Human DNA methyltransferase gene DNMT1 is regulated by the APC pathway

Paul M. Campbell and Moshe Szyf1

Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada H3G 1Y6


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epigenomic changes in DNA methylation patterns are evident in a variety of cancers, including colorectal cancer (CRC). In addition, a large proportion of CRC tumors and cell lines harbor genetic mutations in the APC/ß-catenin/TCF transcription activation pathway. While several target genes have been proposed, a causal downstream agent between APC mutation and cancer has not been fully established. Because previous work implicates DNA methyltransferase (DMNT1) as a critical point in tumorigenesis and recent studies suggest that familial CRC also exhibits epigenetic alterations, we sought to investigate whether this gene might be regulated by APC in CRC. Reconstitution of wild type APC in HT-29 CRC cell lines reduced the expression of both a reporter gene driven by the minimal DNMT1 promoter and DNMT1 mRNA that is independent of cell growth stasis. We also provide evidence for a causal role of DNMT1 in CRC by demonstrating that antisense-driven reduction of DNMT1 mRNA inhibits anchorage-independent growth, an indicator of tumorigenesis, of CRC cells. These data support future consideration of DNMT1 as a target in the treatment of CRC.

Abbreviations: APC, adenomatous polyposis coli; CAT, chloramphenicol acetyltransferase; CRC, colorectal cancer; DNMT1, DNA (cytosine-5) methyltransferase 1; DNTCF, dominant negative N-terminal mutant T-cell factor; FAP, familial adenomatous polyposis; HNPCC, hereditary nonpolyposis colorectal cancer; Lef, lymphocyte enhancer factor; TCF, T-cell factor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The methylation status of DNA and the machinery responsible for it have been implicated in both the initiation and progression of cancer (1). The primary enzyme responsible for adding methyl groups to 5 position of cytosine in a CG dinucleotide motif is DNA (cytosine-5) methyltransferase (DNMT1). Cancer cell lines and tumor biopsies show increased levels of DNMT1 protein and activity (2,3). It has also been demonstrated that promoter region hypermethylation can inhibit the transcription of target genes, including the tumor suppressors p14ARF and p16INK4a (4,5) and BRCA1 (6). Recent evidence expands on the importance of gene silencing via promoter methylation in inherited cancers, where inactivation of the wildtype allele follows the germline mutation (7). In addition to an enzymatic role for DNMT1 in cancer, it appears that protein–protein interaction between DNMT1 and other players can affect cell cycle (8,9). Inhibition of DNMT1 gene or DNMT1 protein by antisense or suicide oligonucleotides respectively, has been shown to suppress the initiation of DNA replication and carcinoma cell growth (10,11). One of these agents reduced the progression of xenograft tumors in LAF mice (12) and is currently in development in clinical trials (52). While another DNMT1 antisense oligo has also shown the ability to stimulate expression of p21 and p16 tumor suppressor genes (13,14), the mechanisms for the plethora of actions of DNMT1 as well as its inhibitors with respect to cancer is currently unknown.

While most forms of CRC are currently designated sporadic, ~80% show mutated versions of APC (15). In hereditary cases of CRC, the Wnt/APC/ß catenin/TCF cascade is implicated in the initiation and progression of the disease. In healthy intestinal epithelia, the APC/ß catenin pathway is involved in the migration of cells from the proliferative zone of the crypt to the villus. When Apc or ß catenin genes are mutated in familial adenomatous polyposis (FAP), these cells do not migrate; rather they continue to divide and thus accumulate, forming polyps. At the same time, there is a disruption in the normal functioning of APC to target ß catenin for degradation by proteosomes, and ß catenin accumulates first in the cytoplasm, then translocates to the nucleus. Here it activates the TCF/Lef (T-cell factor/lymphocyte enhancer factor) family of transcription factors that bind to DNA in a site-specific manner (for review see ref. 16).

The potential target genes of this activation responsible for CRC are now being discovered, and several candidates exist including c-myc, cyclin D1, PPAR{delta} and CD44 (1721). A genetic interaction between DNMT1 and the APC/ß catenin pathway in colorectal cancer in mice has already been put forth by others (22,23). Laird et al. showed that mice with a homologous mutation in APC (24) developed spontaneous intestinal tumors like those in FAP colorectal cancer patients, and when crossed with mice with reduced expression of methyltransferase, the number of tumors and the degree of genetic instability were decreased (22). When the amount of cytoplasmic DNMT1 protein was further reduced using the suicide substrate 5-aza-deoxycytidine, polyp number was additionally decreased.

In this paper we test the hypothesis that APC/ß catenin pathway regulates DNMT1 expression in human cancer cells, thus providing an explanation for the genetic interaction observed in the mouse model. We report here that mutations in the tumor suppressor Apc gene increase the transcriptional activation of DNMT1 promoter and mRNA levels in human cancer cells. We further show that antisense oligonucleotides to DNMT1 mRNA can inhibit anchorage-independent growth of these APC-mutated cells, thus supporting the hypothesis that DNMT1 is a critical mediator in APC-mutation triggered colorectal tumorigenesis. Our results might support use of DNMT1 inhibitors in treatment of FAP.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
The HT-29 Apc–/– human colon carcinoma cells were obtained from the American Type Culture Collection and were maintained in McCoy's 5A medium (Gibco BRL) supplemented with 10% fetal calf serum (Colorado Serum Company) and 0.292 mg/ml glutamine. HT29APC cells, a stable HT-29 transfectant expressing a wild-type APC cDNA under the direction of a metallothionein promoter, and HT29ßGal, and a control transfectant expressing the bacterial ß-galactosidase gene under the direction of the same promoter, were kind gifts of Drs Kinzler and Vogelstein (25). APC or ßGal expression was induced in the respective transfectants by adding 100 µM of ZnCl2 to the culture medium.

To determine cell growth kinetics, 50 000 HT29ßGal or HT29APC cells per well were plated in six well dishes in standard conditions and treated with 100 µM ZnCl2 the following day. Cells were harvested after 0, 6, 12, 24, and 48 h of induction by trypsin and counted. Experiments were performed in triplicate.

To establish the effect of growth inhibition on DNMT1 expression, two methods of cell stasis were used. First, proliferating HT29APC cells were subjected to serum starvation (1% fetal calf serum as opposed to 10%) for 3 days. Cells were then treated with 100 µM of ZnCl2 for 24 h, harvested and total RNA extracted for northern analysis. Second, cell cycle inhibition involved transient transfection of a constitutively expressed human wildtype p53 tumor suppressor gene, a kind gift of Dr Branton (26), into HT29APC cells. Cells were harvested after 48 h and total RNA extracted for northern analysis.

To investigate the effect of wildtype APC on ectopic DNMT1 transcription, transient transfections were performed into HT29APC of a DNMT1 expression construct with a CMV promoter in place of the endogenous reporter (full length DNMT1 (27) in pcDNAHis vector (Invitrogen)). Transfection efficiency and translational control was ascertained by cotransfection of a CMV–GFP cDNA in the same vector, and blotting with anti-GFP antibodies (sc-8334, Santa Cruz Biotechnology, Santa Cruz, CA). Cells were observed for GFP expression, harvested, and proteins extracted and resolved. Western blotting against the Xpress tag of the vector according to the manufacturer's instructions allowed for specific visualization of the exogenous DNMT1 expression.

Antisense oligonucleotide treatments were performed in serum-reduced media (OptiMEM, Gibco BRL) before changing the media back to McCoy's 5A. For anchorage–independent growth assays, treated HT-29 cells were mixed with enriched media (McCoy's 5A containing an additional 10% of fetal calf serum) before adding the agar. For all experiments, cells were maintained at 37°C, 95% O2, 5% CO2.

CAT reporter assays
Cells were plated in six-well plates at a density of 300 000/well. Transfections of reporter constructs took place the following day. CAT reporter assays were performed as has been previously described (28). Briefly, a 0.8 kb genomic fragment containing the 5' DNMT1 minimal promoter was excised with XbaI and BamHI and ligated to a bacterial chloramphenicol acetyltransferase reporter construct (pCAT, Promega) to create pMet-P1-{Delta}XH-CAT ((29), Figure 1AGo). Using standard calcium-mediated transfer protocols (28), 5 µg of pMet-P1-{Delta}XH-CAT reporter was transfected in triplicate either alone or in consort with plasmids containing an inducible wild type Apc or lacZ cassette (5 µg each, Figure 1BGo, pSAR-MT-APC and pSAR-MT-ßGal respectively, generous gifts from Drs B.Vogelstein and K.Kinzler, Johns Hopkins University (25)) into HT-29 human colon carcinoma cells. Additional reporter activation experiments were performed with the same inducible constructs stably transfected into HT-29 cells (HT29APC and HT29ßGal). In both cases, production of full length APC protein was induced with 100 µM ZnCl2.



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Fig. 1. Full-length APC suppresses DNMT1 mRNA levels in HT-29 CRC cells. Either a promoterless CAT construct (pCAT) or a DNMT1 5' minimal promoter CAT construct ((29), pMet-P1-{Delta}XH-CAT) (A) were transfected in triplicate into HT-29, HT29APC or HT29ßGal cells. (B) The physical map of the APC/ßGal expression vector. (C–D) CAT activity was measured 2 days after transfection as described in Materials and methods. The graphs depict the average of three transfections +SEM (C). pMet-P1-{Delta}XH-CAT was transiently transfected into cells harboring a stable (C) or transient (D) APC (pSAR-MT-APC) or ß-galactosidase (pSAR-MT-ßGal) expression vector.

 
Forty-eight hours after transfection, cells were harvested and total cellular extracts were isolated. Fifty µl of the extract was used per assay. DNMT1 promoter-driven CAT gene activation was measured by the enzymatic transfer of tritiated acetyl groups from [3H]acetylcoenzyme A to chloramphenicol. The latter was extracted and 3H counted.

Adenoviral infection and expression
To demonstrate a dependence of DNMT1 transcription on the ß catenin/TCF pathway in human colon cancer cells, 300 000 HT-29 cells were plated in standard conditions. These cells were then infected the following day with 3–300 MOI of an adenovirus expressing either ß-galactosidase or dominant-negative human TCF4 (AdßGal or AdDNTCF4, respectively, generous gifts from Drs B.Vogelstein and K.Kinzler, Johns Hopkins University (25)). Virus stocks were propagated in HEK293 cells to high titers, and then purified in CsCl gradients (18). Cells were harvested 48 h after infection and total RNA was processed for northern blotting.

DNMT1 antisense treatment and anchorage-independent growth assays
The DNMT1 antisense used in this study was previously characterized (11,14). HT-29 cells were plated at a density of 50 000 cells per well in six well dishes 18 h before treatment. Lipofectin (6.25 µl/ml final concentration, Gibco BRL) was mixed with 100 µl of serum-reduced OptiMEM media (Gibco BRL) for 45 min at room temperature. Two hundred nM (final concentration) of MG88F (5'-fluoroscein-tagged) or MG208 4 x 4 hybrid 2'-O-methylphosphorothioate antisense oligonucleotide (14) (5'-AAGC-AATGAGCACCGTTCTCC-3' and 5'-AACGATCAGGACCCTTGTCC-3', respectively, OligosEtc., Wilsonville, OR; underlined bases are 2'-O-methyl modified) was added and incubated for an additional 15 min. The volume was brought to 2 ml and the oligo mixture was layered onto PBS-rinsed cells. Transfection of the oligo into the cells took place over 4 h at 37°C before the media was replaced with normal McCoy's 5A. Antisense treatment was repeated once. Treated cells were trypsinized, and 3000 cells/well were counted and mixed with 3 ml of enriched media and 1 ml of warm (65°C) 1.5% agar and plated onto six-well plates. One ml of standard McCoy's media was added to the top of the gelled matrix and colonies were allowed to grow at 37°C for 21 days. Digital images were captured with a CCD camera (XC-77, Sony) through a Nikkor 55 mm lens (Nikon) to a MCID-M4 image analysis system (Imaging Research, St Catharines, ON). As a control, 300 cells per well were grown in standard conditions to ensure that treated cells were still viable.

Northern and western blot analyses
Cells were lysed directly on the culture plate with 1 ml of Trizol (Gibco BRL) and total RNA prepared as per the manufacturer's instructions. Ten µg of RNA were separated in a 1% denaturing agarose gel, transferred to nitrocellulose (Hybond N+, Amersham Pharmacia Biotech) and crosslinked with 120 mJ of UV radiation (Stratalinker 2400, Stratagene). The northern blots were prehybridized in a phosphate-based cocktail (5 ml 1 M sodium phosphate, pH 6.8, 5 ml dH2O, 20 µl 0.5 M EDTA, 1 ml 2 mg/ml herring sperm, 0.7 g SDS) for 1 h at 68°C, and then were hybridized with a 32P-labelled BamHI–EcoRI cDNA fragment of DNMT1 (Accession number NM_001379, +156–500) for 5 h at 68°C. Membranes were washed 2 x10 min Solution A (3 ml 2 mg/ml herring sperm, 25 g SDS, 20 ml 1 M NaPO4 pH 6.8, 1 ml 0.5 M EDTA) at 65°C, 4 x10 min Solution B (5 g SDS, 20 ml 1 M NaPO4, pH 6.8, 1 ml 0.5 M EDTA) at 65°C and exposed to XAR film (Eastman Kodak). Blots were stripped off the DNMT1 probe and rehybridized to an 18S rRNA-specific 32P-labelled oligonucleotide and exposed again to a film. Films were scanned and the DNMT1 signal was quantified on a MCID-M4 image analysis system (Imaging Research, St Catharines, ON).

Western blot analysis to study expression of full and mutated APC protein was performed using the method described by Smith et al. (30). Proteins (100 µg) from whole cell lysates were separated on 3% agarose/ TBE/0.1% SDS, blotted to Hybond P+ membranes (Amersham). Membranes were blocked in 10% non-fat milk in TBST (Tris-buffered saline + 0.1% Tween-20) for 1 h at room temperature and incubated with 1 µg/ml in 5% milk/TBST of a mouse monoclonal antibody against APC (OP44, Oncogene Research Products, San Diego, CA) overnight at 4°C. An HRP-conjugated goat anti-mouse secondary was used for 1 h at room temperature and the signals were visualized with enhanced chemiluminescence using the standard ECL kit from Amersham.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Wild type APC expression suppresses the activity of the human DNMT1 promoter in a transient transfection assay
To examine the possibility that increased activity of the DNMT1 promoter is a downstream consequence of mutations in the APC/ß-catenin/TCF axis, we utilized a previously characterized reporter construct bearing the bacterial CAT gene under the direction of the 5' minimal promoter of the human DNMT1 gene (10) (Figure 1AGo). When transiently transfected into HT-29APC colon carcinoma cells, the 0.8 kb HindIII–XbaI minimal human DNMT1 promoter stimulates the expression CAT >14-fold over the basal activity of the promoterless construct. Zinc induction of the wildtype, full-length APC protein inhibited the DNMT1-driven CAT activity by >76% (Figure 1CGo; P = 0.011). To ensure that the results were not an artefact of the specific stable APC transfectant, we transiently cotransfected either the Zn-inducible wildtype Apc gene or control ß-Gal vector along with pMet-P1-{Delta}XH-CAT reporter constructs into HT-29 cells. While induction of full length APC reduced DNMT1-dependent CAT activity by 85% (Figure 1DGo; P = 0.00016), the ß-Gal control plasmid had no effect. These data are consistent with the hypothesis that induction of DNMT1 promoter is a downstream consequence of disrupted APC/ß catenin/TCF transcription pathway in CRC, and that the inability of truncated APC protein to cause ß catenin degradation may result in augmented DNMT1 expression.

Full-length APC suppresses DNMT1 mRNA levels in HT-29 CRC cells
Since the mutated APC pathway results in stimulation of DNMT1 promoter activity in a transient transfection reporter assay, we determined whether APC could modulate the expression of the endogenous mRNA in HT-29 cells. HT29APC cells were induced to express wild type APC and DNMT1 mRNA was quantified by northern blot analysis. The DNMT1 signal was normalized to the 18S rRNA signal (Figure 2BGo). As a control we subjected HT29ß-Gal transfectants to the same treatment. Expression of wild type APC following Zn induction of HT29APC cells was verified by a western blot analysis (Figure 3BGo). Our results reveal that after a 9 h exposure to 100 µM ZnCl2, HT29APC Zn-induced cells show 50% diminished levels of DNMT1 mRNA relative to that of HT29ß-Gal cells (Figure 2AGo). Continued 40% suppression of DNMT1 transcription was seen at 12 h following wild type APC induction. Repeated experiments yielded similar results. Later time points of Zn induction were not analyzed for DNMT1 message as global mRNA degradation was evident (data not shown). This may have been a consequence of increasing cell detachment or apoptotic DNA cleavage.



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Fig. 2. Induction of wildtype APC protein reduces DNMT1 mRNA. HT-29 cells stably transfected with either pSAR-MT-APC or pSAR-MT-ßGal (25) were treated with 100 mM ZnCl2 and total RNA was isolated at indicated time points and subjected to a northern blot analysis with a DNMT1 cDNA probe (Accession number NM_001379, +156–500) (A). A0, HT29APC cells noninduced; AZ, ZnCl2 induced HT29APC cells; ß0, HT29ßGal cells noninduced; ßZ, ZnCl2 induced HT29ßGal cells. (B) Quantification of DNMT1/18S RNA ratio, normalized to that of noninduced cells is presented in the graph.

 


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Fig. 3. Wildtype APC protein inhibits growth of colon carcinoma cells. HT29APC or control HT29ßGal cells were treated with 100 mM ZnCl2 and counted at several points after induction (*P < 0.05, #P < 0.0005) (A). Western blot analysis of APC protein in ZnCl2 treated and untreated HT29APC cells (B). The arrow shows induced wildtype APC, the asterix indicates degradation of the full-length APC, and the large arrowhead marks truncated mutant forms of APC.

 
Wildtype APC inhibits cell proliferation
DNMT1 inhibition has been previously shown to inhibit the proliferation of human cancer cells in vitro (10,11). Here we show that the reduction in DNMT1 mRNA following APC induction coincides with inhibition of cell growth. HT29APC colon carcinoma cells supplemented with 100 µM ZnCl2 to induce full length APC (Figure 3BGo) rapidly reach a plateau in proliferation (Figure 3AGo) only 6 h after initiation of Zn induction. Further expression of wildtype APC for 2 days inhibited cell growth by 67%, while ß-galactosidase induction in control cells had little effect on proliferation at either time point. Morin et al. have previously shown that the growth plateau following wildtype APC reconstitution persists for at least 5 days and is the result of apoptosis (25).

Because of the links shown between DNMT1 and elements of cell cycle regulation (31) and DNA replication (32), we sought to discover whether the reduction in DNMT1 expression was a result of the either transcription regulation or the growth effects of differing APC status. We first inhibited HT29APC cell proliferation by limiting exposure to fetal calf serum to one-tenth of its concentration in normal media. We found that this results in cell growth inhibition without overt cell death (data not shown). No differences were seen in the level of DNMT1 transcription for the serum starved cells versus the untreated counterparts (Figure 4AGo). Serum deprived cells were then treated with ZnCl2 to induce wildtype APC. Full-length APC protein expression results in a 61% reduction of DNMT1 mRNA (Figure 4DGo) in serum deprived HT29APC cells.



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Fig. 4. Reduction of DNMT1 is not solely a result of growth inhibition. (A) HT29APC cells were arrested by serum starvation (SS) or human wildtype p53 expression (p53) or left in normal proliferating conditions (untreated, UT). Total RNA was resolved and probed with a radiolabeled DNMT1 probe (upper panel) and 18S (lower panel). (B) Serum starved and p53 transfected HT29APC cells were treated with Zn to induce full-length APC. Total RNA was resolved and probed with similar probes as in (A). (C–D) Quantification of northerns in A and B, respectively. DNMT1 mRNA signal is normalized to 18S rRNA signal. (E) Western blot of p53 treated cells. p53 protein is indicated, arrowheads indicate degradation products of exogenous p53 and mutant p53 isoforms. (F) Western blot showing DNMT1 expression driven by a CMV promoter is not affected by wildtype APC induction. CMV–GFP serves as a transfection/expression control.

 
We then investigated whether the tumor suppressor p53 could modulate the expression of DNMT1, and whether wildtype APC reconstitution was additive. The parent HT-29 cell line harbors mutant p53 (33). Transient transfection of a human wildtype p53 expression vector resulted in a one-third reduction of DNMT1 mRNA (Figure 4A,C). Further reduction of DNMT1 message, to almost 34% of that with p53 expression alone, was effected by induction of wildtype APC (Figure 4B,D). These results indicate that while proliferation status is a factor in the degree of expression of DNMT1 in certain contexts, so too does APC have a role in the transcriptional activation of this methyltransferase protein.

Dominant-negative TCF inhibits the transcription of DNMT1
To further test the hypothesis that DNMT1 is one of the downstream effectors of APC/ß-catenin/TCF signalling, we took advantage of an N-terminal deletion dominant negative mutant of the human transcription factor TCF4 (DNTCF4 (34)). This dominant-negative mutant has been previously shown to suppress transcription of APC/ß catenin/TCF regulated sequences. We infected HT-29 cells with either an adenovirus expressing this N-terminal mutant or a control adenovirus expressing ß-Gal at the indicated MOI. Both viruses express the fluorescent protein GFP which allowed for monitoring the level of infection by fluorescence microscopy. RNA was extracted from the cells 48 h after initiation of infection and was subjected to a northern blot analysis. As seen in Figure 5Go, DNMT1 mRNA levels are diminished with increasing expression of DNTCF4.



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Fig. 5. Inhibition of TCF transcription decreases DNMT1 mRNA. HT-29 cells were treated with increasing multiplicities of infection of an adenovirus expression vector harboring either a dominant negative human TCF4 or a ß-galactosidase cassette. Total RNA was extracted and subjected to northern blot analysis with either a radiolabeled DNMT1 probe (A, upper panel) or 18S rRNA (A, bottom panel). Quantification is depicted in (B) showing DNMT1/18S ratio.

 
To further ensure that reduction of DNMT1 was mediated through TCF activation in cis, we performed transfections of a DNMT1 expression construct that lacks the 5' DNMT1 promoter, driven instead by a CMV promoter lacking TCF consensus sites. In HT29APC cells, activation of APC by Zn had no effect on the expression of the exogenous DNMT1 gene (Figure 3FGo). Transfection and expression efficiency was similarly unaffected for a GFP expression construct driven by the same CMV promoter.

DNMT1 antisense inhibition of colony formation
To test whether increased DNMT1 expression plays a causal role in the pathway leading from APC mutation to cellular transformation, we determined whether an antisense knockdown of DNMT1 in HT-29 cells inhibited the transformed properties of these cells using anchorage-independent growth as an indicator of tumorigenicity (35). We have previously designed and demonstrated the efficacy of DNMT1 antisense oligonucleotides for decreasing cell line tumorigenicity ex vivo and in vivo (12). MG88 is a human DNMT1 antisense that has previously been shown to inhibit DNMT1 mRNA at an EC50 of 80 nM (11,14). Three daily treatments of HT-29 cells with 200 nM doses of either MG88F or the mismatched control MG208 were performed. Colonies were visible in the soft agar matrix after 7–10 days, and were quantified after 21 days in agar. Digital images representative of triplicate wells are shown in Figure 6AGo–B. HT-29 cells treated with MG88F lose the ability to grow in a semi-solid medium, with visible colony number being reduced in MG88F-treated cells by 89% as compared with control treatment (Figure 6CGo). Figure 6DGo shows intracellular localization of the fluorescein-tagged MG88F with intense accumulation in the nucleus 4 h after transfection on Day 1. Treated cells were also plated on plastic cell culture plates to insure that viable contact dependent growth in standard conditions was not inhibited (data not shown).



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Fig. 6. An oligonucleotide antisense to DNMT1 inhibits contact-independent cell proliferation. HT-29 cells were treated with 200 nM antisense (MG88F, A) or control (MG208, B) oligonucleotides for 3 consecutive days. Harvested cells were resuspended in an agar/media gel and colonies >10 cells were counted after 21 days. Bars in (C) represent average +SEM of triplicate experiments. Micrograph shows intracellular localization of fluorescent-tagged MG88F (D); bar represents 10 µm.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The APC mutation-triggered pathway plays an important role in both hereditary and nonhereditary colon carcinoma but the mechanisms initiated by this pathway that are critical for cellular transformation remain to be clarified. The HT-29 colorectal cancer cell line and derivatives thereof were used for these experiments since like the majority of CRC cell lines, HT-29 cells lack the full-length APC protein (36), instead expressing truncated forms of APC that lack the ß-catenin-binding domain. To investigate the role of APC in normal epithelia, or the lack of it in CRC, stable transfection of the wildtype APC was introduced to HT-29 cells such that the full-length gene product could be induced through a zinc-sensitive metallothionein promoter (25). Previous work with these cell lines indicated both detachment of adherent cells and apoptosis after ZnCl2 induction of the full-length APC protein (25). We also found an inhibition of growth of HT-29APC cells within 6 h (38%) through 2 days (66%) of expressing wildtype APC compared with noninduced HT-29APC cells.

In this manuscript we provide evidence that mutations in APC result in an upregulation of DNMT1 promoter activity and DNMT1 mRNA in human colorectal HT-29 cells. First, induction of wild type APC in HT29APC transfectants suppresses DNMT1 promoter-driven CAT activity. Second, cotransfection of wild type APC suppresses DNMT1 promoter driven CAT activity in transient transfection assays. Third, induction of wild type APC in HT29 cells leads to suppression of DNMT1 mRNA levels that is at least partially independent of the cell proliferation inhibition caused by full length APC, and fourth, ectopic expression of a dominant negative mutant of TCF4 suppresses DNMT1 mRNA.

Additionally, our data indicate that this upregulation is critical for the transformation process triggered by APC since knockdown of DNMT1 levels by antisense oligonucleotides reduce the tumorigenicity of these cells.

These data provide an explanation to the previously observed genetic linkage between the APC mutation and DNMT1. In those data, abrogation of the dnmt1 gene within a murine model of FAP with a homologous mutation in Apc (ApcMin/+ (24,37) resulted in fewer spontaneous intestinal polyps. Further reduction of DNA methyltransferase burden by treating these mice with the DNMT1 suicide substrate 5-aza-2'deoxycytidine, inhibited polyp load to an even greater extent. In a recent study, it has been shown that Min mice that are hypomorphic for DNMT1 expression are protected from colon carcinoma. The observation that DNMT1 expression is induced by APC mutation provides a possible explanation for this linkage.

DNMT1 is responsible for maintaining the DNA methylation pattern during DNA replication. It has been previously proposed that DNMT1 overexpression might be triggering cellular transformation by methylation-dependent and independent mechanisms (8). It has been shown that increased DNMT1 activity can lead to hypermethylation of CG islands in both cell culture and in vivo (38). Hypermethylation of CG islands and silencing of tumor suppressor genes is frequently observed in cancer and has been shown to be involved in the silencing of tumor suppressor genes (8). Herman et al. have shown this to be the case for the majority of sporadic CRC tumors with microsatellite instability, indicating hypermethylation of the mismatch repair gene hMLH1 promoter (39). Others have shown this hypermethylation of mismatch repair genes as well (40). There appears to be a positive feedback relationship between APC and its methylation, where in both sporadic and inherited CRC, mutations in Apc lead to increases in DNMT1, which in turn often causes hypermethylation of Apc (7). Evidence exists to indicate an association between either methylation or DNA methyltransferase and CRC. Focal hypermethylation of promoter regions in hMLH1, a mismatch repair gene, has been shown in replication error-prone tumors of both HNPCC and sporadic CRC patients, and as such, a decrease in hMLH1 protein may result (3941). This lack of mismatch repair leads to genomic instability. In addition, DNMT1 can also suppress tumor suppressors such as p21 directly by a mechanism that does not involve DNA methylation (13,42). It is possible that both mechanisms are involved in cellular transformation in response to APC mutation in CRC.

An open question is how does the APC-TCF pathway regulate DNMT1 expression? APC suppresses the transcription factors TCF/LEF whereas mutant APC is not inhibitory. While the promoter element of DNMT1 investigated here lacks consensus TCF/Lef binding sites (43), it is possible that TCF is binding to the DNA at nonconsensus sites, or that activated TCF is an intermediate transcription factor, upregulating another signalling molecule which in turn increases the transcription of DNMT1. AP-1 activity has previously been shown to be induced by activation of TCF/LEF in colorectal cancer cell lines (44). As AP-1 is a ubiquitous transcription factor, with several putative sites within the DNMT1 promoter region, we tested this latter hypothesis. No AP-1 binding was found, however, in the minimal promoter region used in our experiment (data not shown), in agreement with previous results that demonstrated the presence of AP-1 responsive elements outside the 5' minimal promoter region (29). Though the exact mechanisms by which mutations in APC affect the expression of DNMT1 are not yet clear, our data indicate that reconstitution of wildtype APC protein quickly diminishes the transcription of DNMT1 mRNA. Within nine hours of wildtype APC induction in stably transfected HT-29APC cells, DNMT1 mRNA levels were abrogated to at least half of those in the control HT-29ßGal cells. The rapid elimination of DNMT1 message following full-length APC production indicates either transcriptional inhibition or augmented mRNA degradation. However, given that mutant APC has already been shown to be a transcriptional activator through decreased ß-catenin elimination, and the effect that APC has on DNMT1 promoter activity in transient transfection assays, it is likely that DNMT1 transcription is upregulated by the APC mutation. Further experiments are required to identify the cis acting sequences in the DNMT1 promoter that respond to the APC/ß-catenin/TCF pathway.

If augmented expression of DNMT1 is a key element in the tumorigenesis of APC mutated CRC, then it may be possible to suppress the neoplastic activity by inhibiting the production of DNMT1. We have previously developed modified oligonucleotides that are antisense to DNMT1, and shown their efficacy both in vitro and in vivo with the non-small cell lung carcinoma A549 cell line (12). These oligos bind to DNMT1 mRNA and the heterodimer is degraded by RNAse H. Antisense oligos developed to target DNMT1 are now in clinical trials for both renal carcinoma and head and neck squamous cell cancer, but have yet to be tried as a treatment for colon cancer. Our data suggest that this treatment might be relevant for APC-mutated CRC.

A variety of cancers in addition to CRC have abnormal ß-catenin/TCF-driven transcription and these cancers may result from increased production of DNMT1 (4551). As the majority of HNPCC patients exhibit genetic mutations in the APC/ß-catenin/TCF pathway, familial history of colorectal cancer could provide reason to screen for such mutations. Therefore, along with treating CRC patients, the potential exists to employ DNMT1 antisense therapies to mutation-positive individuals in a prophylactic paradigm, and move treatment to the presymptomatic stage.


    Notes
 
1 To whom correspondence should be addressed at: Department of Pharmacology and Therapeutics, 3655 Sir William Osler Promenade, Room 1309, Montreal, Quebec, Canada H3G 1Y6 Email: mszyf{at}pharma.mcgill.ca Back


    Acknowledgments
 
The authors wish to thank Drs Ken Kinzler and Bert Vogelstein of Johns Hopkins University for the DNTCF4 adenovirus, pSAR-MT-APC and pSAR-MT-ßGal plasmids, and HT29APC and HT29ßGal cells. We wish to extend thanks to Dr Phil Branton for the p53 expression plasmid. This work was supported by grants from the National Cancer Institute of Canada. Paul M.Campbell is a grateful recipient of a McGill Centre for Translational Research in Cancer Fellowship sponsored by the Israel Cancer Research Fund – Montreal Chapter.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Szyf,M. (1994) DNA methylation properties: consequences for pharmacology. Trends Pharmacol. Sci., 15, 233–238.[CrossRef][ISI][Medline]
  2. De Marzo,A.M., Marchi,V.L., Epstein,J.I. and Nelson,W.G. (1999) Proliferative inflammatory atrophy of the prostate: implications for prostatic carcinogenesis. Am. J. Pathol., 155, 1985–1992.[Abstract/Free Full Text]
  3. Issa,J.P., Vertino,P.M., Wu,J., Sazawal,S., Celano,P., Nelkin,B.D., Hamilton,S.R. and Baylin,S.B. (1993) Increased cytosine DNA-methyltransferase activity during colon cancer progression. J. Natl Cancer Inst., 85, 1235–1240.[Abstract]
  4. Burri,N., Shaw,P., Bouzourene,H., Sordat,I., Sordat,B., Gillet,M., Schorderet,D., Bosman,F.T. and Chaubert,P. (2001) Methylation silencing and mutations of the p14ARF and p16INK4a genes in colon cancer. Lab. Invest., 81, 217–229.[ISI]
  5. Zheng,H. and Shank,R.C. (1996) Changes in methyl-sensitive restriction sites of liver DNA from hamsters chronically exposed to hydrazine sulfate. Carcinogenesis, 17, 2711–2717.[Abstract]
  6. Collins,N., Wooster,R. and Stratton,M.R. (1997) Absence of methylation of CpG dinucleotides within the promoter of the breast cancer susceptibility gene BRCA2 in normal tissues and in breast and ovarian cancers. Br. J. Cancer, 76, 1150–6.[ISI][Medline]
  7. Esteller,M., Fraga,M.F., Guo,M., et al. (2001) DNA methylation patterns in hereditary human cancers mimic sporadic tumorigenesis. Hum. Mol. Genet., 10, 3001–3007.[Abstract/Free Full Text]
  8. Szyf,M., Knox,D.J., Milutinovic,S., Slack,A.D. and Araujo,F.D. (2000) How does DNA methyltransferase cause oncogenic transformation? Ann. NY Acad. Sci., 910, 156–174; discussion 175–177.[Abstract/Free Full Text]
  9. Szyf,M. (2001) The role of DNA methyltransferase 1 in growth control. Front. Biosci., 6, D599–609.[ISI][Medline]
  10. Bigey,P., Knox,J.D., Croteau,S., Bhattacharya,S.K., Theberge,J. and Szyf,M. (1999) Modified oligonucleotides as bona fide antagonists of proteins interacting with DNA. Hairpin antagonists of the human DNA methyltransferase. J. Biol. Chem., 274, 4594–4606.[Abstract/Free Full Text]
  11. Knox,J.D., Araujo,F.D., Bigey,P., Slack,A.D., Price,G.B., Zannis-Hadjopoulos,M. and Szyf,M. (2000) Inhibition of DNA methyltransferase inhibits DNA replication. J. Biol. Chem., 275, 17986–17990.[Abstract/Free Full Text]
  12. Ramchandani,S., MacLeod,A.R., Pinard,M., von Hofe,E. and Szyf,M. (1997) Inhibition of tumorigenesis by a cytosine-DNA, methyltransferase, antisense oligodeoxynucleotide. Proc. Natl Acad. Sci USA, 94, 684–689.[Abstract/Free Full Text]
  13. Milutinovic,S., Knox,J.D. and Szyf,M. (2000) DNA methyltransferase inhibition induces the transcription of the tumor suppressor p21 (WAF1/CIP1/sdi1). J. Biol. Chem., 275, 6353–6359.[Abstract/Free Full Text]
  14. Fournel,M., Sapieha,P., Beaulieu,N., Besterman,J.M. and MacLeod,A.R. (1999) Down-regulation of human DNA- (cytosine-5) methyltransferase induces cell cycle regulators p16 (ink4A) and p21 (WAF/Cip1) by distinct mechanisms. J. Biol. Chem., 274, 24250–24256.[Abstract/Free Full Text]
  15. Fearon,E.R. and Vogelstein,B. (1990) A genetic model for colorectal tumorigenesis. Cell, 61, 759–767.[ISI][Medline]
  16. Barker,N., Morin,P.J. and Clevers,H. (2000) The Yin-Yang of TCF/beta-catenin signalling. Adv. Cancer Res., 77, 1–24.[ISI][Medline]
  17. Chang,K.W., Lin,S.C., Mangold,K.A., Jean,M.S., Yuan,T.C., Lin,S.N. and Chang,C.S. (2000) Alterations of adenomatous polyposis Coli (APC) gene in oral squamous cell carcinoma. Int. J. Oral Maxillofac. Surg., 29, 223–226.[CrossRef][ISI][Medline]
  18. He,T.C., Sparks,A.B., Rago,C., Hermeking,H., Zawel,L., da Costa,L.T., Morin,P.J., Vogelstein,B. and Kinzler,K.W. (1998) Identification of c-MYC as a target of the APC pathway. Science, 281, 1509–1512.[Abstract/Free Full Text]
  19. He,T.C., Chan,T.A., Vogelstein,B. and Kinzler,K.W. (1999) PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell, 99, 335–345.[ISI][Medline]
  20. Tetsu,O. and McCormick,F. (1999) Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature, 398, 422–426.[CrossRef][ISI][Medline]
  21. Wielenga,V.J., Smits,R., Korinek,V., Smit,L., Kielman,M., Fodde,R., Clevers,H. and Pals,S.T. (1999) Expression of CD44 in Apc and Tcf mutant mice implies regulation by the WNT pathway. Am. J. Pathol., 154, 515–523.[Abstract/Free Full Text]
  22. Laird,P.W., Jackson-Grusby,L., Fazeli,A., Dickinson,S.L., Jung,W.E., Li,E., Weinberg,R.A. and Jaenisch,R. (1995) Suppression of intestinal neoplasia by DNA hypomethylation. Cell, 81, 197–205.[ISI][Medline]
  23. Eads,C.A., Nickel,A.E. and Laird,P.W. (2002) Complete genetic suppression of polyp formation and reduction of CpG-island hypermethylation in Apc (Min/+) Dnmt1-hypomorphic Mice. Cancer Res., 62, 1296–1299.[Abstract/Free Full Text]
  24. Moser,A.R., Pitot,H.C. and Dove,W.F. (1990) A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science, 247, 322–324.[ISI][Medline]
  25. Morin,P.J., Vogelstein,B. and Kinzler,K.W. (1996) Apoptosis and APC in colorectal tumorigenesis. Proc. Natl Acad. Sci. USA, 93, 7950–7954.[Abstract/Free Full Text]
  26. Querido,E., Blanchette,P., Yan,Q., et al. (2001) Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs via a novel mechanism involving a Cullin-containing complex. Genes Dev., 15, 3104–3117.[Abstract/Free Full Text]
  27. Araujo,F.D., Croteau,S., Slack,A.D., Milutinovic,S., Bigey,P., Price,G.B., Zannis-Hajopoulos,M. and Szyf,M. (2001) The dnmt1 target recognition domain resides in the n terminus. J. Biol. Chem., 276, 6930–6936.[Abstract/Free Full Text]
  28. Rouleau,J., Tanigawa,G. and Szyf,M. (1992) The mouse DNA methyltransferase 5'-region. A unique housekeeping gene promoter. J. Biol. Chem., 267, 7368–7377.[Abstract/Free Full Text]
  29. Bigey,P., Ramchandani,S., Theberge,J., Araujo,F.D. and Szyf,M. (2000) Transcriptional regulation of the human DNA methyltransferase (dnmt1) gene. Gene, 242, 407–418.[CrossRef][ISI][Medline]
  30. Smith,K.J., Johnson,K.A., Bryan,T.M., et al. (1993) The APC gene product in normal and tumor cells. Proc. Natl Acad. Sci. USA, 90, 2846–2850.[Abstract]
  31. Szyf,N., Bozovic,V. and Tanigawa,G. (1991) Growth regulation of mouse DNA methyltransferase gene expression. J. Biol. Chem., 266, 10027–10030.[Abstract/Free Full Text]
  32. Leonhardt,H., Page,A.W., Weier,H.U. and Bestor,T.H. (1992) A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell, 71, 865–873.[ISI][Medline]
  33. Karpf,A.R., Moore,B.C., Ririe,T.O. and Jones,D.A. (2001) Activation of the p53 DNA damage response pathway after inhibition of DNA methyltransferase by 5-aza-2'-deoxycytidine. Mol. Pharmacol., 59, 751–757.[Abstract/Free Full Text]
  34. Roose,J. and Clevers,H. (1999) TCF transcription factors: molecular switches in carcinogenesis. Biochim. Biophys. Acta, 1424, M23–37.[CrossRef][ISI][Medline]
  35. Freedman,V.H. and Shin,S.I. (1974) Cellular tumorigenicity in nude mice: correlation with cell growth in semi-solid medium. Cell, 3, 355–359.[ISI][Medline]
  36. von Kleist,S., Chany,E., Burtin,P., King,M. and Fogh,J. (1975) Immunohistology of the antigenic pattern of a continuous cell line from a human colon tumor. J. Natl Cancer Inst., 55, 555–560.[ISI][Medline]
  37. Su,L.K., Kinzler,K.W., Vogelstein,B., Preisinger,A.C., Moser,A.R., Luongo,C., Gould,K.A. and Dove,W.F. (1992) Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science, 256, 668–670.[ISI][Medline]
  38. Biniszkiewicz,D., Gribnau,J., Ramsahoye,B., et al. (2002) Dnmt1 overexpression causes genomic hypermethylation, loss of imprinting and embryonic lethality. Mol. Cell Biol., 22, 2124–2135.[Abstract/Free Full Text]
  39. Herman,J.G., Umar,A., Polyak,K., et al. (1998) Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc. Natl Acad. Sci. USA, 95, 6870–6875.[Abstract/Free Full Text]
  40. Wheeler,J.M., Beck,N.E., Kim,H.C., Tomlinson,I.P., Mortensen,N.J. and Bodmer,W.F. (1999) Mechanisms of inactivation of mismatch repair genes in human colorectal cancer cell lines: the predominant role of hMLH1. Proc. Natl Acad. Sci. USA, 96, 10296–10301.[Abstract/Free Full Text]
  41. Cunningham,J.M., Christensen,E.R., Tester,D.J., Kim,C.Y., Roche,P.C., Burgart,L.J. and Thibodeau,S.N. (1998) Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res., 58, 3455–3460.[Abstract]
  42. Robertson,K.D., Ait-Si-Ali,S., Yokochi,T., Wade,P.A., Jones,P.L. and Wolffe,A.P. (2000) DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat. Genet., 25, 338–342.[CrossRef][ISI][Medline]
  43. Korinek,V., Barker,N., Morin,P.J., van Wichen,D., de Weger,R., Kinzler,K.W., Vogelstein,B. and Clevers,H. (1997) Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC–/– colon carcinoma. Science, 275, 1784–1787.[Abstract/Free Full Text]
  44. Mann,V., Szyf,M., Razin,A., Chriqui-Zeira,E. and Kedar,E. (1986) Characterization of a tumorigenic murine T-lymphoid-cell line spontaneously derived from an IL-2-dependent T-cell line. Int. J. Cancer, 37, 781–786.[ISI][Medline]
  45. Voeller,H.J., Truica,C.I. and Gelmann,E.P. (1998) Beta-catenin mutations in human prostate cancer. Cancer Res., 58, 2520–2523.[Abstract]
  46. Zurawel,R.H., Chiappa,S.A., Allen,C. and Raffel,C. (1998) Sporadic medulloblastomas contain oncogenic beta-catenin mutations. Cancer Res., 58, 896–899.[Abstract]
  47. Rubinfeld,B., Robbins,P., El-Gamil,M., Albert,I., Porfiri,E. and Polakis,P. (1997) Stabilization of beta-catenin by genetic defects in melanoma cell lines [see comments]. Science, 275, 1790–1792.[Abstract/Free Full Text]
  48. Palacios,J. and Gamallo,C. (1998) Mutations in the beta-catenin gene (CTNNB1) in endometrioid ovarian carcinomas. Cancer Res., 58, 1344–1347.[Abstract]
  49. Miyoshi,Y., Iwao,K., Nawa,G., Yoshikawa,H., Ochi,T. and Nakamura,Y. (1998) Frequent mutations in the beta-catenin gene in desmoid tumors from patients without familial adenomatous polyposis. Oncol. Res., 10, 591–594.[ISI][Medline]
  50. de La Coste,A., Romagnolo,B., Billuart,P., et al. (1998) Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas. Proc. Natl Acad. Sci. USA, 95, 8847–8851.[Abstract/Free Full Text]
  51. Chan,E.F., Gat,U., McNiff,J.M. and Fuchs,E. (1999) A common human skin tumour is caused by activating mutations in beta-catenin. Nat. Genet., 21, 410–413.[CrossRef][ISI][Medline]
  52. Davis,A.J., Moore,M.J., Gelmonk,A. et al. (2000) Phase I and phermacodynamic study of human DNA methyltransferase (metase) antisense oligodeoxynucleotide (ODN), MG68, administered as 21-day infusion q4 weekly. Clin. Can. Res., 6, 45175.
Received May 10, 2002; revised September 11, 2002; accepted September 12, 2002.