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Concomitant Changes in Polyamine Pools and DNA Methylation during Growth Inhibition of Human Colonic Cancer Cells
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
243, 319 –325 (1998)
EX984148
Concomitant Changes in Polyamine Pools and DNA Methylation during Growth Inhibition of Human Colonic Cancer Cells Benoıˆt Duranton,* Ge´rard Keith,† Francine Gosse´,* Christian Bergmann,* Rene´ Schleiffer,* and Francis Raul*,1 *CJF INSERM 95-09 et Laboratoire du Controˆle Me´tabolique et Nutritionnel en Oncologie Digestive de l’ULP, IRCAD, 1 Place de l’Hoˆpital, 67091 Strasbourg, France; and †UPR 9002 CNRS, IBMC, 15 rue Descartes, 67084 Strasbourg, France
The effects of CGP 48664 and DFMO, selective inhibitors of the key enzymes of polyamine biosynthesis, namely, of S-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), were investigated on growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. Both inhibitors caused growth inhibition and affected similarly the initial expression of the differentiation marker sucrase. In the presence of the AdoMetDC inhibitor, ODC activity and the intracellular pool of putrescine were enhanced, whereas the spermidine and spermine pools were decreased. In the presence of the ODC inhibitor, the AdoMetDC activity was enhanced and the intracellular pools of putrescine and spermidine were decreased. With both compounds, the degree of global DNA methylation was increased. Spermine and spermidine (but not putrescine) selectively inhibited cytosine–DNA methyltransferase activity. Our observations suggest that spermidine (and to a lesser extent spermine) controls DNA methylation and may represent a crucial step in the regulation of Caco-2 cell growth and differentiation. © 1998 Academic Press Key Words: spermidine; sucrase; ornithine decarboxylase; S-adenosylmethionine decarboxylase; cytosine– DNA methyltransferase.
INTRODUCTION
The polyamines (putrescine, spermidine, and spermine) are ubiquitous, small polycations that are essential for cell growth and differentiation [1, 2]. The two key enzymes involved in polyamine biosynthesis are ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) [2, 3]. ODC catalyzes the formation of putrescine from L-ornithine, and AdoMetDC 1 To whom correspondence and reprint requests should be addressed at CJF INSERM 9509, IRCAD, 1, place de l’hoˆpital, 67091 Strasbourg Cedex, France. Fax: (33)388119097. E-mail: Francis.raul@ ircad.u-strasbg.fr.
decarboxylates S-adenosylmethionine (AdoMet). The product of this reaction, decarboxylated S-adenosylmethionine (dcAdoMet), is the aminopropyl group donor for spermidine and spermine synthesis. AdoMet is a common substrate for numerous methylation reactions including DNA methylation and for polyamine biosynthesis. Therefore, it can be hypothesized that changes in cellular polyamine metabolism may affect the degree of DNA methylation and consequently modify gene expression [4]. Putrescine and spermidine levels and their biosynthetic decarboxylases are generally elevated in cancer cells and in rapidly growing tissues, compared to normal or quiescent cells [5, 6]. In order to assess further the role of polyamine metabolism on the regulation of cellular growth and differentiation, we investigated the changes in polyamine biosynthesis and DNA methylation of the human colon carcinoma cell line Caco-2. This cell line spontaneously undergoes structural and functional enterocytic differentiation at late confluency; phenotypic changes after confluency include formation of brush border membranes and expression of intestinal hydrolases which are markers of functional differentiation normally found in human fetal colon [7–9]. In the present study we examined the effects of a potent inhibitor of AdoMetDC activity, 4-amidinoindan-1-one 29-amidinohydrazone (CGP 48664) [10], and of 2-(difluoromethyl)ornithine (DFMO), an irreversible inhibitor of ODC [11], on cell growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. MATERIALS AND METHODS Cell culture. Caco-2 cells, obtained from the European Collection of Animal Cell Culture (CERDIC, Sophia-Antipolis, France), were cultured in 75-cm2 Falcon flasks containing Dulbecco’s modified Eagle’s medium (DMEM) at 25 mM glucose supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids, 100 U/ml penicillin, and 100 mg/ml streptomycin. The cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and subcultured to preconfluency after trypsinization (0.5% trypsin/2.6 mM EDTA). They were harvested between passage 30 and 45. In all experiments, the cells were seeded at 6 3 105 cells on culture dishes (100 mm in diameter). The culture medium was composed of DMEM
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0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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supplemented with 3% FBS, transferrin (5 mg/ml), selenium (5 ng/ ml), and insulin (10 mg/ml) (TSI-defined medium; Gibco BRL, Life Technologies SARL, France). When needed, the AdoMetDC inhibitor CGP 48664 (Novartis, Basel, Switzerland) was added to the culture medium 48 h after seeding at the LD50 concentration (10 mM) which was determined by the Alamar Blue assay (Interchim, Montluc¸on, France). In another experimental series, the ODC inhibitor, DFMO (Marion Merrell Dow Research Institute, Strasbourg, France), was added at 100 mM to the culture medium 48 h after seeding. In all experimental settings the culture medium was replaced every 48 h and either the AdoMetDC or the ODC inhibitor were freshly added. Cells were harvested after various times, washed three times with PBS (pH 7.2), and kept frozen at 270°C until assays were performed. Brush border sucrase. Cells were homogenized in 4 ml of Tris– mannitol buffer (50 mM mannitol, 2 mM Tris, pH 7.1). Brush border membranes were isolated as described by Schmitz et al. [12]. Sucrase activity was determined according to Messer and Dahlqvist [13]. The enzyme activity was expressed as specific activity (milliunits per milligram of brush border protein). One unit of activity corresponds to one mmol of product formed per minute at 37°C. ODC and SAMDC activities. Cells were homogenized in 100 mM Tris–HCl buffer, pH 7.4 (1 mM EDTA, 1 mM dithiothreitol, 0.5 mM leupeptin, 0.5 mM phenylmethylsulfonyl fluoride). After centrifugation at 33,000g for 25 min at 4°C, the supernatants were collected and ODC and AdoMetDC assays were performed. ODC activity was evaluated by measuring the rate of 14CO2 formation from [1-14C]Lornithine (55 mCi/mmol, Amersham, UK) [14] and AdoMetDC activity was evaluated by measuring the rate of 14CO2 formed from [1-14C]S-adenosylmethionine (60 mCi/mmol, Amersham) [15]. Polyamine determinations. Cells were homogenized in perchloric acid (200 mM), and the homogenates were centrifuged at 3000g for 10 min after standing for 16 h at 12°C. The clear supernatants were diluted with perchloric acid (200 mM) and 200-ml aliquots were applied on a reversed-phase column for separation. The polyamines were determined by separation of the ion pairs formed with n-octanesulfonic acid, reaction of the column effluent with o-phthaldialdehyde/2-mercaptoethanol reagent, and monitoring of fluorescence intensity [16]. Measurements of 5-methyldeoxycytidine (m5C) in DNA samples. Cellular DNA was purified using the Wizard genomic DNA purification kit (Promega Corp., Madison, WI) and m5C was determined by the method of Wilson et al. [17] modified as follows: DNA (10 mg) was digested at 37°C for 3 h, into 39-deoxymonophosphate nucleosides, using micrococcal nuclease (5–10 mg) and spleen phosphodiesterase (1–2 mg) in 10 mM CaCl2 and 20 mM sodium succinate, pH 6.0, buffer. Aliquots of the digest containing the 39-deoxymonophosphate nucleosides were converted in presence of [g-32P]ATP and T4 polynucleotide kinase into 32P-labeled nucleoside 39,59-biphosphate nucleosides (the 32P being attached to the free 59 hydroxyl group). The biphosphate nucleosides were further hydrolyzed into [32P]59-monophosphate deoxynucleosides using nuclease P1 [18]. Finally, the labeled mononucleotides were separated by two-dimensional thinlayer chromatography on cellulose plates (Fig. 1). Quantification of individual nucleotides was carried out by scraping off the spots detected by autoradiography and counting of their radioactivity. The m5C content of DNA was calculated from the radioactivity found in m5dCMP and dCMP by the equation
FIG. 1. Autoradiogram of a 32P-labeled DNA digest of Caco-2 cells on two-dimensional thin-layer cellulose plates. Development in A (first dimension), isobutyric acid/concentrated ammonia/H2O (66/ 1/33, v/v/v); B (second dimension), 0.1 M sodium phosphate pH 6.8/ ammonium sulfate/n-propanol (100/60/2, v/w/v). The four faint spots shown by small arrows correspond clockwise to the five ribonucleotides pG, pA, pC, and pU from contaminating RNA in some DNA samples.
100 mg/ml RNase A). The cell lysate (corresponding to 5 mg protein) was made up in 15 ml of the same buffer and mixed with 0.5 mg of poly(d(I-C).d(I-C)) (synthetic DNA consisting of repeats of inosine– cytosine) (Pharmacia Inc., France) and 1.5 mCi S-adenosyl-L-[methyl3 H]methionine (80 Ci/mmol, Amersham) in a total volume of 23 ml. The mixture was incubated at 37°C for 2 h. The reaction was stopped by adding 300 ml of a solution containing 1% sodium dodecyl sulfate, 2 mM EDTA, 3% 4-aminosalicylate, 5% butanol, 125 mM sodium chloride, 0.25 mg/ml salmon testis DNA, and 1 mg/ml proteinase K. After an additional 30 min of incubation at 37°C, DNA was purified by phenol/chloroform extraction and ethanol precipitation. The precipitate was resuspended in 0.3 M sodium hydroxide and incubated at 37°C for 35 min. This solution was spotted on Whatman 3MM filter paper strips, washed in ice-cold 10% trichloroacetic acid (TCA) for 10 min, 5% TCA for 5 min followed by 70% ethanol. The dried filters were placed in 5 ml of scintillation fluid and counted in a scintillation counter. For exclusion of background protein and RNA methylation, all samples were assayed with a negative control containing the whole cell lysate but no poly(d(I-C).d(I-C)). Statistical analysis. Statistical significance between treated and control cells was assessed by one-way analysis of variance (ANOVA) plus comparison of means. Differences were considered significant at P , 0.05.
% m5dCMP 5 ~m5dCMP 3 100!/~m5dCMP 1 dCMP!.
RESULTS Cytosine–DNA methyltransferase (DNA–MTase) assay. We adapted a microassay technique for DNA–MTase activity [19]. Cells were harvested after 4 days of culture and pelleted. Lysis was achieved by freeze–thawing the cells four times in 50 mM Tris–HCl buffer, pH 7.8 (1 mM EDTA, 1 mM dithiothreitol, 0.01% sodium azide, 6 mg/100 ml phenylmethylsulfonyl fluoride, 10% glycerol, 1% Tween 80, and
Effects of SAMDC and ODC Inhibitors on Caco-2 Cell Growth and Differentiation Growth and differentiation of Caco-2 cells grown in the absence or presence of the AdoMetDC inhibitor CGP
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POLYAMINE POOLS AND DNA METHYLATION IN Caco-2 CELLS
48664, and the ODC inhibitor DFMO, were compared. In the absence of inhibitors, cells reached confluency at day 5 of culture and a plateau at day 9 (Fig. 2), indicating that the cells have entered a stationary phase of growth. When cells were grown 48 h after plating in the presence of 10 mM of the AdoMetDC inhibitor, growth was inhibited by more than 80% and in the presence of 100 mM of the ODC inhibitor a 70% growth inhibition was observed (Fig. 2). Caco-2 cells were also assayed for sucrase activity, which is a characteristic marker of the brush border villi of enterocytes and is indicative of differentiated Caco-2 cells [20]. As shown in Table 1, sucrase activity appeared at confluency and increased progressively reaching highest level at day 15 during the stationary phase. This shows that the cells progressively acquire, after confluency, characteristics of mature enterocytes. In the presence of either the AdoMetDC or the ODC inhibitor, sucrase activity appeared as in control cells, at day 5 of culture despite the fact that in the presence of inhibitors cells did not reached confluency. However, the maximum level of sucrase activity in presence of the inhibitors remained low and reached in CGP 48664-treated cells at day 15 a level corresponding to 10% of control activity (Table 1). In DFMO-treated cells, confluency was reached by day 13 (4 days later than in controls) and the level of sucrase activity remained significantly lower (75%) compared with controls (Table 1). Changes in Polyamine Metabolism during Caco-2 Cell Growth In control Caco-2 cells the activities of ODC and AdoMetDC decreased after confluency, i.e., during the
FIG. 2. Caco-2 cell growth curves. Cells were seeded at 6 3 105 cells on culture dishes (100 mm in diameter) in DMEM medium supplemented with 3% FBS, transferrin (5 mg/ml), selenium (5 ng/ ml), and insulin (10 mg/ml). In the absence (open square) or in the presence of DFMO (100 mM) (closed triangle) or of CGP 48664 (10 mM) (closed square). The inhibitors were added 48 h after seeding, the culture medium was replaced every 48 h, and the inhibitor was freshly added. Values represent means 6 SEM (n 5 4).
TABLE 1 Sucrase Specific Activity in Brush Border Membranes Isolated from Caco-2 Cells Grown in the Absence or Presence of CGP 48664 (10 mM) or DFMO (100 mM) Sucrase activity (mU/mg protein) Days of culture 3 5 9 15
Control ND 0.9 6 0.1a 15 6 0.9b 66 6 11c
CGP48664
DFMO
ND 1.2 6 0.3a 2.2 6 0.2b* 5.6 6 0.6c*
ND 1.0 6 0.1a 3.0 6 0.6b* 17 6 0.9c*
Note. Results are means 6 SEM of three independent experiments performed in triplicate. ND, activity not detected. For each column, a Þ b Þ c, P , 0.05. Control versus CGP 48664 or DFMO, *P , 0.05.
stationary phase, when Caco-2 cells are differentiating (Table 2). The intracellular pools of putrescine and spermidine decreased significantly in parallel to the changes observed for the enzyme activities, reaching their lowest values at day 15. The pool of spermine did not change significantly (Table 3). In Caco-2 cells treated with CGP 48664, ODC activity was 13-fold higher at day 3 of culture and 140-fold higher than in control cells at day 15 (Table 2). In cells treated with DFMO, ODC activity decreased by about 70% during the initial phase of growth (day 3) and remained practically unchanged thereafter. It was 5-fold higher than in controls by day 15 (Table 2). The inhibitory effect of DFMO was evident from the absence of detectable amounts of putrescine (Table 3). Exposure of the cells to DFMO led to a progressive elevation of AdoMetDC activity: a 3-fold increase was observed at day 3 and a 60-fold increase was reached at day 15 when compared to Caco-2 cells grown in the absence of the inhibitor (Table 2). The DFMO-triggered increase of AdoMetDC activity explaines the drop of the spermidine pool (Table 3). DFMO treatment led to a huge elevation in AdoMetDC activity (Table 2). In cells treated with CGP 48664, AdoMetDC activity, as determined in vitro, exhibited first a rapid and significant decrease at day 3 of culture but recovered progressively, reaching even higher values than in control cells at day 5 and 15. This is most probably due to the fact that CGP 48664 is a competitive inhibitor, so that active enzyme may accumulate in the cells which is not inhibited completely under the assay conditions due to dilution. Nevertheless, as shown in Table 3, the intracellular AdoMetDC activity was obviously inhibited by CGP 48664, as is seen from the significant decrease of the intracellular pools of spermidine and spermine. At the same time, putrescine accumulated. By day 5 of culture, cells grown in presence of the AdoMetDC inhibitor exhibited a 100-fold accumulation of putrescine and a decrease of spermidine and spermine contents by 30 and 85%,
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TABLE 2 Ornithine Decarboxylase and S-Adenosylmethionine Decarboxylase Activities in Caco-2 Cells Maintained in Culture in the Absence and Presence of CGP 48664 (10 mM) or DFMO (100 mM) ODC activity (pmol/mg protein/h) Days of culture 3 5 15
SAMDC activity (pmol/mg protein/h)
Control
CGP48664
DFMO
Control
79 6 2a 48 6 8b 5 6 0.6b
996 6 10a* 360 6 19b* 723 6 42c*
22 6 2a* 35 6 4b 29 6 2a,b*
28 6 2a 24 6 0.3a 10 6 0.7b
CGP48664
DFMO
12 6 1a* 32 6 1b* 32 6 0.6b*
76 6 8a* 733 6 43b* 683 6 142b*
Note. Results are means 6 SEM of three independent experiments performed in triplicate. For each column, a Þ b Þ c, P , 0.05, and control versus CGP 48664 or DFMO, *P , 0.05
respectively. The observed changes of the polyamine pools are directly related to the induction of ODC activity, and presumably the reduced availability of dcAdoMet, the aminopropyl donor for spermidine and spermine synthesis. Effects on the DNA Methylation Status As is illustrated in Fig. 3, the amount of m5C in the DNA increased significantly (by 20%) when Caco-2 cells reached stationary phase by 15 days of culture. Growth inhibition by the AdoMetDC inhibitor caused a significant elevation in the m5C content of DNA when compared to nontreated control Caco-2 cells after 3 and 5 days, the proliferation phase of these cells. As shown in Fig. 3, the stimulation of AdoMetDC activity in cells treated with DFMO also led to a similar significant increase of DNA methylation. This result indicates that in Caco-2 cells the activity of AdoMetDC may not be an important factor in DNA methylation. In order to assess whether the changes in the intracellular polyamines affect DNA methylation, we determined the effects of the various polyamines on the cytosine–DNA methyltransferase activity. As shown in Fig. 4, spermidine and spermine but not putrescine inhibited the cytosine–DNA methyltransferase activity of Caco-2 cell extracts obtained from cells during the proliferative phase. Spermine (80 mM) was most effi-
cient and inhibited the enzyme activity by 70%. Spermidine at the same concentration caused a 50% inhibition of the enzyme. Enzyme activity was already significantly (P , 0.05) reduced with lower concentrations (20 and 40 mM) of spermine and spermidine (results not shown). Putrescine was ineffective even at 100 mM (Fig. 4). DISCUSSION
Our results confirm that polyamine biosynthesis is markedly reduced in Caco-2 cells after confluency presumably due to the low ODC activity [21–23]. In addition, AdoMetDC exhibited also reduced activity at this stage as shown in this study. Similar changes in polyamine metabolism and DNA methylation can be observed in differentiated Caco-2 cells and in cells after treatment with CGP 48664 or with DFMO: Under all these conditions polyamine biosynthesis is significantly diminished, the degree of global DNA methylation is enhanced and the differentiation marker sucrase is expressed. Sucrase expression was initiated by inhibiting the growth of Caco-2 cells during the proliferation phase by reducing the intracellular pools of spermidine and spermine. In contrast, control cells expressed sucrase only when cells reached confluency. CGP 48664 and DFMO, which have cytostatic effects
TABLE 3 Intracellular Polyamine Content (pmol/mg protein) of Caco-2 Cells in the Absence and in Presence of CGP 48664 (10 mM) or DFMO (100 mM) Putrescine
Spermidine
Days of culture
Control
CGP48664
DFMO
3 5 15
277 6 23a 133 6 16b 73 6 3c
7487 6 903a* 13793 6 1851b* 16313 6 1061b*
ND ND ND
Control
CGP48664
Spermine DFMO
Control
CGP48664
DFMO
3993 6 254a 4050 6 386a 1707 6 50a* 7430 6 254a 2213 6 432a* 6947 6 424a 3520 6 202a 2503 6 240b* 163 6 7b* 8090 6 135a,b 1180 6 136b* 5177 6 243b* 2525 6 454b 1620 6 72c 97 6 8c* 7200 6 250a,c 570 6 92b* 4743 6 147b*
Note. Results are means 6 SEM of three independent experiments performed in triplicate. ND, activity not detected. For each column, a Þ b Þ c, P , 0.05, and control versus CGP 48664 or DFMO, *P , 0.05.
POLYAMINE POOLS AND DNA METHYLATION IN Caco-2 CELLS
FIG. 3. Changes in DNA methylation of Caco-2 cells grown in the absence (open columns) or presence of CGP 48664 (hatched columns) or of DFMO (dotted columns). DNA samples were digested enzymatically to nucleoside monophosphates which were converted to 32Plabeled nucleosides and separated by two-dimensional thin-layer chromatography (see legend to Fig. 1). The percentage of m5C present in DNA was calculated from the radioactivity found in m5dCMP and dCMP as described under Materials and Methods. Values represent means 6 SEM (n 5 6). Columns not sharing a common superscript letter differ significantly, a Þ b, P , 0.05.
on Caco-2 cells, seem to favor only the early initial step of sucrase expression as is presumed from the fact that maximum sucrase activities were lower in the cells treated with the inhibitors than in confluent cells. This indicates that regulatory mechanisms are triggered through cell to cell interactions at confluency which are necessary for optimal expression of the differentiation marker. This may explain the higher level of sucrase expression observed in the presence of DFMO at the time when cells reached confluency. Cytostatic effects of CGP 48664 have previously been described for several mammalian cell lines [24]. In contrast with a previous report on Caco-2 cells [25], the present study shows that cell growth seemed not supported by ODC and the huge putrescine pool, since growth inhibition by CGP 48664 occurred in the presence of high intracellular putrescine concentrations and a high ODC activity. These data are in accordance with studies suggesting that overexpression of ODC alone is not sufficient to favor cell proliferation [26]. The compensatory increase in ODC activity observed with CGP 48664 treatment is most probably related to the feedback control of this enzyme by polyamines [27]. Likewise, AdoMetDC is induced in the CGP 48664-treated cells, as has been shown in L1210 cells [10]. This is confirmed by the high AdoMetDC activity as measured in vitro in extracts of Caco-2 cells and by the enhanced amount of the AdoMetDC mRNA in the CGP 48664 treated cells (results not shown). Similarly, the ODC activity measured in vitro when cells were treated with DFMO may be explained by the accumulation of active ODC molecules due to the rel-
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atively low concentration of the ODC inhibitor used in the present study. Both DFMO and CGP 48664 exhibited similar effects on Caco-2 cell growth and differentiation, despite completely different mechanisms of action. The AdoMetDC inhibitor caused the accumulation of putrescine, and increased ODC activity, the depletion of spermine and of spermidine, and presumably a decreased concentration of dcAdoMet. In contrast, the ODC inhibitor provokes an increase in AdoMetDC activity, the depletion of putrescine and spermidine concentrations, and as is shown from the literature [28, 29] the enhanced formation of dcAdoMet. These results are in contrast with those obtained in F9 teratocarcinoma stem cells where the inhibition of ODC by DFMO but not the inhibition of AdoMetDC with MGBG, a competitive inhibitor of this enzyme, structurally related to CGP 48664, led to cell differentiation [30]. In this study DFMO-induced differentiation was related to excessive accumulation of dcAdoMet which competes with AdoMet in DNA methylation reactions, but not to polyamine depletion [30]. In the human colonic cancer cell line Caco-2, the only common feature produced by both inhibitors was the extensive depletion of spermidine, indicating that spermidine, and not putrescine or dcAdoMet, plays an important role in the regulation of Caco-2 cell growth and differentiation. Polyamine biosynthesis is closely related to the DNA methylation status [4]; this affects the expression of genes that may have a key role in regulating growth [31]. The decreased amount of spermidine measured in “differentiated” Caco-2 cells after confluency and in Caco-2 cells treated either with DFMO or with CGP 48664 may enhance cytosine-DNA methyltransferase activity [32]. Our present data suggest that changes in Caco-2 cell growth correlate with changes in DNA
FIG. 4. Effects of polyamines on DNA–methyltransferase activity of Caco-2 cell extracts. Cell extracts were obtained after 4 days of culture and assayed in the absence (open column) or in the presence of 80 mM polyamines: putrescine (dotted column), spermidine (hatched column), or spermine (column with vertical strips). Values represent means 6 SEM of three independent experiments performed in triplicate. DPM, disintegrations per minute. Columns not sharing a common superscript letter differ significantly, a Þ b Þ c, P , 0.05.
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methylation. Thus, enhanced DNA methylation was associated with growth arrest due to contact inhibition in confluent Caco-2 cells and in cells after growth inhibition by both AdoMetDC and ODC inhibitors. These results are in accordance with several reports showing that global genomic levels of DNA methylation seemed to be lower in highly proliferative cancer cells than in normal tissues [33, 34]. Furthermore, we show that spermine and spermidine (but not putrescine) inhibit cytosine–DNA methyltransferase activity of Caco-2 cells. This favors the idea that the depletion of the spermidine pools by DFMO or CGP 48664 enhances DNA methylation. In conclusion, our data suggest that polyaminecontrolled changes in DNA methylation might be a crucial step in the regulation of Caco-2 growth, but further investigations are needed to obtain more insights into the molecular mechanisms that control the expression of individual growth and/or differentiation-related genes. The authors would like to thank le Comite´ De´partemental du Haut-Rhin de la Ligue contre le Cancer for financial support, and Dr. N. Seiler for stimulating discussion and reviewing the manuscript. C.G.P. 48664 was kindly provided by Dr. H. Mett, Novartis, Basel, Switzerland.
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Herold, G., Besemer, F., Rogler, D., and Stange, E. F. (1993). Polyamine deficiency impairs proliferation and differentiation of cultured enterocytes (Caco-2). Z. Gastroenterol. 31, 120 –128.
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Bey, P. (1978). Substrate-induced irreversible inhibition of a-aminoacid decarboxylases: Application to glutamate, aromatic L-a-aminoacic and ornithine decarboxylases. In “Enzyme-
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POLYAMINE POOLS AND DNA METHYLATION IN Caco-2 CELLS 27.
Heby, O., and Persson, L. (1990). Molecular genetics of polyamine synthesis in eukaryotic cells. Trends Biochem. Sci. 15, 153–158. 28. Mamont, P. S., Danzin, C., Wagner, J., Siat, M., Joder-Ohlenbusch, A. M., and Claverie, N. (1982). Accumulation of decarboxylated S-adenosyl-L-methionine in mammalian cells as a consequence of the inhibition of putrescine biosynthesis. Eur. J. Biochem. 123, 499 –504. 29. Pegg, A. E., Poso, H., Shuttleworth, K., and Bennett, R. A. (1982). Effect of inhibition of polyamine synthesis on the content of decarboxylated S-adenosylmethionine. Biochem. J. 202, 519 –526. 30. Frostesjo¨, L., Holm, I., Grahn, B., Page, A. W., Bestor, T. H., and Heby, O. (1997). Interference with DNA methyltransferase activity and genome methylation during F9 teratocarcinoma Received January 16, 1998 Revised version received April 30, 1998
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stem cell differentiation induced by polyamine depletion. J. Biol. Chem. 272, 4359 – 4366. 31.
Wainfan, E., and Poirier, L. A. (1992). Methyl groups in carcinogenesis: Effects on DNA methylation and gene expression. Cancer Res. 52, 2071S–2077S.
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Ruiz-Herrera, J., Ruiz-Medrano, R., and Dominguez, A. (1995). Selective inhibition of cytosine-DNA methylases by polyamines. FEBS Lett. 357, 192–196.
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243, 319 –325 (1998)
EX984148
Concomitant Changes in Polyamine Pools and DNA Methylation during Growth Inhibition of Human Colonic Cancer Cells Benoıˆt Duranton,* Ge´rard Keith,† Francine Gosse´,* Christian Bergmann,* Rene´ Schleiffer,* and Francis Raul*,1 *CJF INSERM 95-09 et Laboratoire du Controˆle Me´tabolique et Nutritionnel en Oncologie Digestive de l’ULP, IRCAD, 1 Place de l’Hoˆpital, 67091 Strasbourg, France; and †UPR 9002 CNRS, IBMC, 15 rue Descartes, 67084 Strasbourg, France
The effects of CGP 48664 and DFMO, selective inhibitors of the key enzymes of polyamine biosynthesis, namely, of S-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), were investigated on growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. Both inhibitors caused growth inhibition and affected similarly the initial expression of the differentiation marker sucrase. In the presence of the AdoMetDC inhibitor, ODC activity and the intracellular pool of putrescine were enhanced, whereas the spermidine and spermine pools were decreased. In the presence of the ODC inhibitor, the AdoMetDC activity was enhanced and the intracellular pools of putrescine and spermidine were decreased. With both compounds, the degree of global DNA methylation was increased. Spermine and spermidine (but not putrescine) selectively inhibited cytosine–DNA methyltransferase activity. Our observations suggest that spermidine (and to a lesser extent spermine) controls DNA methylation and may represent a crucial step in the regulation of Caco-2 cell growth and differentiation. © 1998 Academic Press Key Words: spermidine; sucrase; ornithine decarboxylase; S-adenosylmethionine decarboxylase; cytosine– DNA methyltransferase.
INTRODUCTION
The polyamines (putrescine, spermidine, and spermine) are ubiquitous, small polycations that are essential for cell growth and differentiation [1, 2]. The two key enzymes involved in polyamine biosynthesis are ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) [2, 3]. ODC catalyzes the formation of putrescine from L-ornithine, and AdoMetDC 1 To whom correspondence and reprint requests should be addressed at CJF INSERM 9509, IRCAD, 1, place de l’hoˆpital, 67091 Strasbourg Cedex, France. Fax: (33)388119097. E-mail: Francis.raul@ ircad.u-strasbg.fr.
decarboxylates S-adenosylmethionine (AdoMet). The product of this reaction, decarboxylated S-adenosylmethionine (dcAdoMet), is the aminopropyl group donor for spermidine and spermine synthesis. AdoMet is a common substrate for numerous methylation reactions including DNA methylation and for polyamine biosynthesis. Therefore, it can be hypothesized that changes in cellular polyamine metabolism may affect the degree of DNA methylation and consequently modify gene expression [4]. Putrescine and spermidine levels and their biosynthetic decarboxylases are generally elevated in cancer cells and in rapidly growing tissues, compared to normal or quiescent cells [5, 6]. In order to assess further the role of polyamine metabolism on the regulation of cellular growth and differentiation, we investigated the changes in polyamine biosynthesis and DNA methylation of the human colon carcinoma cell line Caco-2. This cell line spontaneously undergoes structural and functional enterocytic differentiation at late confluency; phenotypic changes after confluency include formation of brush border membranes and expression of intestinal hydrolases which are markers of functional differentiation normally found in human fetal colon [7–9]. In the present study we examined the effects of a potent inhibitor of AdoMetDC activity, 4-amidinoindan-1-one 29-amidinohydrazone (CGP 48664) [10], and of 2-(difluoromethyl)ornithine (DFMO), an irreversible inhibitor of ODC [11], on cell growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. MATERIALS AND METHODS Cell culture. Caco-2 cells, obtained from the European Collection of Animal Cell Culture (CERDIC, Sophia-Antipolis, France), were cultured in 75-cm2 Falcon flasks containing Dulbecco’s modified Eagle’s medium (DMEM) at 25 mM glucose supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids, 100 U/ml penicillin, and 100 mg/ml streptomycin. The cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and subcultured to preconfluency after trypsinization (0.5% trypsin/2.6 mM EDTA). They were harvested between passage 30 and 45. In all experiments, the cells were seeded at 6 3 105 cells on culture dishes (100 mm in diameter). The culture medium was composed of DMEM
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supplemented with 3% FBS, transferrin (5 mg/ml), selenium (5 ng/ ml), and insulin (10 mg/ml) (TSI-defined medium; Gibco BRL, Life Technologies SARL, France). When needed, the AdoMetDC inhibitor CGP 48664 (Novartis, Basel, Switzerland) was added to the culture medium 48 h after seeding at the LD50 concentration (10 mM) which was determined by the Alamar Blue assay (Interchim, Montluc¸on, France). In another experimental series, the ODC inhibitor, DFMO (Marion Merrell Dow Research Institute, Strasbourg, France), was added at 100 mM to the culture medium 48 h after seeding. In all experimental settings the culture medium was replaced every 48 h and either the AdoMetDC or the ODC inhibitor were freshly added. Cells were harvested after various times, washed three times with PBS (pH 7.2), and kept frozen at 270°C until assays were performed. Brush border sucrase. Cells were homogenized in 4 ml of Tris– mannitol buffer (50 mM mannitol, 2 mM Tris, pH 7.1). Brush border membranes were isolated as described by Schmitz et al. [12]. Sucrase activity was determined according to Messer and Dahlqvist [13]. The enzyme activity was expressed as specific activity (milliunits per milligram of brush border protein). One unit of activity corresponds to one mmol of product formed per minute at 37°C. ODC and SAMDC activities. Cells were homogenized in 100 mM Tris–HCl buffer, pH 7.4 (1 mM EDTA, 1 mM dithiothreitol, 0.5 mM leupeptin, 0.5 mM phenylmethylsulfonyl fluoride). After centrifugation at 33,000g for 25 min at 4°C, the supernatants were collected and ODC and AdoMetDC assays were performed. ODC activity was evaluated by measuring the rate of 14CO2 formation from [1-14C]Lornithine (55 mCi/mmol, Amersham, UK) [14] and AdoMetDC activity was evaluated by measuring the rate of 14CO2 formed from [1-14C]S-adenosylmethionine (60 mCi/mmol, Amersham) [15]. Polyamine determinations. Cells were homogenized in perchloric acid (200 mM), and the homogenates were centrifuged at 3000g for 10 min after standing for 16 h at 12°C. The clear supernatants were diluted with perchloric acid (200 mM) and 200-ml aliquots were applied on a reversed-phase column for separation. The polyamines were determined by separation of the ion pairs formed with n-octanesulfonic acid, reaction of the column effluent with o-phthaldialdehyde/2-mercaptoethanol reagent, and monitoring of fluorescence intensity [16]. Measurements of 5-methyldeoxycytidine (m5C) in DNA samples. Cellular DNA was purified using the Wizard genomic DNA purification kit (Promega Corp., Madison, WI) and m5C was determined by the method of Wilson et al. [17] modified as follows: DNA (10 mg) was digested at 37°C for 3 h, into 39-deoxymonophosphate nucleosides, using micrococcal nuclease (5–10 mg) and spleen phosphodiesterase (1–2 mg) in 10 mM CaCl2 and 20 mM sodium succinate, pH 6.0, buffer. Aliquots of the digest containing the 39-deoxymonophosphate nucleosides were converted in presence of [g-32P]ATP and T4 polynucleotide kinase into 32P-labeled nucleoside 39,59-biphosphate nucleosides (the 32P being attached to the free 59 hydroxyl group). The biphosphate nucleosides were further hydrolyzed into [32P]59-monophosphate deoxynucleosides using nuclease P1 [18]. Finally, the labeled mononucleotides were separated by two-dimensional thinlayer chromatography on cellulose plates (Fig. 1). Quantification of individual nucleotides was carried out by scraping off the spots detected by autoradiography and counting of their radioactivity. The m5C content of DNA was calculated from the radioactivity found in m5dCMP and dCMP by the equation
FIG. 1. Autoradiogram of a 32P-labeled DNA digest of Caco-2 cells on two-dimensional thin-layer cellulose plates. Development in A (first dimension), isobutyric acid/concentrated ammonia/H2O (66/ 1/33, v/v/v); B (second dimension), 0.1 M sodium phosphate pH 6.8/ ammonium sulfate/n-propanol (100/60/2, v/w/v). The four faint spots shown by small arrows correspond clockwise to the five ribonucleotides pG, pA, pC, and pU from contaminating RNA in some DNA samples.
100 mg/ml RNase A). The cell lysate (corresponding to 5 mg protein) was made up in 15 ml of the same buffer and mixed with 0.5 mg of poly(d(I-C).d(I-C)) (synthetic DNA consisting of repeats of inosine– cytosine) (Pharmacia Inc., France) and 1.5 mCi S-adenosyl-L-[methyl3 H]methionine (80 Ci/mmol, Amersham) in a total volume of 23 ml. The mixture was incubated at 37°C for 2 h. The reaction was stopped by adding 300 ml of a solution containing 1% sodium dodecyl sulfate, 2 mM EDTA, 3% 4-aminosalicylate, 5% butanol, 125 mM sodium chloride, 0.25 mg/ml salmon testis DNA, and 1 mg/ml proteinase K. After an additional 30 min of incubation at 37°C, DNA was purified by phenol/chloroform extraction and ethanol precipitation. The precipitate was resuspended in 0.3 M sodium hydroxide and incubated at 37°C for 35 min. This solution was spotted on Whatman 3MM filter paper strips, washed in ice-cold 10% trichloroacetic acid (TCA) for 10 min, 5% TCA for 5 min followed by 70% ethanol. The dried filters were placed in 5 ml of scintillation fluid and counted in a scintillation counter. For exclusion of background protein and RNA methylation, all samples were assayed with a negative control containing the whole cell lysate but no poly(d(I-C).d(I-C)). Statistical analysis. Statistical significance between treated and control cells was assessed by one-way analysis of variance (ANOVA) plus comparison of means. Differences were considered significant at P , 0.05.
% m5dCMP 5 ~m5dCMP 3 100!/~m5dCMP 1 dCMP!.
RESULTS Cytosine–DNA methyltransferase (DNA–MTase) assay. We adapted a microassay technique for DNA–MTase activity [19]. Cells were harvested after 4 days of culture and pelleted. Lysis was achieved by freeze–thawing the cells four times in 50 mM Tris–HCl buffer, pH 7.8 (1 mM EDTA, 1 mM dithiothreitol, 0.01% sodium azide, 6 mg/100 ml phenylmethylsulfonyl fluoride, 10% glycerol, 1% Tween 80, and
Effects of SAMDC and ODC Inhibitors on Caco-2 Cell Growth and Differentiation Growth and differentiation of Caco-2 cells grown in the absence or presence of the AdoMetDC inhibitor CGP
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48664, and the ODC inhibitor DFMO, were compared. In the absence of inhibitors, cells reached confluency at day 5 of culture and a plateau at day 9 (Fig. 2), indicating that the cells have entered a stationary phase of growth. When cells were grown 48 h after plating in the presence of 10 mM of the AdoMetDC inhibitor, growth was inhibited by more than 80% and in the presence of 100 mM of the ODC inhibitor a 70% growth inhibition was observed (Fig. 2). Caco-2 cells were also assayed for sucrase activity, which is a characteristic marker of the brush border villi of enterocytes and is indicative of differentiated Caco-2 cells [20]. As shown in Table 1, sucrase activity appeared at confluency and increased progressively reaching highest level at day 15 during the stationary phase. This shows that the cells progressively acquire, after confluency, characteristics of mature enterocytes. In the presence of either the AdoMetDC or the ODC inhibitor, sucrase activity appeared as in control cells, at day 5 of culture despite the fact that in the presence of inhibitors cells did not reached confluency. However, the maximum level of sucrase activity in presence of the inhibitors remained low and reached in CGP 48664-treated cells at day 15 a level corresponding to 10% of control activity (Table 1). In DFMO-treated cells, confluency was reached by day 13 (4 days later than in controls) and the level of sucrase activity remained significantly lower (75%) compared with controls (Table 1). Changes in Polyamine Metabolism during Caco-2 Cell Growth In control Caco-2 cells the activities of ODC and AdoMetDC decreased after confluency, i.e., during the
FIG. 2. Caco-2 cell growth curves. Cells were seeded at 6 3 105 cells on culture dishes (100 mm in diameter) in DMEM medium supplemented with 3% FBS, transferrin (5 mg/ml), selenium (5 ng/ ml), and insulin (10 mg/ml). In the absence (open square) or in the presence of DFMO (100 mM) (closed triangle) or of CGP 48664 (10 mM) (closed square). The inhibitors were added 48 h after seeding, the culture medium was replaced every 48 h, and the inhibitor was freshly added. Values represent means 6 SEM (n 5 4).
TABLE 1 Sucrase Specific Activity in Brush Border Membranes Isolated from Caco-2 Cells Grown in the Absence or Presence of CGP 48664 (10 mM) or DFMO (100 mM) Sucrase activity (mU/mg protein) Days of culture 3 5 9 15
Control ND 0.9 6 0.1a 15 6 0.9b 66 6 11c
CGP48664
DFMO
ND 1.2 6 0.3a 2.2 6 0.2b* 5.6 6 0.6c*
ND 1.0 6 0.1a 3.0 6 0.6b* 17 6 0.9c*
Note. Results are means 6 SEM of three independent experiments performed in triplicate. ND, activity not detected. For each column, a Þ b Þ c, P , 0.05. Control versus CGP 48664 or DFMO, *P , 0.05.
stationary phase, when Caco-2 cells are differentiating (Table 2). The intracellular pools of putrescine and spermidine decreased significantly in parallel to the changes observed for the enzyme activities, reaching their lowest values at day 15. The pool of spermine did not change significantly (Table 3). In Caco-2 cells treated with CGP 48664, ODC activity was 13-fold higher at day 3 of culture and 140-fold higher than in control cells at day 15 (Table 2). In cells treated with DFMO, ODC activity decreased by about 70% during the initial phase of growth (day 3) and remained practically unchanged thereafter. It was 5-fold higher than in controls by day 15 (Table 2). The inhibitory effect of DFMO was evident from the absence of detectable amounts of putrescine (Table 3). Exposure of the cells to DFMO led to a progressive elevation of AdoMetDC activity: a 3-fold increase was observed at day 3 and a 60-fold increase was reached at day 15 when compared to Caco-2 cells grown in the absence of the inhibitor (Table 2). The DFMO-triggered increase of AdoMetDC activity explaines the drop of the spermidine pool (Table 3). DFMO treatment led to a huge elevation in AdoMetDC activity (Table 2). In cells treated with CGP 48664, AdoMetDC activity, as determined in vitro, exhibited first a rapid and significant decrease at day 3 of culture but recovered progressively, reaching even higher values than in control cells at day 5 and 15. This is most probably due to the fact that CGP 48664 is a competitive inhibitor, so that active enzyme may accumulate in the cells which is not inhibited completely under the assay conditions due to dilution. Nevertheless, as shown in Table 3, the intracellular AdoMetDC activity was obviously inhibited by CGP 48664, as is seen from the significant decrease of the intracellular pools of spermidine and spermine. At the same time, putrescine accumulated. By day 5 of culture, cells grown in presence of the AdoMetDC inhibitor exhibited a 100-fold accumulation of putrescine and a decrease of spermidine and spermine contents by 30 and 85%,
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TABLE 2 Ornithine Decarboxylase and S-Adenosylmethionine Decarboxylase Activities in Caco-2 Cells Maintained in Culture in the Absence and Presence of CGP 48664 (10 mM) or DFMO (100 mM) ODC activity (pmol/mg protein/h) Days of culture 3 5 15
SAMDC activity (pmol/mg protein/h)
Control
CGP48664
DFMO
Control
79 6 2a 48 6 8b 5 6 0.6b
996 6 10a* 360 6 19b* 723 6 42c*
22 6 2a* 35 6 4b 29 6 2a,b*
28 6 2a 24 6 0.3a 10 6 0.7b
CGP48664
DFMO
12 6 1a* 32 6 1b* 32 6 0.6b*
76 6 8a* 733 6 43b* 683 6 142b*
Note. Results are means 6 SEM of three independent experiments performed in triplicate. For each column, a Þ b Þ c, P , 0.05, and control versus CGP 48664 or DFMO, *P , 0.05
respectively. The observed changes of the polyamine pools are directly related to the induction of ODC activity, and presumably the reduced availability of dcAdoMet, the aminopropyl donor for spermidine and spermine synthesis. Effects on the DNA Methylation Status As is illustrated in Fig. 3, the amount of m5C in the DNA increased significantly (by 20%) when Caco-2 cells reached stationary phase by 15 days of culture. Growth inhibition by the AdoMetDC inhibitor caused a significant elevation in the m5C content of DNA when compared to nontreated control Caco-2 cells after 3 and 5 days, the proliferation phase of these cells. As shown in Fig. 3, the stimulation of AdoMetDC activity in cells treated with DFMO also led to a similar significant increase of DNA methylation. This result indicates that in Caco-2 cells the activity of AdoMetDC may not be an important factor in DNA methylation. In order to assess whether the changes in the intracellular polyamines affect DNA methylation, we determined the effects of the various polyamines on the cytosine–DNA methyltransferase activity. As shown in Fig. 4, spermidine and spermine but not putrescine inhibited the cytosine–DNA methyltransferase activity of Caco-2 cell extracts obtained from cells during the proliferative phase. Spermine (80 mM) was most effi-
cient and inhibited the enzyme activity by 70%. Spermidine at the same concentration caused a 50% inhibition of the enzyme. Enzyme activity was already significantly (P , 0.05) reduced with lower concentrations (20 and 40 mM) of spermine and spermidine (results not shown). Putrescine was ineffective even at 100 mM (Fig. 4). DISCUSSION
Our results confirm that polyamine biosynthesis is markedly reduced in Caco-2 cells after confluency presumably due to the low ODC activity [21–23]. In addition, AdoMetDC exhibited also reduced activity at this stage as shown in this study. Similar changes in polyamine metabolism and DNA methylation can be observed in differentiated Caco-2 cells and in cells after treatment with CGP 48664 or with DFMO: Under all these conditions polyamine biosynthesis is significantly diminished, the degree of global DNA methylation is enhanced and the differentiation marker sucrase is expressed. Sucrase expression was initiated by inhibiting the growth of Caco-2 cells during the proliferation phase by reducing the intracellular pools of spermidine and spermine. In contrast, control cells expressed sucrase only when cells reached confluency. CGP 48664 and DFMO, which have cytostatic effects
TABLE 3 Intracellular Polyamine Content (pmol/mg protein) of Caco-2 Cells in the Absence and in Presence of CGP 48664 (10 mM) or DFMO (100 mM) Putrescine
Spermidine
Days of culture
Control
CGP48664
DFMO
3 5 15
277 6 23a 133 6 16b 73 6 3c
7487 6 903a* 13793 6 1851b* 16313 6 1061b*
ND ND ND
Control
CGP48664
Spermine DFMO
Control
CGP48664
DFMO
3993 6 254a 4050 6 386a 1707 6 50a* 7430 6 254a 2213 6 432a* 6947 6 424a 3520 6 202a 2503 6 240b* 163 6 7b* 8090 6 135a,b 1180 6 136b* 5177 6 243b* 2525 6 454b 1620 6 72c 97 6 8c* 7200 6 250a,c 570 6 92b* 4743 6 147b*
Note. Results are means 6 SEM of three independent experiments performed in triplicate. ND, activity not detected. For each column, a Þ b Þ c, P , 0.05, and control versus CGP 48664 or DFMO, *P , 0.05.
POLYAMINE POOLS AND DNA METHYLATION IN Caco-2 CELLS
FIG. 3. Changes in DNA methylation of Caco-2 cells grown in the absence (open columns) or presence of CGP 48664 (hatched columns) or of DFMO (dotted columns). DNA samples were digested enzymatically to nucleoside monophosphates which were converted to 32Plabeled nucleosides and separated by two-dimensional thin-layer chromatography (see legend to Fig. 1). The percentage of m5C present in DNA was calculated from the radioactivity found in m5dCMP and dCMP as described under Materials and Methods. Values represent means 6 SEM (n 5 6). Columns not sharing a common superscript letter differ significantly, a Þ b, P , 0.05.
on Caco-2 cells, seem to favor only the early initial step of sucrase expression as is presumed from the fact that maximum sucrase activities were lower in the cells treated with the inhibitors than in confluent cells. This indicates that regulatory mechanisms are triggered through cell to cell interactions at confluency which are necessary for optimal expression of the differentiation marker. This may explain the higher level of sucrase expression observed in the presence of DFMO at the time when cells reached confluency. Cytostatic effects of CGP 48664 have previously been described for several mammalian cell lines [24]. In contrast with a previous report on Caco-2 cells [25], the present study shows that cell growth seemed not supported by ODC and the huge putrescine pool, since growth inhibition by CGP 48664 occurred in the presence of high intracellular putrescine concentrations and a high ODC activity. These data are in accordance with studies suggesting that overexpression of ODC alone is not sufficient to favor cell proliferation [26]. The compensatory increase in ODC activity observed with CGP 48664 treatment is most probably related to the feedback control of this enzyme by polyamines [27]. Likewise, AdoMetDC is induced in the CGP 48664-treated cells, as has been shown in L1210 cells [10]. This is confirmed by the high AdoMetDC activity as measured in vitro in extracts of Caco-2 cells and by the enhanced amount of the AdoMetDC mRNA in the CGP 48664 treated cells (results not shown). Similarly, the ODC activity measured in vitro when cells were treated with DFMO may be explained by the accumulation of active ODC molecules due to the rel-
323
atively low concentration of the ODC inhibitor used in the present study. Both DFMO and CGP 48664 exhibited similar effects on Caco-2 cell growth and differentiation, despite completely different mechanisms of action. The AdoMetDC inhibitor caused the accumulation of putrescine, and increased ODC activity, the depletion of spermine and of spermidine, and presumably a decreased concentration of dcAdoMet. In contrast, the ODC inhibitor provokes an increase in AdoMetDC activity, the depletion of putrescine and spermidine concentrations, and as is shown from the literature [28, 29] the enhanced formation of dcAdoMet. These results are in contrast with those obtained in F9 teratocarcinoma stem cells where the inhibition of ODC by DFMO but not the inhibition of AdoMetDC with MGBG, a competitive inhibitor of this enzyme, structurally related to CGP 48664, led to cell differentiation [30]. In this study DFMO-induced differentiation was related to excessive accumulation of dcAdoMet which competes with AdoMet in DNA methylation reactions, but not to polyamine depletion [30]. In the human colonic cancer cell line Caco-2, the only common feature produced by both inhibitors was the extensive depletion of spermidine, indicating that spermidine, and not putrescine or dcAdoMet, plays an important role in the regulation of Caco-2 cell growth and differentiation. Polyamine biosynthesis is closely related to the DNA methylation status [4]; this affects the expression of genes that may have a key role in regulating growth [31]. The decreased amount of spermidine measured in “differentiated” Caco-2 cells after confluency and in Caco-2 cells treated either with DFMO or with CGP 48664 may enhance cytosine-DNA methyltransferase activity [32]. Our present data suggest that changes in Caco-2 cell growth correlate with changes in DNA
FIG. 4. Effects of polyamines on DNA–methyltransferase activity of Caco-2 cell extracts. Cell extracts were obtained after 4 days of culture and assayed in the absence (open column) or in the presence of 80 mM polyamines: putrescine (dotted column), spermidine (hatched column), or spermine (column with vertical strips). Values represent means 6 SEM of three independent experiments performed in triplicate. DPM, disintegrations per minute. Columns not sharing a common superscript letter differ significantly, a Þ b Þ c, P , 0.05.
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methylation. Thus, enhanced DNA methylation was associated with growth arrest due to contact inhibition in confluent Caco-2 cells and in cells after growth inhibition by both AdoMetDC and ODC inhibitors. These results are in accordance with several reports showing that global genomic levels of DNA methylation seemed to be lower in highly proliferative cancer cells than in normal tissues [33, 34]. Furthermore, we show that spermine and spermidine (but not putrescine) inhibit cytosine–DNA methyltransferase activity of Caco-2 cells. This favors the idea that the depletion of the spermidine pools by DFMO or CGP 48664 enhances DNA methylation. In conclusion, our data suggest that polyaminecontrolled changes in DNA methylation might be a crucial step in the regulation of Caco-2 growth, but further investigations are needed to obtain more insights into the molecular mechanisms that control the expression of individual growth and/or differentiation-related genes. The authors would like to thank le Comite´ De´partemental du Haut-Rhin de la Ligue contre le Cancer for financial support, and Dr. N. Seiler for stimulating discussion and reviewing the manuscript. C.G.P. 48664 was kindly provided by Dr. H. Mett, Novartis, Basel, Switzerland.
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