Transformation of immortalized colorectal crypt cells by microcystin involving constitutive activation of Akt and MAPK cascade
Yongliang Zhu 1,
Xian Zhong 2,
Shu Zheng 2, *,
Zhen Ge 2,
Qin Du 1 and
Suzhang Zhang 2
1 Department of Gastroenterology and 2 Cancer Institute, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
* To whom correspondence should be addressed. Tel: +86 571 87784501; Fax: +86 571 87214404; Email: zhengshu{at}zju.edu.cn
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Abstract
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It has been shown by epidemiological and animal studies that microcystin is an important exogenous factor involved in the carcinogenesis of colorectal cancer (CRC). However, details of the mechanism remain unclear. Transformation of colorectal cells is an important initial step in carcinogenesis. Whether microcystin is capable of transforming immortalized colorectal crypt cells, and what the mechanism might be, was investigated. In the present study, we demonstrated that immortalized colorectal crypt cells could be transformed by microcystin. Transformed colorectal crypt cells showed an anchorage-independent growth phenotype, and the proliferation activities of microcystin-transformed cells were also greater than that of immortalized colorectal crypt cells. The Akt and the p38, JNK of mitogen-activated protein kinase (MAPK) pathways in microcystin-transformed cells were found to be constitutively activated. In microcystin-transformed cells, PI3K, MAPKAPK2, Akt, cyclin D1 and cyclin D3 in the Akt pathway; IQGAP-2, RabGTPase, Rap1GAP, RasGAP, R-Ras, Krev-1 and TC21 of the Ras GTP/GDP protein family; and A-Raf, B-Raf and PAK in the Ras/MAPK pathway were all markedly upregulated. However, in positive control cells, dimethylhydrazine-transformed cells, only the Akt pathway was activated by PI3K, and no evidence of alteration of any molecules of the Ras superfamily was observed. Inhibition of Akt, p38 and JNK activation led to a reduced proliferation of microcystin-transformed cells. This implies that the constitutive activation of Akt and the p38, JNK of MAPK pathways in microcystin-transformed cells may be the mechanism by which this important external factor acts in the carcinogenesis of CRC.
Abbreviations: 17-AAG, 17-(allylamino)-17-demethoxy-geldanamycin; CRC, colorectal cancer; DMH, 1,2-dimethylhydrazine; DMSO, dimethyl sulfoxide; FAK, focal adhesion kinase; GSK-3, glycogen synthase kinase-3; IVT, in vitro transcription; MAPK, mitogen-activated protein kinase; MAPKAPK2, MAPK activated protein kinase 2; MCLR, Microcystin LR; MTT, methyl thiazolyl tetrazolium; PI3K, phosphatidylinositol 3 kinase; PP1, serine/threonine protein phosphatase 1; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole; SP600125, 1,9-pyrazoloanthrone; SV40 LT, SV 40 large T antigen
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Introduction
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Colorectal cancer (CRC) is one of the most common malignant tumours in China (1,2). CRC typically develops over decades and involves multiple genetic events during carcinogenesis. It is generally believed that one of the initiating steps in colorectal carcinogenesis is a mutation in stem cells or its early progenitor, whereby colorectal stem/progenitor cells are modified by internal and external factors, and where cancer develops after a series of events, including tumour initiation, promotion and progression (3,4). External factors play an important role in the carcinogenesis of CRC (5). In our previous study, the high risk factors found in China were identified through a series of epidemiological studies. The incidence of CRC was decreased by 31.7%, after population prevention and in a 10 year follow-up, which also indicated an important role for external factors in the carcinogenesis and development of CRC (6). Among the numerous other high risk factors, the contamination of drinking water by microcystin LR (MCLR) is considered to be an important external factor in the carcinogenesis of CRC in some rural areas of China. The fatality rates for CRC were found to increase when there was a contamination of drinking water by MCLR in epidemiological studies performed in Haining county, China, where the incidence of CRC is the highest (7,8). Animal studies also indicate that the presence of aberrant crypt foci in the mouse colon is caused by MCLR (9). Although it was shown in these results that MCLR is an important factor in CRC, the pathogenesis remains unclear.
MCLR is a cyclic hepatotoxin peptide released by blue-green algae (cyanobacteria), which is toxic to animals and humans. MCLR has been shown to promote hepatocarcinoma in experimental animals and is significant in the carcinogenesis of human liver cancer (10,11). MCLR is a potent inhibitor of the serine/threonine protein phosphatase 1 (PP1), PP2a (12). It has been shown in recent studies that MCLR could interfere with cellular signalling pathways, one of the important properties of this toxin, through the activation of protein kinase C (13). The inhibition of PP2a by MCLR activates tyrosine kinase-coupled G protein receptors, leading to phospholipase C activation and to the production of diacylglycerol from phosphatidyl inositol 4,5-diphosphate. The diacylglycerol activates protein kinase C and then upregulates mitogen-activated protein kinse (MAPK) cascades (13,14). However, to date, the alteration of the genes upstream of MAPK has remained poorly understood.
In the development of normal intestinal mucosa, through epithelial proliferation, benign adenoma, adenocarcinoma, to metastatic cancer, CRC originates from stem cell/progenitor or crypt cells (3,4,15,16). This has lead to colorectal crypt cells becoming classed as an ideal target for the investigation of the molecular alteration events of carcinogenesis. Transformation of colorectal cells is an important initial step in carcinogenesis (17,18). Whether MCLR is capable of transforming normal colorectal crypt cells in vitro and what the mechanism might be after cell transformation remains unknown. Normal colorectal crypt cells are difficult to culture; therefore, a conditionally immortalized normal human colorectal crypt epithelial cell line (NCC), with the same phenotype as normal crypt cells, which was successfully established in our previous study, was employed as a target (19). It was shown that the colorectal crypt cells transformed by MCLR possess a greater proliferation activity and that this is associated with a constitutive activation of the Akt and the MAPK pathways.
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Materials and methods
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Cell line
The NCC cell line, a conditionally immortalized normal human colorectal crypt epithelial cell line, was established by the ectopic expression of human telomerase reverse transcriptase (hTERT) (gift from Professor Robert A. Weinberg, Whitehead Institute, MA, USA) and SV40 large T (SV40 LT) antigen, as described previously (19). Briefly, colorectal epithelial cells were separated from a human fetus by dispase I digestion and were subsequently cultured on a Matrigel matrix (Becton & Dickinson). The primary cultured cells were then transfected with recombinant virus, containing either hTERT or SV40 LT. No evidence of tumorigenesis was found in nude mouse experiments or in an in vitro soft agarose (Sigma) colony test. This study was approved by the Review Board of Zhejiang University School of Medicine.
Transformation of NCC cells by MCLR
NCC cells were seeded (1 x 104 cells) in 35 mm culture dishes, in which the medium was changed to fresh RPMI 1640 medium (without antibiotic) (Gibco BRL), including MCLR and 1,2-dimethylhydrazine (DMH) (as a positive control) (Sigma) at different concentrations (1, 0.1, 0.01, 0.001 and 0.0001 µg/ml and 1, 0.1, 0.01, 0.001 and 0.0001 µg/ml, respectively). Meanwhile, as a negative control, NCC cells were cultured in fresh RPMI 1640 medium. All cell dishes were incubated under 5% CO2 at 32.5°C for 24 h. Cells were washed with serum-free RPMI 1640 medium three times the following day. The cells were mixed with 0.4% agarose (1x RPMI 1640 medium, containing 10% fetal calf serum and 100 U/ml gentamicin), pre-heated at 42°C after being digested by 0.25% trypsin with 0.02% EDTA. In each group, three wells of six-well plates (covered with 1 ml of 0.4% agarose) were inoculated and were then incubated at 4°C for 15 min. After agarose solidification, the plates were covered with 0.5 ml of 0.4% agarose and 0.5 ml medium and cultured under 5% CO2 at 32.5°C overnight. After being hermetically cultured in the above conditions for 28 days, vigorous colonies were screened in six-well plates and washed by serum-free medium twice to remove the free agarose. The cells were then cultured in fresh RPMI 1640 medium with 20% fetal calf serum and the medium was replaced every 3 days until the clones grew sufficiently for further culturing. MCLR and DMH-transformed cells are termed here MTC (MCLR-transformed cell) and DTC (DMH-transformed cell), respectively.
Analysis of cellular proliferation activity
MCLR, DMH (MTC, DTC) and NCC cells were seeded in 96-well plates after cell synchronization and cultured with 5% CO2 at 32.5°C for 72 h, 10 µl methyl thiazolyl tetrazolium (MTT) dye was added, and the mixture was incubated under 5% CO2 at 32.5°C for 4 h. After centrifugation at 1000 r.p.m. (low-speed plate centrifuge, LDZ5-2) for 10 min, the supernatants were removed and the precipitate was mixed with 300 µl dimethyl sulfoxide (DMSO). The absorbencies at 570 nm were then measured with an enzyme reader (
9600 reader).
Determination of serine/threonine protein phosphatase PP2a, PP2b and PP2c activities of transformed cells
All procedures were carried out according to the manufacturer's protocols (Promega). Cells in a 50 ml culture bottle were digested and centrifuged and then washed with saline three times. About 1.8 ml TrisEDTA buffer, pH 7.5, containing 1% Triton X-100 was added to induce lysis. After centrifugation at 13 000 r.p.m., the supernatants were collected and the protein concentrations determined. Five hundred microliters of supernatant for each group was then passed through Sephadex G-25 and eluted with 100 ml with the following buffers: for PP2a, 5x buffer (250 mM imidazole, 1 mM EGTA, 0.1% ß-mercaptoethanol and 0.5 mg/ml BSA); for PP2b, 5x buffer (250 mM imidazole, 1 mM EGTA, 50 mM MgCl2, 5 mM NiCl2, 250 µg/ml calmodulin and 0.1% ß-mercaptoethanol); and for PP2c, 5x buffer (250 mM imidazole, 1 mM EGTA, 25 mM MgCl2, 0.5 mg/ml BSA and 0.1% ß-mercaptoethanol). Thirty-five microliters of each elution buffer was added to 96-well plates containing 10 µl 5x reaction buffer and 1 µl peptide phosphate and the mixture was incubated at room temperature for 10 min, with a standard curve being prepared. Finally, 50 µl molybdate dye/additive mixture (100:1) was added. Then the plates were incubated at room temperature for 10 min and the absorbency of each well was determined at 630 nm.
Genechip analysis
The total RNA from MTC, DTC and NCC cells was extracted and purified using RNeasy mini kits (Qiagen). Total RNA (10 µg) was converted into double-stranded cDNA by reverse transcription using the T7T24 primer set (Affymetrix) and subjected to phenolchloroformisoamyl extraction. For cRNA conversion, the in vitro transcription (IVT) MEGAscriptTM T7 kit (Ambion), along with biotinylated nucleotides, was used. The IVT product was purified using RNeasy mini columns and fragmented. Fragmented IVT product was then hybridized to an U95Av2 Genechip (Affymetrix) and washed, as suggested by the manufacturer. Each hybridized Affymetrix Genechip array was scanned with an argon-ion laser scanner (Affymetrix) at 570 nm. Initial absolute and comparative analysis of the data images was performed with Affymetrix custom image analysis software (Genechip version 3.1). The difference between immortalized colorectal crypt cells and transformed cells were compared by unilateral and bilateral ANOVA statistical analysis. The detection specificity of the genechip was 1/100 000 copies. Expressions of the target genes were judged to increase if the value was
1.5 times than that of the immortalized colorectal crypt cells and to decrease if it was
0.5 times than that of the colorectal crypt cells. The gene analytic data were from the GenBank database (http://www.ncbi.nim.nih.gov) or the Gene Cards database (http://bioinf.weizmann.ac.il/cards/index.html), except as indicated in the references. Quantitative real-time PCR was performed to verify B-Raf, R-Ras, Rb and procollagen 1
, which were altered significantly, and were used to evaluate the reliability of the Genechip detection. The results were consistent with that expected for Genechip detection (data not shown).
Western blot assay
Subconfluent MTC, DTC and NCC cells were harvested by treating with 0.25% trypsin containing 0.1% EDTA in Hank's saline (Gibco BRL), the digest being terminated by the addition of complete medium. Collected cells were washed three times with 10 mM PBS (pH 7.4). Cells were then lysed with lysis buffer at 100°C for 10 min, and centrifuged at 13 000 r.p.m. for 30 min, respectively. After the protein concentrations were adjusted to the same volume, the cell lysates were subjected to SDSPAGE and were subsequently transferred onto polyvinyl difluoride membranes (Bio-Rad). The blots were blocked with 1% BSA in Tris-buffered saline with 0.05% Tween-20 at room temperature for 2 h, diluted primary antibody added (p38 1:100, JNK 1:200, Akt 1:200, cyclin D1 1:200, HER2 1:100 and inter-reference SV40 T antigen monoclonal antibody 1:500, respectively; all antibodies were purchased from Santa Cruz Biotechnology) at 37°C for 2 h, followed by an anti-rabbit/goat/mouse secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology). Visualization was carried out using the ECL detection reagent (Santa Cruz Biotechnology), exposing the blots to Fuji medical X-ray film, and developing the film using an automatic developer.
In vitro Akt, p38, JNK and MAPKAPK2 kinase assays
Ten micrograms of cell lysate from MTC, DTC or NCC cells was incubated with 10 µl of assay dilution buffer (20 mM MOPS, pH 7.2, 25 mM glycerophosphate, 20 mM MgCl2, 5 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol and 1 mM sodium vanadate) containing 2 µg of substrates, respectively. Substrates used were as follows: for Akt, glycogen synthase kinase-3 (GSK-3) fusion protein (Cell Signaling Technology); for P38, ATF2 (Cell Signaling Technology); for JNK, soluble GST-C-jun (Cell Signaling Technology); and for MAPK activated protein kinase 2 (MAPKAPK2), the HSP27 peptide (RRLNRQLSVA). Five micrometers of cAMP-dependent protein kinase inhibitor peptide was used, and the reaction was initiated by the addition of 5 µl of ATP (250 µM ATP and 1 µCi (
-32P]ATP) followed by incubation at 30°C for 10 min. Subsequently, 20 µl of the reaction mixture was spotted onto Whatman P81 filter paper, washed extensively with 1% phosphoric acid, and then analysed in a scintillation counter after the addition of 250 µl of scintillation fluid. Each assay was performed in duplicate and repeated twice (20).
Phosphatidylinositol 3 kinase (PI3K) assay
Briefly, cells were lysed in 137 mM NaCl, 20 mM Tris, pH 7.5, 1 mM MgCl2, 10% glycerol, 1% Triton X-100, 10 mM phenylmethylsulfonyl fluoride and 10 µM each of leupeptin, aprotinin and soybean trypsin inhibitor. Detergent lysates were immunoprecipitated with the p85-PI3K antibody for 3 h, the beads were then washed twice with lysis buffer and three times with TrisHCl (pH 7.4). Subsequently, the precipitated immunocomplex was prepared by drying with nitrogen and resuspending in 10 µl of 30 mM HEPES. This was added to wash the beads, and the tube was then left on ice for 10 min. Forty microliters of kinase buffer (30 mM HEPES, 30 mM MgCl2, 50 µM ATP, 200 µM adenosine and 10 µCi [
-32P]ATP) was then added to each tube and the reaction allowed to proceed at room temperature for 15 min. The reaction was stopped with 0.1 N HCl and the lipids extracted with 200 µl of chloroform/methanol (1:1). The products were separated on potassium oxalate pre-treated TLC plates by developing with chloroform, methanol, water and 30% ammonium hydroxide (112:88:19:6, by vol). After drying, the plates were exposed to autoradiography and the phosphorylated products quantified by excising the spot and scintillation counting (20).
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Results
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NCC cells were transformed by MCLR or DMH
NCC cells were cultured in monolayer and displayed obvious intercellular contact inhibition. Transformed cell colonies emerged at the 28th day in soft agarose plates, when the cells were processed by MCLR or DMH for 24 h (Figure 1). However, the relationship between the number of cell colonies transformed by MCLR and the MCLR concentration was unclear. This may have been on account of the Rb and P53 proteins of NCC cells being captured by the SV40 LT antigen, increasing the sensitivity of NCC cells to carcinogens (21). The transformed cell colonies have been continuously cultured, with >40 passages to date. Compared with immortalized NCC cells, the transformed cells grow vigorously in multilayers (anchorage-independent growth), with no intercellular contact inhibition and with colonies in soft agarose plates surviving multiple passaging, indicating the stability of the transformed phenotype of these cells.

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Fig. 1. NCC cells were transformed by MCLR or DMH. A total of 1 x 104 cells were used for a colony test, as described in Materials and methods. The panels show the photographs of the transformed cell colonies in soft agarose plates. (A) No transformed cell colonies emerged at day 28 (at x20 magnification). (B) NCC cells were transformed by MCLR (at x20 magnification). (C) NCC cells were transformed by DMH (DTC) (at x40 magnification).
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Determination of cell proliferation and serine/threonine protein phosphatase activities in MTC, DTC and NCC cells
The proliferation activities of transformed cells, which grew anchorage independently, were checked to see if they differed from that of the immortalized cells. After cell synchronization, the proliferation activities of MTC and DTC cells at the fifth passage were greater than that of immortalized NCC cells, as detected by MTT assay (MTC versus NCC, DTC versus NCC, P < 0.05, respectively, Student's t-test) (Figure 2A).

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Fig. 2. Determination of cell proliferation and serine/threonine protein phosphatase activities in MTC, DTC and NCC cells. (A) Determination of cell proliferation activities in MTC, DTC and NCC cells. Cells were cultured to the fifth passage, MTC and DTC cells were seeded in 96-well plates at 1 x 103 cells/well and cultured for 72 h. The cell proliferation activities of the transformed cells, detected by MTT assay, were greater than the value for the immortalized NCC cells (MTC versus NCC, DTC versus NCC, P < 0.05, respectively, Student's t-test). (B) Determination of serine/threonine protein phosphatase activities in MTC, DTC and NCC cells. The PP2a, PP2b and PP2c activities of MTC and DTC cells (1 x 106 cells) were determined using a chemical substrate assay (see Materials and methods). No statistical difference between MTC or DTC cells and NCC cells was found (all P > 0.05, Student's t-test).
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We next determined if the enhanced proliferation activities of transformed cells was related to an inhibition of serine/threonine protein phosphatases (PP2), leading to the promoted cellular proliferation activity. The PP2a, PP2b and PP2c activities of NCC and transformed cells were compared using a chemical chromogenic substrate assay. No statistical differences between the respective values of MTC or DTC and NCC cells were found (MTC versus NCC, P > 0.05; DTC versus NCC, P > 0.05, Student's t-test) (Figure 2B), consistent with PP2a, PP2b and PP2c activities being not inhibited in MTC cells.
Analysis of alteration of cell signalling pathways in MTC and DTC cells
The phenotypes and proliferation activities of MTC cells altered markedly compared with NCC cells. We, therefore, examined which signalling pathways might be responsible. The results were determined using an U95Av2 Genechip. It was found that Akt, and the p38, JNK of MAPK and focal adhesion kinase (FAK) signalling pathway molecules in MTC cells were upregulated, compared with NCC cells (Akt and MAPK pathways showing the greatest change). However, the Akt pathway was also upregulated in DTC cells (Figure 3A), suggesting that it plays a role in NCC cell transformation.

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Fig. 3. Akt/cyclin D pathway was activated in MTC cells. (A) Activation of signalling pathways in MTC cells. The total RNA of MTC, DTC and NCC cells was isolated and processed for gene expression by Genechip analysis. The results showed that MAPK, PI3K and FAK were increased in MTC, but only PI3K increased in DTC cells. (B) Alteration of the molecules in Akt/cyclin D pathway. The analysis by gene expression showed that MAPKAPK2, Akt, HER2, PI3K, cyclin D1 and cyclin D3 mRNA in MTC cells were markedly higher than that observed in NCC cells (all P < 0.001, ANOVA). It is noted that PI3K, cyclin D1 and cyclin D3 mRNA in the DTC cells increased significantly. (C) Alteration of protein expression. The cellular lysates from the different cell types were processed for protein levels by western blot analysis. SV40 LT protein levels were used as a loading control. HER2, Akt, cyclin D1 and cyclin D3 were increased in MTC and cyclin D1 and cyclin D3 were upregulated in DTC compared with NCC cells. (D) Determination of enzyme activity. For the determination of MAPKAPK2 and Akt activity, cell lysates from MTC, DTC or NCC cells along with substrate on HSP27 and GSK-3, respectively, with [ -32P]ATP. Subsequently, the reaction mixture was spotted onto a filter paper and washed extensively, and analysed in a scintillation counter. For the determination of PI3 kinase activity, cell lysates from the different cells was immunoprecipitated with the p85-PI3-K antibody and washed twice. Subsequently, procedures were as described in Materials and methods. Each assay was performed in duplicate and repeated on two separate occasions. The results showed that MAPKAPK2, PI3 and Akt kinase activities were increased in MTC compared with NCC cells.
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The Akt pathway plays an important role in promoting cell proliferation. We examined the expression alteration of the components of the Akt/cyclin D pathway using the Genechip, kinase assays and western blots. It was found that MAPKAPK2, PI3K, Akt, cyclin D1 and cyclin D3 in the Akt/cyclin D pathway levels were significantly increased (all P < 0.001, ANOVA) (Figure 3B). The protein expression levels of HER2, Akt, cyclin D1 and cyclin D3 in MTC cells were also increased, as shown by western blot analysis (Figure 3C). MAPKAPK2, PI3K and Akt kinase activities of MTC cells were much greater compared with NCC cells (MAPKAPK2 and Akt, all P < 0.01; and PI3K, P < 0.05, Student's t-test) (Figure 3D). However, in positive control cells (DTC), only mRNA and the activities of PI3K kinase showed a significant increase (PI3K, P < 0.001, ANOVA; PI3K, kinase, P < 0.01, Student's t-test), Akt kinase activity was also much greater compared with NCC cells (P < 0.05, Student's t-test) (Figure 3B and D). This implies that Akt was activated in a different manner in MTCs and in DTCs. Because activated Akt might inactivate downstream GSK-3ß kinase activity through phosphorylation, thus prolonging the half-time of cyclin D1 and cyclin D3, the alterations of cyclin D1, cyclin D3 expression probably resulted from a cooperative effect of MAPKAPK2, HER2 and PI3K on Akt in MTC cells. Increased cyclin D1, cyclin D3 can accelerate the course of the cell through G1 phase, which is probably related to the enhanced proliferation activity of MCLR transformed cells.
In the MAPK pathway of MTC cells, the mRNA expression of IQGAP-2, RabGTPase, Rap1GAP, RasGAP, R-Ras, Krev-1 and TC21 of the Ras GTP/GDP protein family, of A-Raf, B-Raf, PAK and the p38, JNK of MAPK were all increased significantly (all P < 0.001, ANOVA). Of these, B-Raf showed the greatest change. In DTC cells, only R-Ras mRNA was upregulated and no evidence of alteration of any molecules of the Ras superfamily was observed (Figure 4A). In MTC cells, the protein levels of p38 and JNK altered consistently with mRNA, as shown by the western blot analysis (Figure 4B). This leads to the question of whether the increases of mRNA and protein levels of p38 and JNK resulted in further increases of p38 and JNK MAP kinase activities. The activities for MTC cells were increased more than for DTC and NCC cells (MTC versus NCC P < 0.05, DTC versus NCC P > 0.05, Student's t-test), assessed by kinase activity analyses (Figure 4C). In conclusion, the increased activities of p38 and JNK kinases in MTC cells was attributed to the extensive activation of molecules of the Ras GTP/GDP proteins. In the FAK pathways of MTC cells, R-Ras and FAK altered markedly; however, the upstream integrins and downstream effectors of FAK were unchanged (Figures 3A and 4A).

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Fig. 4. Ras/MAPK pathway was activated in MTC cells. (A) Analysis of gene expression. Genechip analysis showed that IQGAP-2, RabGTPase, Rap1GAP, RasGAP, R-Ras, Krev-1 and TC21 and of the Ras GTP/GDP protein family and of A-Raf, B-Raf in the Raf family, PAK and the p38, JNK of MAPK were all increased significantly with B-Raf showing the greatest change in MTC cells. Only R-Ras mRNA in the positive control cells (DTC) increased significantly. (B) Expression of p38 and JNK2 protein. The cellular lysates from MTC, DTC and NCC cells were examined by western blot analysis for protein levels. SV40 LT protein levels were used as a loading control. p38 and JNK2 were markedly increased in MTC. (C) Determination of p38 and JNK kinase activity. Assays were performed by incubating cell lysates from MTC, DTC and NCC cells with substrates, respectively, P38 substrate was ATF2, JNK substrate was soluble GST-C-jun and [ -32P]ATP, the reaction mixture was spotted onto filter paper and washed extensively, and then analysed in a scintillation counter. Each assay was performed in duplicate and repeated twice. The results showed that p38 and JNK kinase activities were increased in MTC compared with DTC or NCC cells.
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Constitutive activation of Akt, p38 and JNK kinase activities in MCLR transformed cells
The above results indicated markedly increased activities of Akt, and the p38, JNK MAP kinase in MCLR transformed cells. The duration of the elevated activities were next examined. The Akt, p38 and JNK kinase activities in MTC cells did not alter significantly at population doubling time of 15 (the fifth passage) and 30 (the tenth passage) (P > 0.05, Student's t-test), which indicated a constitutive activation of Akt, p38 and JNK kinases in MTC cells (Figure 5).

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Fig. 5. Constitutive activation of Akt, P38 and JNK in MTC cells. Activation of Akt, p38 and JNK MAP kinase, determined by kinase analysis, in lysed MTC cells at (1 x 106 cells) did not alter significantly at cell population doubling time (PDs) of 15, 21 or 30 (all P > 0.05, Student's t-test).
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Inactivation of Akt, p38 and JNK inhibited the proliferation of MTC cells
The results above demonstrated that Akt, p38 and JNK kinase activities in MTC cells were constitutively activated. However, it was not known if Akt, p38 and JNK kinase activities were the cause or consequence of cell transformation. Therefore, we sought to determine whether the inhibition of Akt, p38 and JNK kinase activities could inhibit the proliferation of MTC cells. The medium was supplemented with 50 nM 17-(allylamino)-17-demethoxy-geldanamycin (17-AAG), which is a potent inhibitor of Akt activation, 10 nM 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), as a selective inhibitor of p38 kinase and 10 nM 1,9-pyrazoloanthrone (SP600125) to selectively inhibit JNK kinase. By this means, it was found that the proliferation of MTC could be significantly decreased by each agent added separately compared with MTC treated with 10 nM DMSO (control) (17-AAG and SB203580, all P < 0.01; SP600125, P < 0.05, Student's t-test), as detected by MTT assay, and to an even greater extent if 17-AAG and SB203580 were added together (Figure 6). This suggests that Akt, p38 and JNK activities may be required for cell transformation and, therefore, these kinases may be useful therapeutic targets for blocking the carcinogenesis of colorectal epithelial cells induced by MCLR.

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Fig. 6. Inactivation of Akt, p38 and JNK MAP kinase inhibited the proliferation of MTC cells. A total of 0.5 x 103 cells were used for a colony test supplemented with 50 nM 17-AAG, 10 nM SB203580 and/or SP600125 in medium. Then, the cellular proliferation of MTCs and DTCs were determined by MTT assay. It was found that the proliferation of MTC could be significantly decreased by each agent added separately compared with MTC treated with 10 nM DMSO and to an even greater extent if 17-AAG and SB203580 were added together.
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Discussion
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The results of the present study indicate that MCLR, one of the important external factors in the carcinogenesis of CRC, induces transformation of immortalized NCC cells, an observation that concurs with the results of epidemiological reports (8,9). The main effect of a tumour promoting agent is its ability to induce an alteration of gene expression, resulting in aberrant cell differentiation, proliferation and apoptosis through intervention with various cell signalling pathways (22,23). An abnormal activation of the MAPK signalling pathway, controlling cell growth and apoptosis, etc. is common in tumours and this could promote cell proliferation (24,25). MCLR could result in tumours through its tumour promoting effects (13). In addition, MCLR also has a gene-toxic effect, producing reactive oxygen species and oxidative DNA damage, genomic DNA fragmentation and apoptosis (2628).
Immortalized NCC cells, with anchorage-independent growth, as employed in the present study, could be transformed after exposure to MCLR for 24 h. However, the concentration of MCLR was not simply related to the colony number of the transformed cells (data not shown). It may be that the sensitivity of SV40 LT immortalized NCC cells to external carcinogens was increased, since the checkpoint function was lost owing to the capture of p53 and Rb proteins by large T antigen (21). Moreover, it may be that the concentration of MCLR was higher than the transformation threshold or that there was a lack of an obvious threshold.
MTC cells, growing vigorously in multilayers, were subcultured continuously and colonized in soft agarose plates, and could even be passaged to the 17th passage, indicating the stability of the transformed phenotype in these cells. MCLR is able to interfere with cell signalling (13); therefore, the pathways that were modified in transformed MTC and DTC cells compared with NCC cells was investigated. Akt, the p38, JNK of MAPK and FAK signalling pathway molecules of MTC cells were activated, as determined by Genechip, western blot and kinase activity analyses; however, only Akt was activated in DMH-transformed cells, while the p38, JNK MAPK and FAK pathways did not alter markedly. These results indicate that different agents transform cells by different mechanisms and also that Akt, p38, JNK and FAK pathways participate in the transformation of NCC cells by MCLR.
Akt kinase was activated by MAPKAPK2, HER2 and PI3K in MTC cells but only by PI3K in DTC cells. MAPKAPK2, one of the substrates of p38 MAPK is related to stress stimulation, such as oxygen free radicals and heat shock. However, the kinase activity is also increased in tumours (20,29). Activated Akt could phosphorylate the downstream GSK-3ß kinase that resulted in the loss of GSK-3ß kinase activity. Cyclin D is dephosphorylated by inactivated GSK-3 and the half-life of dephosphorylated cyclin D is prolonged significantly. The protein levels of cyclin D1 and cyclin D3 in MTC cells were clearly higher than that of NCC cells, as determined by western blot. Increased cyclin D activates CDK4 molecules. Rb protein, as one of the substrates of CDK4, released E2F transcriptional factor, after being phosphorylated by CDK4, which promotes cell proliferation by accelerating the progress of cell cycle G1 phase (30). Nevertheless, since the Rb in immortalized NCC cells was captured by SV40 LT, Rb and E2F transcriptional factors could not be further investigated. Activated Akt could also phosphorylate several downstream substrates, such as Bad, nuclear factor-
B, mdm2 and the transcriptional factor FKH-TFs (3133). It was also reported that Akt is activated by phosphorylation (at Ser-473), and that the protein kinase Myt1 of the Wee family, is inactivated by Akt phosphorylation, which alters the balance between cdc25c and Myt1, and which impelled cells to the mitosis phase (34). In addition, recent studies show that Akt plays an important role in carcinogenesis (35).
In MTC cells, molecules of the Ras GTP/GDP protein family (members of the Ras superfamily), and B-Raf, A-Raf in the Ras/MAPK pathway, were activated, B-Raf to the greatest degree. This indicated that the sustained activation of the MAPK pathway is attributable to the extensive activation of Ras GTP/GDP proteins in MTC cells. The p38 and JNK MAP kinase pathway were selectively activated by the upregulation of Ras GTP/GDP proteins, which is presumably associated with the activation of PAK (36,37). Although B-Raf can activate Erk1/2 MAP kinase (38), it did not obviously affect the MAPK (Erk1/2) in MTC cells (data not shown). Activation of p38 MAPK is relevant to the cell stress response, and results in inflammation and apoptosis, as indicated in some studies (39,40). However, it was shown in recent investigations that the activation of p38 MAPK induced proliferation in certain cells and promoted cell survival and differentiation. It has also been suggested that the p38 MAPK is activated specifically in the development of primary human erythroid cells (41). p38 MAPK has been shown elsewhere (42) to be a therapeutic target for treating follicular lymphoma. The JNK pathway participates in broad biological processes, such as cell development, apoptosis and survival and the occurrence of inflammation, similar to p38, but dependent on the cell line and function. The JNK signalling pathway mainly acts in stress responses, as induced by toxins, inflammatory cytokines, ultraviolet irradiation and osmotic stress (43,44). It has been shown in recent experiments that Fas-mediated cell apoptosis is inhibited by the upregulation of JNK (45). Meanwhile, JNK is apparently capable of operating on phenotype transformation (46). It was also shown that JNK activity and transformation induced by the Ras is blocked by negative SEK1 transfection, and the transforming capacity of Ras was enhanced by wild SEK1 transfection without Erk participation. The transforming capability of JNK is relevant to its effect on cell cycle elements. The SV40 small T antigen is able to induce cell transformation through stimulating the cyclin D1 promoter, which would be blocked by negative SEK1, indicating that the JNK pathway possesses the ability to promote cell transformation (47).
Although there is
50% homology of R-Ras with H, N and K-Ras, the biological functions differ. Sustained activation of R-Ras resulted in the phosphorylation of FAK and p130Cas. PI3K, Raf, Ral-GDS, Rif, Rgl and Nore1 are the targets of R-Ras (4851). Therefore, upregulation of R-Ras had a direct effect on PI3K kinase activity and the activation of R-Ras also led to the activation of FAK, a signalling pathway of integrins. However, in this study, with MTC cells, where the transcription of related genes of R-Ras was investigated, including upstream integrins, no marked change was found (data not shown). In addition, to explore the activities of Akt, p38 and JNK in cell transformation, we used 17-AAG, SB203580 and SP600125 inhibitors, respectively, in the medium. It was found that each compound could inhibit the proliferation of MTC cells. Thus, Akt, p38 and JNK could be therapeutic targets of cells that are transformed by MCLR.
Activation of p38 and JNK in MTC cells was derived from the activation of upstream Ras GTP/GDP proteins, but it remains unclear how MCLR activated these protein molecules. It was reported that the 12th base of K-Ras in RSa cells mutated on day 6, after being treated with MCLR (52). Although MCLR is considered to have mutagenic capability, the 12th codon of the K-Ras gene was not found to mutate in the present study (data not shown), but this remains to be further investigated. The present study addresses the transformation of immortalized NCC cells by MCLR and the possible mechanism, thus providing a theoretical foundation for effective selection of molecular targets in the future. Interestingly, DMH, as a mutagen, was widely used to induce colon cancer in animal (rat or mouse) model, but we found that DMH could also activate the Akt pathway by PI3K (53). How DMH activated PI3K molecule needs to be further investigated.
In conclusion, Akt and the p38, JNK of MAPK pathways appear to participate in the transformation of NCC cells. Activation of Akt and p38, JNK of MAPK pathways accelerated cell proliferation. NCC cells, transformed by MCLR, could grow in soft agarose medium in an anchorage-independent manner that was maintained by constitutively activated Akt and p38, JNK of MAPK. Constitutive activation of Akt and the p38, JNK of MAPK pathway, in MCLR transformed immortalized colorectal crypt cells, may be one of the mechanisms of pathogenesis induced by MCLR, the important external factor in the carcinogenesis of CRC.
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Acknowledgments
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We thank Professor Jiayi Ding for generously providing U95Av2 Genechip and technical support. We also thank Mr Wenzhi Jiang for his technical assistance and the entire laboratory for fruitful discussions. This work was partly supported by grant from National Basic Research Program "973 Program" (G1998051200, 2004CB518707) and Health Bureau of Zhejiang Province (2002ZD008).
Conflict of Interest Statement: None declared.
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Received December 3, 2004;
revised February 2, 2005;
accepted March 8, 2005.