too numerous to be referenced here. When mentioned, the selecting agent was always present in the cultures of the stably transformed cells. In studies concerning the effect of the absence or overexpression of proteins potentially involved in the differentiation of these cells, SI, because it is exclusively expressed in the brush border of the intestine (13, 27), has been regularly taken as a control marker of the differentiation state of Caco-2 cells, as exemplified in Ref. 8. In these studies, metabolic properties and, more particularly, the rates of glucose consumption and lactic acid production of the transformed cells, were never reported. In view of the dramatic increase of glucose consumption in hygromycin-treated cells and the demonstrated repressive effect of glucose on the transcription of the SI gene (25), it is clear that not taking into account these parameters may lead to false interpretations.

This, of course, does not preclude the utilization of hygromycin B as a selection agent in transfection experiments. It requires, however, that, when the selection is completed, cells should be subcultured in the absence of the drug. In such drug-free conditions, the episomal plasmid is stably maintained in the transformed cells, as initially reported in other systems and cell types (32), and, as soon as the first passage, the normal metabolic status of the cells is restored. In contrast, when the drug is removed after confluence, the reversibility of the metabolic modifications and the restoration of gene expression, as exemplified by the increase in sucrase activity, are very slow. Whether these differences depend on the more rapid turnover of membrane constituents in dividing, compared with postconfluent cells, is questionable. Therefore, in culturing selected transformed cells in the absence of the selecting agent from the day of seeding and after, one should avoid misleading interpretation of results, such as assigning to the transfected cDNA or promoter an effect, which, in fact, could be a consequence of drug-induced modifications of the metabolic status of the cells.

    ACKNOWLEDGEMENTS

A. Rodolosse was supported by a fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche. This work was supported in part by grants from the Association pour la Recherche sur le Cancer (Grant no. 1393) and Université Paris XI.

    FOOTNOTES

Address for reprint requests: M. Rousset, INSERM U 178, 16 Ave. Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France.

Received 19 August 1997; accepted in final form 16 January 1998.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Gastroint Liver Physiol 274(5):G931-G938
0193-1857/98 $5.00 Copyright © 1998 the American Physiological Society




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