Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Altered phenotype of HT29 colonic adenocarcinoma cells following expression of the DCC gene

Abstract

On 18q, frequently deleted in late stage colorectal cancers, a gene, Deleted in Colon Cancer (DCC), has been identified and postulated to play a role as a tumor suppressor gene. DCC is retained in the majority of mucinous tumors, which produce high levels of mucins, and seems to be preferentially expressed in intestinal goblet cells. To investigate whether DCC is related to mucin expression and can modulate the transformed phenotype, we introduced a full-length DCC cDNA into HT29 cells, which can be induced in vitro to express MUC2, the gene that encodes the major colonic mucin. Expression of DCC did not modulate constitutive or induced expression of MUC2, nor did DCC induce a mature goblet cell phenotype. However, HT29 clones expressing high and low levels of DCC protein showed a significant decrease in cell proliferation and tumorigenicity. Furthermore, increased shedding and an elevated rate of spontaneous apoptosis were associated with higher levels of expression of DCC. In summary, while restoration of DCC expression in a human colon carcinoma cell line did not influence expression of differentiation markers, DCC expression did affect the growth and tumorigenic properties of the cells suggesting that DCC can modulate the malignant phenotype of colon cancer.

Introduction

Seventy per cent of colorectal cancers show LOH at chromosome 18q (Fearon and Vogelstein, 1990; Vogelstein et al., 1988). On a 18q a gene, Deleted in Colon Cancer (DCC) has been identified and postulated to play a role as a tumor suppressor gene (Fearon et al., 1990). The DCC gene encodes a transmembrane protein with homology to the neuronal cell adhesion molecule family (N-CAM) (Fearon et al., 1990), and shares similarity with neogenin, a recently identified gene which is expressed in the developing chicken nervous system (Vielmetter et al., 1994). In addition, there is evidence that DCC can bind netrin-1, an axonal chemo-attractant involved in the guidance of developing axons (Chen et al., 1996; Keino-Masu et al., 1996; Kolodziej et al., 1996). In agreement with a postulated role of DCC in the developing nervous system, DCC is expressed at higher levels in the nervous system (Fearon et al., 1990; Hedrick et al., 1994; Reale et al., 1994), and mice with an inactivated Dcc gene show defects in axonal projections (Fazell et al., 1997).

Although Dcc null mice do not present obvious alterations in the growth, differentiation, morphogenesis and tumorigenesis of the intestine, a previous report has suggested an association of DCC with the differentiation of goblet cells (Hedrick et al., 1994), which in the colon are responsible for synthesizing and secreting mucus (Colony, 1989). Consistent with a role of DCC in mucin expression, mucinous tumors that are characterized by a large production of mucus, show a lower frequency of LOH at 18q (Hedrick et al., 1994), suggesting that DCC may be required for mucin production.

Our previous work has utilized HT29 cells to study the regulation and structure of a human gastrointestinal mucin gene, MUC2 (Velcich and Augenlicht, 1993; Velcich et al., 1997), which encodes the major form of intestinal mucins and is expressed predominantly in goblet cells (Chang et al., 1994; Gum et al., 1994; Gendler and Spicer, 1995). We have shown that in HT29 cells MUC2 gene expression can be induced (Velcich and Augenlicht, 1993), however, the cells do not acquire a fully differentiated goblet phenotype, as they fail to polarize and secrete a mucus gel (Velcich et al., 1995). In addition, we have shown that the MUC2 gene is deregulated in mucinous tumor cell lines (Velcich and Augenlicht, 1993).

In this report, we investigated potential mechanistic links between DCC expression and the differentiation pathway of goblet cells: we ascertained whether expression of DCC in HT29 cells could modulate regulation of the MUC2 gene and elaboration of a fully mature goblet cell phenotype. We show that expression of DCC did not modulate the expression of differentiation markers of the absorptive or goblet cell lineage, but both high and low levels of DCC affected the growth and tumorigenic properties of the cells, and are associated with elevated rates of cell shedding and apoptosis. Therefore, are data, which demonstrate that expression of DCC in a human colon carcinoma cell line alters features of the transformed phenotype, suggest that, in vivo, DCC may act as a modulator of the cancer cell phenotype. This finding is consistent with the fact that 18q LOH is linked clinically with colon tumors that progress more rapidly, but 18q LOH is neither necessary nor sufficient for progression of the disease.

Results

Phenotypic characteristics of stably transfected HT29 cells expressing the DCC gene

We transfected HT29 cells, which do not express detectable levels of DCC, as determined by RT – PCR and Western blot analysis (Figure 1a,b), with an eukaryotic expression vector encoding the full-length human DCC cDNA under control of the cytomegalovirus (CMV) promoter/enhancer elements (CMV – DCC) (Pierceall et al., 1994). This vector also contains the neomycin gene that confers resistance to G418. A control vector, harboring only the neo gene (CMV-neo), was also used. We were able to establish several clonal cell lines in which DCC expression was detected, and two of these, DCC11 and DCC3, were chosen for our studies, as they expressed low and high levels of DCC mRNA and protein (Figure 1a,b).

Figure 1
figure1

Level of expression of DCC in two clones of HT29 cells. (a) Total RNA was isolated from parental HT29 and two independently isolated clones of HT29 cells DCC3 and DCC11 stably transfected with the CMV – DCC expression vector. 1 μg of RNA was analysed by RT – PCR reaction for the presence of DCC and GAPDH mRNA, as detailed in Materials and methods. Levels of DCC mRNA are expressed relative to the levels of GAPDH. (b) Western blot analysis of DCC in cytoplasmic cell extracts from the indicated cell lines was performed using the antibody AB-1 in conjunction with the ECL kit as described in Materials and methods. The DCC protein migrates with apparent molecular weight between 170 – 185 kd. The position and weight (in kilodaltons) of markers are also indicated

Morphology of the DCC+ cells is shown in Figure 2. Both DCC3 and DCC11 clones showed highly vacuolated monolayers, with this phenotype being more pronounced in the DCC11 cells. Vacuoles were absent from control Neo+ cultures, but were present, fewer and smaller, in confluent cultures of parental HT29 cells.

Figure 2
figure2

Morphology of DCC+ clones of HT29 cells. Confluent cultures of HT29, DCC3 and DCC11 cells, grown on plastic, were photographed. Large vacuoles are present in the cultures of DCC+ clones. Magnification 20×

DCC expression and goblet cell differentiation

The normal function of DCC in the intestine has not yet been elucidated, although it has been suggested that DCC may play a role in the differentiation of the goblet cell lineage (Hedrick et al., 1994). Therefore, we investigated whether expression of DCC was mechanistically linked to the elaboration of the goblet cell phenotype. We have previously shown that HT29 cells can be induced to express a high level of MUC2 mRNA and protein, which represents the major form of colonic mucin, in response to forskolin and TPA (Velcich and Augenlicht, 1993). Thus, we determined whether the DCC+ clones expressed MUC2 mRNA constitutively, or whether DCC could potentiate the responses to treatment with forskolin and TPA. We compared the basal, forskolin and TPA induced levels of MUC2 mRNA in parental (HT29) and control (Neo+) cells and in the DCC3 and DCC11 clones. As shown in Figure 3 no difference was detected among the different cell groups both in the basal level or induced level of MUC2 expression.

Figure 3
figure3

MUC2 expression is not affected by DCC levels. HT29, Neo+, DCC3 and DCC11 cells were not treated or treated with forskolin (F) or TPA for 8 h, as previously reported (Velcich and Augenlicht, 1993). Total RNA was isolated and levels of MUC2 mRNA were determined by Northern blot analysis. Also shown are the levels of GAPDH mRNA for comparative purposes. The polydispersity of the MUC2 RNA is a characteristic of many mucin mRNAs (Velcich et al., 1997)

Forskolin and TPA increase MUC2 expression in HT29 cells without inducing a fully differentiated goblet cell phenotype. In fact, HT29 cells fail to elaborate and secrete fully mature glycosylated mucins (Velcich et al., 1995). Thus, we investigated whether DCC could overcome this block towards a more differentiated phenotype. However, parental, control Neo, and DCC3 and DCC11 cultures stained negative with Alcian Blue, a dye commonly used to detect glycosylated acidic mucins (data not shown; Velcich et al., 1995).

We have previously shown that in HT29 cells different inducers can modulate different aspects of intestinal cell differentiation characterized by the induction of markers specific for distinct cell lineages (Velcich et al., 1995). Treatment of HT29 cells with NaB induces aspects of differentiation along the absorptive cell lineage, as well as apoptosis (Heerdt et al., 1994). Since the DCC11 clone showed an increased rate of spontaneous apoptosis, we investigated whether expression of DCC could modulate the kinetics and extent of the response to NaB treatment. Analysis of NaB induction of alkaline phosphatase and CEA, which represent markers for the enterocyte cell lineage, did not reveal any difference among the different cell types (data not shown).

DCC expression affects the growth properties of HT29 cells

Despite the lack of modulation of differentiation pathways in the DCC+ clones, it was clear that DCC affected the growth properties of the cells. To confirm and expand this observation, we compared the growth kinetics of parental, control Neo+ and DCC+ clones. As shown in Figure 4, both DCC2 and DCC11 had slower growth kinetics characterized by a longer lag period after plating, and slower exponential growth rates as compared to parental HT29 and control Neo+ cells. A decrease in growth kinetics has been reported in CoKFu cells transformed with the entire human chromosome 18, which harbors DCC (Tanaka et al., 1991).

Figure 4
figure4

Growth curve of control and DCC+ clones. The growth rate of parental HT29, control Neo+, DCC3 and DCC11 cells was determined by counting the number of adherent cells, at the indicated time points after plating

As a higher number of cells shed into the supernatant was observed in the DCC11 cultures, we evaluated whether expression of DCC increased rate of shedding of DCC+ cells. The fraction of shed cells versus the total number of cells, determined by the DNA concentration in each fraction (Materials and methods) for each cell line as a function of time is shown in Figure 5a. Clearly, DCC11 cultures shed twice as many cells over time than control HT29 cells. DCC3 cells, which express much lower levels of DCC protein than DCC11, had a ratio of floating versus adherent cells which was similar to controls.

Figure 5
figure5

Kinetics of cell shedding and apoptosis in cultures of DCC+ cells. (a) The analysis of cell shedding in HT29, Neo+, and DCC+ cell cultures was determined by calculating the amount of DNA corresponding to cells collected from the conditioned medium relative to the total amount of DNA in each culture at the indicated time points following addition of fresh medium. The amount of DNA in the supernatant and in the adherent fraction was determined by the DPA reaction, as detailed in Materials and methods. (b) DNA was extracted from adherent cells at the indicated time points after addition of fresh medium, as high and low molecular weight fractions. The percentage of low molecular weight fraction relative to total DNA recovery was determined as index of fragmentation. (c) Cells were scraped 24 h after refeeding, pelleted, resuspended in 1 ml of hypotonic PI solution and analysed by FACScan as described in Materials and methods. The percentage of the subG0 fraction was taken as a measure of apoptotic cells. *P<0.05

This altered shedding activity is suggestive of cells undergoing apoptosis (Heerdt et al., 1994); in addition, a recent report establishes that transient expression of DCC in 293T embryonic kidney cells induces high levels of apoptosis (Mehelen et al., 1998). Thus, we investigated whether the expression of DCC caused an increase in the rate of apoptosis in the monolayer of DCC+ cultures compared to parental and control cells. The kinetics of apoptosis in DCC+, Neo+ and parental HT29 cells, were determined by analysing the percentage of fragmented DNA in the monolayer. As shown in Figure 5b, a modest, but statistically significant (Anova, P<0.05), increased level of DNA fragmentation is present in the monolayer of DCC11 cells at 24 and 48 h after refeeding, indicating an elevated number of cells undergoing apoptosis. Similar results (Student t-test P<0.05) were obtained by flow cytometric measurement of the percentage of apoptotic nuclei (sub G0 fraction) after Propidium Iodide staining in cultures 24 h after refeeding (Figure 5c). The excellent correlation we found between the flow cytometric analysis and the colorimetric method for measuring apoptotic cells confirms previous reports (Nicoletti et al., 1991; Heerdt et al., 1996).

DCC expression suppresses tumorigenicity bothin vitro andin vivo

The DCC gene was originally isolated as a candidate tumor suppressor gene involved in the development of colon cancer. In view of the decreased growth characteristics of the DCC+ clones and increased rate of apoptosis, whose inhibition has been reported during development of colorectal cancer (Bedi et al., 1995), we investigated the properties of DCC as a tumor suppressor gene, and determined whether the expression of wild type DCC gene altered the transformed phenotype of HT29 cells. First, we evaluated the ability of control HT29, Neo+ and DCC+ cells to form colonies in soft agar. The inability of cells to grow in soft agar is generally an indication of a more normal phenotype. Figure 6a shows that both stably transformed clones, DCC3 and DCC11, failed to generate colonies when plated in soft agar. Conversely, both parental and control Neo+ cells were capable of forming large size colonies under the same conditions. The results shown in Figure 6a were highly reproducible in three independent experiments, each performed in six replicas. To confirm and expand these results, we tested the ability of the cells in each group (HT29, Neo+, DCC3 and DCC11), to grow as xenografts in immunodeficient mice. Nude mice were injected subcutaneously, as detailed in Materials and methods. The kinetics of tumor growth, as determined in bi-weekly measurements, is shown in Figure 6b. DCC+ cells gave rise to much smaller tumors than those obtained with the parental and Neo+ cells. Similar results were obtained in three separate experiments and linear regression analysis shows that the mean square root of tumor volume between DCC+ cells and controls (HT29 and Neo+) were significantly different (P<0.01). The reduced tumorigenicity of DCC3 and DCC11 cells is consistent with the slower growth rate of the cells as well as their lack of growth in soft agar.

Figure 6
figure6

DCC+ clones express a less tumorigenic phenotype. (a) HT29, Neo+, DCC3 and DCC11 cells were tested for their ability to form colonies in soft agar. 103 cells per well were seeded in soft agar (0.5% bottom layer and 0.33% top layer) and cultured for 2 weeks. Colonies were visualized by staining with a vital tetrazolium dye, as described in Materials and methods, and photographed. Three independent experiments were performed, each in six replicas, and representative wells for each cell line are shown. (b) To determine tumor growth kinetics of DCC+ clones 106 cells, per cell line, were injected subcutaneously in nu/nu females. At the indicated time points after injection two dimensional measurements were taken and volume was calculated with the following formula ((L×W)×3.14)/6. Linear regression analysis shows that the mean square root of tumor volume between DCC+ cells and controls (HT29 and Neo+) were significantly different (P<0.01)

Discussion

It has been suggested that DCC may play a role in the differentiation of the goblet cell lineage (Hedrick et al., 1994). In the colon, goblet cells are responsible for synthesizing and secreting mucus. Consistent with a role of DCC in mucin expression, mucinous tumors, that are characterized by a large production of mucus, show a lower frequency of LOH at 18q than do common colonic tumors (Hedrick et al., 1994), suggesting that DCC may be required for mucin production. In an extension of our previous work showing that HT29 cells can be induced to express high levels of MUC2 mRNA and protein, but fail to acquire a fully differentiated phenotype (Velcich et al., 1995), we investigated whether expression of DCC was mechanistically linked to the elaboration of the goblet cell phenotype. Our data show that DCC does not modulate the expression of MUC2 constitutively or MUC2 induced by TPA and forskolin, nor does DCC confer to HT29 cells the ability to elaborate a mature goblet cell phenotype in response to agents which induce expression of MUC2. In sum, our data argue that, at least in this in vitro system, DCC is not required for mucin production. In addition, DCC did not alter the induction of CEA or alkaline phosphatase by sodium butyrate.

DCC+ clones have slower growth kinetics and, accordingly, the DCC+ clones show a decreased ability to form colonies in soft agar, and decreased tumorigenesis in nude mice. Thus, our results in colonic cells correlate restoration of DCC expression, at both low or higher level, with the suppression of aspects of the transformed phenotype and are in agreement with previous work demonstrating that the reintroduction of the entire chromosome 18 in a DCC colorectal cancer cell line, or expression of the DCC cDNA in DCC transformed keratinocytes, suppress cell tumorigenicity (Tanaka et al., 1991; Klingelhutz et al., 1995). Furthermore, restoration of DCC expression increased shedding and rate of spontaneous apoptosis in DCC11 cells, which express the higher levels of DCC protein. These data are in agreement with a recent work demonstrating that transient expression of DCC in 293 cells induces apoptosis (Meheleh et al., 1998). The discrepancy between high levels of apoptosis in 293 embryonic kidney cells and the more modest, but statistically significant, increase we detect in HT29 cells, may be due to the different cell systems or the assay of transient transfectants in the 293 cells versus stable transformants in our work, since the selection of such stable transformants would, of necessity, select against cells which can efficiently progress to the end stage of apoptosis.

Increased apoptosis may represent an aspect of the attenuated tumorigenic phenotype associated with restoration of DCC expression. Interestingly, this modulation is not observed in the DCC3 clone which expresses a very low level of DCC protein, despite the fact that DCC3 cells display an attenuated tumorigenic phenotype, both in vitro and in vivo, comparable to that of DCC11 cells. There are several possible explanations for these observations: either these two processes, increased shedding and rate of apoptosis, and decreased tumorigenicity, may be controlled by distinct mechanisms that can be differentially modulated by levels of DCC expression; or small alterations in the rate of apoptosis may be induced by low levels of DCC and, although difficult to detect because of the transient nature of the process, may still be highly significant in altering tumor growth (Bursch et al., 1990). In addition, our results are consistent with in vivo data showing that only a small percentage of colonic cells in situ can be identified at the terminal stage of apoptosis.

An attenuated transformed phenotype is also inferred by the cell morphology: DCC clones are characterized by the presence of large vacuoles, which may be similar to the intracellular lumina, a distinct feature of cells unable to develop cell polarity (Laboisse, 1989). Interestingly, similar structures have been described during epithelia morphogenesis in fetal rat colon (Colony and Neutra, 1983).

The growth suppressive effects, increased rate of apoptosis and the presence of vacuoles could be due to a toxic effect of the DCC protein expressed at elevated levels from a constitutive promoter. However, the fact that we were able to establish several independent clones which have different levels of DCC, yet show a similar, less tumorigenic, phenotype, and the presence of similar, though less prominent, vacuoles in parental cultures, argue against this possibility.

Our results demonstrating that DCC can modulate the tumorigenicity of a colonic carcinoma cell line, are consistent with the role attributed to DCC in colorectal carcinogenesis. DCC was originally cloned as the candidate target gene of 18q LOH. This cytogenetic abnormality is detected in the majority of primary colon carcinomas and almost 100% in liver metastasis, suggesting that 18q LOH is associated with tumor progression (Vogelstein et al., 1988; Ookawa et al., 1993). Although somatic mutations in the remaining DCC allele have been detected in few cases (Cho et al., 1994), there is marked reduction or absence of DCC expression in colorectal cancer (Cho and Fearon, 1995; Fearon et al., 1990; Itoh et al., 1993), and recent reports correlate the loss of DCC expression with poor prognosis (Jen et al., 1994; Shibata et al., 1996). In addition, a role for DCC in tumor progression has been substantiated by numerous reports describing alterations in DCC in tumors derived from different organs (Fearon, 1996; Rieger-Christ et al., 1997), including the brain, where DCC is expressed at its highest levels, and, as in the case for colorectal cancer, 18q LOH is associated with a propensity of tumor cells to metastasize (Reale et al., 1996; Reyes Mugica et al., 1997, 1998).

Additional candidate genes at 18q12-21, include DPC4 and MADR2/JV18 (Hahn et al., 1996; Eppert et al., 1996; Riggins et al., 1996), now renamed Smad4 and Smad2 (Derynk et al., 1996). These two loci show inactivating mutations in 16 and 6% of colon cancers, respectively. Further evidence for the role of these genes in colorectal cancer comes from a recent report that inactivation of the murine Smad4 gene accelerates tumor formation in the genetically initiated Apc+/− mice (Takaku et al., 1998). However, a careful analysis of a panel of colorectal tumor xenografts determined that, although DPC4 and JV18 showed inactivation in a subset of colon tumors, the frequency of alteration in the panel was not sufficiently high to make these genes the sole candidate targets for inactivation (Eppert et al., 1996; Thiagalingam et al., 1996). Conversely, all the tumors studied in detail showed aberrant expression of DCC (Thiagalingam et al., 1996), and absence of DCC expression was correlated with poor prognosis (Shibata et al., 1996).

The controversy on the role of 18q21 LOH in general (Carethers et al., 1998; Martinez-Lopez et al., 1998) and loss of DCC expression in particular (Shibata et al., 1996; Gotley et al., 1996) in colon tumorigenesis has been further fueled by the finding that targeted inactivation of the mouse Dcc gene does not play a detectable role in the process of tumor formation in the gastrointestinal tract (Fazell et al., 1997), even in the genetically initiated Min mouse which harbors a mutated Apc allele (Moser et al., 1990). However, there are two important aspects of these findings that need to be kept in proper context: the extent to which findings in the mouse can be extrapolated to human disease; and the phenotype one would expect to observe based on the clinical observations regarding 18q LOH. First, the sequence of events that cause tumor formation in the mouse may be different from human. In this regard, tumors which develop in the Apc+/− animal models do not accumulate ras and p53 mutations (Shoemaker et al., 1997; Smits et al., 1997), and the min phenotype is not potentiated in mice which also have targeted expression of kras and p53 mutant transgenes in the intestine (Kim et al., 1993). In contrast, in human, ras and p53 mutations play a role in tumor progression, being present in approximately 50 and 75% of colorectal carcinoma (Fearon and Vogelstein, 1990).

The discrepancy between the mouse and human data for several loci may in part be attributed to the fact that DCC (as well as p53) inactivation is a late event in the progression of human colon cancer, generally associated with the increased propensity of tumor cells to metastasize, while in mice tumors of the gastrointestinal tract vary rarely metastasize. Thus, late events in tumor progression in human, which are dependent upon a great complexity of changes in gene structure and expression (Augenlicht et al., 1991; Zhang et al., 1997), seem to be difficult to reproduce in mouse. In this regard it will be interesting to evaluate whether the absence of Dcc in the Smad3−/− mice which develop metastatic colonic tumors (Zhu et al., 1998), would facilitate the appearance of metastasis. Second, multiple target loci are present in 18q which may have a role in progression by modulating the tumor phenotype; however, since 18q LOH is not sufficient nor necessary for human colon tumorigenesis, expectation that individual target genes on the chromosome could initiate or suppress tumorigenesis, may be simplistic. Instead, our finding that DCC expression can modulate the transformed phenotype of colonic carcinoma cells is entirely consistent with the clinical observation that 18q LOH status is linked to prognosis, but that even those patients who retain 18q still exhibit progressive, although less aggressive, disease.

Materials and methods

Cell culture and transfection protocol

HT29 cells were maintained in minimal essential medium supplemented with non-essential amino acids and 10% fetal calf serum. For transfection, 1×106 cells were seeded in 100 mm dishes 2 days prior to transfection. Cells were transfected using 50 μg of the plasmid CMV – DCC (a generous gift of B Vogelstein), and 50 μl of lipofectAMINE (Gibco/BRL) following the manufacturer's instructions. Incubation of cells with the liposome – DNA complex was for 5 h followed by the addition of fresh medium. Forty-eight hours later, cells were split into 6×100 mm dishes in complete medium containing 800 μg/ml of G418. Individual colonies were isolated, expanded and further characterized. Induction of cells with TPA and forskolin was for 8 h as previously described (Velcich and Augenlicht, 1993).

RNA isolation and analysis, and immunoblots

RNA was isolated, from DCC+ and control cells, as previously described (Velcich and Augenlicht, 1993), and analysed by RT – PCR. For first strand cDNA synthesis 1 μg of total RNA was used in 20 μl of RT reaction using an RNA – PCR kit (Perkin Elmer) according to the manufacturer's instructions. Ten μl of the RT reactions were amplified using the DCC primers described by Fearon et al. (1990). The other 5 μl were amplified using primers specific for GAPDH. These conditions were chosen after pilot experiments were carried out to establish that reactions were performed in the linear range.

PCR products were analysed by electrophoresis on agarose gel, followed by blotting and hybridization to DCC and GAPDH specific probes. The intensity of the bands was determined using a Phosphoimager (Molecular Dynamics). MUC2 expression was determined as previously described by Northern blot analysis (Velcich and Augenlicht, 1993).

For Western blot analysis, total cell extracts from the cell lines were prepared as previously described (Velcich et al., 1995). Equal amounts of protein, as determined by the Bradford microassay (BioRad), were diluted with an equal volume of 2× sample buffer, boiled for 5′ and fractionated by electrophoresis on a 8% precasted polyacrylamide gel, followed by electrotransfer to nitrocellulose. After transfer, filters were stained with Ponceau S to control for equal loading and transfer. The DCC specific band was detected by using monoclonal antibody DCC Ab-1 (Oncogene Research Products) following the supplier's instructions. Immune complexes were visualized using the ECL kit (Amersham) following the manufacturer's instructions. Positive control for the DCC protein was represented by cell extract from the IMR32 cells, a human neuroblastoma cell line which expresses wild type DCC (Pierceall et al., 1994).

Cell proliferation, cell growth in soft agar, and tumor xenograft

Kinetics of cell growth were determined by seeding 103 cells per well in 96 well plates for each cell group. At the indicated time points cells, in triplicate, were trypsinized and counted using a Coulter counter.

For the soft agar colony assay, one 6 well plate was used per cell type. 5×106 cells in 1 ml of top agar (0.33%) in complete medium were seeded on a layer of 3 ml of bottom agar (0.5%). After 1 week cells were fed with 1 ml of complete medium, with care taken not to disturb the top layer. Colonies were visualized 2 weeks after plating by staining with vital tetrazolium dye (Schaeffer and Friend, 1976) and photographed. In vivo tumorigenicity was evaluated by injecting subcutaneously 106 cells in nude/nude BalbC female, 4 – 6-week-old, mice. Tumor growth was recorded twice weekly.

Cellular shedding, DNA fragmentation and flow cytometric analysis

DNA fragmentation was determined as the fraction of low molecular weight DNA from the monolayer of cell cultures (Heerdt et al., 1996). Briefly, cells were seeded at 5×104 per well in 24 well plates. Three days later fresh medium was added and, at the indicated time points, cells in the supernatant and monolayer were harvested, washed twice in phosphate buffered saline solution (PBS), resuspended in 100 μl of lysis buffer (10 mM Tris pH 7.2, 1 mM EDTA, 0.2% Triton X-100) and kept on ice for 10 min. The high molecular weight DNA fraction was pelleted and the supernatant, containing the low molecular weight DNA, transferred to a clean tube. The high molecular weight DNA fraction was resuspended in 100 μl of lysis buffer. DNA from the three different fractions (shed, low and high molecular weight DNA from monolayer) was precipitated with 12.5% (final concentration) trichloroacetic acid (TCA) overnight at 4°C. DNA pellets were extracted in 80 μl of 5% TCA by heating at 90°C for 10 min. Perchloric acid was added to a final concentration of 0.5 N. DNA concentration was determined by the diphenylamine method (DPA). 200 μl of freshly prepared reagent (150 mg DPA, 150 μl of H2SO4, and 50 μl of acetaldehyde (16 mg/ml)/10 ml of glacial acetic acid) were added to the DNA samples. The color reaction was developed at 30°C overnight and samples were read at 590 nm along with a standard curve prepared from salmon sperm DNA. The fraction of DNA from cells in the supernatant, expressed as percentage of total DNA, is an index of cell shedding.

The flow cytometric analysis of propidium iodide stained nuclei was performed as described by Nicoletti et al. (1991). The fluorescence of individual nuclei was measured and the recorded data analysed using a FACScan flow cytometer and software (Becton Dickinson) respectively.

References

  1. Augenlicht L, Taylor J, Anderson L and Lipkin M. . 1991 Proc. Natl. Acad. Sci. USA 88: 3286–3289.

  2. Bedi A, Pasricha PJ, Akhatar AJ, Barber JP, Bedi JC, Giadiello FM, Zehnbauer BA, Hamilton SR and Jones RJ. . 1995 Cancer Res. 55: 1811–1816.

  3. Bursch W, Paffe S, Putz B, Barthel G and Schulte-Hermann R. . 1990 Carcinogenesis 11: 847–853.

    CAS  Article  Google Scholar 

  4. Carethers JM, Hawn JK, Greenson JK, Hitchcock CL and Boland CR. . 1998 Gastroenterology 114: 1188–1195.

    CAS  Article  Google Scholar 

  5. Chang S-K, Dohrman A, Basbaum C, Ho S, Tsuda T, Toribara N, Gum J and Kim YS. . 1994 Gastroenterology 107: 28–36.

    CAS  Article  Google Scholar 

  6. Chen SS-Y, Zheng H, Su M-W, Wilk R, Killeen MT, Hedgecock EM and Culotti JG. . 1996 Cell 87: 187–195.

    CAS  Article  Google Scholar 

  7. Cho KR and Fearon ER. . 1995 Curr. Opin. Genet. Dev. 5: 72–78.

  8. Cho KR, Oliner JD, Simons JW, Hedrick L, Fearon ER, Preisinger AC, Hedge P, Silverman GA and Vogelstein B. . 1994 Genomics 19: 525–531.

    CAS  Article  Google Scholar 

  9. Colony P. . 1989 In: Cell and molecular biology of colon cancer. L. Augenlicht (ed.) CRC Press: Boca Raton, Florida pp.1–25.

    Google Scholar 

  10. Colony P and Neutra M. . 1983 Dev. Biol. 97: 349–363.

  11. Derynck R, Gelbart WM, Harland RM, Heldin C-H, Kern SE, Massague J, Melton DA, Mlodzick M, Padgett RW, Roberts AB, Smith J, Thomsen GH, Vogelstein B and Wang X-F. . 1996 Cell 87: 173.

    CAS  Article  Google Scholar 

  12. Eppert K, Scherer SW, Ozcelik K, Pirone R, Hoodless P, Kim H, Tsue L, Bapat B, Gallinger S, Andrulis IL, Thompsen GH, Wrana JL and Attisano L. . 1996 Cell 86: 543–552.

    CAS  Article  Google Scholar 

  13. Fazell A, Dickinson SL, Hermiston ML, Tighe RB, Steen RG, Small CG, Stoeckli ET, Keino-Masu K, Masu M, Rayburn H, Simons J, Bronson RT, Gordon JI, Tessier-Lavigne M and Weinberg RA. . 1997 Nature 386: 796–804.

    CAS  Article  Google Scholar 

  14. Fearon ER. . 1996 Biochem. Biophys. Acta 1288: M17–M23.

  15. Fearon ER, Cho KR, Nigro JM, Kern SE, Simons JW, Ruppert JM, Hamilton SR, Preisinger AC, Thomas G, Kinzler KW and Vogelstein B. . 1990 Science 247: 49–56.

    CAS  Article  Google Scholar 

  16. Fearon ER and Vogelstein B. . 1990 Cell 61: 759–767.

    CAS  Article  Google Scholar 

  17. Gendler SJ and Spicer AP. . 1995 Ann. Rev. Physiol. 57: 607–634.

  18. Gotley DC, Reeder J, Fawcett J, Walsh M, Bates P, Simmons D and Antalis T. . 1996 Oncogene 13: 787–795.

  19. Gum J, Hicks J, Toribara N, Siddiki B and Kim YS. . 1994 J. Biol. Chem. 269: 2440–2446.

  20. Hahn SA, Schutte M, Shamsul Hoque ATM, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, Fisher A, Yeo CJ, Hruban RH and Kern SE. . 1996 Science 271: 350–353.

    CAS  Article  Google Scholar 

  21. Hedrick L, Cho KR, Fearon ER, Wu TC, Kinzler KW and Vogelstein B. . 1994 Genes Dev. 8: 1174–1183.

  22. Heerdt B, Houston M and Augenlicht L. . 1994 Cancer Res. 54: 3288–3294.

  23. Heerdt B, Houston M, Rediske J and Augenlicht L. . 1996 Cell Growth & Diff. 7: 101–106.

  24. Itoh F, Hinoda Y, Ohe M, Ohe Y, Ban T, Endo T, Imai K and Yachi A. . 1993 Int. J. Cancer 53: 260–263.

    CAS  Article  Google Scholar 

  25. Jen J, Kim H, Piantadosi S, Liu Z, Levitt RC, Sistonen P, Kinzler KW, Vogelstein B and Hamilton SR. . 1994 N. Engl. J. Med. 331: 213–221.

  26. Keino-Masu K, Masu M, Hinck L, Leonardo ED, Chan S S-Y, Culotti JG and Tesier-Lavigne M. . 1996 Cell 87: 175–185.

    CAS  Article  Google Scholar 

  27. Kim H, Roth KA, Moser AR and Gordon JI. . 1993 J. Cell Biol. 123: 877–893.

  28. Klingelhutz AJ, Hedrick L, Cho KR and McDougal JK. . 1995 Oncogene 10: 1581–1586.

  29. Kolodziej PA, Timpe LC, Mitchell KJ, Fried SR, Goodman CS, Jan LY and Jan YN. . 1996 Cell 87: 197–204.

    CAS  Article  Google Scholar 

  30. Laboisse C. . 1989 In: Cell and molecular biology of colon cancer. L. Augenlicht (ed.) CRC Press: Boca Raton. pp.28–43.

    Google Scholar 

  31. Martinez-Lopez E, Abad A, Font A, Monzo M, Ojanguren I, Pifarre A, Sanchez JJ, Martin C and Rossell R. . 1998 Gastroenterology 114: 1180–1187.

    Article  Google Scholar 

  32. Mehelen P, Rabizadeh S, Snipas S, Assa-Munt N, Salvesen G and Bredesen D. . 1998 Nature 395: 801–804.

    CAS  Article  Google Scholar 

  33. Moser AR, Pitot HC and Dove WF. . 1990 Science 247: 322–324.

    CAS  Article  Google Scholar 

  34. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F and Riccardi C. . 1991 J. Immunol. Meth. 139: 271–279.

  35. Ookawa K, Sakamoto M, Hirohashi S, Yoshida Y, Sugimura T, Terada M and Yokota J. . 1993 Int. J. Cancer 53: 382–387.

    CAS  Article  Google Scholar 

  36. Pierceall WE, Cho KR, Getzenberg RH, Reale MA, Hedrick L, Vogelstein B and Fearon ER. . 1994 J. Cell. Biol. 124: 1017–1027.

  37. Reale MA, Hu G, Zafer AL, Getzemberg RH, Levine SM and Fearson ER. . 1994 Cancer Res. 54: 4493–4501.

  38. Reale MA, Reyes-Mugica M, Pierceall WE, Rubinstein MC, Hedrick L, Cohn SL, Nakagawara A, Brodeur GM and Fearon ER. . 1996 Cancer Res. 2: 1097–1102.

  39. Reyes-Mugica M, Rieger-Christ K, Ohgaki H, Ekstrand BC, Helie M, Kleinman G, Yahanda A, Fearon ER, Kleihues P and Reale MA. . 1997 Cancer Res. 382–386.

  40. Reyes-Mugica M, Lin P, Yokota J and Reale M. . 1998 Laboratory Invest. 78: 669–675.

  41. Riefer-Christ KM, Brierly KL and Reale MA. . 1997 Front Biosci. 15: 438–448.

  42. Riggins GJ, Thiagalingam S, Rozenblum E, Weinstein CL, Kern SE, Hamilton S, Wilson JK, Markowitz SD, Kinzler KW and Vogelstein B. . 1996 Nature Genet. 13: 347–349.

  43. Schaeffer, WI and Friend K. . 1976 Cancer Lett. 1: 259–262.

  44. Shibata D, Reale MA, Lavin P, Silverman M, Fearon ER, Steele G, Jessup JM, Loda M and Summerhayes IC. . 1996 N. Engl. J. Med. 335: 1727–1732.

  45. Shoemaker AR, Luongo C, Moser AR, Marton LJ and Dove NF. . 1997 Cancer Res. 57: 1999–2006.

  46. Smits R, Kartheuser A, Jagmohan-Changur S, Leblanc V, Breukel C, de Vries A, van Kranen H, van Krieken JH, Williamson S, Edelman W, Kucherlapati R, Khan PM and Fodde R. . 1997 Carcinogenesis 18: 321–327.

  47. Takaku K, Oshima M, Miyoshi M, Matsui M, Seldin M and Taketo M. . 1998 Cell 92: 645–656.

    CAS  Article  Google Scholar 

  48. Tanaka K, Oshimura M, Kikuchi R, Seki M, Hayashi T and Miyaki M. . 1991 Nature 349: 340–342.

    CAS  Article  Google Scholar 

  49. Thiagalingam S, Langauer C, Leach FS, Schutte M, Hahn SA, Overhauser J, Willson JK, Markovitz SA, Hamiltion SR, Kern SE, Kinzler KW and Vogelstein B. . 1996 Nature Genet. 13: 343–346.

  50. Velcich A and Augenlicht L. . 1993 J. Biol. Chem. 268: 13956–13961.

  51. Velcich A, Palumbo L, Jarry A Laboisse C, Racevskis J and Augenlicht L. . 1995 Cell Growth & Diff. 6: 749–757.

  52. Velcich A, Palumbo L, Selleri L, Evans G and Augenlicht L. . 1997 J. Biol. Chem. 272: 7968–7976.

    CAS  Article  Google Scholar 

  53. Vielmetter J, Kayyem JF, Roman JM and Dreyer WJ. . 1994 J. Cell. Biol. 127: 2009–2020.

  54. Vogelstein B, Fearon ER, Hamilton S, Kern S, Preisinger AC, Leppert M, Nakamura Y, White S, Smits A and Bos J. . 1988 N. Engl. J. Med. 319: 525–532

  55. Zhang L, Zhou W, Velculescu V, Kern SE, Hruban RH, Hamilton SR, Vogelstein B and Kinzler KW. . 1997 Science 276: 1268–1272.

  56. Zhu Y, Richardson J, Parada L and Graff J. . 1998 Cell 94: 703–714.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank Dr B Vogelstein for the generous gift of the CMV – DCC expression plasmid, and critical reading of the manuscript. This work was supported by Grants CA-55913, CA-68965 and P30-13330 from the National Cancer Institute.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Anna Velcich.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Velcich, A., Corner, G., Palumbo, L. et al. Altered phenotype of HT29 colonic adenocarcinoma cells following expression of the DCC gene. Oncogene 18, 2599–2606 (1999). https://doi.org/10.1038/sj.onc.1202610

Download citation

Keywords

  • DCC
  • MUC2 expression
  • colon cancer

Further reading

Search

Quick links