Effects of the garlic compound diallyl disulfide on the metabolism, adherence and cell cycle of HT-29 colon carcinoma cells: evidence of sensitive and resistant sub-populations
Véronique Robert,
Béatrice Mouillé,
Camille Mayeur,
Marie Michaud and
François Blachier,1
Laboratoire de Nutrition et Sécurité Alimentaire, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France
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Abstract
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Diallyl disulfide (DADS) is a major organosulphur compound present in garlic with an anti-mitotic potential against colon neoplasic lesions in vivo and colon tumour cell growth in vitro. Using the human colon adenocarcinoma HT-29 Glc/+ cell line we identified sub-populations of tumoural cells with markedly different characteristics in terms of metabolic capacities, adhesion properties and distribution in the cell cycle phases. After 1 and 2 days treatment with 100 µM DADS HT-29 cells were largely released into the culture medium. These floating cells accumulated in the G2/M phase and were characterized by a 5-fold reduction in cell capacity for de novo protein synthesis. Polyamine metabolism, which is necessary for intestinal epithelial cell attachment and growth, was also severely affected, since 3-fold reductions in polyamine biosynthesis and net accumulation of putrescine were measured after DADS treatment. However, oxidation of L-glutamine, the main precursor of the tricarboxylic acid cycle in these cells, and de novo synthesis of glutathione, a tripeptide involved in tumoural cell chemoresistance, were not affected by DADS treatment. In contrast, the adherent sub-population of HT-29 cells, although partially accumulated in G2/M phase, were characterized by unaffected metabolic capacities when compared with control cells except for putrescine accumulation, which was transiently decreased, and L-glutamine oxidation, which was increased 2-fold. DADS-resistant cells selected within 5 days were then able to proliferate at a similar rate to control untreated cells. The DADS-induced changes in HT-29 metabolic capacities, adhesion properties and the cell cycle are discussed from a causal perspective.
Abbreviations: DADS, diallyl disulfide; DFMO, difluoromethylornithine; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethylsulphoxide; DPP IV, dipeptidylpeptidase IV; LDH, lactate dehydrogenase; ODC, ornithine decarboxylase; TCA, trichloroacetic acid.
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Introduction
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Environment is considered to play a major role in causing sporadic colorectal cancer (1). Among environmental factors it is mainly dietary compounds and related metabolites that are believed to modify colorectal cancer risk. Convincing epidemiological evidence strongly suggests that diets rich in vegetables protect against cancers of the colon and rectum (2). Among the numerous biologically active phytochemicals present in vegetables (3), organosulphur compounds contained in plants belonging to the genus Allium have received attention due to their wide spectrum of biological effects (4), particularly diallyl disulfide (DADS), a major organosulphur compound present in garlic which is produced by stepwise enzymatic conversion of the precursor S-alk(en)yl-L-cysteine sulphoxide followed by non-enzymatic reactions (5). In animal models of chemically induced carcinogenesis DADS was shown to decrease colon neoplasic lesions (6,7) and to reduce tumour volume after colon tumour cell xenografts (8). Furthermore, in other in vivo experiments DADS was reported to increase levels of the phase II detoxification enzyme glutathione transferase in various parts of the gastrointestinal tract (9,10). In in vitro experiments DADS was able to decrease the proliferation of human colon tumour cells, an effect related to a transient increase in cells in the G2/M phase of the cell cycle and a decrease in p34 cdc2 kinase activity (11), alterations in cell calcium homeostasis (12) and apoptosis induction (13).
In this context the aim of the present work was to further document the mechanism of action by which DADS inhibits cell growth, with special attention placed on nitrogen metabolism. Using the human colon adenocarcinoma cell line HT-29 Glc/+ (14) we have studied L-glutamine oxidative metabolism, since this amino acid, in contrast to D-glucose which is almost completely oxidized in the pentose phosphate pathway, is a major precursor of the tricarboxylic acid cycle in this cell line (15). We also studied the effect of DADS on de novo protein synthesis, which is related to cell division and to cell progression through the different cell cycle phases (1619). Polyamine biosynthesis from the precursor L-ornithine and accumulation of the diamine putrescine were studied, since these metabolites are necessary for cell attachment and growth (2022). Lastly, the effects of DADS on the capacity of HT-29 cells to synthesize glutathione was investigated, because cell sensitivity to anti-mitotic drugs has been reported to be dependent on cellular glutathione content (2326). We identified sub-populations of tumoural cells with markedly different characteristics in terms of metabolic capacities, adhesion properties and distribution in the phases of the cell cycle.
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Materials and methods
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Materials
DADS was purchased from Aldrich (St Quentin Fallavier, France). Dimethylsulphoxide (DMSO), Gly-Pro-p-nitroanilide, cycloheximide, putrescine dihydro- chloride and N-acetyl-L-cysteine were purchased from Sigma chemicals (St Louis, MO). L-[U-14C]leucine, L-[1-14C]ornithine, L-[U-14C]glutamine, [U-14C]glycine, [glycine 2-3H]glutathione, [1,4-14C]putrescine and L-[1-14C]glucose were purchased from New England Nuclear (Boston, MA). D,L-
-Difluoromethyl- ornithine (DFMO) was a gift from the Marion Merrell Dow Research Institute (Strasbourg, France).
Cell culture
The human adenocarcinoma cell line HT-29 was established in permanent culture in 1975. The HT-29 Glc/+ cells used in this study were selected by Zweibaum et al. (27) from parental cells by growing them in a glucose-free medium for 36 passages, then leaving them to grow at 37°C under a 10% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) foetal bovine serum and containing 4 mM L-glutamine, 25 mM D-glucose, 100 U/ml penicillin, 100 µg/ml streptomycin and 1 µg/ml amphotericin B. HT-29 Glc/+ cells were used between passage 37 and passage 72 (one passage per week) and were seeded at a density of 0.1x106 cells/ml on day 0. The culture medium was changed every day with freshly prepared DADS solutions obtained by dilution of a commercial preparation of DADS dissolved in DMSO in order to obtain DADS stocks solutions (i.e. 100 mM) and then diluted in the culture medium. At each step of dilution the DADS solution was treated for 1 min with ultrasonic waves in order to obtain a fully homogeneous solution. In control experiments involving vehicle alone DMSO was diluted in the culture medium in order to reach the same DMSO concentration (i.e. 0.1%) as used in the DADS experiments. Adherent cell isolation was performed with phosphate-buffered saline containing 1 g/l EDTA and 0.25 g/l trypsin. Adherent and floating cells were counted on a haemacytometer.
Measurement of membrane integrity
Membrane integrity was estimated by release of the cytosolic enzyme lactate dehydrogenase (LDH) into the culture medium. Briefly, after 24 h culture the culture medium, on the one hand, and adherent cells recovered by scraping, on the other, were used for LDH activity measurement (28). The percentages of cells retaining membrane integrity were calculated using the LDH activity associated with the scraped cells and expressed as a percentage of total LDH (i.e. the sum of LDH released into the culture medium and present in the scraped cells).
Flow cytometry of HT-29 cells
Adherent and floating cells (12x106) were recovered after different times following DADS addition to the culture medium, centrifuged at 1000 g for 4 min and the cell pellets washed thrice with phosphate-buffered saline. Cells were fixed with LPR solution and stained with propidium iodide solution containing RNase, using the DNA preparation Coulter Reagent kit (Coulter, Miami, FL). Flow cytometry analysis was performed on a FACScan (Becton-Dickinson, San Jose, CA) using Lysis II software. For each time point 20x103 cells were counted and propidium fluorescence was displayed on a linear scale using a FL2 photodetector. The DNA profile indicates the relative abundances of G0/G1, S and G2/M phase populations.
Dipeptidylpeptidase IV (DPP IV) activity measurement
DPP IV activity associated with adherent and floating HT-29 cells was measured after sonication using the method of Nagatsu et al. (29) on scraped cells using 1.5 mM Gly-Pro-p-nitroanilide as the substrate in a glycine (71 mM, pH 8.7) buffer containing 1% Triton X-100. The protein content of the samples was determined using the Lowry procedure (30).
Measurement of HT-29 cell metabolic capacities
For measurement of the DADS-treated adherent and floating HT-29 cell capacity to synthesize proteins, cells were recovered after different time of exposure to DADS or vehicle alone and resuspended in DMEM gassed with a mixture of O2/CO2 (19:1 v/v) containing 10 mg/ml bovine serum albumin. The net incorporation of radioactive L-leucine into cellular proteins was measured by incubating HT-29 cells for 90 min at 37°C in 120 µl of the gassed DMEM containing 0.8 mM L-[U-14C]leucine both in the presence and absence of 0.2 mM cycloheximide and by measuring the cycloheximide-sensitive presence of radioactivity in the trichloroacetic acid (TCA)-precipitable material as described (31). The flux of L-ornithine through the reaction catalysed by ornithine decarboxylase was measured after resuspension of the DADS-treated cells in KrebsHenseleit bicarbonate-buffered medium (pH 7.4) saturated with a mixture of O2/CO2 (19:1 v/v) containing 10 mM HEPES, 1.3 mM CaCl2, 2 mM MgCl2 and 10 mg/ml bovine serum albumin. The polyamine production capacity was determined by incubating HT-29 cells for 90 min at 37°C in 120 µl of incubation medium containing 1 mM L-[1-14C]ornithine both in the presence and absence of 10 mM DFMO, known to completely inhibit polyamine synthesis in HT-29 cells (32). The radioactive CO2 was trapped in methylbenzethonium hydroxide and quantified by liquid scintillation.
The capacity of HT-29 cells to oxidize L-glutamine was determined by incubating the cells for 90 min at 37°C in 120 µl of incubation medium (see above) containing 2 mM L-[U-14C]glutamine and measuring the radioactive CO2 released by HT-29 cells. For measurement of HT-29 cell capacity for net de novo glutathione synthesis, cells were incubated for 90 min at 37°C in incubation medium containing 1 mM [U-14C]glycine, 1 mM L-glutamate and 1 mM N-acetylcysteine. At the end of incubation 10 µl of dithiothreitol (final concentration 10 mM) and 5 µl of a cocktail of protease inhibitors (0.4 mg/ml AEBSF, 5 mg/ml EDTA-Na2, 1 µg/ml leupeptin and 1 µg/ml pepsatin) were added to the samples which were kept at 80°C up to separation of radioactive glutathione from radioactive glycine by reversed phase HPLC. For this purpose samples were mixed with 20 µl of perchloric acid (14%), centrifuged and the metabolites contained in the supernatants were separated on a C18-Luna column (Phenomenex, Torrance, CA) using a buffer flow rate of 0.2 ml/min. The elution buffer was 0.1 M sodium acetate buffer, pH 3.8. The amount of radioactive glutathione eluted from the column was corrected for recovery of internal standard amounts of [glycine 2-3H]glutathione.
Net accumulation of putrescine in HT-29 cells
Net accumulation of putrescine in DADS-treated cells was measured by cell centrifugation through an oil layer as described (31). Briefly, 0.3x106 cells were incubated for 90 min at 37°C in 120 µl of incubation medium containing 10 µM [1,4-14C]putrescine and then centrifuged (12 000 g for 5 min) through 150 µl of a silicon oil layer (Nyosil M20; NYE Lubricants, New Bedford, MA) and recovered in 50 µl of an aqueous solution containing 0.64 M CsCl and 0.05 M HCl. Measurement of cell pellet-associated radioactivity was by liquid scintillation. In all experiments net accumulation of putrescine was corrected for extracellular contamination using 30 µM L-[1-14C]glucose. After 1 and 2 day treatments with 100 µM DADS the L-glucose space was similar in adherent and floating cells averaging, respectively, 0.56 ± 0.12 and 0.84 ± 0.11 µl/106 cells (n = 4). In control experiments (i.e. vehicle alone) the L-glucose space averaged, respectively, 0.38 ± 0.05 and 0.50 ± 0.21 µl/106 cells (n = 3) after 1 and 2 days.
Data analysis
The production of radioactive metabolites was calculated by reference to the specific activity of precursors in the incubation medium. The results are expressed as means ± SEM of individual experiments performed with HT-29 cells isolated during different passages. The statistical significance of differences between means was assessed by the Student t-test.
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Results
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Effect of DADS on HT-29 cell proliferation
When 100 µM DADS was added to the culture medium 1 day after cell seeding this agent was found to severely reduce the number of adherent cells in the culture flask up to day 6 of cell culture when compared with cells cultured in the presence of vehicle alone (i.e. DMSO), which by itself has no effect on cell growth (Figure 1
). However, thereafter the number of adherent cells recovered under DADS treatment, increased between days 6 and 17 of cell culture. In contrast, lower DADS concentrations (i.e. 50 and 75 µM) failed to exert any detectable effect on cell proliferation (Figure 1
), leading us to use 100 µM DADS in all further experiments.

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Fig. 1. Effect of DADS on HT-29 cell proliferation. DADS or the vehicle alone (i.e. DMSO) was added 1 day after cell seeding and the culture medium was changed every day. Results are expressed as means ± SEM and represent four or five individual experiments.
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Effect of DADS on HT-29 cell detachment and membrane integrity
When the cells were treated with 100 µM DADS and the culture medium, which was replaced every day, was examined under a light microscope it was found that a significant proportion of HT-29 cells were released into the culture medium up to 5 days after treatment. In contrast, 6 days post-treatment and thereafter no floating cells could be recovered in the culture medium. Indeed, after a 1 day treatment with 100 µM DADS it was found, using a haemacytometer, that 52 ± 4% (n = 11) of the total cells were released into the culture medium. In the next 24 h (i.e. after a 2 day treatment with DADS) the percentage of floating cells averaged 47 ± 3% (n = 7). This DADS-induced cell release into the culture medium did not coincide with any loss of cell viability in terms of membrane integrity. Indeed, whatever the experimental situation (i.e. standard culture medium, DMSO or 100 µM DADS) the percentages of cells retaining membrane integrity were similar, representing after 1, 2, 3, 5 and 10 days, respectively, 83 ± 2 (n = 5), 88 ± 3 (n = 4), 90 ± 2 (n = 3), 96 ± 1 (n = 4) and 98 ± 1% (n = 3). When the proportion of floating cells was more accurately estimated using the measurement of LDH activity associated with adherent and floating cells it was found that after a 1 day treatment with 100 µM DADS 49 ± 7% (n = 4) of the whole cells were released into the culture medium, this percentage being similar to that calculated using cell counting with a haemacytometer (see above). Using measurement of LDH activity associated with adherent and floating cells we determined that the percentages of floating cells measured every day, although high in the first days of DADS treatment, represented not more than 14% after 5 days treatment and only 1% after 10 days treatment (Figure 2
). In control experiments performed with HT-29 cells cultured with or without vehicle alone (i.e. DMSO) the percentages of floating cells did not represent more than 8% (Figure 2
).

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Fig. 2. Effect of DADS on HT-29 cell detachment. DADS (100 µM) or vehicle alone (i.e. DMSO) was added 1 day after cell seeding and the culture medium was changed every day. The relative numbers of floating cells in the flask was calculated after different lengths of DADS treatment by measuring the LDH activity associated with floating and adherent cells. The number of floating cells recovered in the flask was expressed as a percentage of the total amount of cells (i.e. floating and adherent cells). Results are expressed as means ± SEM and represent between three and five individual experiments.
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Effect of DADS on HT-29 cell cycle
When 100 µM DADS was added to the HT-29 cells 1 day after cell seeding this agent was able to modify the proportion of HT-29 cells in the different cell cycle phases after 1 and 2 days treatment in both adherent and floating cells (Figure 3
) when compared with controls (vehicle alone). Almost all recovered floating cells accumulated in the G2/M phase after both 1 and 2 days DADS treatment, at the expense of the G0/G1 and S phases (Table I
). In contrast, when the same analysis was performed with adherent cells it was found that these cells only partially accumulated in the G2/M phase. Incidentally, 50 µM DADS did not modify the proportion of adherent HT-29 cells in the different cell cycle phases after 1 and 2 days treatment when compared with control experiments (data not shown). After 3 and 4 days treatment with 100 µM DADS the percentages of adherent and floating HT-29 cells accumulated in the G2/M phase were similar to that found after 2 days treatment, averaging 69 ± 4 (n = 4) and 79 ± 1% (n = 4), respectively.

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Fig. 3. Effect of DADS on HT-29 cell cycle distribution. DADS (100 µM) or vehicle alone (i.e. DMSO) was added 1 day after cell seeding and the culture medium was changed every day. Adherent and floating cells were analysed (after fixation and staining with propidium iodide) by flow cytometry using a Becton Dickinson FACScan.
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Attachment capacity of DADS treated-floating cells
When HT-29 cells were treated for 1 day with 100 µM DADS and the floating cells (i.e. 0.96 ± 0.28x106cells) were seeded in culture medium without DADS 40 ± 6% (n = 3) of the floating cells were able to re-attach to the culture flask after a subsequent 24 h culture period. After a 2 day culture in medium without DADS the percentages of attached cells were even higher, averaging 66 ± 3% (n = 3) of the total number of cells (i.e. 0.60 ± 0.16x106 cells). In contrast, when the floating cells were recovered after a 2 day treatment with 100 µM DADS and placed in culture medium not containing DADS, although 32 ± 3% (n = 3) of the seeded cells (i.e. 0.31 ± 0.06x106 cells) were able to adhere to the flask after 1 day, no adherent cells could be detected after 2 days culture.
DPP IV activity associated with adherent and floating cells
In order to assess whether the HT-29 cell phenotype (in terms of enterocyte-like differentiation) was different in the floating and adherent cells recovered after 1 and 2 days treatment with 100 µM DADS we measured DPP IV activity associated with both cellular populations. Since the measured DPP IV activities were very similar after 1 and 2 days DADS treatment the results were pooled for analysis.
It was determined that DPP IV activity in the adherent cells (24.2 ± 3.2 nmol/min/mg protein, n = 4) was very close to the DPP IV activity associated with floating cells (26.5 ± 3.8 nmol/min/mg protein, n = 4).
Effect of DADS on HT-29 cell metabolic capacities
Different parameters of HT-29 cell metabolic capacities were measured in both adherent and floating cells recovered at the end of the 100 µM DADS treatment. As judged from the incorporation of radioactive L-leucine into cellular proteins, it was found that floating cells were characterized after both 1 and 2 days treatment by a dramatic decrease in their capacity to synthesize proteins when compared with both adherent cells and control cells cultured in the presence of vehicle alone (Table II
). The second metabolic parameter measured was the flux of L-ornithine through the reaction catalysed by ornithine decarboxylase (ODC), which allows the stepwise synthesis of the polyamines putrescine, spermidine and spermine. As indicated in Table II
, floating cells recovered after 1 and 2 days treatment with 100 µM DADS were found to possess a drastically diminished capacity for polyamine biosynthesis when compared with DADS-treated adherent cells and cells treated with vehicle alone. This effect was not related to any short-term effect of DADS on polyamine biosynthesis. Indeed, 100 µM DADS exerted no effect after 30 min incubation at 37°C on the flux of L-[1-14C]ornithine through ODC in HT-29 cells when compared with control experiments (i.e. incubation with DMSO; data not shown). DADS, in addition to its effect on polyamine biosynthesis, was able to severely reduce net accumulation of exogenous putrescine in HT-29 cells (Figure 4
). Indeed, after 24 h treatment with DADS net accumulation of putrescine inside HT-29 cells was decreased ~3-fold in both floating and adherent cells. In contrast, after 2 days treatment the capacity of HT-29 cells to accumulate putrescine was much more affected in floating than in adherent cells (Figure 4
).

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Fig. 4. Effect of DADS on net accumulation of exogenous putrescine in HT-29 cells. DADS (100 µM) or vehicle alone (i.e. DMSO) was added 1 day after cell seeding and the culture medium was changed every day. Adherent and floating cells were then studied for their capacity to accumulate 10 µM [1,4-14C]putrescine after 90 min incubation at 37°C. Net accumulation was corrected for the L-[1-14C]glucose space. Putrescine net accumulation in control experiments represented 99.5 ± 16.3 and 149.4 ± 33.9 pmol/106 cells/90 min after 1 and 2 days treatment, respectively (n = 4). Results are expressed as means ± SEM and represent four individual experiments. aP < 0.025 versus control; bP < 0.05 versus control.
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In contrast, the capacity of HT-29 cells to oxidize L-glutamine was not significantly modified after a 1 day treatment with 100 µM DADS when comparing adherent and floating cells (Table II
). After a 2 day treatment with DADS it was found that adherent cells were characterized by an increased L-glutamine oxidation capacity when compared with both floating cells and with cells cultured in the presence of vehicle alone. Lastly, HT-29 cell metabolic capacity for de novo glutathione synthesis was not significantly affected by treatment with DADS (Table II
).
Effect of exogenous putrescine on DADS-induced inhibition of HT-29 cell proliferation
When 10 µM putrescine was added to the culture medium together with 100 µM DADS it failed to reverse the growth inhihitory effect of the organosulphur compound towards HT-29 cells after both 24 and 48 h treatment (Figure 5
).

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Fig. 5. Effect of exogenous putrescine on DADS-induced inhibition of HT-29 cell proliferation. DADS (100 µM), putrescine (10 µM) or vehicle (i.e. DMSO) was added 1 day after cell seeding and the number of adherent cells was measured after 1 and 2 days treatment. Results are expressed as means ± SEM and represent four individual experiments.
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Discussion
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Our data clearly demonstrate that the slow down in HT-29 Glc/+ cell growth induced by 100 µM DADS was due to extensive cell release into the culture medium and to transient and partial accumulation of adherent cells in the G2/M phase of the cell cycle. Indeed, after a 1 day treatment with this compound half of the cells present in the culture flasks were floating cells. Taking into account that the medium was replaced every day and that the percentages of detached cells gradually decreased throughout treatment our results are compatible with a selection process which was almost complete after 5 days. The DADS-resistant sub-population of HT-29 cells selected after that period of time were then able to proliferate at a similar rate to the control untreated cells. This treatment allowed us to identify and separately study various sub-populations of tumoural cells with different sensitivities to DADS, referred to as floating and adherent cells, respectively. No inhibitory effects of DADS on adherent cell metabolic capacities (i.e. de novo protein, polyamine and glutathione synthesis) could be measured when compared with control experiments, with the exception of accumulation of the diamine putrescine, which was decreased 3-fold after a 1 day treatment. In addition, adherent HT-29 cells were characterized by a marked accumulation in the G2/M phase after 14 days treatment with DADS. In the second part of this work we studied the sub-population of floating HT-29 cells and found some spectacular phenotypic differences when compared with the adherent sub-population. First, the floating sub-population was characterized after a 1 day treatment by a >75% decrease in de novo protein synthesis capacity when compared with either the control or DADS-resistant cells. This drastic fall was very likely induced by DADS and was not a consequence of mere cell sorting of pre-existing HT-29 cell sub-populations, since the cellular capacity of control cells to synthesize proteins was not equal to the mean capacity of sensitive and resistant cells after correction for their respective numbers. Furthermore, the effect of DADS on protein biosynthesis was not a consequence of a cytotoxic effect of the agent upon HT-29 cells, since other metabolic pathways (such as L-glutamine oxidation and glutathione biosynthesis) were not decreased by DADS treatment. The fact that membrane integrity was also not affected by the treatment reinforces this view. The second metabolic parameter which was severely affected after a 1 day treatment with DADS was polyamine metabolism in floating HT-29 cells. Both net accumulation of putrescine and de novo polyamine biosynthesis were decreased ~3-fold. These metabolic phenomenona, which coincided with almost complete accumulation of floating HT-29 cells in the G2/M phase and with loss of cell anchorage, posed the question of a possible causal link(s) between the different effects of DADS.
Detachment of HT-29 cells following DADS treatment cannot be directly related to accumulation in the G2/M phase since adherent cells also markedly accumulated in the G2/M phase. Similarly, the metabolic alterations recorded in the floating HT-29 cell sub-population (i.e. protein and polyamine metabolism inhibition) were probably not primarily directly linked to accumulation in the G2/M phase, since adherent cells also accumulated in that phase but presented no alterations in these metabolic parameters. In other words, it is likely that the effects of DADS on HT-29 cell metabolic capacities and on cell cycle modification occur independently. In contrast, a causal link may be suspected between the effects of DADS on HT-29 cell polyamine metabolism and on adhesion properties. Indeed, a recent work demonstrated that inhibition of polyamine biosynthesis in epithelial intestinal cells prevented cell attachment to plastic and to different proteinaceous matrices (20).
However, addition of putrescine in addition to DADS to the culture medium did not prevent the effect of the organosulphur compound on HT-29 cell proliferation, indicating that the drastic fall in cell capacity for putrescine accumulation induced by DADS may limit the supply of putrescine required for cell adhesion and proliferation. It should be noted that this lack of effect of added putrescine on cell growth may also be due to other effects of DADS on HT-29 cells (e.g. protein synthesis inhibition). Lastly, DADS did not appear to affect cell differenciation, as judged by measurement of brush border-associated DPP IV activity (33). This result is in contrast to results obtained with erythroleukaemia cells (34), showing that DADS acts as a growth inhihitory and pro-differentiating agent. This suggests that although DADS appears to be an anti-mitotic agent in numerous cancer cell types (12,35,36), the pro-differentiating effect of the agent is dependent on cell type.
Taken as a whole, the data obtained in the present study demonstrate that DADS is able to extensively modify metabolic capacities, adherence properties and cell cycle distribution without affecting cell viability in HT-29 cells. Although in vitro DADS treatment appears to select for a resistant sub-population with a close to normal proliferation rate, this agent represents an interesting anti-mitotic agent, particularly with regard to its marked inhitory effect on both putrescine synthesis (through the oncogene product ODC; 37) and on intracellular accumulation of this diamine. Indeed, agents which inhibit polyamine metabolism are considered to have substantial potential for colorectal cancer prevention and treatment (21,38).
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Notes
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1 To whom corrrespondence should be addressed Email: blachier{at}jouy.inra.fr 
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Acknowledgments
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The authors gratefully acknowledge Sandrine Boguais for secretarial help.
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Received February 2, 2001;
revised April 17, 2001;
accepted April 18, 2001.