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Annals of Clinical & Laboratory Science 39:263-269 (2009)
© 2009 Association of Clinical Scientists

Restoration of DLC1 Gene Inhibits Proliferation and Migration of Human Colon Cancer HT29 Cells

Ping-ping Wu1, Yue-ling Jin2, Yan-fang Shang2, Zhi Jin2, Peng Wu2 and Pei-lin Huang2
1 Departments of Oncology and 2 Pathology, Zhongda Hospital and School of Clinical Medicine, Southeast University, Nanjing, China

Address correspondence to Professor Pei-lin Huang, Department of Oncology, Medical College, Southeast University, Nanjing 210009, China; tel 86 25 8379 2919; fax 86 25 8339 2919; e-mail huangpeilin2002{at}yahoo.com.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
DLC1 (deleted in liver cancer-1) is a new candidate tumor suppressor gene, which is inactive in various types of human cancers including colon cancer. To study the function of DLC1, we constructed a pcDNA3.1 vector containing the DLC1 gene and transfected it into HT29 colon cancer cells that were deficient in DLC1 expression. The restoration of DLC1 expression in HT29 cells significantly inhibited cell proliferation and migration. Flow cytometry showed that DLC1 transfection into HT29 cells induced apoptosis and that the cell cycle was arrested at S-phase. Additionally, cyclinD1 mRNA and protein expression were down-regulated while p21 expression was increased in pcDNA3.1-DLC1-HT29 cells compared to wild HT29 cells. These results confirm the role of DLC1 gene as a tumor suppressor, which may be manifested by regulation of p21 and cyclinDl. The DLC1 gene has a potential therapeutic role in inhibiting the development of colon cancer.

Keywords: colon cancer, DLC1 (deleted in liver cancer-1) gene, cyclinD1, p21, apoptosis


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The incidence of colorectal cancer has been rising on a global scale. It is now the second most prevalent cause of cancer mortality in western countries and the third or fourth in China [1]. Many tumor suppressor genes are involved in carcinogenesis of the colon and rectum. The DLC1 (deleted in liver cancer-1) gene, first identified from a primary hepatocellular carcinoma (HCC) [2], is located on chromsome 8p21.3–22, a region that is frequently deleted in tumors, and encodes a Rho GTPase-activating protein (RhoGAP). The human DLC1 gene is believed to be a negative regulator of the Rho protein family of small GTPases [3]. Rho family GTPases serve as molecular switches in various cellular functions, including cell cycle progression, cytoskeletal organization, malignant transformation, cell migration, and cell adhesion to the extracellular matrix (ECM). It is clear that DLC1 functions as a bona fide tumor suppressor gene, given that DLC1 expression is downregulated by genomic deletions or DNA methylation in several types of human cancers such as HCC, breast, colon, prostate, stomach cancer, and lymphoma [4–6]. Reactivation of DLC1 function results in suppression of tumor cell proliferation and induces caspase-3-mediated apoptosis in vitro and it also abolishes or reduces tumorigenicity in vivo [7]. Our previous data suggest that the DLC1 gene is associated with human colon cancer LoVo cell proliferation, migration, and cell cycle redistribution [8], but the molecular mechanisms underlying these effects remain unclear. Thus, we transfected DLC1 cDNA into the HT29 cell line, which is deficient in DLC1 gene expression, in order to study the function of DLC1 and to clarify the possible role of DLC1 in the development of colorectal cancer (CRC).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines.  HT29 colon cancer cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were cultured at 37°C and humidified 5% CO2 in RPMI 1640 medium (Sigma, St. Louis, MO, USA), supplemented with 10% fetal bovine serum and 100 units/ml of penicillin and streptomycin.

Construction of pcDNA3.1-DLC1.  Using pCMV/DLC1 plasmid as template, the 3.3 kb full-length DLC1 cDNA was amplified by PCR. The primers included Xba I and BamH I. After digestion with Xba I and BamH I, the PCR product was inserted into the Xba I and BamH I site of the pcDNA3.1 vector.

Transfection Assay.  Two ml of HT29 cells were plated at a density of 2x105cells/ml. When the cells had grown to 70% confluence in 6-well plates, pcDNA3.1 and pcDNA3.1-DLC1 vectors were transfected. After 8 hr, the transfection medium was replaced with RPMI 1640 medium for 24 hr. Selection for transfected cells was carried out in medium containing 600 mg/ml G418 (Geneticin; Nitrogen, USA). HT29 cells stably transfected with pcDNA3.1 and pcDNA3.1-DLC1 vectors were used for assays. The cells were divided into 3 groups: (1) pcDNA3.1-DLC1-HT29 (transfected with pcDNA3.1-DLC1 vector), (2) pcDNA3.1-HT29 (transfected with pcDNA3.1 vector alone), and (3) wild HT29 cells.

RNA Extraction and RT–PCR.  Total RNA extraction was performed using Trizol reagent (Takara, Shiga, Japan). The reverse transcription (RT) reaction was performed using 2 µg of total RNA with a first strand cDNA kit (Takara), according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was run in a 25 µl volume containing 2 µl of cDNA template, 10x buffer, 0.15 mM dNTP, 0.1 mM of each primer, and 0.5 U of Ex Taq Hot Start Version (Takara). The primers used in the PCR reaction and the amplification conditions are listed in Table 1Go. The final products were identified in 1.7% agarose gel and stained with ethidium bromide.


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  		Table 1. List of primer sequences and amplification conditions used in RT-PCR.
 
Western Blotting.  Each group of cells was washed with PBS twice, lysed for 10 min in hot water, and centrifuged for 10 min. Total proteins (10 µl) were separated by 5% SDS-PAGE, and then transferred onto a polyvinylidene fluoride (PVDF) membrane. After immersion in 10 ml of 5% non-fat milk in TBST solution for 1 hr, the membrane was incubated with primary antibodies and second antibody. Human monoclonal anti-DLC1 antibody (1:300 v/v; clone 3; BD Biosciences PharMingen), monoclonal anti-cyclinD1 antibody (1:1000), monoclonal anti-p21 antibody (1:1000), and β-actin antibody (1:500) were used as primary antibodies and bovine anti-mouse IgG (1:2500; Santa Cruz, CA) as the second antibody. Finally, results were photographed with ECL substrate.

MTT Assay.  For cell proliferation assays, each group of cells was plated in triplicate in 96-well plates at a density of 1x104 cells/well and grown for 24, 48, 72, and 108 hr, respectively, Twenty µl of 5 mg/ml MTT was added. After incubation for 4 hr, the number of metabolically active cells was quantified.

Colony Formation Assay.  Cells (1x103) were seeded into 6-well plates with 2 ml of culture medium. After incubation for 2 weeks in RPMI 1640 medium, supplemented with 10% fetal bovine serum at 37°C and 5% CO2, the colonies were washed twice with PBS, stained with Giemsa, counted, visualized microscopically, and photographed.

Transwell Assay.  For migration assays, NIH3T3 cells were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum at 37°C and 5% CO2. When the cells had grown to 80% confluence, they were incubated for 24 hr in medium without fetal bovine serum. The cell culture supernates were collected and preserved at –20°C for further use as epidermal growth factor (EGF). The undersides of Transwell Chamber membranes (BD Biosciences PharMingen) were coated with 250 µl Matrigel gels mixed with 250 µl of RPMI 1640 medium. HT29 cells from the transfected and control groups (1x105 cells) respectively were seeded on the Transwell Chamber. Then 800 µl of NIH3T3 EGF (prepared before) was added to the 6-well plates. After 24 hr, Matrigel gel on the upper sides of the membranes was removed using cotton swabs. The Transwell Chamber membranes were fixed in 95% ethanol for 15 min. The cells that had migrated to the undersides of the membranes were stained with H&E and counted by microscopy (200x). Results were determined by averaging the cell counts in 5 fields.

Flow Cytometry.  Each group of cells was washed with PBS and centrifuged (12,000 rpm) 3 times for 10 min. After adjustment to a density of 1x107 cells/ml, the cells were fixed in 70% ethanol at –20°C for 24 hr and incubated for 30 min at 37°C with RNase A. The cells were stained with propidium iodide (50 mg/ml, Sigma) and the cell cycle distribution was analyzed within 1 hr by flow cytometry with a FACSort instrument (Becton-Dickinson, Boston, MA).

Statistics.  Data were analyzed by the SPSS software program (v 11.0). Nonparametric tests were made using independent samples. Means were compared by one-way ANOVA. A p value <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Restoration of DLC1 Expression in pCDNA3.1-DLC1-HT29 Cells.  HT29 cells transfected with pcDNA3.1-DLC1 could effectively express DLC1 gene as indicated by RT-PCR (Fig. 1Go) and Western blot (Fig. 2Go), while the control groups of pcDNA3.1-HT29 and wild HT29 cells were deficient in DLC1 expression.


Figure 1
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  		Fig. 1. mRNA expression of DLC1 gene in HT29 cells was examined by semiquantitive RT-PCR. β-actin was analysed as a positive control. HT29 cells transfected with pcDNA3.1-DLC1 effectively expressed the DLC1 gene while the control groups of pcDNA3.1-HT29 and HT29 cells were deficient in DLC1 expression.

 

Figure 2
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  		Fig. 2. Protein expression of DLC1 gene in HT29 cells was examined by Western blot using human monoclonal anti-DLC1 antibody diluted 1:300. Compared to the control groups, restoration of DLC1 was detected in pcDNA3.1-DLC1-HT29 cells.

 
Restoration of DLC1 Expression in HT29 Cell Line Inhibited Cell Proliferation.  The MTT assay showed that the proliferation of cells transfected with pcDNA3.1-DLC1 was significantly slower than that of the pcDNA3.1-HT29 cells and the controls, especially at 60 hr of seeding (p <0.05) (Fig. 3Go). No significant difference was seen between pcDNA3.1-HT29 and wild HT29 cells (p >0.05). To examine whether transfection of DLC1-cDNA affects long-term cell growth, we tested the ability of colony formation in different cell groups. A significant decrease in colony number was observed in pcDNA3.1-DLC1-HT29 with smaller volume (Fig. 4Go). The mean colony forming efficiency of the transfected cells was 20.20 ± 0.85%, whereas the colony forming efficiencies of pcDNA3.1-HT29 and wild HT29 cells averaged 27.63 ± 0.87% and 28.37 ± 0.11%, respectively (p<0.05).


Figure 3
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  		Fig. 3. Cell proliferation was assessed by MTT. Cells seeded in 96-well plates were grown for 24, 48, 72, or 108 hr, and then incubated with 20 µl of 5mg/ml MTT for 4 hr. The quantification of metabolically activity in the cells transfected with pcDNA3.1-DLC1 was significantly lower than the cells of control groups, especially after 60 hr.

 

Figure 4
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  		Fig. 4. Inhibition of colony formation in HT29 cells transfected with DLC1 gene: 1x103 cells seeded in 6-well plates were cultured for 2 wk; colonies were stained with Giemsa, counted, and photographed. A significant decrease in colony number and volume was seen in pcDNA3.1-DLC1-HT29 cells (C), compared to the HT29 cells (A) and the pcDNA3.1-HT29 cells (B).

 
Restoration of DLC1 Expression in HT29 Cells Inhibited Migration.  The ability of cells to migrate toward Transwell Chamber membranes was tested using transwell migration assays. Compared to wild HT29 cells and pCDNA3.1-HT29 cells, the number of pCDNA3.1-DLC1-HT29 cells that migrated into the lower Transwell Chamber membranes was markedly decreased (p <0.05) (Fig. 5Go), which shows that DLC1 inhibited the migration of HT29 cells.


Figure 5
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  		Fig. 5. Detection of HT29 cell invasiveness in vitro by transwell assay. Cells were evaluated for their ability to migrate across a matrigel-coated membrane in an invasion chamber. After incubation for 20 hr at 37°C, the number of cells that had migrated across the membrane was determined. Invasive cells were stained, photographed, and counted under a microscope (magnification x200). The number of pcDNA3.1-DLC1-HT29 cells (C) that migrated into the lower chamber membrane was markedly less, compared to the HT29 cells (A) and the pcDNA3.1-HT29 cells (B).

 
Restoration of DLC1 Expression in HT29 Cells Disturbed the Cell Cycle and Induced Apoptosis.  We examined the effect of DLC1 expression on the cell cycle by flow cytometric analysis. In the control group, the percentages of HT29 cells in G2-phase, S-phase, and apoptotic cells were 5.76 ± 0.98%, 34.76 ± 1.40%, and 0.17 ± 0.04%, respectively; in the pCDNA3.1-HT29 group, the percentages were 6.35 ± 0.78%, 34.34 ± 0.61%, and 0.21 ± 0.05%; whereas in the pCDNA3.1-DLC1-HT29 cells, the percentages were 1.06 ± 0.26%, 45.60 ± 1.41%, and 5.74 ± 0.24% (p <0.05) (Fig. 6Go). These results indicate that DLC1 restoration leads to HT29 cell cycle arrest at S-phase and induces cell apoptosis.


Figure 6
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  		Fig. 6. Effects of DLC1 gene on cell cycle distribution in HT29 cells detected by flow cytometry. Cell cycle distribution was analyzed within 1 hr using a FACSort flow cytometer. The percentages of pcDNA3.1-DLC1-HT29 cells (C) in S-phase and in apoptosis were higher than those of HT29 cells (A) or pcDNA3.1-HT29 cells (B).

 
DLC1 Transfection Affects the Expression of p21 and CyclinD1.  DLC1 gene transfection induced marked increase of p21 mRNA (Fig. 7Go) and protein (Fig. 8Go) expression in HT29 cells, while cyclinD1 expression was greatly decreased (p <0.05).


Figure 7
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  		Fig. 7. Effects of DLC1 gene on p21 and cyclinD1 mRNA expression in HT29 cell lines. Restoration of DLC1 gene in pcDNA3.1-DLC1-HT29 cells induced markedly increased expression of p21 mRNA, while cyclinD1 mRNA expression was substantially decreased.

 

Figure 8
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  		Fig. 8. Effects of DLC1 gene on p21 and cyclinD1 protein expression in HT29 cells (human monoclonal anti-p21 antibody and anti-cyclinD1 diluted 1:1000). Restoration of DLC1 gene in pcDNA3.1-DLC1-HT29 cells induced markedly increased expression of p21 protein, while cyclinD1 protein expression was substantially decreased.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The identification of genes that contribute to the development of colon cancer is critical to the understanding of molecular mechanisms of carcinogenesis and may provide new strategies for clinical therapy. A new candidate tumor suppressor gene, DLC1, first identified as a rat p122RhoGAP homolog [2], is diminished or silenced in various types of human cancer, as well as in metastatic cells compared to non-metastatic cells [10,11]. Ectopic expression of DLC1 was found to suppress cell proliferation, migration, and colony formation in several cancer cell lines [12–14], which suggests that DLC1 plays a role as a tumor suppressor and has potential therapeutic value in cancer.

Investigations of DCL1 in colorectal cancer are relatively limited. Therefore, based on our previous work, we studied the functions of DLC1 further. The present report shows that transfection of DLC1 cDNA into the HT29 cell line causes significant inhibition of cell growth and cell migration, which confirms that DLC1 acts as a tumor suppressor gene in colon cancer. The molecular mechanism whereby DLC1 plays its suppressor role has not been fully elucidated. That DLC1 negatively regulates the activity of the Rho protein family, especially of RhoA, through the RhoGAP domain, seems likely to be responsible for DLC1-mediated antitumor activity [15,16]. A recent study found that DLC1 repressed cell movement in HCC cells by negatively regulating Rho/Rock-mediated cytoskeletal rearrangement, a well known downstream effector of RhoA [17]. Significantly, the inhibitory effects of DLC1 depended on its RhoGAP activity [18]. Since the Rho/Rock signal transduction pathway has an important role in regulation of cellular dynamics, it is possible that transfection of the DLC1 gene into HT29 cells resulted in inhibition of cell migration ability via the downstream Rho/Rock signal transduction pathway.

Our results showed that DLC1 restoration induced a marked increase of p21 expression and a decrease of cyclinD1 expression in HT29 cells. Further, flow cytometry showed that expression of DLC1 blocked the HT29 cell cycle at S-phase, which indicates that the DLC1 gene induces cell-cycle arrest and inhibits cell proliferation by regulating p21 and cyclinD1. The cell cycle is an ordered set of events, whose disregulation may lead to tumor formation. CyclinD1 belongs to G1-phase cyclin, which is a key protein that pushes the cell from G1-to S-phase and subsequently causes cell proliferation, while p21 is one of the cyclin-dependent kinases inhibitors (CDKI) [19] that have extensive inhibitory effects. Previous studies have shown that p21 can combine with most of cyclin-CDK complexes, such as cyclinD-CDK4/CDK6, cyclinE-CDK2, and cyclinA-CDK2, and that over-expression of p21 induces cell cycle arrest in G1-, G2-, or S-phase [20]. Of note, the RhoGAP domain of DLC1 is thought to be essential to the regulation of p21 and cyclinD1 expression. Rho family GTPases have important roles in regulation of the cell cycle [21]; Rho A protein can down-regulate the expression of p21, and increase the cyclinD1 promoter activity via EGF and Ras signals [22]. Therefore, DLC1 may reduce the activity of Rho GTPase, which in turn may affect cell cycle-related proteins, and suppress the proliferation of HT29 cells.

Flow cytometry showed that restoration of the DLC1 gene in HT29 cells induced apoptosis, which contributed to inhibiting cell proliferation and migration as well. Transfection and retrieval of DLC1 expression in HCC cell lines also caused the induction of apoptosis associated with cleavage of caspase-3 and a reduced level of Bcl-2 [8]. It is unclear which mechanism is involved in the apoptosis after restoration of the DLC1 gene. Studies have shown that p21 promotes apoptosis under certain conditions [23,24]. Perhaps the upregulation of p21 expression and S-phase arrest partly account for the apoptosis of pcDNA-DLC1-HT29 cells in our study.

In lung cancer cells, the Rho-GAP domain of DLC1 has recently been reported to stimulate the GTPase activity of RhoA as well as that of RhoB, RhoC, and Cdc42 and to inhibit cell proliferation by both Rho-GAP domain-dependent and domain-independent mechanisms [25]. DLC1 has also been shown to be located in focal adhesions and to interact with members of the tensin family of focal adhesion proteins [26,27]. Additionally, the sterile alpha-motif (SAM) domain [28] and the focal adhesion-targeting (FAT) domain [29] of DLC1 have been suggested to be essential to cell motility and morphology. Therefore, the mechanism of DLC1 as a suppressor gene appears to be more complicated than previously suspected; the detailed molecular pathways by which DLC1 affects the proliferation and migration of colon cancer cells need further study.

In conclusion, the present study shows that restoration of the DLC1 gene in HT29 human colon cancer cells results in inhibition of cell proliferation and migration and induces cell-cycle arrest. The DLC1 gene may function as a tumor suppressor via regulation of p21 and cyclinDl. Our results confirm that silencing of DLC1 signaling is an important event in the progression and metastasis of colon cancer.


   Acknowledgments
 
This work was supported by the National Natural Science Foundation of China (No. 30471937) and the Research Fund for the Doctoral Program of Higher Education of China (No. 200802860040).


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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