Correlations of partial and extensive methylation at the p14ARF locus with reduced mRNA expression in colorectal cancer cell lines and clinicopathological features in primary tumors

Shichun Zheng, Pengchin Chen, Alex McMillan, Amalia Lafuente, Maria Jose Lafuente, Antonio Ballesta, Manuel Trias and John K. Wiencke

Laboratory for Molecular Epidemiology, Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, CA 94143-0560, USA,
1 Department of Clinical Biochemistry, Hospital Clinic, University of Barcelona and
2 Department of Surgery, Hospital St Pablo, Barcelona, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
p14ARF is a putative tumor suppressor gene thought to modify the levels of p53. CpG sites within the 5'-flanking region and exon 1ß of p14ARF are targets of aberrant methylation and transcriptional silencing in human colorectal cancer (CRC). Here we have developed methylation-specific polymerase chain reaction (MSPCR) methods to detect methylation of CpG sites in p14ARF in CRC cell lines and primary CRC tumors, and correlated p14ARF mRNA expression with methylation in CRC cell lines using competitive quantitative reverse transcription–polymerase chain reaction methods. Ten CRC cell lines were studied; three (DLD-1, HCT15 and SW48) showed extensive methylation and six (Colo320, SW480, HT29, Caco2, SW837 and WiDr) were unmethylated; the other cell line, LoVo, showed partial methylation that affected exon 1ß but not the immediate upstream CpG sites. p14ARF mRNA was expressed at extremely low levels in fully methylated cell lines and at 104- to 105-fold higher levels in unmethylated cell lines. p14ARF expression in the partially methylated LoVo cell line was intermediate. Treatment of LoVo cells with 2 µM 5-aza-2'-deoxycytidine for 72 h was associated with marked (100-fold) induction of mRNA levels. Of 119 primary CRCs, 18% contained p14ARF methylation, although partial methylation was the most common pattern observed (in 67% of methylated tumors). Methylation of p14ARF was often accompanied by p16INK4a methylation; however, 50% of p14ARF methylated tumors contained unmethylated p16INK4a. Methylation at p14ARF was associated with female gender, greater age, proximal anatomic location and poor differentiation, but not stage at diagnosis. A two-step MSPCR method for assaying p14ARF methylation in human tumors is described.

Abbreviations: ARF, alternative reading frame; AZA, 5-aza-2'-deoxycytidine; CRC, colorectal cancer; MSPCR, methylation-specific polymerase chain reaction; RT–PCR, reverse transcription–polymerase chain reaction


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aberrant methylation is now considered an important epigenetic alteration in colorectal cancer (CRC); examples of loci affected by methylation and transcriptional silencing include the DNA repair genes hMLH1 (13) and MGMT (4) and the cyclin D/cdk regulator p16INK4a (5,6) on chromosome 9p21. In a previous study, we used a multiplex methylation-specific polymerase chain reaction (MSPCR) method based on primers designed by Herman et al. (7) to detect p16INK4a methylation. We reported that 18% of a consecutive case series of sporadic CRCs contained methylation of the 5'-flanking region of p16INK4a and observed associations of methylation with clinicopathological features and patient gender (8). Also residing on chromosome 9p21 is p14ARF (alternative reading frame; p19ARF in mice) gene, which shares a portion of the p16INK4a coding region (i.e. exon 2) but has a unique promoter and first exon (exon 1ß) located ~20 kb upstream of p16INK4a (9,10). Mice deficient in p19ARF are susceptible to a variety of spontaneous and induced neoplasms (11,12). In addition, p14ARF is frequently deleted in human cancers; thus, the ARF locus is considered a potential human tumor suppressor (13). p19ARF has recently been ascribed a role in modifying the levels of p53 by interacting with MDM2 and inhibiting MDM2-mediated p53 degradation via the ubiquitin/proteosome pathway (14). Wild-type p53 itself down-regulates p19ARF expression, indicating a `self-regulating feedback' loop between the two components. Further, p19ARF has not been shown to affect p53 levels after DNA damage but is required for induction of p53 in response to hyperproliferative signals such as c-Myc, E1A and Ras (1517). Our aim was to develop MSPCR methods to detect p14ARF methylation in human tumor specimens. To maximize efficiency we explored multiplex MSPCR. During the course of these experiments we encountered an anomalous PCR result with our multiplex MSPCR method in the LoVo cell line that led to the discovery of a pattern of methylation characterized by `partial' methylation within the untranslated region of exon 1ß of p14ARF. This partial methylation pattern was associated with reduced levels of p14ARF mRNA and represents the predominant pattern of p14ARF methylation in primary sporadic CRCs.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell lines and DNA/RNA isolation
Human CRC cell lines were obtained from the American Type Culture Collection (ATCC) and were cultured according to conditions recommended by the ATCC. DNA was isolated by standard methods including proteinase K, RNase, chloroform–isoamyl alcohol extraction and ethanol precipitation. DNA was quantified by Hoescht 33258 fluorimetry (Hoefer Scientific Instruments, San Francisco, CA). Total RNA was isolated using RNAzol B (BiotecX, Houston, TX); mRNA was then isolated using Qiagen (Valencia, CA) Oligotex spin column protocols. cDNA was prepared using MMLV reverse transcriptase and random hexamer primers (BRL, Gaithersburg, MD) according to the manufacturer's directions. A 360 bp fragment containing a distal segment of exon 1ß and a portion of exon 2 (bp +173 to +533; accession no. U38945) was amplified using the following primers: sense, 5'-TTCTTGGTGACCCTCCGGATT-3'; antisense, 5'-CAGGCATCGCGCACGTCCAGC-3'. All chemical reagents were HPLC grade.

Primary colorectal tumors
Tumor specimens were obtained from a consecutive case series of resected CRCs as previously described (8). Briefly, tumors were obtained from patients (aged 35–90 years) with sporadic CRC undergoing surgery at the University of Barcelona Hospital Clinic. Primary tumors were surgically dissected and immediately frozen at -80°C. Non-tumorous colon tissue was also collected. Patients provided signed informed consent; all procedures were approved by the Hospital Clinic's institutional review board. Information was collected on patient characteristics (age and gender) and tumor characteristics (stage at diagnosis, anatomical location and degree of differentiation).

MSPCR
Methylated CpG sites within the 5' region of the p14ARF gene were detected using MSPCR methods. For all assays, 1.0 µg of purified DNA was diluted in 36 µl H2O, to which was added 4 µl of 3.0 M NaOH and the DNA denatured at 37°C for 15 min. The samples were then treated with 416 µl of 3.6 M sodium bisulfite solution (pH 5.0) and 24 µl of 10 mM hydroquinone. Both bisulfite and hydroquinone solutions were prepared fresh for each analysis. Samples were incubated at 55°C for 16 h; 100 µl of mineral oil was layered on top of the solution to prevent evaporation. After incubation, the solution was cooled to –80°C for 10 min, after which the unfrozen mineral oil was removed without disturbing the bisulfite–DNA solution.

Bisulfite-modified DNA was purified with the Wizard DNA Clean-Up System and Vacuum Manifold (Promega, Madison, WI) according to the manufacturer's instructions. DNA was eluted with a total volume of 30 µl TE buffer (pH 7.8). The final step of the cytosine->uracil conversion reaction was achieved by treatment with alkali (NaOH; final concentration 0.3 M) at 37°C for 15 min followed by neutralization with ammonium acetate (pH 7.0; final concentration 3.0 M) and ethanol precipitation. Human peripheral blood lymphocyte DNA treated with methylase SssI (New England BioLabs, Beverly, MA) was used as a positive control.

For each cell line and tumor specimen, both monoplex and multiplex PCR amplification of bisulfite-treated DNA was carried out with primers (BRL) specific for methylated (M-primer) and unmethylated (U-primer) CpG sites within the p14ARF promoter. Previous studies with p16INK4a showed complete concordance of multiplex and monoplex results. A region including ~150 bp upstream and downstream of the p14ARF transcription initiation site was chosen for assay development. For p16INK4a and hMLH1, primers were used as previously described (3,6).

The PCR mixture contained GeneAmp PCR buffer (Perkin-Elmer Corp., Foster City, CA), 1.5 mM MgCl2, dimethylsulfoxide (DMSO; 5% final concentration), 200 µM of each deoxynucleotide triphosphate), 0.4 µM of each primer, 50 ng modified DNA templates and 2.5 U of AmpliTaq with TaqStart antibody (Clontech, Palo Alto, CA) in a total volume of 50 µl. The PCR reaction was repeated for 35 cycles on a GeneAmp 9600 thermal cycler (Perkin-Elmer Corp.) under the following conditions: preheat at 94°C for 2 min, 94°C for 30 s, 65°C for 10 s, 72°C for 30 s and a final extension at 72°C for 10 min. Aliquots (15 µl) of PCR products were loaded on to 2.5% agarose gels stained with ethidium bromide and visualized under UV light.

To assess the sensitivity of the MSPCR method, methylated and unmethylated cell line DNA samples were mixed in different ratios; a single unambiguous p14ARF methylated band was detectable when methylated template was present at >1:32 (3%) of the total DNA. All analyses were carried out on coded samples without knowledge of the cell lines' p14ARF expression status or of patients' clinical status.

DNA cloning and sequencing
After bisulfite treatments and methylation-specific amplification, PCR products were gel purified (Qiagen) and ligated into the PCR 2.1-TOPO plasmid vector using the TA cloning system (Invitrogen, Carlsbad, CA). Plasmid-transformed Escherichia coli were cultured overnight and plasmid DNA was isolated (Qiagen). Purified plasmid DNA containing p14ARF sequence was sequenced in both directions using an ABI 373 automated sequencer with dye primer chemistry and standard M13 primers.

Quantitative competitive PCR and p14ARF expression
Expression of p14ARF mRNA in CRC cell lines was measured by quantitative competitive PCR as previously described (18). In this method, target cDNA is amplified in the presence of a known concentration of a competitor sequence that has the same primer binding sites and the same amplification efficiency as the target sequence. By serial dilution of the competitor, the equivalence point (i.e. the point at which the ratio of target to competitor is 1:1) is determined. At the equivalence point, the amount of target cDNA can be quantified. Equal amounts of total cDNA were added to each competitive PCR reaction by normalizing cDNA with the concentration of HPRT cDNA; HPRT cDNA levels were estimated in each sample by competitive PCR as described (19). PCR conditions were identical to those for MSPCR except in this instance 5% DMSO was not added. The p14ARF competitor amplicon was constructed to contain primer-binding sites that span exons 1ß and 2 of the p14ARF cDNA, and to contain an 80 bp insert to make the competitor and target amplicons distinguishable by agarose gel electrophoresis. The 80 bp fragment was inserted at the unique XmaI restriction site within p14ARF exon 2. The number of copies of p14ARF cDNA present at the equivalence point was calculated using the competitor DNA concentration and dilution factor at the equivalence point, and the molecular weight of the competitor. Visual inspection followed by UV densitometry was used to carry out the serial dilutions and to measure equivalence of PCR bands. For 5-aza-2'-deoxycytidine (AZA) treatments, cells were passaged and, 24 h later, treated with freshly prepared 2 µM AZA for 72 h; they were then harvested for RNA isolation.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Monoplex and multiplex MSPCR
In our initial experiments, we designed two sets of methylation-sensitive primers (M-primers and U-primers) to amplify CpG-rich regions that are 5' flanking to exon 1ß and within the initial portion of exon 1ß (Figure 1Go). The primers were designed so that the unmethylated product would share a common 5' location but the 3' U-primer would bind downstream of the 3' M-primer (i.e. primers A/D and B/C) (Figure 1Go). This combination produces methylated and unmethylated PCR products of different sizes in multiplex reactions containing both M- and U-primers. We compared monoplex and multiplex amplifications using bisulfite-treated DNA isolated from normal lymphocyte control, methylase-treated and 10 different CRC cell lines. Figure 2aGo shows the results of monoplex (M or U) and multiplex (M and U) reactions using primer pairs A/D (U-primers) and B/C (M-primers). For each cell line except LoVo, a single band was detected in multiplex or monoplex reactions; in SW480, SW837, HT29, WiDr, Colo320 and Caco2 the band was amplified only with U-primers, and in HCT15, DLD-1, SW48 it was amplified only with M-primers. In multiplex reactions LoVo produced a methylated product, but not in monoplex PCR reactions with M-primers only. As shown in Figure 2aGo, row D, monoplex PCR with the U- and M-primer pair amplified a product in LoVo but not in any other cell line. Also shown in Figure 2bGo is a sensitivity analysis indicating that methylation within the region can be detected when the methylated template is present at >3% of total template. The most striking result of these experiments is the discordant results when LoVo cells were examined by MSPCR in the monoplex versus multiplex format. The target sequence was amplified poorly from LoVo cells with either monoplex M- or U-primers but a very robust methylation band was produced when both sets of primers were used. Our results with monoplex PCR using a 5' U-primer and 3' M-primer (primers A/C; Figure 2aGo, row D) are consistent with the formation of a hybrid product formed in the multiplex reaction. This explanation would require that the sequence chosen for multiplex PCR amplification would span a boundary region that was unmethylated at one end and methylated at the other.



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Fig. 1. (a) Sequence of the p14ARF 5' flanking region and exon 1ß with CpG sites numbered for reference to sequencing results. {triangledown}, Start of exon 1ß of p14ARF. {blacktriangledown}, Start of translation of p14ARF. Open arrows indicate the position of primers specific for unmethylated DNA and closed arrows indicate the position of primers specific for methylated DNA. (b) Sequences of primers A–E.

 



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Fig. 2. (a) Multiplex and monoplex PCR of p14ARF methylation in CRC cell lines. Row A: multiplex results with primers A/D and B/C; the upper band is the unmethylated product and the lower band the methylated product. Rows B and C: monoplex MSPCR reactions with primers A/D (row B) or B/C (row C). Note the concordance of results between multiplex and monoplex results for all cell lines except LoVo. Row D: result of monoplex MSPCR using partial methylation MSPCR primers A/C; these primers only amplified a product in DNA from LoVo cells. (b) Multiplex MSPCR amplification of serially diluted lymphocyte DNA treated with methylase SssI. A methylated band was detectable at 3% methylated template. Unmethylated lymphocyte DNA was added and the total amount of template kept constant at 50 ng/reaction.

 
Bisulfite sequencing
We next amplified the putative boundary region with primers downstream of the previous M-primers (i.e. using primer A/E) (Figure 1Go), an area that contains 15 CpG sites and the previous M-primer annealing site of primer C. Sequencing results are shown for multiple clones of LoVo in Table IGo. Primers A and E in Figure 1Go were used to amplify the region. CpG positions are indicated relative to the transcription start site. Nine independent clones were sequenced; CpG sites in the 5'-flanking region of p14ARF were unmethylated but sites +32 and +35 were extensively methylated. These latter sites lie within the methylated primer for the multiplex reaction and occur within the untranslated region of exon 1ß. These sites correspond to CpG sites 6 and 7 in Figure 1Go. The results confirm the MSPCR data indicating that the anomalous multiplex product arose by the U-primer annealing upstream and the M-primer annealing downstream. Also consistent with this interpretation was the fact that the reverse combination of primers [i.e. 5' M-primer (B) and 3' U-primer (D)] never produced a PCR product in any cell line. Extensive methylation of all 15 CpG sites in the region was confirmed by sequencing in SW48 and DLD-1 cells and in the methylase-treated positive control, whereas no CpG site was methylated in Colo320 cells (data not shown). The partial pattern of methylation was also found in another cell line, HCT116. Because the sequencing primers were designed to be sensitive to the methylation status of individual CpG sites, it should be noted that these sequencing studies and those described in primary tumors do not give an unbiased picture of the distribution of CpG sites in this region. Instead they were carried out to confirm the interpretation of the multiplex result and provide evidence for the existence of the two patterns of methylation (i.e. partial and extensive).


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Table I. Bisulfite sequencing of cloned PCR products from the LoVo cell line
 
p14ARF mRNA expression
The expression of p14ARF was estimated for each cell line and the relative concentrations of p14ARF cDNA are shown in Table IIGo and Figure 3aGo. Expression was normalized to the cell line with the lowest level of expression (i.e. DLD-1), which was assigned an arbitrary value of 1 unit of p14ARF cDNA. Methylated cell lines had very low levels of p14ARF expression (1–230 relative units; mean, 79), whereas unmethylated cell lines expressed an average of 1760-fold higher levels (i.e. mean = 139 000). LoVo cells expressed intermediate levels of p14ARF, with 33-fold higher levels than in methylated cell lines and 54-fold lower levels than in unmethylated cell lines.


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Table II. Methylation-specific PCR of p14ARF, p16INK4a, hMLH1 and p14ARF expression in CRC cell lines
 



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Fig. 3. (a) Quantitative competitive PCR analysis of p14ARF expression in CRC cell lines. Competitor products contain an 80 bp insert and are seen as the upper (higher molecular weight) band. Row A: PCR results at the equivalence point, following dilution of the competitor DNA to a level where p14ARF cDNA and competitor bands are of equal intensity. Row B: relative intensity of competitor and target (p14ARF) before serial dilution of the competitor. Note the weak or undetectable wild-type p14ARF cDNA bands in the methylated cell lines DLD-1, HCT15 and SW48 and the stronger bands in the unmethylated cell lines Caco2, HT29, SW480, WiDr and SW837. Row C: normalization of total cDNA by dilution to a standard HPRT competitor concentration. (b) Reactivation of p14ARF after 72 h treatment with 2 µM AZA. Row A: results of competitive PCR at the equivalence point after dilution of the competitor. Row B: results of competitive PCR at equal total cDNA levels before dilution of the competitor; note the marked increase in intensity of the wild-type cDNA band after AZA treatment in LoVo cells and lack of change in the unmethylated cell line SW837. Before dilution of the competitor there appears to be no detectable increase in expression following AZA treatment in DLD-1; however, an ~10 000-fold lower dilution factor was required to achieve the equivalence point (row A), indicating a marked reactivation of p14ARF in DLD-1.

 
To confirm that the intermediate and low mRNA levels of p14ARF in LoVo cells were associated with CpG methylation, LoVo cells were cultured for 72 h in the presence of AZA. p14ARF levels were induced 100-fold (26 x 104 relative units) by AZA in LoVo cells (Figure 3bGo). The methylated cell line DLD-1 also showed a 10 000-fold increase in p14ARF transcripts after AZA treatment but still expressed much lower levels of p14ARF than LoVo or unmethylated cell lines (1 x 104 units). Expression of p14ARF in the unmethylated cell line SW837 was unaffected by the AZA treatment (10.5 x 104 units, or a 15% decrease).

Prevalence of p14ARF methylation in primary tumors
A total of 119 primary cancers were assayed using five different MSPCR protocols: monoplex for extensive methylation, multiplex, monoplex with 5' U-primer and 3' M-primer specific for the partial methylation, and monoplex with 5' M-primer and 3' U-primer (Figure 4Go). No PCR amplification was detected in any primary tumor using the 5' M-primer and 3' U-primer combination. Similarly, this combination of primers failed to amplify product in all CRC cell lines. Twenty-one tumors were methylated in multiplex reactions; however, monoplex reactions showed that only seven of these were extensively methylated. Most methylated primary tumors were found to contain the partial pattern of methylation (Table IIIGo). In addition, methylation was associated with increased age, female gender, proximal location and poor differentiation. In Figure 5Go we present a graphical representation of these clinicopathological correlations; we calculated the Kendall correlation coefficient to test for trends in p14ARF methylation (i.e. unmethylated, partial methylation or extensive methylation) with each of the patient variables. The Kendall correlation is a non-parametric approach used here to test for trends in methylation with selected patient characteristics. A significant correlation implies that the numerical value of a variable in patients with partially methylated tumors is intermediate between the values for patients with unmethylated and extensively methylated tumors. As is evident from Figure 5Go, a significant Kendall correlation was observed for the proportion of female patients, increased age and proximal location but not for stage or differentiation. Hence, these data suggest an intermediate phenotype (age, gender and anatomical location) associated with the partial pattern of methylation. It is also clear from the data that extensive but not partial methylation is associated with poor differentiation. This is reflected in the statistically significant association of extensive methylation and differentiation in Table IIIGo but a non-significant Kendall correlation in Figure 5Go. Several tumors demonstrating methylation and one unmethylated tumor were sequenced using the A and E sequencing primers. The partial methylation by MSPCR was confirmed by sequencing and was similar to the pattern observed in LoVo cells. All CRC tumor specimens contained unmethylated products, which probably represent non-tumorous elements in the surgical specimen. Sequencing unmethylated products yielded unmethylated CpG sites. In non-tumorous colon, p14ARF methylation was never detected. We also found the partial pattern of methylation in a second CRC cell line, HCT116. Thus, LoVo is not the only cell line exhibiting this pattern of p14ARF methylation.



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Fig. 4. MSPCR of p14ARF in primary colorectal tumors. The results from six different tumors using four MSPCR protocols are shown. Row A: results of multiplex MSPCR using the A/D and B/C primer combination. Rows B and C: monoplex MSPCR reactions using primers A/D or B/C, respectively. Row D: results with the partial methylation primers A/C. Tumors 180 and 227 are unmethylated, 567 and 124 are methylated, and 137 and 570 are partially methylated. See Figure 1Go for primer sequences.

 

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Table III. Prevalence of p14ARF methylation by patient gender and age, and anatomical location, stage and differentiation of tumor
 


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Fig. 5 . Selected patient characteristics displayed according to the methylation status of the patient's tumor (unmethylated, partially methylated or extensively methylated). Kendall's correlation was used to test for trends between methylation status and patient characteristics. (A) Significant correlation of methylation status with female gender ({tau} coefficient 0.21; P = 0.02); (B) marginally significant association with age at diagnosis ({tau} coefficient 0.14; P = 0.06); (C) non-significant association with advanced stage cancer (i.e. greater than Duke's stage B) ({tau} coefficient 0.08; P = 0.34); (D) significant association with proximal anatomic location ({tau} coefficient 0.27; P = 0.003) in which proximal includes the cecum and the ascending and transverse segments of the colon; (E) significant association of proximal anatomic location ({tau} coefficient 0.25; P = 0.006) in which proximal includes the cecum and the ascending, transverse and descending segments of the colon; (F) non-significant trend for methylation and percentage of tumors displaying poor differentiation. Analyses included 119 patients except for differentiation, which only included 111, because of missing data.

 
Among the 119 primary tumors there was a significant correlation of p14ARF and p16INK4a methylation ({chi}2 =16.8; P < 0.001 for any p14ARF methylation); the association between p16INK4a methylation and partial p14ARF methylation was much less pronounced ({chi}2= 4.2; P = 0.04). Only five (36%) of 14 tumors with partially methylated p14ARF contained p16INK4a methylation compared with six of seven extensively methylated tumors. Overall, 11 of 22 (50%) of p16INK4a methylated tumors were also methylated at p14INK4a. Conversely, 11 of 98 (11%) of p14ARF unmethylated tumors contained p16INK4a methylation.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, we evaluated several MSPCR protocols for detecting CpG methylation associated with down-regulation of the p14ARF gene in CRC cell lines. Our results indicate two patterns of CpG methylation within the region we examined. CRC cell lines displaying very low levels of p14ARF mRNA showed extensive methylation throughout the 5' flanking region near exon 1ß, whereas LoVo cells, which display 33-fold higher levels of mRNA expression, contain a partially methylated CpG island that spans the exon 1ß transcription site. Our sequencing results indicate that CpG sites immediately upstream of the transcription start site are unmethylated in partially methylated DNA from LoVo cells. MSPCR analysis using a 5' U-primer and a 3' M-primer readily detected this unique pattern. We never observed the opposite pattern (i.e. 5'-methylated and 3' unmethylated) in any cell line or primary tumor. It is of interest that the two methylated CpG sites positioned within the 3' M-primer are in the untranslated region of exon 1ß; these sites were previously reported to be located within a potential binding site for members of the E2F family of transcription factors. In addition, the p14ARF promoter was found to be highly responsive to E2F1 expression (20). Our results showing a 100-fold induction of p14ARF mRNA levels in LoVo cells after AZA treatment are consistent with this site being a functioning promoter element in p14ARF. This hypothesis will require further study, however, because CpG sites downstream of the translation start signal were also heavily methylated. Generally, CpG methylation within coding regions of exons is thought to be permissive in transcriptional regulation (21). In addition, a previous study using restriction digestion and Southern blots indicated that further upstream sites in the 5'-flanking region of LoVo cells were also methylated.

It is now thought that p16INK4a and p14ARF are regulated independently. Our finding that all cell lines contained p16INK4a methylation, but only four of the 10 were methylated at p14ARF is consistent with this idea. Our data showing discordance between p16INK4a and p14ARF methylation in primary tumors also confirms the independence of methylation events within the respective promoters of these two genes. Our MSPCR results were also consistent with the previously reported methylated and unmethylated alleles within hMLH1 in HT29; the score methylation pattern was detected in WiDr, which is derived from HT29 (22). SW48 also showed methylation of the hMLH1. Among the unmethylated cell lines, we observed a 45-fold variation in p14ARF expression levels. The significance of this variation is unknown. The unmethylated cell lines showing the lowest p14ARF expression were Colo320, SW480 and HT29. Other researchers have shown that c-myc is amplified 35-, 4- and 1.8-fold in Colo320, SW480 and HT29, respectively (23). No other cell lines in our series are known to contain c-myc amplification. Expression of p14ARF may be reduced in cell lines containing c-myc amplification through the action of trans-acting factors. For example, down-regulation of p14ARF by the transcriptional repressor bmi-1 has recently been shown to be involved in cell immortalization by c-myc (24). Further investigation of the variations in p14ARF expression we have observed may provide clues concerning methylation-independent mechanisms modifying p14ARF expression.

To examine whether the two patterns of methylation found in established CRC cell lines were detectable in primary human tumors, we applied MSPCR methods to a series of 119 primary sporadic CRCs. About 18% of CRCs showed methylation using multiplex MSPCR. Of the methylated tumors, however, the majority of tumors (14/21; 67%) showed the partial methylation pattern observed in the LoVo cell line. Interestingly, we observed significant trends between the three patterns of methylation (unmethylated, partially methylated, extensively methylated) and female gender, proximal tumor location and older age. These data indicate that partial methylation, which showed intermediate in vitro mRNA expression in cell lines, may be associated with an intermediate clinical phenotype in vivo. In contrast, the degree of tumor differentiation was inversely associated with extensive but not partial methylation. This latter result suggests that the more complete silencing of p14ARF expression by extensive methylation compared with partial methylation may be required to promote the poorly differentiated tumor growth pattern.

The associations of p14ARF methylation with female gender and proximal tumor location are similar to our previous studies of p16INK4a in this series of patients (8). The clustering of both of these methylation events within CRCs arising in the proximal colon is consistent with the hypothesis that there exists a hypermethylation phenotype for a subset of CRCs. Our data, however, also show that the methylation status of 50% of CRCs were discordant at the two loci. Thus, our findings emphasize that even among CRC exhibiting hypermethylation, different genetic pathways can be targeted in individual cases. Further research is needed to elucidate the mechanisms that give rise to the specific constellation of genes affected by aberrant methylation. The significance of these differences in methylation patterns with respect to the etiology of CRC, or their potential to improve diagnosis or prognosis, also remains to be determined.

It is evident that extensive methylation of p14ARF is not common in sporadic CRC. Similarly, p14ARF is seldom extensively methylated in bladder cancer (25). However, the partial methylation pattern we found associated with relatively low levels of p14ARF in the LoVo cell line appears relatively common in CRC (i.e. 12%). Both extensive and partial methylation can be detected by three PCR reactions; for example, a multiplex MSPCR to identify methylated tumors, followed by monoplex PCR reactions on methylated tumors using the 5' U-/3' M-primer or 5' M-/3' M-primer combinations to confirm the presence of the partial and extensive methylation products, respectively. Because p14ARF is not methylated in most tumors, screening with this approach involves about half the number of PCR reactions compared with a monoplex scheme.

LoVo cells, which show partial CpG methylation and intermediate expression of p14ARF, carry a wild-type p53 gene, as do the more heavily methylated SW48 and DLD-1. All of the unmethylated cell lines studied contain mutant p53. Only HCT15 is methylated and has been reported to carry a p53 mutation (i.e. codon 241 TCC->TTC transition) (26). It is not known whether the intermediate expression of p14ARF observed in LoVo cells or the related partially methylated p14ARF in primary CRCs affects the levels or function of p53.


    Notes
 
1 To whom correspondence should be addressed E-mail: wiencke{at}itsa.ucsf.edu Back


    Acknowledgments
 
We thank David Corry and Richard Locksley for providing the human HPRT competitor construct. This study was supported by the Foundation Marato (A.L., M.J.L.) and the National Institutes of Environmental Health Sciences, NIH Grant P42-ES04705 with funds provided by the Environmental Protection Agency (J.K.W., S.Z., C.G.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Kane,M.F., Loda,M., Gaida,G.M. et al. (1997) Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res., 57, 808–811.[Abstract]
  2. Thibodeau,S.N., French,A.J., Cunningham,J.M. et al. (1998) Microsatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hMLH1. Cancer Res., 58, 1713–1718.[Abstract]
  3. 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]
  4. Esteller,M., Hamilton,S.R., Burger,P.C. et al. (1999) Inactivation of the DNA repair gene O6-methylguanine–DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res., 59, 793–797.[Abstract/Free Full Text]
  5. Merlo,A., Herman,J.G., Mao,L. et al. (1995) 5' CpG island methylation is associated with transcriptional silencing of the tumour suppressor P16/CDKN2/MTS1 in human cancers. Nature Med., 1, 686–692.[ISI][Medline]
  6. Herman,J.G., Merlo,A., Mao,L. et al. (1995) Inactivation of the CDKN2/P16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res., 55, 4525–4530.[Abstract]
  7. Herman,J.G., Graff,J.R., Myohanen,S. et al. (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl Acad. Sci. USA, 93, 9821–9826.[Abstract/Free Full Text]
  8. Wiencke,J.K., Zheng,S., Lafuente,A. et al. (1999) Aberrant methylation of p16INK4a in anatomic and gender specific subtypes of sporadic colorectal cancer. Cancer Epidemiol. Biomarkers Prev., 8, 501–506.[Abstract/Free Full Text]
  9. Mao,L., Merlo,A., Bedi,G. et al. (1995) A novel p16INK4a transcript. Cancer Res., 55, 2995–2997.[Abstract]
  10. Sidransky,D. (1996) Two tracks but one race? Cancer genetics. Curr. Biol., 6, 523–525.[ISI][Medline]
  11. Kamijo,T., Zindy,F., Roussel,M.F. et al. (1997) Tumor suppression at the mouse INK4a locus mediated by the alternate reading frame product p19ARF. Cell, 91, 649–659.[ISI][Medline]
  12. Kamijo,T., Bodner,S., van de Kamp,E. et al. (1999) Tumor spectrum in ARF-deficient mice. Cancer Res., 59, 2217–2222.[Abstract/Free Full Text]
  13. Chin,L., Pomerantz,J., and De Pinho,R.A. (1998) The INK4a/ARF tumor suppressor: one gene, two products, two pathways. Trends Biochem Sci., 23, 291–296.[ISI][Medline]
  14. Kamijo,T., Weber,J.D., Zambetti,G. et al. (1998) Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc. Natl Acad. Sci. USA, 95, 8292–8297.[Abstract/Free Full Text]
  15. Zindy,F., Eischen,C.M., Randle,D.H. et al. (1998) Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization. Genes Dev., 12, 2424–2433.[Abstract/Free Full Text]
  16. de Stanchina,E., McCurrach,M.E., Zindy,F. et al. (1998) E1A signaling to p53 involves the p19(ARF) tumor suppressor. Genes Dev., 12, 2434–2442.[Abstract/Free Full Text]
  17. Palmero,I., Pantoja,C. and Serrano,M. (1998) p19ARF links the tumour suppressor p53 to Ras. Nature, 395, 125–126.[ISI][Medline]
  18. Wang,Z., Zheng,S., Corry,D.B. et al. (1994) Interferon-{gamma} independent effects of interleukin-12 administered during acute or established infection due to Leishmania major. Proc. Natl Acad. Sci. USA, 91, 12932–12936.[Abstract/Free Full Text]
  19. Corry,D.B. and Locksley, R.M. (1999) Construction of polycompetitors for competitive PCR. In: Kochanowski,B. and Reischl U. (Eds), Methods in Molecular Medicine, Vol. 26: Quantitative Protocols. Humana Press Inc., Totowa, NJ, pp. 253–264.
  20. Robertson,K.D. and Jones,P.A. (1998) The human ARF cell cycle regulatory gene promoter is a CpG island which can be silenced by DNA methylation and down-regulated by wild-type p53. Mol. Cell. Biol., 18, 6457–6473.[Abstract/Free Full Text]
  21. Jones,P.A. (1999) The DNA methylation paradox. Trends Genet., 15, 34–37.[ISI][Medline]
  22. Chen,T.R., Drabkowski,D., Hay,R.J. et al. (1987) WiDr is a derivative of another colon adenocarcinoma cell line, HT-29. Cancer Genet Cytogenet., 27, 125–134.[ISI][Medline]
  23. Augenlicht,L.H., Wadler,S., Corner,G., Richards,C., Ryan,L., Mulatani,A.S., Pathak,S., Benson,A., Haller,D. and Heerdt,B.G. (1997) Low-level c-myc amplification in human colonic carcinoma cell lines and tumors: a frequent, p53-independent mutation associated with improved outcome in a randomizd multiinstitutional trial. Cancer Res., 57, 1769–1775.[Abstract]
  24. Jacobs,J.J.L., Kieboom,K., Marino,S. et al. (1999) The oncogene and polycomb-group gene bmi-1 regulates cell proliferation and senescence through the INK4a locus. Nature, 397, 164–168.[ISI][Medline]
  25. Gonzalgo,M.L., Hayashida,T., Bender,C.M. et al. (1998) The role of DNA methylation in expression of the p19/p16 locus in human bladder cancer cell lines. Cancer Res., 58, 1245–1252.[Abstract]
  26. Cottu,P.H., Muzeau,F., Estreicher,A. et al. (1996) Inverse correlation between RER+ status and p53 mutation in colorectal cancer cell lines. Oncogene, 13, 2727–2729.[ISI][Medline]
Received May 10, 2000; revised July 18, 2000; accepted July 27, 2000.