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From the Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In normal tissues, methylation of the promoter region CpG islands is associated with transcriptional silencing of imprinted alleles and genes on the inactive X chromosome.12-14 These patterns of methylation are often altered in tumors. Indeed, aberrant DNA methylation of the promoter region CpG islands can serve as an alternative to mutations in the coding region for the inactivation of tumor suppressor genes, including the retinoblastoma gene (Rb), p16INK4A, and p15INK4B.15-19 Hypermethylation of the p16 CpG island promoter correlated with transcriptional silencing, whereas treatment with 5-aza-2'-deoxycytidine (AZAdC) reactivated transcription in some tumors and cell lines.13,18,19 Thus, multiple mechanisms for inactivation of the remaining p16 allele in primary tumors with 9p21 loss of heterozygozity have been reported, but the frequency of these mechanisms varies with different tumor types. Promoters silenced by methylation can be reactivated in many cases by treatment with the drug AZAdC, which is a well established inhibitor of DNA methylation.12-19 Moreover, DNA methylation abnormalities in neoplastic cells is associated with an apparent increase in DNA methyltransferase (DNA-MTase) activity.20,21
To determine whether methylation of the p27 gene is involved in loss of expression of p27 protein in GH3 and GHRH-CL1 cells, we used bisulfite-modified genomic sequencing to provide a detailed map of the methylation patterns in the p27 gene in normal rat pituitary and in various pituitary tumor cell lines and methylation-specific polymerase chain reaction (PCR) to detect methylation sites in exon 1 of the p27 gene. DNA-MTase mRNA expression and its activity in NP and pituitary tumor cells were also measured. Our results showed that DNA methylation of the p27 gene is an important mechanism regulating p27 expression in pituitary tumor cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NPs were obtained from female 60-day-old Wistar-Furth rats (Harlan, Indianapolis, IN). Total RNA was extracted with the TRIzol reagent kit (Life Technologies, Gaithersburg, MD) as specified by the manufacturer. Genomic DNA was prepared by previously reported.22 The monoclonal antibody to p27 was purchased from Transduction Laboratories (Lexington, KY). The prolactin (PRL) antibody was from the National Pituitary Agency (Bethesda, MD).
Reverse Transcription PCR
p27 expression was examined by reverse transcription (RT)-PCR and immunocytochemistry (ICC).1 DNA-MTase gene transcripts (mRNA) were measured by semiquantitative RT-PCR as previously described.1,23 DNA-MTase and GAPDH were co-amplified in the same reaction. Primer sequences of DNA-MTase were as follows: sense, 5'-GGA AAG CAG AAA TGG CAG ACT-3' (296 to 316), and the antisense, 5'-GAT TCA TTT GCG TTT CCC TTT-3' (495 to 515).24
Cell Culture and Treatment with AZAdC
The rat GH3 cell line secreting PRL and growth hormone (GH) derived from the rat MtT W5 pituitary tumor and the AtT20 mouse pituitary cell line secreting adrenocorticotrophin were obtained from the American Type Culture Collection (Rockville, MD). The mouse GHRH-CL1 cell line was developed from a GH-releasing hormone transgenic mouse pituitary tumor.1,23 HP75 human pituitary tumor cell line was generated in our laboratory.25 Cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 15% horse serum, 2.5% fetal calf serum, 1 µg/ml insulin, and 1% antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone). GH3 and GHRH-CL1 cells were treated with different concentrations of AZAdC (Sigma Chemical Co., St. Louis, MO; 0.5 to 30 µmol/L) for 1 to 7 days and then harvested, aliquots of cells were placed on slides by cytocentrifugation (1 x 105 cells per slide), and the remainder (10 x 106 cells per group) was used for RNA and DNA preparations.1,22 Cell proliferation analyses and ICC were done as previously reported.1,23
Bisulfite Genomic Sequencing
Denatured genomic DNA was modified by reaction with bisulfite
under conditions that convert all unmethylated cytosines to uracils.
Genomic DNA from NP and pituitary tumor cells was modified by bisulfite
using standard procedures.26
Briefly, 2 µg of genomic DNA
was denatured in a final volume of 29 µl, with freshly prepared NaOH
at a final concentration of 0.3 mol/L for 15 minutes at 37°C, and 208
µl of freshly prepared 2.2 mol/L sodium metabisulfite (Sigma)/0.5
mmol/L hydroquinone (Sigma) mixture (pH 5.0) was added to each
denatured DNA sample and incubated under mineral oil at 50°C for at
least 16 hours. Bisulfite-modified DNA was recovered using the Wizard
resin (Promega, Madison, WI) and desulfonated before PCR amplification
in 50 µl of H2O followed by treatment with NaOH at a
final concentration of 0.3 mol/L for 20 minutes at 37°C. The reaction
was neutralized with 30 µl of 10 mol/L ammonium acetate. Each DNA
sample was purified by ethanol precipitation and resuspended in 50 µl
of H2O. The PCR reaction was performed in a volume of 100
µl.1
PCR primers (Figure 1)
were designed to be complementary to the converted DNA sequences with a
minimum of CpG dinucleotides in the corresponding region of the
original unconverted DNA. Each DNA sample was analyzed four times for
bisulfite conversion reaction. To ensure that the bisulfite conversion
was complete, a control digestion was performed with Hsp
92II enzyme, which has a recognition sequence (CATG) that should be
destroyed by the bisulfite conversion. Any cleavage by Hsp
92II indicated either non-CpG DNA methylation or incomplete sodium
bisulfite conversion.27
PCR-generated DNA fragments were
ligated into the Topo TA cloning vector using standard protocols
(Invitrogen, Carlsbad, CA). Cloning of PCR products amplified from
bisulfite-modified DNA was performed with at least four plasmid clones,
which were then sequenced using forward and reverse M13 primers that
yielded sequences from both strands. Sequence data analyses were
performed using the GCG program (University of Wisconsin, Madison, WI).
The sequence of the p27 gene from NP and tumor cell lines before and
after bisulfite modification was compared with the original sequence in
the GenBank database (accession numbers D86924, U09968, and U10906 for
rat, mouse, and human, respectively). Automated sequencing was done in
the Molecular Biology Core at the Mayo Clinic using an ABI PRISM 377
DNA sequencer (Perkin Elmer, Norwalk, CT).
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A PCR-based method was used to confirm p27 gene methylation
patterns from the bisulfite DNA sequencing. Genomic DNA (500 ng) was
digested with methylation-sensitive enzymes under conditions specified
by the manufacturer, and 50 ng of DNA was used for amplification with
the exon 1 primer sets of p27 (Figure 1)
flanking the restriction
sites. Identical restriction sites could be examined in exon 1 of p27,
which included SmaI, HhaI, and AvaI.
Controls included p27 gene completely digested by PstI,
undigested p27 gene incubated with HindIII, and DNA without
enzyme digestion. The PCR conditions used were as described
previously.1,23
To rule out the possibility of incomplete
digestion, all samples were digested with each of the enzymes in two
independent experiments. PCR amplifications from each of the duplicate
digests were repeated at least twice. To prevent overcycling, the cycle
number was determined for each primer set by performing a cycle curve
number using undigested template and template digested with
PstI restriction enzyme, which did not produce any signal.
DNA-MTase Activity Assay
The activity of DNA-MTase was determined as described by Adams et al.20,28 NP and tumor cells were homogenized on ice in 500 µl of lysis buffer (50 mmol/L Tris, pH 7.8, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 0.01% sodium azide, 6 mg/100 ml phenylmethylsulfonyl fluoride, 10% glycerol, and 1% Tween 80). This suspension was then passed twice through a 25-gauge needle, frozen at -70°C, and then thawed at 37°C. The freeze-thaw cycle was repeated three times. The protein concentration in the extracts was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA). Five micrograms of cellular protein was mixed with 500 ng of poly(dI-dC)·poly(dI-dC) (Pharmacia Biotechnology, Piscataway, NJ) and 3 µCi of [methyl-3H]S-adenosyl-L-methionine (75 Ci/mmol; Amersham, Arlington Heights, IL) in a total volume of 20 µl, and this solution was incubated at 37°C for 2 hours. The reaction was stopped by adding 300 µl of a solution containing 1% sodium dodecyl sulfate, 2 mmol/L EDTA, 3% 4-aminosalicylate, 5% butanol, 125 mmol/L sodium chloride, 0.25 mg/ml carrier salmon testis DNA, and 1 mg/ml proteinase K. After an additional 30 minutes of incubation at 37°C, the mixture was then extracted twice with phenol/chloroform/isoamyl alcohol (25:24:1) and ethanol precipitation. RNA was removed by resuspension in 0.3 mol/L sodium hydroxide and incubation at 37°C for 30 to 45 minutes, and the final solution was spotted on a Whatman GF/C filter and then dried at 80°C for 5 minutes, washed in ice-cold 5% trichloroacetic acid followed by 70% ethanol, placed in 5 ml of scintillation fluid (Beckman Instrument, Fullerton, CA), and counted in a Beckman scintillation counter. Negative control reactions lacked poly(dI-dC)·poly(dI-dC). Results are expressed as cpm per 5 µg of protein per 2 hours incubation minus the value of the negative control.
Statistical Analysis
A minimum of three independent experiments using three or more replicates per treatment group were done. Statistical analysis was done with the Student's t-test. Results are expressed as the mean ± SE.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After immunostaining with the anti-p27 antibody, p27 protein was
readily detected in NP cells, as nuclear staining (Figure 2
, left), but not in GH3
cells (Figure 2
, middle). GH3 cells were exposed for
different times (range, 1 to 7 days) with various concentrations of
AZAdC between 0.5 and 30 µmol/L. Treatment with 10 µmol/L AZAdC for
7 days produced the maximal induction of p27 with the lowest toxicity.
p27 expression evaluated by ICC was localized in the nucleus of
GH3 cells. After 10 µmol/L AZAdC treatment for 7 days,
approximately 50% of GH3 cells were positive for p27 by
ICC (Figure 2
, right). GHRH-CL1 cells also were negative for p27
staining. After AZAdC treatment for 7 days, up to 40 to 50% of
GHRH-CL1 cells were stained with anti-p27 antibody as strong nuclear
positive. AtT20 and HP75 cells normally expressed p27 with 60 to 70%
and 30 to 40% positive cells detected by ICC, respectively (data not
shown).
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. Duplex RT-PCR using GAPDH as an internal
standard in the same reaction was used to confirm the mRNA quality
(Figure 3)
. Three weeks after
discontinuation of AZAdC treatment, 40 to 50% of GH3 cells
remained positive for p27. There was more than a twofold increase in
PRL expression (control, 9.8 ± 1.1%; treated, 22.5 ±
1.6%; P < 0.01) by ICC in AZAdC-treated cells. AZAdC
inhibited GH3 cell proliferation as shown by
[3H]thymidine incorporation ([3H]thymidine
labeling index: control, 25.6 ± 0.8%; treated, 8.2 ±
0.5%; P < 0.001).
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Sequencing analysis of exon 1 and exon 2 (primers 3 and 6 in
Figure 1
) showed that p27 gene mutations were not present in
GH3 tumor cells. The splicing sites for exons 1 and 2 in
the rat p27 gene were confirmed by sequencing of intron 1 (data not
shown).
To elucidate the specific location of DNA methylation in the p27 gene,
we used the bisulfite genomic sequencing method, which allows
unambiguous identification of sites of methylated cytosines in clones
derived from individual DNA strands. Bisulfite DNA sequencing showed
complete conversion of the control enzyme restriction site
(Hsp 92II). PCR products were not cleaved by the
Hsp 92II, which confirmed the bisulfite sequencing data. To
eliminate the possibility that our analyses were affected by selection
bias introduced by the cloning procedures, after cloning of the each
PCR reaction product in Topo TA plasmid, at least four randomly
selected clones with the correct size inserts were sequenced. Detailed
sequence data are shown for seven different representative samples
covering bases 34 to 153 (Figure 4)
. A
total of eight methylated cytosines in GH3 cells and seven
methylated cytosines in GHRH-CL1 cells were found in this region, but
no methylated cytosines were present in the NP and AtT20 cells in this
region.
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, the methylation
pattern of the p27 gene in treated GH3 and GHRH-CL1 cells
was different from that of the untreated GH3 and GHRH-CL1
cells. AZAdC treatment had decreased numbers of methylated cytosines in
different plasmid clones as measured by bisulfite genomic sequencing in
exon 1 of p27 gene ranging from 60 to 90%.
A 98% sequence similarity in p27 gene between mouse and rat allowed
the use of same primers (sense primer 1 was identical, and antisense
primer 2 had one mismatch between rat and mouse) for the bisulfite
analysis. However, because there was only 88% sequence homology
between rat and human, we modified the primers to match the human
sequence for HP75 human cell analyses. The mouse GHRH-CL1 cell line
showed similar changes in methylation as the rat GH3 cell
line. A total of 28 methylated cytosines were found in exon 1 of the
p27 gene in both GH3 and GHRH-CL1 cells. One methylated
cytosine located at 104 in GH3 cells shifted to 382 in
GHRH-CL1 cells. There were decreased methylation and increased p27
protein expression after AZAdC treatment. In contrast, the mouse AtT20
cell line, which expressed abundant p27 protein with approximately 60%
of cells stained with anti-p27 antibody without AZAdC treatment,
contained only one methylated cytosine at position 410 in exon 1
(Figure 5)
. Human HP75 cells showed a
similar methylation pattern with AtT20 cells. One methylated cytosine
was found in HP75 cells with variable location in the different plasmid
clones, but it disappeared after AZAdC treatment (data not shown).
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. The results from SmaI,
HhaI, and AvaI cleavage supported the conclusion
that p27 methylation patterns of exon 1 of the p27 gene between NP and
GH3 cells were very different (Figure 5)
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There was an increase in the mRNA expression of DNA-MTase in
GH3 and GHRH-CL1 cells compared with NP and AtT20 cells
detected by semiquantitative RT-PCR (Figure 7)
. DNA-MTase activity was elevated
approximately fourfold in GH3 and twofold in GHRH-CL1 cells
compared with NP and AtT20 cells. DNA-MTase activity decreased
significantly after GH3 and GHRH-CL1 cells were treated
with 10 µmol/L AZAdC for 7 days (Figure 8)
. DNA-MTase mRNA expression and
activity correlated with DNA methylation of the p27 gene in NP and
pituitary tumor cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The methylation pattern of the p27 gene was analyzed by bisulfite genomic sequencing and by the cleavage of genomic DNA with methylation-sensitive enzymes, including SmaI, HhaI, and AvaI. Bisulfite genomic sequencing showed that the normally unmethylated CpG in exon 1 in NP was extensively methylated in GH3 cells. All SmaI, HhaI, and AvaI enzyme sites of the p27 gene at exon 1 were methylated in GH3 cells but not in NP. The results obtained with both techniques were similar.
Because exon 2 (120 bp) of the p27 gene contains the methylation-sensitive restriction sites located at the 3' end, it was difficult to design primers to amplify this region. The present study was focused on exon 1 (475 bp) of the p27 gene. Genomic sequencing of bisulfite-treated DNA has several advantages. 1) It is easy to evaluate because it gives a positive display of the position of the methylated cytosine in a DNA sequence, which is distinct from the absence of all unmethylated cytosines. 2) Unlike methods depending on restriction enzyme specificity, it enables the methylation status of all cytosines in a target DNA sample to be determined irrespective of nearest neighbor compositions, which is critical in studying the potential silencing of genes involved in development and imprinting and in cancer. 3) The PCR products can be cloned and sequenced to provide methylation maps of single DNA molecules. 4) It allows the identification of hemi-methylated sites not normally detected with restriction enzymes.26 High levels of both p27 protein and mRNA expressions in NP were detected by immunoblotting and RT-PCR in this study and in our previous report.1 In the present study, GH3 and GHRH-CL1 cells had undetectable p27 protein, although these cells had very low levels of p27 mRNA as detected by RT-PCR.1
The possible role of p27 as a tumor suppressor gene has been analyzed by various investigators.1-4,10,11,29 Analysis of over 500 tumors to date has shown only a few mutations, including point mutations in 2 of 36 primary breast carcinomas.5-9 When an astrocytoma cell line was transfected with p27 there was decreased malignant behavior, supporting the concept that loss of p27 expression may be associated with tumor progression.30 There is aberrant expression of p27 protein in neoplasms with a marked decrease of p27 during progression from normal to benign and malignant tumors.5-8,29 p27 protein has been shown to be degraded by the ubiquitin-proteasome pathway, and the degradation is increased in some tumor tissues.8,11 Although ubiquitination is a principal post-translational regulatory path for p27 protein, our study shows that DNA methylation can also regulate p27 expression in rodent pituitary cells. We have also observed that ubiquitination is one of the regulatory mechanism for p27 expression in the immortalized HP75 human pituitary cell line (unpublished observation). This cell line was also used in the current study and showed a low level of methylation of the p27 gene.
Cancer cells often show altered patterns of DNA methylation. This change may contribute to genomic instability.31 In some tumors, the normally unmethylated CpG islands in the promoter region of critical genes can become highly methylated, and the associated transcriptional silencing becomes an epigenetic alternative to coding region mutations in causing loss of tumor suppressor gene function.12-19
The methylation patterns are generated by DNA-MTase, although the actual mechanism involved is still unclear. One of the characterized DNA-MTases in mammalian cells has a pronounced sequence selectivity for the methylation of cytosine in hemi-methylated CG sequences..12-14 DNA-MTase activity is generally severalfold higher in various transformed cells compared with normal cultured murine and human cells.20,21 Our study has shown that DNA-MTase mRNA levels and enzyme activity are much higher in GH3 and GHRH-CL1 cells than in NP and AtT20 cells. Because AZAdC is a cytidine analog and causes a powerful inhibition of DNA-MTase, GH3 and GHRH-CL1 cells treated with AZAdC had increased expressed p27 and had decreased DNA-MTase activity. The present study suggests that the level of increased DNA-MTase activity could involve increased methylation of regional or specific genes. Changes in DNA methylation may be accompanied by changes in tumor cell phenotype and induction of differentiation in vitro,12-14,22,32 which was observed in the GH3 cells,;ie, these cells had increased PRL expression and decreased proliferation after AZAdC treatment.
Treatment of GH3 and GHRH-CL1 cells with AZAdC for 7 days in vitro decreased the number of methylated cytosines and induced expression of both p27 protein and mRNA. Variable methylation patterns of the p27 gene were detected in AZAdC-treated GH3 and GHRH-CL1 cells, because these cells were heterogeneous with respect to DNA methylation after treatment. DNA methylation correlated inversely with the expression of the p27 gene in NP, GH3, and GHRH-CL1 cells.
The mouse GHRH-CL1 cell line, which produces PRL and GH, was very similar to the rat GH3 cell line, which also produces PRL and GH, with respect to p27 expression and methylation of exon 1 of the p27 gene. However, the AtT20 mouse pituitary cell line, which produces adrenocorticotrophic hormone (ACTH), had abundant p27 protein and few methylated cytosines in exon 1. These observations substantiate our findings about the inverse relationship of p27 expression and DNA methylation and suggest that the methylation regulatory mechanisms in these cell lines may be related to the specific phenotype of the pituitary cell line.
In the p27 knockout models, the ACTH cells from the intermediate lobe developed ACTH cell hyperplasia, suggesting a greater regulatory role of p27 on ACTH cells.2-4 Additional studies are needed to analyze the relationship of pituitary cell phenotype and p27 gene methylation.
In summary, this is the first demonstration that the p27 gene in some rodent pituitary cell lines, including GH3 and GHRH-CL1, have increased methylation that results in p27 gene silencing. These results offer a molecular explanation for p27 aberrant gene inactivation in some pituitary tumors and possibly in other types of neoplasms.
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Footnotes |
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Supported in part by NIH grants CA 37231 and CA 42951.
Accepted for publication July 24, 1998.
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, methylated
cytosines; 
, unmethylated cytosines.
