Long-term persistence of acquired resistance to 5-fluorouracil in the colon cancer cell line SW620

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Research Article

Long-term persistence of acquired resistance to 5-fluorouracil in the colon cancer cell line SW620 I.K. Tentes a,⁎, W.M. Schmidt b , G. Krupitza c , G.G. Steger d , W. Mikulits d , A. Kortsaris a , R.M. Mader d a

Department of Biochemistry, Medical School, Democritus University of Thrace, 6th km Alexandroupolis-Komotini (Dragana), 68100 Alexandroupolis, Greece b Center for Anatomy & Cell Biology, Währinger Strasse 13, 1090 Vienna, Austria c Institute of Clinical Pathology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria d Department of Medicine I, Medical University of Vienna, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria

A R T I C L E I N F O R M A T I O N

A B S T R A C T

Article Chronology:

Treatment resistance to antineoplastic drugs represents a major clinical problem. Here, we

Received 16 March 2010

investigated the long-term stability of acquired resistance to 5-fluorouracil (FU) in an in vitro colon

Revised version received

cancer model, using four sub-clones characterised by increasing FU-resistance derived from the

2 September 2010

cell line SW620.

Accepted 4 September 2010

The resistance phenotype was preserved after FU withdrawal for 15 weeks (~ 100 cell divisions)

Available online 21 September 2010

independent of the established level of drug resistance and of epigenetic silencing. Remarkably, resistant clones tolerated serum deprivation, adopted a CD133+ CD44− phenotype, and further

Keywords:

exhibited loss of membrane-bound E-cadherin together with predominant nuclear β-catenin

Colon cancer

localisation.

SW620, chemoresistance

Thus, we provide evidence for a long-term memory of acquired drug resistance, driven by multiple

5-fluorouracil

cellular strategies (epithelial–mesenchymal transition and selective propagation of CD133+ cells).

Stem cell

These resistance phenomena, in turn, accentuate the malignant phenotype.

Epigenetics

© 2010 Elsevier Inc. All rights reserved.

Transdifferentiation

Introduction Post-surgery management of colon cancer patients often involves the use of chemotherapy in order to combat residual disease. In advanced disease, administration of chemotherapy including 5fluorouracil (FU) or its prodrug capecitabine is mandatory. However, after long-term exposure to the drug, individual cancer cell sub-clones may arise resulting eventually in a chemoresistant phenotype. In a previous study, our group has investigated resistance with particular emphasis to the progression and staging of the process. The model substance used was FU addressing low, intermediate, ⁎ Corresponding author. Fax: + 30 2551030502. E-mail address: [email protected] (I.K. Tentes). 0014-4827/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2010.09.003

and high-resistance phenotypes. The sub-clones, derived from the metastatic colon cancer cell line SW620 by long-term selection in the presence of the drug, are characterised by stage-dependent gene regulations affecting major cellular functions such as cytoskeleton, cell–cell communication, apoptosis, signal transduction and cell cycle control [1]. Noteworthily, different degrees of chemoresistance address different cellular mechanisms, thus providing to the cancer cell the necessary flexibility to cope with different levels of cytotoxic stress. Amongst differentially expressed genes in the progression of resistance, several genes emerged indicative for specific phenotypic alterations, namely the down-regulation of E-cadherin or the up-regulation of the

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prominin gene encoding CD133 or genes encoding ATP-binding drug transporters. Besides transcriptional control mechanisms, epigenetic regulations are driving forces in malignant processes starting from the very beginning in the form of cancer progenitor cells [2]. Thoroughly described in different phases of tumourigenesis, it has been recently shown that cytotoxics directly interact with the methylation promoter status [3]. In agreement with these observations an epigenetic involvement in chemoresistance is therefore plausible. Considering the temporal resolution of the process of chemoresistance, relatively little attention has been paid to the endurance of resistance phenomena. Noteworthy, the stability of the resistance phenotype at different resistance levels is still an open question. This might be of particular interest after long-term selection in the presence of FU. In the present report, we address several of these questions by studying the stability of the chemoresistance phenotype after withdrawal of the cytotoxic agent. In particular, we investigated a potential epigenetic effect aiding the maintenance of FU chemoresistance, the putative epithelial–mesenchymal transition, and the propagation of tumour side population cells with stem cell characteristics. Our results show that chemoresistance is still maintained after 15 weeks of culturing FU-resistant cell lines in the absence of the drug, and that this effect is not optional to the DNA methylation status. These findings bring forth the issue of memory of chemoresistance as a parameter augmenting the malignant phenotype of cancer cells, thus simulating a therapeutic situation by an in vitro model of clinical significance.

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SW620 + 125 μM FU) were cultivated in parallel in the presence (control experiment) and in the absence of FU, over a period of 15 weeks in total.

Assessment of IC50 for FU for SW620 resistant sub-clones cultivated after withdrawal of the drug Cell proliferation was periodically assessed at 2–3 week intervals using the Cell Titer 96 Non-Radioactive Cell proliferation assay, with MTT (3-4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) from Promega (Madison, WI, USA). Upon splitting, 5000 cells were exposed to log-concentrations of FU (range: 0.01– 10,000 μM). After six days of exposure, the read-out of the proliferation assay was used to generate a dose–response curve. All assays were performed in triplicate.

Serum deprivation

Materials and methods

The parent cell lines SW480 and SW620, as well as the four 5-FUresistant sub-clones were seeded in duplicate T25 flasks at a density of 250,000 cells. On the second day after seeding, serum was removed from half of the flasks, and maintained on the homologous duplicates (controls). All cell lines were maintained with regular changes of media for 30 days whereupon floating cells from the resistant sub-clones were collected, seeded in 96well plates at a density of 2500 cells per well and cultured in a) RPMI 1640 in the absence of serum, b) RPMI supplemented with 0.1% BSA, c) RPMI supplemented with 10% charcoal-stripped serum, d) RPMI supplemented with 10% serum or e) cultured with RPMI in the absence of serum in collagen-coated 96-well plates. After six days of culturing, cell viability was tested by the MTT assay as above.

Cell culturing

5-aza-2′-deoxycytidine treatment of cell lines

The colon cancer cell lines SW480 (the primary adenocarcinoma of the colon, also known as ATCC CCL228) and the corresponding lymph node metastasis SW620 (also known as ATCC CCL227) were obtained from ATCC (Rockville, MD, USA). Standard maintenance conditions involved RPMI supplemented with Glutamax-I, 10% heat-inactivated foetal calf serum (FCS) and 50 μg gentamycin/ml medium (all reagents from Gibco, Paisley, U.K.) at 37 °C in a humidified atmosphere of 5% CO2:95% air. For experimental purposes, cells were harvested at their logarithmic growing phase. Four sub-clones of SW620, resistant to 5, 25, 75 or 125 μM FU were used in the study, previously generated by continuous exposure of the tumour cells to increasing concentrations of FU over a period of more than two years. Starting with the addition of 1 μM FU under standard cell culture conditions, this concentration was gradually enhanced when the growth rate of the exposed cells was similar to that of FU naive cells. 15–31 cell passages were necessary before proceeding to the next higher dose level. The initially observed enhanced rate of cell mortality returned to baseline levels observed under standard cell culture conditions. This adaptation was completed between approximately 10–15 cell passages. Cells grown under continuous exposure to FU still remained susceptible to the compound at very high concentrations [1,4]. For the purpose of the present study, the resistant sub-clones (SW620 + 5 μM FU, SW620 + 25 μM FU, SW620 + 75 μM FU and

The FU-resistant SW620 sub-clones (1 × 106 cells) were seeded into 25 cm2 flasks in the appropriate culture medium and treated with 2 μM 5-aza-2′-deoxycytidine (5-aza-dC) that was added from a freshly-made PBS (1000×) stock. After 48 h incubation, the cells were washed with PBS, and incubated for a further 48 h in medium containing fresh 2 μM 5-aza-dC. Then, they were washed again with PBS, and incubated in medium containing fresh 2 μM 5-azadC. After 48 h incubation, they were finally washed with PBS and fresh medium without 5-aza-dC was replaced [5]. After a final 48 h incubation, they were detached with trypsin and transferred to a 96-well plate at a density of 5000 cells per well, for MTT assay as described above.

Methylation-specific PCR Genomic DNA isolated from tumour cells was subjected to bisulphite modification prior to evaluation by MS-PCR as described previously [6] and purified using the Wizard DNA clean-up system (Promega, Madison, WI, USA). Promoters from five genes encoding enzymes relevant to the fluoropyrimidine pathway were considered: ribonucleotide reductase (large subunit M1), uracil-DNA glycosylase, orotate phosphoribosyltransferase, thymidylate synthase and thymidine phosphorylase (oligonucleotides and sequence data listed in Supplementary Table 1). For cycling, 30 ng DNA (either unmodified or bisulphite treated) were

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subjected to PCR analysis (conditions listed in Supplementary Table 2) and visualised by ethidium bromide staining after agarose gel electrophoresis (2% agarose in TBE buffer). It should be noted here that for testing the epigenetics hypothesis we focused on DNA methylation and designed the 5deaza-dC treatment according to a protocol established for our parent colon cell line in order to avoid any deleterious effect of the agent on the treated cells [5]. Similarly, the epigenetic promoter methylation was done according to an accepted standard procedure [6].

Determination of cell death by cellular staining To measure rates of apoptosis and necrosis, cells were double stained with Hoechst 33258/propidium iodide (HOPI). Cells were seeded at a density of 1 × 104 in 24-well plates and cultivated under serum deprivation. Hoechst 33258 dye and propidium iodide (both from Sigma, St. Louis, MO, USA) were added directly to the culture medium (final concentrations of 5 μg/ml and 2 μg/ ml, respectively) for 1 h at 37 °C. Stained cells were examined under a Zeiss Axiovert 35 fluorescence microscope equipped with a DAPI filter, photographed (Kodak Ektachrome P1600 film; Eastman Kodak Company, Rochester, NY, USA), analysed and counted as previously described [7,8]. Experiments were done in triplicate. After penetration of intact membranes, Hoechst 33258 stains nuclear chromatin thereby allowing the monitoring of nuclear changes associated with apoptosis. Propidium iodide is excluded from viable and early apoptotic cells. Consequently, propidium iodide uptake indicates loss of membrane integrity, one of the characteristics of necrotic and late apoptotic cells. In combination with fluorescence microscopy, double staining with these two dyes allows the differentiation between viable cells with apoptotic phenotypes and non-apoptotic (necrotic) phenotypes.

The effect of 5-aza-2′-deoxycytidine treatment on cell death The 5-FU-resistant cell sub-clones were treated with 5-aza-2′deoxycytidine as before, and then cultured with RPMI 1640 medium without serum for seven days. A duplicate control experiment included homologous cells (also treated with 5-aza2′-deoxycytidine) cultured in RPMI 1640 supplemented with 10% FCS. A second control experiment was also done in parallel with the 5-FU-resistant sub-clones (not treated with 5-aza-2′-deoxycytidine), cultured in the absence of FCS. Cell death was estimated by double staining with Hoechst 33258/propidium iodide (HOPI) as described before.

β-catenin immunostaining For immunostainings, cells were grown on glass slide flasks to different degrees of confluence (<70% confluence or complete confluence). Cells were washed 2–3 times with PBS, fixed with 3.7% formalin in PBS for 30 min at room temperature. Fixation was stopped in NH4Cl (125 mg/50 ml water) for 5 min and washed 5 min with PBS before staining. Cells were subsequently incubated with primary antibody diluted in PBS/0.2% gelatine for 60 min at room temperature. Dilutions of primary antibodies: anti-Ecadherin (Transduction Laboratories (TL), Lexington, UK), 1:100; anti-active β-catenin, clone 8E7 (Upstate Biotechnology, Lake

Placid, NY, USA), 1:100. Cye-dye conjugated secondary antibodies (Jackson Laboratories, West-Grove, USA) were applied for 30 min at room temperature at a dilution of 1:150 in PBS/0.2% gelatine. Imaging of cells was performed with a TCS-SP confocal microscope (Leica, Heidelberg, Germany). Nuclei were visualised using ToPRO3 at a dilution of 1:500 (Invitrogen, Carlsbad, USA).

FACS analysis Cells were trypsinised, counted and washed three times with standard buffer in aliquots of 1 × 106 cells and incubated with fluorescence labelled antibody for 30 min on ice in the dark using the following antibodies: CD44 FITC-conjugated anti-human monoclonal antibody (BD Biosciences, Belgium) and CD133 PElabelled human antibody (Miltenyi Biotec, Germany). On the BD FACS Calibur System, a total of 10,000 cells were counted prior to data analysis with BD CellQuest Pro Software. By an analogue approach, CD44 or CD133 labelled side populations were obtained using a fluorescence activated cell sorter (BD FACS Aria) and seeded on microtiter plates for functional studies.

Statistics The IC50 for FU was calculated by fitting the data to a sigmoid dose– response curve (nonlinear fitting with the software package GraphPad, San Diego, CA, USA). Using the same software, linear regression analysis was performed to assess the trend of chemosensitivity over time.

Results The acquisition of chemoresistance to FU is a stepwise process, involving a wide molecular repertoire. Previous work from our group has shown that the malignant cell is particularly challenged at the beginning of this process, whereas acquisition of the highresistance phenotype seems to be less demanding [1]. The different genes addressed during this process point at different mechanisms underlying the kinetics of chemoresistance as a strongly time-dependent process. In order to extend our current understanding of chemoresistance, we interpreted the results obtained so far by addressing the involvement of phenotypic characteristics together with the stability of chemoresistance as a functional read-out at the cellular level.

Memory of chemoresistance Having outlined the impact of the resistance phenotype on the tumour biologic phenotype in our model, the stability of the resistance phenotype was interrogated by a comparative analysis of sensitivity to FU after long-term culture of the resistant subclones in the absence of cytotoxic stress. Resistance was a stable phenotype over the whole observation period of 15 weeks as proven by very similar results obtained when comparing cellular proliferation in the presence and the absence of FU (Table 1). This memory of chemoresistance was independent of the degree of resistance and included all four investigated levels, i.e. low (SW620 + 5 μM FU), intermediate (SW620 + 25 μM FU), and high resistance to FU (SW620 + 75 μM FU and SW620 + 125 μM FU). For the period studied, there was no

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Table 1 – Comparison of the IC50 in chemoresistant cells cultured either in the absence or in the presence of FU. Cell line CCL227 + 5 μM FU CCL227 + 25 μM FU CCL227 + 75 μM FU CCL227 + 125 μM FU

p IC50 of cells cultured IC50 of cells cultured without FU [μM] with FU [μM] value 724 ± 289

667 ± 332

n.s.

1107 ± 288

1221 ± 411

n.s.

2533 ± 589

2222 ± 483

n.s.

2920 ± 708

2737 ± 1035

n.s.

Values are given as mean ± standard deviation considering the whole observation period of 15 weeks; the statistical significance of the IC50 with and without FU was calculated by the Wilcoxon test.

significant difference in the IC50 between sub-clones cultured in the presence or in the absence of FU (Fig. 1). In order to assess possible alterations of the resistance with time, a trend analysis for the chemoresistant sub-clones of SW620 was performed for a total of 15 weeks corresponding to ~ 100 cell divisions in the presence and in the absence of FU. The linear regression analysis showed no significant deviation from slope 0, neither in the presence nor the absence of the drug. During the maximally reasonable period in culture, there was no sign of attenuation of the acquired resistance phenotype in these cells. This observation was evident in all investigated sub-clones thus covering the whole range of low, intermediate, and high resistance to FU in colon cancer in vitro.

Serum deprivation In order to test for autonomous cell survival or cell growth, serum was removed from the cell culture system. Serum withdrawal led to the complete eradication of the parent cell line SW480 within two weeks, whereas fractions of SW620 were viable for more than 30 days in culture. In none of the parent cell lines re-growth was observed. After an initial growth arrest, cells were considerably reduced in cell number, mainly by necrosis but also by apoptosis down to a surviving fraction of approximately 15%–20% of the initial cell count, with a considerable number of cells surviving as floating in suspension (data not shown). Thereafter, all resistant sub-clones restarted to grow and reached confluence within 31 days after serum deprivation. Resumption of proliferation was independent of the degree of chemoresistance.

Fig. 1 – Trend analysis for the chemoresistant CCL227 cell lines, cultured for a total of 15 weeks in the absence (A) and in the presence (B) of 5-fluorouracil.

Anchorage-independent growth Epigenetic mechanisms Floating cells harvested from all four 5-FU-resistant sub-clones cultured in the absence of serum for 30 days, resumed growth when the culture medium was supplemented with serum (Fig. 2). No floating cells were available from the two parental cell lines SW480 and SW620 (refer to previous paragraph). Both FCS as well as charcoal-stripped FCS supported re-growth in all resistant cell sub-clones. BSA also supported re-growth of the high resistance SW620 + 125 μΜ cell sub-clone, whereas the low resistance SW620 + 5 μM sub-clone also showed growth enhancement on a collagen-coated plate.

The potential effect of the DNA methylation pattern on FU resistance was also investigated using 5-aza-dC as a demethylating agent. The corresponding ratio of IC50 (untreated control vs. 5-aza-dC-treated) for the low (5 μM FU) resistant SW620 cells was 1.25, for the intermediately (25 μM FU) resistant it was 1.23 and for the highly (75 and 125 μM FU) resistant SW620 sub-clones the ratios were 1.0 and 1.27 respectively. It was therefore evident that there was no significant change of FU resistance in the cell lines investigated due to an epigenetic silencing by DNA-methyltransferases. To underpin

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The effect of 5-aza-dC treatment on cell death

Fig. 2 – The effect of cell re-growth brought about by different media (RPMI 1640, RPMI 1640 supplemented with 10% FCS, RPMI 1640 + charcoal-filtered FCS, RPMI 1640 + 0.1% BSA and RPMI 1640 in collagen-coated plates) observed on floating cells harvested from the 5-FU-resistant sub-clones (SW620 + 5 μM FU, SW620 + 25 μM FU, SW620 + 75 μM FU and SW620 + 125 μM FU) that were cultured in the absence of serum for 30 days. For more details, refer to “Materials and methods”.

these results, we investigated the promoter methylation status of five known key enzymes relevant to the fluoropyrimidine pathway. Comparing the chemoresistant sub-clones with the parent cell line, there was no differential DNA methylation at the promoter site (Fig. 3). Epigenetic silencing may be an issue for one of the investigated genes, i.e. thymidine phosphorylase, but was regulated independently from the level of FU resistance.

Treatment of the 5-FU-resistant cell sub-clones with 5-aza-dC, increased the apoptotic death rate brought about by serum deprivation by 3.56-fold in the low (5 μM FU) resistant SW620 cells, 4.78-fold for the intermediately (25 μM FU) resistant and 5.9and 2.74-fold for the highly (75 and 125 μM FU) resistant SW620 sub-clones respectively (Fig. 4). This effect was uniformly observed in all 5-FU-resistant cell sub-clones and was not correlated with the level of chemoresistance. Comparison of apoptotic death rate seen in the 5-FU-resistant cell sub-clones in the absence of both serum and 5-aza-dC to the rate observed in the absence of serum in the presence of 5-aza-dC, revealed that 5-aza-dC brought a marked increase by 3.32-fold in the low (5 μM FU) resistant SW620 cells, 3.87-fold for the intermediately (25 μM FU) resistant and 5.14- and 4.64-fold for the highly (75 and 125 μM FU) resistant SW620 sub-clones respectively (Fig. 4.). This effect was uniformly observed in all 5FU-resistant cell sub-clones and was not correlated with the level of chemoresistance.

Epithelial–mesenchymal transition Starting from previous expression profiling data, the progressive down-regulation of E-cadherin suggested the involvement of transdifferentiation, i.e. epithelial–mesenchymal transition (EMT). In order to test this hypothesis, immunocytochemical stainings for E-cadherin and β-catenin were performed considering different cell densities and the distribution of β-catenin between cytoplasm and nucleus. Indeed, there was a progressive down-regulation of E-cadherin at the cell membrane with a complete absence of the protein in the intermediate and the high-resistant phenotype, whereas the cellular localisation of βcatenin was predominantly nuclear with a small fraction of activated β-catenin in the cytoplasm (Fig. 3). This nuclear localisation of β-catenin was already observed in the parent cell lines SW480 and SW620, suggesting that in our model nuclear β-catenin might not be an informative marker of EMT.

Fig. 3 – Promoter methylation status of key enzymes of the fluoropyrimidine pathway. A = orotate phosphoribosyltransferase; B = ribonucleotide reductase (large subunit); C = uracil-DNA glycosylase; D = thymidylate synthase; E = thymidine phosphorylase; Lanes M: marker, 1: CCL 228, 2: CCL 227, 3–6: resistant sub-clones of CCL 227 (3: 5 μM, 4: 25 μM, 5: 75 μM, 6: 125 μM), 7: positive control, 8: negative control.

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120

100

80

60

40

20

0

-FCS/-5aza-dC

+ 10% -FCS/+5- -FCS/-5FCS/+5- aza-dC aza-dC aza-dC

SW620 + 5µM FU

'+ 10% -FCS/+5- -FCS/-5FCS/+5- aza-dC aza-dC aza-dC

SW620 + 25µM FU

Total apoptotic cells

'+ 10% -FCS/+5- -FCS/-5FCS/+5- aza-dC aza-dC aza-dC

SW620 + 75µM FU

Necrosis

'+ 10% -FCS/+5FCS/+5- aza-dC aza-dC

SW620 + 125µM FU

Cells Alive

Fig. 4 – The effect of DNA demethylation on apoptotic and necrotic death of 5-FU-resistant sub-clones derived from the cell line SW620. All cells were treated with 5-aza-2′-deoxycytidine; FCS was then withdrawn for a period of seven days, whereupon cell death was estimated with double Hoechst/propidium iodide staining. For comparison, homologous cells (also treated with 5-aza-2′-deoxycytidine) were cultured in the presence of 10% FCS, and a control experiment was done, showing the 5-FU-resistant sub-clones (not treated with 5-aza-2′-deoxycytidine), cultured in the absence of FCS. All methods are described in “Materials and methods”.

These observations were independent of cell density (70% confluence vs. complete confluence of cells in culture; data not shown). Taken together, this data support the assumption that EMT by loss of E-cadherin could be a decisive determinant of resistance to FU at the cellular level.

Expansion of a CD133-positive cell population Previous gene expression data indicated the sharp up-regulation of the prominin gene encoding the CD133 molecule. In order to follow up this finding, cells were stained and analysed by FACS. Again, the array data were confirmed at the protein level with a strong CD133 positive phenotype at the intermediate and the high-resistance sub-clone (Table 2). Remarkably, the low expression of CD133 on the primary adenocarcinoma was accom-

Table 2 – Expression of CD44 and CD133 on naïve and chemoresistant colon cancer cell lines. Cell line CCL228 CCL227 CCL227 + 5 μΜ FU CCL227 + 25 μΜ FU CCL227 + 75 μΜ FU CCL227 + 125 μΜ FU

+

CD44

cells [%]

58.4 ± 9.2 9.1 ± 1.3 13.2 ± 2.6 18.5 ± 2.8 2.7 ± 0.1 1.9 ± 0.2

+

CD133

panied by a discrete staining in the lymph node metastasis (32.6% positive). In parallel, the colon stem cell marker CD44 decreased from 58.4% in SW480 to 9.1% in SW620 with an almost complete down-regulation in the high-resistant phenotype (Table 2). The relationship between CD44 and CD133 expression and degree of resistance was assessed by sorting of cells into CD133 negative and positive cells. These experiments were performed in SW480 (isolation of CD44-positive cells), SW620 (isolation of CD133positive cells and double negative cells) and the sub-clone resistant to 125 μM FU (isolation of double-positive cells). CD133-positive cells either single or CD44/CD133 doublepositive cells were significantly less sensitive to FU when compared with the unsorted cell populations (mean factor: 2.03; p < 0.01). Given that our cellular models exhibit levels of resistance up to a factor of 190, this ratio was considered to be of minor influence. The expansion of a CD133-positive and a CD44negative cell pool, however, parallels the progression of resistance to FU (Fig. 5).

Time kinetics of CD44 and CD133 regulation

cells [%]

9.1 ± 0.1 32.6 ± 1.7 86.3 ± 1.1 95.3 ± 2.4 93.5 ± 6.4 98.0 ± 0.6

Values are given as mean±standard deviation from individual experiments.

In order to learn more about the kinetics of CD133 induction during exposure to FU, naive cells were incubated with 1 μM FU and analysed up to 8 days for CD44 and CD133 by FACS. Within this period, on the surface of SW620 or SW480 no significant changes of the examined antigens from baseline levels were observed thus excluding an imminent contribution of CD133 regulation to the acute phase of cellular stress response or a selection of a CD133 positive side population (data not shown).

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Fig. 5 – E-Cadherin (A) and β-catenin (B) stainings in chemoresistance. Disassembly of epithelial cell-to-cell junctions and nuclear β-catenin accumulation after treatment with different concentrations of FU. (A) Confocal immunofluorescence images after staining with specific antibodies against E-cadherin. (B) Confocal immunofluorescence images after staining against active β-catenin (active β-cat). Nuclei were visualised using To-PRO3.

Discussion In the present study, the time dependence of chemoresistance was evaluated in four cell lines derived from the metastatic SW620 colon cancer cell line under continuous exposure to low (5 μM), intermediate (25 μM) and high (75 and 125 μM) concentrations of FU. Considering the population doubling times (range: 20.3–25.8 h), all cell sub-clones are characterised by unrestricted proliferation rates in the presence of the corresponding concentrations of FU. As a consequence, cell cycle effects are excluded as a study bias, which allows for the direct comparison of the reported data. This is relevant when considering results from other groups, which repeatedly reported cell cycle perturbations in acquired resistance to FU. These alterations were characterised by impaired transition through the G1/S phase of the cell cycle [9] with a subsequent decrease of the mitotic activity in combination with down-regulation of the corresponding genes [10]. Similar observations were also made under hypoxic conditions, where resistance to FU was associated with the up-regulation of cell cycle inhibitors [11]. The ability of these sub-clones still maintaining chemoresistance after prolonged culturing in the absence of the drug was examined. Despite drug withdrawal, there was no statis-

tically significant difference in drug sensitivity assessed as IC50 by comparison to the homologous control cultures for a period of 15 weeks. According to a trend analysis performed by linear regression (Fig. 1), the IC50 values for all chemoresistant cell sub-clones cultured in the absence of FU were not significantly altered for the whole period of 15 weeks. The observation period of 30 passages (~ 100 cell divisions) corresponds practically to the natural lifespan of the cells, when cells in culture still maintain their initial phenotype. Therefore, the observed long-term resistance in the absence of FU can be considered as a memory effect acquired previously under longterm exposure. This conjecture is further supported by the fact that CD133+ clones did not develop in consequence of a CD133+ subpopulation but through de novo acquisition of CD133 positivity correlating with resistance. The observed long-term memory as a stand-by survival phenotype points at a hereditary base, which is also compatible with the stability of chemoresistance or months after removal of the cytotoxic stress. In contrast to FU, another inhibitor of the folate metabolism, i.e. methotrexate, triggered the amplification of the dihydrofolate reductase gene thus escaping the drug's effect [12]. This amplification, however, was not stable over time and was lost after withdrawal of the drug indicating that methotrexate-

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resistant cells may become responsive to the same anticancer agent after a wash-out period even if there is a genetic determinant of resistance such as double minutes [13]. In search for an explanation of this long-term phenomenon, potential epigenetic changes during the process of acquired drug resistance were considered as part of the aetiology of the longterm memory of this survival phenotype in analogy to previous studies of our group focusing on the role of DNA-methyltransferases during colorectal tumourigenesis [14]. Such changes have been observed in the case of the DNA synthesis inhibitor 5-aza-dC inducing genome-wide DNA hypermethylation and low frequency silencing of thymidine kinase as a consequence of drug resistance in cell culture [15]. It was therefore anticipated that the contribution of epigenetic changes to drug resistance could be a possible link between the genetic information, its expression and the resulting (chemoresistant) phenotype. According to previous data from the expression profiling studies on the same FU-resistant cell lines, the majority of genes were down-regulated as a result of FU resistance thus suggesting that DNA demethylation could restore the expression patterns of these genes and hence their former functions, eventually affecting cell sensitivity to FU [14]. To this end, we tested drug sensitivity after treating the FU-resistant SW620 sub-clones with 5-aza-dC as a known demethylating agent. Apparently though, there was no significant change to FU sensitivity following demethylation in any of the tested CCL 227 sub-clones. As a paradigm, we analysed the promoter methylation status of five key enzymes of the fluoropyrimidine pathway. Again, there was no epigenetic silencing associated specifically with the level of drug resistance. In order to link epigenetics to the described hereditary base of resistance, alterations in histone modification and/or chromatin remodelling as alternative to DNA methylation provide an attractive hypothesis. Initial expression profiling results [1] indeed revealed resistance-associated differential regulation among histone cluster genes as well as the specific up-regulation of the SMARCA1 gene (also known as SNF2L or NURF140), encoding a member of the SWI/SNF family of transcriptional regulators that alter the chromatin structure around target genes. Thus, although not tested herein, FU-resistance related gene expression changes lend support to this hypothesis. The investigated enzymes were chosen as landmarks in the 5FU conversion pathway, in the full knowledge that they represent late events in the course of the escalation of drug resistance in our model system [14]. A recent report [21], has provided evidence that chromatin modification is responsible for a drug-tolerant state in cancer cell subpopulations. Chromatin modification is a key mechanism underlying the phenomenon, requiring histone demethylation. This finding although studied in cell lines other than colon cancer, is supportive of our observation that DNA demethylation does not affect drug resistance. On the other hand, in the present study we provide indirect evidence that specific/targeted promoter methylation is nevertheless aiding survival of the 5-FU-resistant cell sub-clones. Apoptosis is optional to epigenetic regulation through promoter methylation of pro-apoptotic genes (caspase-8, Apaf-1) aiding survival of cancer cells [22]. In the resistant cell sub-clones tested herein, seven pro-apoptotic genes (BIRC5\survivin, Caspase 6, Chemokine rec CXCR4, Nbk\Bik, Perforin-1, Galectin-1, EMP3) sustained down-regulation as a result of 5-FU resistance [1], and the resistant sub-clones survive culturing in

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the absence of serum for more than 30 days. Under this perspective, the 5-FU-resistant sub-clones could survive in the absence of serum by evading apoptotic cell death through promoter methylation, a trait acquired as a result of drug resistance. We have observed that 5-deazadC treatment abolished survival by increasing the apoptotic death rate of all 5-FU-resistant sub-clones cultured in the absence of serum. Apoptosis brought about by demethylation in the 5-FU-resistant cell sub-clones validated our previous observation from expression profiling [1]. This effect had no correlation to the degree of 5-FU resistance, possibly supporting the postulate that most critical changes relating to the acquisition of the resistant phenotype are installed early, at the onset of the phenomenon of chemoresistance. The abolishment of apoptosis by an epigenetic mechanism as a result of 5-FU resistance may highlight an alternative or accessory mechanism for maintaining the drug-tolerant state of the colon cancer cell sub-clones tested. Considering the matter of the long-term chemoresistance, we sought to validate previous data from gene arrays showing that chemoresistance correlated with down-regulation of E-cadherin in the resistant sub-clones. In doing so, we addressed the issue that chemoresistance could affect cell–cell adhesion, compromise epithelial integrity and lead to a more invasive cancer cell phenotype. Results obtained in our immunostaining study validated E-cadherin down-regulation at the cell membrane, and further showed that in the absence of E-cadherin active β-catenin is distributed in the nucleus as well as in the cytoplasm and that this intracellular distribution is independent of the degree of chemoresistance. In the present case, both the naïve SW480 and SW620 cell lines express a truncated form of the APC protein, which nevertheless is still able to bind β-catenin in the cytoplasm, and does not seem to affect per se its subcellular distribution [16]. Therefore, the genetic aberrations in both cell lines would not preclude the observations associated with an EMT phenotype, namely the loss of β-catenin membrane localisation due to decreased E-cadherin binding in the high-resistance phenotype. This phenomenon may also be observed by altered binding affinity of β-catenin to E-cadherin described previously in SW480 [17]. In this investigation, the authors showed an impact of cellular confluence on the interaction between truncated APC, β-catenin, and E-cadherin. This interaction was excluded as experimental bias by reproduction of our results in both dividing and confluent colon cancer cells. Finally, in combination with our previous expression profiling data, E-cadherin down-regulation in the resistant cell lines was paralleled by concomitant up-regulation of the Zeb-1 E-cadherin promoter repressor in the resistant cell lines, and loss of the epithelial marker Mplz2 (Eva1). According to a recent report by Arumungam et al. [18], these events relate to patterns of sensitivity of cancer cells (pancreatic) to conventional chemotherapeutic agents (5-FU) and the mutually exclusive expression pattern of E-Cadherin and Zeb-1 could be considered as a key point in developing potential therapeutic strategies. Also by reference to our expression profiling data, fibronectin up-regulation was observed, supporting EMT as an early event correlating with acquisition of 5-FU resistance [1]. Loss of ZO-1 (TJP1) was also observed, as well as down-regulation of 5 cytokeratin genes (KRT5, KRT7, KRT81 and KRT86) [1]. Under this perspective, we have indeed observed some of the hallmarks of transdifferentiation in our system. Whether this phenomenon is a single element in the course of the propagation

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of chemoresistance or a more integral part in a plethora of mechanisms, is a point that certainly deserves further investigation. Being discussed in association with the cancer stem cell phenotype, the positive correlation of the CD133 positive phenotype with the intermediate and the high-resistance subclone was an observation in line with previous data. Compared with the low expression of CD133 in the primary adenocarcinoma SW480, the lymph node metastasis SW620 was positive to a higher percentage. In parallel, the colon stem cell marker CD44 sharply decreased from 58.4% in SW480 to 9.1% in SW620 and was hardly detectable in the high-resistant phenotype. This counteregulation was, however, of little influence when assessed at the functional level. Although the IC50 of FU in the sorted side populations differed significantly when compared with the unsorted cell populations by a mean factor of 2.03, we tend not to over interpret this finding. This was also supported by the time kinetics of the CD133 induction in our model, as FU exposure of naive SW620 and SW480 cells did not significantly change within 8 days undermining the necessity of these antigens as an immediate cellular stress response. Nevertheless, the expansion of a CD133-positive and a CD44-negative cell pool occurs in parallel to the progression of resistance to FU and may therefore serve as a biomarker for other functionally related genes, which need to be determined so far. Possible candidate molecules related to cell signalling in CD133 or CD44 positive cells have been individualised in PI3K survival signalling [19] and insulin-like growth factor receptor-I mediated signal transduction [20]. In contrast to our model, in HT29 exposed to oxaliplatin and FU a CD44 positive side population was expanded. The positivity for CD133 was similar to that of the resistant CW620 sub-clones employed in our investigations. In these investigations, the functional role of CD133 and CD44 has not been addressed [19]. Another feature of the 5-FU-resistant sub-clones is their ability to manifest anchorage-independent growth and survive as single dividing cells floating in suspension when challenged with longterm (30 days) serum deprivation. The floating cells so obtained, can re-grow when the serum or even part of its constitution (charcoal-filtered serum) is restored; collagen or surrogate substrates facilitating adhesion (BSA) were also partially effective in restoring growth. The low resistance SW620 sub-clone (grown at 5 μM FU) displayed growth enhancement on a collagen-coated plate, and BSA supported re-growth of the high-resistance SW620 + 125 μΜ cell sub-clone. Anchorageindependent growth was therefore observed as a feature emerging in coherence with 5-FU resistance, also ceding an increasing degree of autonomy to the 5-FU-resistant sub-clones for survival in the absence of survival factors. This is a feature exclusive of the resistant sub-clones, as both SW480 and SW620 cell lines cannot withstand serum deprivation. If a pool of resistant cells is present within, and maintained in the absence of serum, then this pool may further increase and be encouraged to grow independently of anchorage, having evaded the process of anoikis. As a conclusion, the observed in vitro FU resistance of the four SW620 cell clones is most possibly due to de novo acquisition of an altered genetic background and its sui generis acquired patterns of expression established as a result of long-term exposure to the drug. This genetic background could enable trained cells to withstand cytotoxic stress without any growth impairment for at least 100 cell generations. It is therefore very likely that similar phenomena may

also be observed in clinical situations, e.g. in the form of residual disease of chemoresistant cancer cells, which may explain relapses and the often observed poor host's response to chemotherapy. At the cellular level, FU resistance seems to be related to overlapping mechanisms, which are observed concomitantly in our model. Those reflect the overlapping of mechanisms usually attributed to transdifferentiation in the form of EMT or the expression of CD133 as a marker associated to the stem cell phenotype. It is fair to say that our findings refer to chemoresistant subclones derived from a single cell line. Thus, the present study certainly needs further validation, possibly in similar cancer cell models in order to evaluate whether the potential mechanisms reflect generic molecular consequences of chemoresistance or individual cell-specific effects brought about by natural selection.

Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.yexcr.2010.09.003.

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