Carcinogenesis

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Carcinogenesis, Vol. 20, No. 11, 2045-2049, November 1999
© 1999 Oxford University Press

Accelerated Papers

Introduction of full-length APC modulates cyclooxygenase-2 expression in HT-29 human colorectal carcinoma cells at the translational level

Linda C. Hsi, Julie Angerman-Stewart and Thomas E. Eling¹

National Institute of Environmental Health Sciences, Eicosanoid Biochemistry Section, Laboratory of Molecular Carcinogenesis, PO Box 12233, Research Triangle Park, NC 27709, USA

	Abstract

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Mutation of the adenomatous polyposis coli (APC) gene is associated with the earliest stages of colorectal tumorigenesis and appears to be responsible for the hereditary condition familial adenomatous polyposis (FAP). Evidence indicates that cyclooxygenase-2 (COX-2) is induced and at elevated levels in human colorectal cancers and in the polyps of mouse FAP models. We have used HT-29 cells, a human colorectal carcinoma cell line with a mutant carboxy-truncated APC gene, in which intact APC gene has been introduced under the control of an inducible promoter. These HT-29-APC cells provide a suitable model system to examine how COX-2 expression becomes dysregulated after loss of APC function. Induction of full-length APC causes the HT-29-APC cells to undergo apoptosis. However, differentiation, as measured by alkaline phosphatase activity, is not induced upon expression of full-length APC. Full-length APC protein has been shown to bind the intracellular protein ß-catenin and, as a result, the Lef/Tcf transcription factors are down-regulated. Analysis of APC immunoprecipitates demonstrate a time-dependent increase of ß-catenin interacting with full-length APC. Thus, the Lef/Tcf signaling pathway is intact at this point in these cells. Furthermore, upon expression of full-length APC, COX-2 protein expression is down-regulated while COX-2 mRNA levels remain the same. These data indicate that APC plays a role, either directly or indirectly, in the translational regulation of COX-2. Treatment of the HT-29-APC cells with sodium butyrate, an inducer of apoptosis, does not alter COX-2 protein expression. Thus, COX-2 down-regulation appears to be APC specific and not just due to apoptotic induction. APC appears to uniquely regulate COX-2 expression. The mechanism by which COX-2 protein expression is down-regulated in the HT-29-APC cells is under investigation.

Abbreviations: APC, adenomatous polyposis coli; COX, cyclooxygenase; FAP, familial adenomatous polyposis; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; NaBT, sodium butyrate.

	Introduction

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Cyclooxygenase (COX) is the key enzyme in arachidonate metabolism and catalyzes the conversion of arachidonic acid to prostaglandin H₂, the precursor for prostaglandins and thromboxanes (1). Two isoforms of the COX enzyme exist, COX-1 and COX-2. COX-1 is constitutively expressed in many tissues (1). The second isozyme, COX-2, which was discovered in 1991, is induced in many inflammatory reactions (1,2). Studies on the regulation of COX-2 expression are numerous and these studies indicate that COX-2 is regulated at the transcriptional level. Many growth factors, cytokines and inflammatory agents all appear to enhance the expression of COX-2 by interacting with various regulatory sequences in the promoter region of this gene (3).

Evidence for the involvement of COXs in cancer was suggested from pharmacologic analysis of prostaglandins. Various animal and human tumor tissues, including human colon cancer, have been reported to contain high concentrations of prostaglandins (4–6). The increased levels of prostaglandins in tumors provided the rational for use of non-steroidal anti-inflammatory drugs (NSAIDs) as potential chemoprevention agents. Accumulating evidence indicates that NSAIDs can reduce the incidence of colorectal cancers in human and experimental animals and can reduce the number and size of polyps in patients with the hereditary condition familial adenomatous polyposis (FAP) (7). Recently, evidence has been presented that COX-2 is induced and at elevated levels in human colorectal cancers, azoxymethane-induced mouse tumors and in the polyps of mouse FAP models (8–12). Moreover, the expression of COX-2 appears to be linked to mutation of the APC gene since COX-2 expression was observed in the intestinal tract of APC mutant mice (13). Perhaps the most striking evidence implicating COX-2 in carcinogenesis is the finding that a null mutation for COX-2 markedly reduced the number and size of intestinal tumors in APC⁷¹⁶ knockout mice, a murine model of FAP (12). Induction of COX-2 is a very early event in the sequence of polyp formation to colon carcinogenesis and suggests that COX-2 plays a critical role in polyp development itself. When the COX-2 gene is inactivated in FAP-model mice, both the size and number of polyps are reduced drastically (12). In addition, selective inhibitors of COX-2 cause a reduction in the number and size of polyps similar to that caused by the COX-2 gene knockout mutations (12). These results provide direct genetic evidence that COX-2 plays a key role in colorectal tumorigenesis resulting from mutation of the APC gene.

Although mutations of the APC gene are associated with the earliest stages of colorectal carcinogenesis and the expression of COX-2, the molecular processes that lead to the expression of COX-2 are not well understood. In this study, we have utilized HT-29-APC cells which express full-length APC under the control of an inducible promoter (14). These cells provide a suitable model system to examine how COX-2 expression becomes dysregulated after loss of APC function.

	Materials and methods

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Cell culture
The human colorectal cell lines HT-29 and HCT-116 were obtained from the American Type Culture Collection (ATCC). HT-29-APC and HT-29-ßgal cells were the generous gift of P.J.Morin and B.Vogelstein (14). HT-29 cells were cultivated in McCoy's 5A media (Gibco BRL) supplemented with 10% fetal bovine serum (Summit) and L-glutamine (Gibco BRL). HT-29-APC and HT-29-ßgal cells were cultivated in the same media as the normal HT-29 cells plus the addition of 0.6 mg/ml hygromycin. HCT-116 cells were cultivated in McCoy's 5A media supplemented with 10% fetal bovine serum. The media for the cells also contained gentamicin (1 mg/100 ml; Life Technologies, Inc.). Unless otherwise indicated, HT-29-APC cells were induced with 100 µM ZnCl₂ (Sigma) for the times indicated. Unless otherwise indicated, HT-29-APC cells were treated with 5 mM sodium butyrate (NaBT) (Sigma) for the times indicated.

SDS–PAGE
For western analysis, remaining attached cells following treatment were harvested, lysed, normalized and then Laemmli sample buffer was added to the samples. The samples were then sheared through a 25 gauge needle and boiled. For APC, proteins were separated by 3% low melting point agarose gel and transferred onto polyvinyldifluoride membrane (Immobilon-P; Millipore) essentially as described previously (15). For COX-2, proteins were separated by 8% SDS–PAGE and transferred onto Hybond-enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham).

Immunoblot analysis
Blots were blocked with 10% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and washed. The blots were then incubated in 1% milk in TBS-T with an anti-appropriate specific antibody. The following antibodies were used: PG27 (Oxford Biochemical Research) for COX-2, APC (Santa Cruz), ß-catenin (Santa Cruz) or actin (Santa Cruz). After washing, blots were incubated with anti-rabbit IgG horseradish peroxidase-linked secondary antibody (Amersham) for COX-2 and APC or anti-goat IgG horseradish peroxidase-linked secondary antibody (Santa Cruz) for ß-catenin and actin respectively. After reacting by chemiluminescence (Amersham ECL detection system), bands were detected by exposure to Hyperfilm-MP (Amersham).

Immunoprecipitation
Cells were lysed in MEBC lysis buffer [50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 0.5% Nonidet P-40] containing protease inhibitors [0.2% (w/v) 4-(2-amino-ethyl)benzenesulfonylfluoride (Calbiochem) and 0.01 mg/ml each of chymostatin, leupeptin, antipain and pepstatin A (all from Sigma)] and phosphatase inhibitors (0.1 mM Na₃VO₄, 50 mM NaF). The samples were equalized for the amount of protein. Samples were pre-cleared with normal mouse or rabbit serum (Accurate Chemical and Scientific Corp.). The samples were then immunoprecipitated with either protein A–Sepharose (Pharmacia Biotech) or protein G–Sepharose (Santa Cruz) beads with the appropriate antibody. The following antibodies were used: mouse monoclonal FE-9 and IE1 (Oncogene Science) for the N- and C-termini of APC, respectively, or rabbit polyclonal APC (Santa Cruz).

Northern analysis
Total RNA from cells was extracted by using TRI Reagent (Sigma). RNA samples were normalized and separated by electrophoresis in a formaldehyde–1% agarose gel. The RNA was transferred in 10x SCC by capillary action onto nylon membrane (Schleicher & Schull) and UV-cross-linked with a Stratalinker UV light source (Stratagene). Human COX-2 (Oxford Biochemical Research) and human glyceraldehyde-3-phosphate dehydrogenase (G3PDH; Clontech) cDNA probes were labeled with [-³²P]dCTP (Amersham Pharmacia Biotech) using the Prime-IT-II random prime kit (Stratagene). After hybridization and washes, the blots were then exposed to X-ray film (Amersham) for autoradiography.

	Results

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Detection of full-length APC
HT-29 is a colorectal cancer cell line which is widely used for experimental studies. These cells retain many biochemical and physiological features of normal colorectal epithelial cells (16). HT-29 cells have a mutant form of the APC gene and express two C-terminal-truncated APC proteins of ~100 and 200 kDa. Introduction of intact APC gene into HT-29 cells under the control of an inducible promoter permits a regulated expression of full-length APC protein and induction of apoptosis (14). HT-29-APC cell lysates were examined for expression of intact APC at various times after treatment with zinc. The expression of full-length APC (300 kDa) was observed (Figure 1). The HT-29-APC cells express full-length APC after 12 h of zinc induction and maintain such expression for at least 4 days (14). In contrast, only the endogenous mutant APC (100 and 200 kDa) could be detected prior to induction (Figure 1). HCT-116 cells, a human colorectal cancer cell line with an intact APC gene that expresses full-length APC protein, were used as a positive control (Figure 1). HT-29 cells transfected with the ß-galactosidase gene in place of the APC gene were also utilized as a control and demonstrate only mutant APC protein after treatment with zinc (Figure 1). These results confirm inducible regulation of full-length APC protein by zinc treatment in the HT-29-APC cells.

View larger version (78K): [in this window] [in a new window]   Fig. 1. Expression of full-length APC in HT-29 cells. Western analysis of cell lysates demonstrating HT-29-APC cells inducible expression of full-length APC (F.L.-APC) when treated for the indicated times with zinc (100 µM) and endogenous mutant APC (MT.-APC). The data shown represent one of three separate experiments with similar results. Forty micrograms of total protein were loaded per lane. Lanes 1 and 8, HCT-116 cells; lanes 2–4, HT-29-APC cells collected at the indicated times, respectively, after treatment with zinc (100 µM); lanes 5–7, HT-29-ßgal cells collected at the indicated times after treatment with zinc (100 µM).

 
Cell growth and apoptosis
The HT-29-APC cells do not reach confluence in the presence of induction, even after long periods of culture, while zinc has no apparent effect on cell density of the HT-29-ßgal cells (14). The HT-29-APC cells gradually round up and detach. Upon expressing full-length APC, HT-29-APC cells also undergo apoptosis as determined by cell counting, TUNEL assay and Hoechst staining (14). The proportion of TUNEL-positive cells increased 10-fold to 3% positive cells after induction compared with uninduced cells (14). We tested and confirmed that following induction of full-length APC, increased apoptosis does occur over a time course from 0 to 96 h (data not shown). Furthermore, we examined whether differentiation occurs following expression of full-length APC as determined by alkaline phosphatase assay. Alkaline phosphatase activity is a marker of cell differentiation (17). We found no increase in alkaline phosphatase activity over the time course from 0 to 96 h following induction (data not shown) indicating no increase in cellular differentiation.

Binding of APC to ß-catenin
Intact APC has been shown to bind the intracellular protein ß-catenin (18,19) and thus down-regulate the Lef/Tcf signaling pathway (20,21). Full-length APC was immunoprecipitated from cell lysates, before and after induction with zinc, with antibodies specific for the C-terminus of APC. The resulting APC immunoprecipitates were analyzed for the presence of ß-catenin by western analysis with an antibody specific to ß-catenin (Figure 2). Analysis of the APC immunoprecipitates demonstrates a time-dependent increase of ß-catenin interacting with full-length APC protein. This shows that binding of full-length APC to ß-catenin does occur and that the Lef/Tcf signaling pathway is presumably active in these cells but is down-regulated as the full-length APC protein is expressed.

View larger version (24K): [in this window] [in a new window]   Fig. 2. ß-Catenin binding to full-length APC. Western analysis of APC immunoprecipitates with a ß-catenin specific antibody. The data shown represent one of four separate experiments with similar results. Twenty micrograms of total protein were loaded per lane. Lanes 1–5, APC immunoprecipitates from HT-29-APC cells collected at the indicated times after treatment with zinc (100 µM); lane 6, APC immunoprecipitate from HCT-116 cells.

 
COX-2 expression
Since HT-29 cells express COX-2, we next determined whether COX-2 expression in the HT-29-APC cells is altered following induction of full-length APC. We performed western analysis of cell lysates both prior to and after treatment with zinc from 0 to 96 h. COX-2 protein was expressed prior to induction and subsequently decreased at early time points following expression of full-length APC (Figure 3). By 48 h following treatment with zinc, COX-2 protein was no longer observed. Furthermore, treatment of wild-type HT-29 cells with zinc has no effect on COX-2 expression (data not shown) demonstrating that zinc itself is not the cause of the loss of COX-2. HCT-116 cells, which do not express any detectable COX-2 protein, were used as negative control. Actin expression was used to demonstrate similar protein loading. Thus, it appears that as full-length APC is expressed, COX-2 protein is down-regulated.

View larger version (0K): [in this window] [in a new window]   Fig. 3. Expression of COX-2 in HT-29-APC cells. Western analysis of HT-29-APC cells treated with zinc (100 µM) and harvested at the times indicated. Top, COX-2; bottom, actin. The data shown represent one of four separate experiments with similar results. Thirty micrograms of total protein loaded per lane. Lane 1, COX-2 standard (10 µg); lanes 2–6, HT-29-APC cells collected at the indicated times after treatment with zinc (100 µM); lane 7, HCT-116 cells.

 
To test whether COX-2 down-regulation is due merely to induction of apoptosis, we next treated the HT-29-APC cells with NaBT, a known inducer of apoptosis. NaBT-dependent induced apoptosis and APC-dependent apoptosis were similar in extent and time course (data not shown). Interestingly, NaBT did not cause down-regulation of COX-2 expression (Figure 4). Actin expression was used to demonstrate similar protein loading. APC appears to uniquely regulate COX-2 expression.

View larger version (0K): [in this window] [in a new window]   Fig. 4. Expression of COX-2 in NaBT-treated HT-29-APC cells. Western analysis of HT-29-APC cells treated with zinc (100 µM) and NaBT (5 mM) harvested at the times indicated. Top, COX-2; bottom, actin. The data shown represent one of two separate experiments with similar results. Thirty-five micrograms of total protein were loaded per lane. Lane 1, COX-2 standard (10 µg); lanes 2–6, HT-29-APC cells collected at the indicated times after treatment with zinc (100 µM) and NaBT (5 mM).

 
COX-2 mRNA expression
We next measured COX-2 mRNA levels both prior to and following expression of full-length APC in the HT-29-APC cells from 0 to 96 h. The COX-2 mRNA levels remained relatively the same despite induction of full-length APC (Figure 5). G3PDH was used as a control for the amount of mRNA loaded. The ratio of COX-2 to G3PDH was unchanged indicating the level of COX-2 mRNA expression was not altered as full-length APC protein was expressed. Thus, the transcriptional control of COX-2 is not effected by expression of full-length APC.

View larger version (0K): [in this window] [in a new window]   Fig. 5. Expression of COX-2 mRNA in HT-29-APC cells. Northern blot of COX-2 (4.6 kb) and G3PDH. The data shown represent one of three separate experiments with similar results. Each lane contains 20 µg of total isolated RNA. G3PDH was used as a control for the amount of RNA loaded. Lanes 1–5, RNA isolated from HT-29-APC cells collected at the indicated times after treatment with zinc (100 µM); lane 6, RNA isolated from HT-29 cells treated with TPA (50 ng/ml) for 24 h as a positive control for COX-2. Dositometry of the bands was performed and expressed as the ratio COX-2/G3PDH.

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
Previous studies on the regulation of COX-2 expression have indicated that COX-2 is regulated at the transcriptional level. Many growth factors, cytokines and inflammatory agents all appear to enhance the expression of COX-2 by interacting with various regulatory sequences in the promoter region of this gene (3). However, the regulation of COX-2 expression in colorectal tissue and colorectal cells in culture as a result of mutation in the APC gene is not understood. In the HT-29-APC cells, our results clearly indicate that critical regulation of COX-2 expression occurs at the translational level. HT-29 cells, which have a mutant APC gene, constitutively express COX-2 mRNA and protein. Upon expression of full-length APC protein in HT-29-APC cells, COX-2 mRNA levels remain constant but there is a time-dependent decrease in the expression of COX-2 protein. The decrease in expression of COX-2 protein appears to correlate with the association of the full-length APC protein to ß-catenin. The expression of full-length APC is also accompanied by the induction of apoptosis in the HT-29-APC cells, as expected for a functional APC protein. The induction of apoptosis and a decrease in COX-2 expression are in agreement with recent findings in the literature which report that COX-2 over-expression results in a loss of apoptosis (22). We tested whether the COX-2 down-regulation in the HT-29-APC cells was due to induction of apoptosis. However, treatment of the HT-29-APC cells with NaBT, a known inducer of apoptosis, had no effect on COX-2 expression. Thus, COX-2 down-regulation appears to be APC specific and not merely due to apoptotic induction.

ß-Catenin transmits signals from the E-cadherin adhesion proteins to the cell's interior (23–25). ß-Catenin interacts with the Tcf and Lef transcription factors. HTcf-4, the Lef/Tcf transcription factor active in gut tissue, transactivates transcription only when associated with ß-catenin (20,21). Full-length APC protein has been shown to bind the intracellular protein ß-catenin and, as a result, the Lef/Tcf transcription factors are down-regulated (18,19). In cells lacking a full-length APC protein, ß-catenin accumulates in the nucleus, where it gets attached to hTcf-4 and activates transcriptional events. Our results suggest that COX-2 mRNA is translated and COX-2 protein expression is observed. However, as full-length APC binds to ß-catenin, it acts to down-regulate the Lef/Tcf-4 pathway which subsequently down-regulates COX-2 protein expression.

The regulation of COX-2 expression in colorectal cells has not been extensively investigated. Kutchera et al. (26) report that HCT-116 cells have increased mRNA for COX-2 as a result of constitutive transcription controlled by the COX-2 promoter. They also transfected their HCT-116 cells to overexpress the APC gene and found no change in the increased level of COX-2 mRNA. COX-2 protein levels, however, were not examined in this study. Kutchera et al. (26) do warn that HCT-116 cells may represent a minority of colon carcinomas as it has an intact APC gene. In HT-29 cells, the APC gene is mutated and expresses truncated APC protein, similar to that observed in most colorectal carcinomas. Our results suggest that COX-2 mRNA is constitutively expressed in HT-29 cells as Kutchera et al. observed in HCT-116 cells (26), but down-regulation of the ß-catenin pathway by full-length APC protein disrupts translation of the COX-2 mRNA to protein.

Our findings in HT-29-APC cells indicate that as full-length APC is expressed, COX-2 protein is down-regulated while COX-2 mRNA levels remain unchanged. Translational regulation of COX-2 is defective in this human colon cancer cell line. The mechanism by which COX-2 expression is lost is currently under investigation. It appears that APC uniquely regulates, either directly or indirectly, COX-2 expression. This phenomenon is likely to occur in other human colon cancer cell lines which have a mutant APC gene.

	Acknowledgments

 
We thank Drs P.J.Morin, B.Vogelstein and K.W.Kinzler for the generous gift of the HT-29-ßgal and HT-29-APC cells.

	Notes

 
¹ To whom correspondence should be addressedEmail: eling{at}niehs.nih.gov

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Received June 17, 1999; revised August 10, 1999; accepted August 16, 1999.

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