Association of H19 Promoter Methylation with the Expression of H19 and IGF-II Genes in Adrenocortical Tumors
ZHI-HE GAO*, SUVIKKI SUPPOLA*, JIANQI LIU*, PÄIVI HEIKKILÄ, JUHANI JÄNNE, AND RAIMO VOUTILAINEN
Department of Pathology, University of Helsinki (Z .- H.G., J.L., P.H., R.V.), FIN-00014 Helsinki, Finland; and Department of Pediatrics, Kuopio University Hospital (Z .- H.G., J.L., R.V.), and A. I. Virtanen Institute for Molecular Sciences, University of Kuopio (S.S., J.J.), FIN-70211 Kuopio, Finland
Low H19 and abundant IGF-II expression may have a role in the development of adrenocortical carcinomas. In the mouse, the H19 promoter area has been found to be methylated when transcription of the H19 gene is silent and unmethylated when it is active. We used PCR-based methylation analysis and bisulfite genomic sequencing to study the cytosine methyl- ation status of the H19 promoter region in 16 normal adrenals and 30 pathological adrenocortical samples. PCR-based anal- ysis showed higher methylation status at three HpaII-cutting CpG sites of the H19 promoter in adrenocortical carcinomas and in a virilizing adenoma than in their adjacent normal adrenal tissues. Bisulfite genomic sequencing revealed a sig- nificantly higher mean degree of methylation at each of 12 CpG sites of the H19 promoter in adrenocortical carcinomas than in normal adrenals (P < 0.01 for all sites) or adrenocor- tical adenomas (P < 0.01, except P < 0.05 for site 12 and P > 0.05 for site 11). The mean methylation degree of the 12 CpG sites was significantly higher in the adrenocortical carcino-
mas (mean ± SE, 76 ± 7%) than in normal adrenals (41 ± 2%) or adrenocortical adenomas (45 ± 3%; both P < 0.005). RNA anal- ysis indicated that the adrenocortical carcinomas expressed less H19 but more IGF-II RNAs than normal adrenal tissues did. The mean methylation degree of the 12 H19 promoter CpG sites correlated negatively with H19 RNA levels (r = - 0.550; P < 0.01), but positively with IGF-II mRNA levels (r = 0.805; P < 0.001). In the adrenocortical carcinoma cell line NCI-H295R, abundant IGF-II, but minimal H19, RNA expression was de- tected by Northern blotting. Treatment with a cytosine meth- ylation inhibitor, 5-aza-2’-deoxycytidine, increased H19 RNA expression, whereas it decreased IGF-II mRNA accumulation dose- and time-dependently (both P < 0.005) and reduced cell proliferation to 10% in 7 d. Our results suggest that altered DNA methylation of the H19 promoter is involved in the ab- normal expression of both H19 and IGF-II genes in human adrenocortical carcinomas. (J Clin Endocrinol Metab 87: 1170-1176, 2002)
H UMAN H19 AND IGF-II genes, mapped contiguously on chromosome 11p15.5, are expressed in a parental origin-specific manner, a phenomenon known as genomic imprinting (1). H19, a gene whose transcript is not translated (2), is supposed to be involved in tumor suppression (3). IGF-II is an important mitogen, playing a role in normal fetal and postnatal growth and in tumorigenesis (4). H19 and IGF-II have been found to show coordinate, reciprocal reg- ulation in a tissue-specific and developmentally regulated manner. H19 is not expressed in choroid plexus or leptome- ninges, where IGF-II is expressed biallelically in mouse and man (5). IGF-II is also expressed biallelically in H19 knockout mice (6, 7). In methyltransferase-deficient mice the normally silent paternal allele of the H19 gene is activated, whereas the normally active paternal allele of the IGF-II gene is re- pressed (8).
Aberrant cytosine methylation has been found to be as- sociated with altered gene expression in tumors (9-11). Cer- tain regions (often located at the 5’-ends of genes) rich in CpG dinucleotides are known as CpG islands (12). The promoter CpG islands are usually unmethylated in normal tissues, but the imprinted genes form an exception, with one of the pa- rental alleles being often methylated (9). In the mouse, the H19 promoter has been found to be methylated when tran-
scription is silent and unmethylated when it is active (13). Normal methylation patterns are frequently disrupted in tumor cells with region-specific hypermethylation (11). In Wilms’ tumors, DNA hypermethylation in the H19 promoter is associated with remarkable down-regulation of H19 ex- pression and loss of imprinting of the IGF-II gene (14). In addition, aberrant methylation of the differentially methyl- ated region upstream of the H19 gene seems to be necessary, but not sufficient, for loss of imprinting (15). In Wilms’ tu- morigenesis, H19 inactivation has been considered a pre- neoplastic event (16, 17). Therefore, hypermethylation of the H19 promoter appears to be an important factor leading to low H19 and high IGF-II expression.
Both H19 and IGF-II genes are supposed to be involved in the normal development and tumorigenesis of human adre- nals. Human fetal adrenals express abundantly both H19 and IGF-II genes, and the regulation of these two genes is parallel and multifactorial, suggesting common regulatory mecha- nisms for these adjacent genes (18). In normal human adult adrenals IGF-II expression is low (19, 20), but H19 expression remains quite high (21). H19 and IGF-II genes are expressed at about the same level in benign adrenocortical neoplasms as in normal adrenal glands, whereas adrenocortical carci- nomas show very low H19 and high IGF-II expression (21, 22). Gicquel et al. (23) found that about 80% of the adreno- cortical tumors with high IGF-II expression exhibited LOH
Abbreviation: Azad, 5-Aza-2’-deoxycytidine.
(loss of maternal allele and duplication of the paternal one), which correlated with the abrogation of H19 expression. Thus, this loss of H19 expression in conjunction with high IGF-II expression may be associated with malignant behav- ior in adrenocortical carcinomas. However, the mechanisms for this phenomenon remain unknown. To determine whether DNA methylation plays a role, we used PCR-based methylation analysis and bisulfite genomic sequencing to assess the methylation status of H19 promoter in different adrenal tumors compared with that in normal adrenals. We then explored the association of the expression of H19 and IGF-II genes with the methylation status of the H19 promoter.
Materials and Methods
Tissues
Normal adrenals (removed during nephrectomy for kidney tumors, n = 7) and pathological adrenal glands were obtained from patients during operations performed at the Department of Surgery, Helsinki University Central Hospital. The pathological adrenocortical tissues in- cluded 1) Cushing’s (n = 6), virilizing (n = 2), and nonfunctional (hor- monally inactive, n = 2) carcinomas; 2) Cushing’s (n = 5), Conn’s (n = 5), virilizing (n = 2), and nonfunctional (n = 4) adenomas; and 3) hyperplastic adrenals (two patients with bilateral adrenal hyperplasia due to Cushing’s disease and two nodularly hyperplastic adrenals). Tumor-adjacent normal adrenal tissues (n = 9) were also used. The tissue specimens were sent to the Department of Pathology, where the diagnoses were established based on both clinical and histopathological data. The study protocol was approved by the local ethical committees.
Cell cultures
Human adrenocortical carcinoma NCI-H295R cells from American Type Culture Collection (Manassas, VA) were grown in a 1:1 mixture of DMEM and Ham’s F-12 medium (Sigma, St. Louis, MO) supplemented with 1% ITS+1 liquid medium supplement (Sigma), 2% Ultroser SF (Biosepra, Marlborough, MA), 2 mmol/liter L-glutamine (Life Technol- ogies, Inc., Paisley, UK), and antibiotics (125 µg/ml streptomycin and 125 IU/ml penicillin; Orion Pharmaceutical Co., Espoo, Finland) at 37 C in a 5% CO2 atmosphere. The medium was refreshed every other day, and the cells were split at a ratio of 1:3 with trypsin after reaching confluence. Treatment with a cytosine methylation inhibitor, 5-aza-2’- deoxycytidine (Azad; Sigma), was initiated on the second day after reseeding the cells. Triplicate dishes were used for the experiments, which were repeated at least three times.
RNA analysis
Total RNA was isolated from frozen tissues by ultracentrifugation through a cesium chloride cushion (24). Cytoplasmic RNA was extracted from the cultured cells (25). Northern blotting and hybridizations were performed as previously described (21). The relative intensities of au- toradiographic signals were quantified by densitometric scanning. All of the RNA data shown here were normalized with the respective 28S rRNA values.
PCR-based methylation analysis
PCR analysis based on the inability of HpaII to cut methylated CCGG sequence (26) was used to analyze the H19 promoter containing three HpaII recognition sites (Fig. 1, GenBank accession no. AF125183). Genomic DNA was isolated as reported previously (27). DNA (500 ng) was digested for 6 h with 10 U of the HpaII enzyme (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacturer’s rec- ommendations. Optimal PCR conditions were found to produce a clean 462-bp PCR product from undigested genomic DNA, but no product from DNA digested with MspI (a methylation-insensitive isoschizomer of HpaII): denaturing at 95 C for 5 min; 30 cycles of 95 C for 60 sec, 57 C (annealing temperature) for 30 sec, and 72 C for 30 sec; and then final extension at 72 C for 8 min. The PCR reaction volume was 50 ul, containing 0.2 mmol/liter of each dNTP, 0.5 µmol/liter of each primer, 1 X reaction buffer, 2.5 mmol/liter MgCl2, 1.5 U Taq DNA polymerase (Fermentas Tamro Corp., Vantaa, Finland), and 50 ng template DNA. The primer set was 5’-AGG TGA TGG GGC AAT GCT CA-3’ (sense, P1 in Fig. 1) and 5’-CCT ACT CCA CAC TCC TCA CT-3’ (antisense, P2). Exon 9 of IGF-II gene (GenBank accession no. X07868) was used as the internal control for the DNA amount because this region has no HpaII/ MspI cutting site. The primer set for IGF-II was 5’-CTT GGA CTT TGA GTC AAA TTG G-3’ (sense) and 5’-GGT CGT GCC AAT TAC ATT TCA-3’ (antisense). The PCR products were resolved on 1.8% agarose gels. PCR-based analyses were performed at least twice to ensure re- producibility of the results.
Bisulfite sequencing methylation analysis
A previously described DNA methylation analysis using bisulfite- induced modification and genomic sequencing (28) with minor modi- fications was used to assess the methylation status of all 14 CpG sites in the H19 promoter region, including the first 2 CpG sites after the tran- scription start site (Fig. 1). Briefly, 3 µg genomic DNA were denatured with NaOH and deaminated with sodium bisulfite to convert all un- methylated cytosines to uracils. The outer PCR reaction was carried out with primers 5’-TTG GTA GGT AGG GAG TAG TAG GTA TG-3’ (sense) and 5’-AAC CCA TCA/G TCC CCA ACT AAT AT-3’ (antisense), cho- sen from the area containing as few CpGs as possible. The PCR was performed for 40 cycles of 96 C for 15 sec, 56 C for 30 sec, and 72 C for 90 sec. The inner PCR was carried out with primers 5’-GTAAAAC- GACGGCCAGT-GGG AGG TGA TGG GGT AAT GTT TA-3’ (sense, P3 in Fig. 1) and 5’-ACC TAC TCC ACA CTC CTC ACT AAC CT-3’ (antisense, P4). The antisense primer was biotinylated to separate and isolate the strands of PCR products with the aid of streptavidin-coated magnetic beads, and the sense primer had in its 5’ a universal oligo- nucleotide for sequencing. The PCR was carried out for 33 cycles of 96 C for 15 sec, 61 C for 20 sec, and 72 C for 90 sec. Sequencing reactions were carried out according to the instructions of the AutoRead 200 DNA sequencing kit (Pharmacia Biotech, Uppsala, Sweden), using fluores- cently labeled primers. The methylation status was categorized with 25% accuracy (28) into 0%, 25%, 50%, 75%, and 100%.
IGF-II imprinting analysis
IGF-II ApaI polymorphism of all carcinoma DNAs and cDNAs was analyzed by PCR amplification (29) using the primers mentioned above in the description of the PCR-based methylation analysis.
-303
1
23
4
5
6 7
8 9 10
11 12
13
14
+222
Transcription
P4
P3
P1
60 bp
P2
adjacent tissue
carcinoma (virilizing)
carcinoma (Cushing’s)
DNA ladder
adjacent tissue
carcinoma (virilizing)
adjacent tissue
adjacent tissue
carcinoma (virilizing)
adjacent tissue
adenoma (virilizing)
adjacent tissue
adenoma (Cushing’s) adjacent tissue
adenoma (Conn’s)
1
1
1
1
2
2
3
3
4
4
5
5
6
6
500 bp —
— H19
400 bp —
300 bp —
— IGF-II
250 bp —
Hpall + + - - + + + + + + + + +
+
Mspl
-
-
+
+
-
-
-
-
-
-
-
-
-
-
Immunocytochemical staining for cell proliferation analysis
Proliferating cells were demonstrated immunocytochemically in NCI-H295R cell cultures with a commercial cell proliferation kit (Am- ersham Pharmacia Biotech, Little Chalfont, UK) according to the man- ufacturer’s instructions. In this assay, 5-bromo-2’-deoxyuridine was in- corporated into replicating DNA, localized with a specific monoclonal antibody, and stained with a peroxidase-based detection system. The culture area was photographed, and the nuclei were counted from each culture dish. The proliferation percentage was calculated on the basis of positively stained nuclei relative to all cells.
Statistical analyses
Nonparametric Kruskal-Wallis test was used to reveal differences in the methylation status (analyzed by bisulfite sequencing) and in the expression levels of the H19 and IGF-II genes among the different groups of adrenal samples. If significant differences were found, a Mann-Whit- ney U test was subsequently used for statistical evaluation. A Mann- Whitney U test was also used to evaluate the changes in H19 and IGF-II expression and in the proliferation of the cultured cells. Simple regres- sion analysis was used to determine the correlation of H19 or IGF-II RNA levels with the degree of H19 promoter methylation. The level of sig- nificance was chosen as P < 0.05.
Results
The primer set P1 and P2 (Fig. 1) amplifies an H19 pro- moter region across three HpaII sites, located at -210, -69, and -16 from the transcription initiation site. Because of the digestion of unmethylated CCGG sequence, only indigest- ible methylated DNA will give a PCR product 462 bp in size. As shown in Fig. 2, this PCR product was more abundant in adrenocortical carcinoma DNA samples than in tumor- adjacent adrenal tissues, suggesting a higher degree of meth- ylation in the carcinoma samples. We saw a clear difference in the intensity of the 462-bp product between carcinomas and their adjacent normal tissues (three pairs). No consistent difference was found between adrenocortical adenomas (Conn’s and Cushing’s syndrome) and their adjacent normal tissues (three pairs). However, PCR-based analysis sug- gested higher promoter methylation in a virilizing adenoma
than in its adjacent normal tissue (Fig. 2), which was verified by sequencing analysis (data not shown).
To clarify the methylation status of the H19 promoter in more detail, we applied automated fluorescent genomic se- quencing after bisulfite-induced modification of genomic DNA. Among normal adult adrenal and tumor-adjacent nor- mal adrenal tissues, there was not much variation in the methylation status of each CpG site in the H19 promoter; sites 5, 7, and 8 showed the least variation (mostly 50%, 25%, and 50% methylation, respectively). No significant difference in the average degree of methylation of the first 12 CpG sites in the H19 promoter was found between the normal adrenals (mean ± SE, 41 ± 2%) and hyperplasias (43 + 5%; P = 0.962) or adenomas (45 ± 3%; P = 0.471). However, the average degree of methylation was significantly higher in carcinomas (76 ± 7%) than in normal adrenals and adenomas (both P < 0.005) or hyperplasias (P < 0.05; Fig. 3).
The methylation degree of each 12 CpG site of the H19 promoter in the hyperplastic adrenals and adenomas was similar to that in the normal adrenals, except for site 11, which showed higher methylation in the adenomas than in the normal adrenals (P < 0.01). Compared with the normal adrenals, each 12 CpG site showed hypermethylation in the carcinomas (P < 0.01; Fig. 4). However, site 11 did not show significantly higher methylation in the carcinomas than in the adenomas (P = 0.094), whereas the other sites did (P < 0.01 for sites 1-10, P < 0.05 for site 12). The methylation status of sites 13 and 14 (the first 2 after the transcription start site, Figs. 1 and 4) was not different in hyperplasias, adenomas, or carcinomas from that in normal adrenals.
As reported previously (21), H19 expression was signifi- cantly lower and IGF-II higher in adrenocortical carcinomas than in normal adrenals (a representative Northern blot in Fig. 5A). There was a negative correlation between the H19 RNA levels and the mean methylation degree of the 12 CpG sites of the H19 promoter (r = - 0.550; P < 0.01; Fig. 5B) and,
on the other hand, a positive correlation between the IGF-II mRNA levels and the mean degree of methylation of the H19 promoter (r = 0.805; P < 0.001; Fig. 5C).
Of the 10 available carcinomas, 3 were heterozygous at the IGF-II ApaI site. Of these informative tumors, 1 demonstrated loss of imprinting, i.e. biallelic IGF-II expression (data not shown).
To confirm the association of DNA methylation with the H19 and IGF-II gene expression levels, we modified DNA methylation of the adrenocortical carcinoma cell line NCI- H295R with Azad, a demethylating agent. Northern blot analysis showed hardly any H19 RNA expression in these cells before treatment. Azad treatment induced a remarkable increase in the H19 RNA content (Fig. 6; P < 0.005, pooled data from six experiments). H19 induction was detectable at 1 µmol/liter Azad after 3 or 7 d, and the maximal increase
90
*
Cytosine methylation %
60
30
0
Normal
Hyperplasia
Adenoma
Carcinoma
appeared at 3 umol/liter. Time-course experiments demon- strated that H19 RNA increased within 2 d, and the aug- mentation continued until 7 d of treatment (Fig. 6). Bisulfite sequencing methylation analysis showed that the methyl- ation of CpG sites 9, 10, and 11 in the H19 promoter was indeed decreased by Azad treatment (Table 1) consistent with PCR-based methylation analysis (data not shown). In- terestingly, the induction of H19 gene expression was ac- companied by a clear decrease in IGF-II expression (P < 0.005, pooled data from six experiments). This reduction of IGF-II mRNA accumulation during Azad treatment was also dose and time dependent (Fig. 6). In addition, treatment with Azad inhibited proliferation of the cells. The proliferation rate of the cells treated with Azad (measured at 30 pmol/liter Azad for 7 d) was less than 10% of the control (P < 0.05, three separate experiments; Fig. 7). Total DNA amount harvested from Azad-treated cells (from two independent experiments) was about 30% of that from the control cultures. Azad treat- ment also induced a morphological change in the cells; the cells and the nuclei enlarged, and the cells flattened (Fig. 7). The cells treated with Azad looked healthy, with cell viability more than 98%, measured by trypan blue exclusion (data not shown). In addition, the functional viability of the cells was preserved during Azad treatment on the basis of well main- tained cortisol secretion (data not shown).
Discussion
Molecular mechanisms leading to adrenocortical tumori- genesis are not yet fully understood, partly due to the dif- ficulty in obtaining human adrenal samples (30, 31). It has been suggested that the loss of the putative H19 tumor sup- pressor activity and the gain of the IGF-II mitotic effect may be involved in the pathogenesis of adrenocortical neoplasms (21-23). If an epigenetic error is the cause of the H19 silencing in adrenocortical carcinomas, this error will most likely occur in the regions that control H19 gene expression. Our meth- ylation analysis revealed that the CpG sites in the H19 pro- moter are hypermethylated in most carcinomas, whereas the two CpG sites after the transcription start site are not. Given the negative correlation of the H19 promoter methylation
100
*
*
*
*
Cytosine methylation %
*: P < 0.01
*
*
*
80
*
*
-Carcinoma
*
*
60
*
0
40
-Hyperplasia
-Normal
20
o-Adenoma
1
2
3
4
5
6
7
8
9
10
11
12
13
14
CpG site
Transcription start site
Normal adrenal
Normal adrenal
Adenoma (virilizing)
A
Carcinoma
Hyperplasia
Hyperplasia
H19
IGF-II
28S
B
Relative H19 RNA level
160
r = - 0.55
120
P < 0.01
80
40
4
0
A
T
A
A
A
25
50
75
100
H19 promoter methylation (%)
C
Relative IGF-II RNA level
2000
A
1500
r = 0.805
A
P < 0.001
1000
A
A
4
4
A
500
0
0
25
50
75
100
H19 promoter methylation (%)
with the H19 RNA expression levels, our results are consis- tent with previous reports showing the H19 promoter to control H19 expression in mice during development (13) and
in other neoplasms, such as Wilms’ tumor and hepatoblas- toma (14, 16, 32). On the other hand, our present study did not tell much about the significance of the loss of IGF-II imprinting in adrenocortical carcinomas, as only three of the carcinomas were informative.
Similar H19 promoter methylation in hyperplastic and normal adrenals suggests that the methylation status in this area has no role in the development of benign hyperplastic changes in human adrenals. No single 1 of the 12 CpG sites in the H19 promoter seems to be more important than the others in determining H19 expression in adrenocortical car- cinomas. However, site 11 may become initially methylated in adrenal tumorigenesis, because its degree of methylation in adenomas was between normal adrenals and carcinomas. It has previously been suggested that H19 hypermethylation might be an early epigenetic error that occurs at the onset of Wilms’ tumor development (16). All adrenocortical carcino- mas are monoclonal in tissue composition, whereas adreno- cortical adenomas can be either monoclonal or polyclonal with various intermediate forms (33). Therefore, heteroge- neous H19 promoter methylation may occur in benign ad- renocortical tumors. The difference in the methylation status between a virilizing adenoma and its adjacent normal ad- renal supports the hypothesis that the development mech- anism of this type of adenoma may be different from that of other adrenal adenomas (34, 35).
The NCI-H295R cell line is a unique model for studying the clustered imprinted genes on chromosome 11p15, as the cells have a deletion of 11p in one allele (36, 37). On the basis of the absent H19 and high IGF-II expression together with hypermethylation of the H19 promoter, the deleted allele is probably the maternal one. Inhibition of CpG methylation of the H19 promoter with Azad treatment indeed activated H19 expression in these cells, confirming the effect of the meth- ylation status of the H19 gene on its expression (38). As the inhibition of methylation by Azad treatment occurs only on de novo synthesized DNA (39), the decrease in DNA meth- ylation of the proliferating cells is probably more significant than that detected in the DNA isolated from all cells in this study. Considering both the reduced methylation of CpG sites 9, 10, and 11 in the cells caused by Azad treatment and the increasing methylation of site 11 from normal adrenals to adenomas (and further to carcinomas), site 11 may be a sensitive point for epigenetic errors. In contrast to the in- duced H19 expression due to the relief of the suppressed allele, IGF-II mRNA expression decreased with Azad treat- ment, fitting to the general opposite expression pattern of these two genes from a single allele (13, 40). NCI-H295R cells express IGF type 1 and 2 receptor genes as well as IGF- binding proteins, which allows the cells to respond to en- dogenous and/or exogenous IGFs (41). The reduced prolif- eration rate after Azad treatment is thus probably explained by the altered expression of the H19 and IGF-II genes.
In this study we did not analyze methylation of the IGF-II gene, which has been shown to be associated with IGF-II expression in some tumors. The methylation status of an IGF-II upstream repressor element plays a role in IGF-II imprinting in mouse (42). A significantly increased IGF-II mRNA content in pediatric adrenocortical tumors was as- sociated with significant IGF-II gene (exon 7) demethylation
A
B
control
Azad 100 µM
Azad 30 µM
Azad 10 µM
Azad 3 µM
Azad 1 µM
control 1d
Azad 1d
control 2d
Azad 2d
control 3d
Azad 3d
control 4d
Azad 4d
control 7d
Azad 7d
— 28S
— 28S
— 18S
— 18S
H19
H19
— 28S
— 28S
— 18S
— 18S
IGF-II
IGF-II
— 28S
— 28S
28S RNA
28S RNA
| CpG sites (1-14) and their methylation status (%) | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | |
| Control | 100 | 100 | 75 | 25 | 25 | 50 | 0 | 75 | 100 | 100 | 100 | 100 | 0 | 0 |
| Azad | 100 | 100 | 75 | 25 | 25 | 50 | 0 | 75 | 75 | 75 | 75 | 100 | 0 | 0 |
Genomic sequencing after bisulfite modification of genomic DNA was used to estimate the methylation status as described in Materials and Methods.
A
B
25 um
compared with normal adrenals (43). However, the changes in IGF-II CpG methylation may be secondary to the inacti- vation of H19 (44). It has been reported that the presence or absence of the active H19 transcription unit affects IGF-II allelic expression to some extent independently of IGF-II
DNA methylation (6, 13, 45). Methylation of the H19 gene regulates the imprinted expression of both H19 and IGF-II genes through mediating methylation-sensitive, enhancer- blocking activity at the H19/IGF-II locus (46-48).
In summary, our results indicate that altered DNA meth- ylation of the H19 promoter may be involved in the abnormal expression of H19 and IGF-II genes in human adrenocortical carcinomas and in the pathogenesis of these neoplasms.
Acknowledgments
Ms. Merja Haukka, Ms. Eija Heiliö, Ms. Arja Korhonen, and Ms. Anne Karppinen are thanked for their technical assistance, and Ms. Sanna Myöhänen, Ph.D., for her methodological advice.
Received August 8, 2001. Accepted December 10, 2001.
Address all correspondence and requests for reprints to: Raimo Vou- tilainen, M.D., Department of Pediatrics, Kuopio University Hospital,
P.O. Box 1777, FIN-70211 Kuopio, Finland. E-mail: raimo.voutilainen@ uku.fi.
This work was supported by the Emil Aaltonen Foundation, the Jalmari and Rauha Ahokas Foundation (to J.L.), the Academy of Finland, the Sigrid Juselius Foundation, and Kuopio University Hospital (to R.V.).
* Z .- H.G., S.S., and J.L. contributed equally to this paper.
References
1. Feinberg AP 1999 Imprinting of a genomic domain of 11p15 and loss of imprinting in cancer: an introduction. Cancer Res 59:1743-1746
2. Brannan CI, Dees EC, Ingram RS, Tilghman SM 1990 The product of the H19 gene may function as an RNA. Mol Cell Biol 10:28-36
3. Hao Y, Crenshaw T, Moulton T, Newcomb E, Tycko B 1993 Tumour- suppressor activity of H19 RNA. Nature 365:764-767
4. Stewart CEH, Rotwein P 1996 Growth, differentiation, and survival: multiple physiological functions for insulin-like growth factors. Physiol Rev 76:1005- 1026
5. Ohlsson R, Hedborg F, Holmgren L, Walsh C, Ekström TJ 1994 Overlapping patterns of IGF2 and H19 expression during human development: biallelic IGF2 expression correlates with lack of H19 expression. Development 120: 361-368
6. Leighton PA, Ingram RS, Eggenschwiler J, Efstratiadis A, Tilghman SM 1995 Disruption of imprinting caused by deletion of the H19 gene in mice. Nature 375:34-39
7. Ripoche MA, Kress C, Poirier F, Dandolo L 1997 Deletion of the H19 tran- scription unit reveals the existence of a putative imprinting control element. Genes Dev 11:1596-1604
8. Li E, Beard C, Jaenisch R 1993 Role for DNA methylation in genomic im- printing. Nature 366:362-365
9. Feinberg AP 2000 DNA methylation, genomic imprinting and cancer. Curr Top Microbiol Immunol 249:87-99
10. Momparler RL, Bovenzi V 2000 DNA methylation and cancer. J Cell Physiol 183:145-154
11. Robertson KD, Jones PA 2000 DNA methylation: past, present and future directions. Carcinogenesis 21:461-467
12. Antequera F, Bird A 1999 CpG islands as genomic footprints of promoters that are associated with replication origins. Curr Biol 9:R661-R667
13. Srivastava M, Hsieh S, Grinberg A, Williams-Simons L, Huang SP, Pfeifer K 2000 H19 and Igf2 monoallelic expression is regulated in two distinct ways by a shared cis acting regulatory region upstream of H19. Genes Dev 14:1186- 1195
14. Steenman MJ, Rainier S, Dobry CJ, Grundy P, Horon IL, Feinberg AP 1994 Loss of imprinting of IGF2 is linked to reduced expression and abnormal methylation of H19 in Wilms’ tumor. Nat Genet 7:433-439
15. Cui H, Niemitz EL, Ravenel JD, Onyango P, Brandenburg SA, Lobanenkov VV, Feinberg AP 2001 Loss of imprinting of insulin-like growth factor-II in Wilms’ tumor commonly involves altered methylation but not mutations of CTCF or its binding site. Cancer Res 61:4947-4950
16. Cui H, Hedborg F, He L, Nordenskjold A, Sandstedt B, Pfeifer-Ohlsson S, Ohlsson R 1997 Inactivation of H19, an imprinted and putative tumor re- pressor gene, is a preneoplastic event during Wilms’ tumorigenesis. Cancer Res 57:4469-4473
17. Okamoto K, Morison IM, Taniguchi T, Reeve AE 1997 Epigenetic changes at the insulin-like growth factor II/H19 locus in developing kidney is an early event in Wilms tumorigenesis. Proc Natl Acad Sci USA 94:5367-5371
18. Voutilainen R, Ilvesmäki V, Ariel I, Rachmilewitz J, De Groot N, Hochberg A 1994 Parallel regulation of parentally imprinted H19 and insulin-like growth factor-II genes in cultured human fetal adrenal cell. Endocrinology 134:2051- 2056
19. Voutilainen R, Miller WL 1988 Developmental and hormonal regulation of mRNAs for insulin-like growth factor II and steroidogenic enzymes in human fetal adrenals and gonads. DNA 7:9-15
20. El-Badry OM, Helman LJ, Chatten L, Steinberg SM, Evans AE, Israel MA 1991 Insulin-like growth factor II-mediated proliferation of human neuroblas- toma. J Clin Invest 87:648-657
21. Liu J, Kahri AI, Heikkila P, Ilvesmaki V, Voutilainen R 1995 H19 and insulin-like growth factor-II gene expression in adrenal tumors and cultured adrenal cells. J Clin Endocrinol Metab 80:492-496
22. Ilvesmäki V, Kahri AI, Miettinen PJ, Voutilainen R 1993 Insulin-like growth factors (IGFs) and their receptors in adrenal tumors: high IGF-II expression in functional adrenocortical carcinomas. J Clin Endocrinol Metab 77:852-858
23. Gicquel C, Raffin-Sanson ML, Gaston V, Bertagna X, Plouin PF, Schlum- berger M, Louvel A, Luton JP, Le Bouc Y 1997 Structural and functional abnormalities at 11p15 are associated with the malignant phenotype in spo-
radic adrenocortical tumors: study on a series of 82 tumors. J Clin Endocrinol Metab 82:2559-2565
24. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299
25. Voutilainen R, Tapanainen J, Chung B, Matteson KJ, Miller WL 1986 Hor- monal regulation of P450scc (20,22-desmolase) and P450c17 (17a-hydroxy- lase/17,20-lyase) in cultured human granulosa cell. J Clin Endocrinol Metab 63:202-207
26. Singer-Sam J, Grant M, LeBon JM, Okuyama K, Chapman V, Mong M, Riggs AD 1990 Use of a HpaII-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-chro- mosome inactivation. Mol Cell Biol 10:4987-4989
27. Liu J, Voutilainen R, Kahri AI, Heikkila P 1997 Expression patterns of the c-myc gene in adrenocortical tumors and pheochromocytomas. J Endocrinol 152:175-181
28. Myöhänen S, Wahlfors J, Jänne J 1994 Automated fluorescent genomic se- quencing as applied to the methylation analysis of the human ornithine de- carboxylase gene. DNA Sequence 5:1-8
29. Tadokoro K, Fujii H, Inoue T, Yamada M 1991 Polymerase chain reaction (PCR) for detection of Apal polymorphism at the insulin like growth factor II gene (IGF2). Nucleic Acids Res 19:6967
30. Latronico AC, Chrousos GP 1997 Extensive personal experience: adrenocor- tical tumors. J Clin Endocrinol Metab 82:1317-1324
31. Schulick RD, Brennan MF 1999 Adrenocortical carcinoma. World J Urol 17:26-34
32. Li X, Kogner P, Sandstedt B, Haas OA, Ekström TJ 1998 Promoter-specific methylation and expression alterations of igf2 and h19 are involved in human hepatoblastoma. Int J Cancer 75:176-180
33. Gicquel C, Leblond-Francillard M, Bertagna X, Louvel A, Chapuis Y, Luton JP, Girard F, Le Bouc Y. 1994 Clonal analysis of human adrenocortical car- cinomas and secreting adenomas. Clin Endocrinol (Oxf) 40:465-477
34. Gicquel C, Bertagna X, Schneid H, Francillard-Leblond M, Luton JP, Gi- rard F, Le Bouc Y 1994 Rearrangements at the 11p15 locus and overexpression of insulin-like growth factor-II gene in sporadic adrenocortical tumors. J Clin Endocrinol Metab 78:1444-1453
35. Liu J, Kahri AI, Heikkila P, Voutilainen R 1997 Ribonucleic acid expression of the clustered imprinted genes, p57KIP2, insulin-like growth factor II, and H19, in adrenal tumors and cultured adrenal cells. J Clin Endocrinol Metab 82:1766-1771
36. Gazdar AF, Oie HK, Shackleton CH, Chen TR, Triche TJ, Myers CE, Chrou- sos GP, Brennan MF, Stein CA, La Rocca RV 1990 Establishment and char- acterization of a human adrenocortical carcinoma cell line that expresses mul- tiple pathways of steroid biosynthesis. Cancer Res 50:5488-5496
37. Dohna M, Reincke M, Mincheva A, Allolio B, Solinas-Toldo S, Lichter P 2000 Adrenocortical carcinoma is characterized by a high frequency of chromo- somal gains and high-level amplifications. Genes Chromosomes Cancer 28: 145-152
38. Barletta JM, Rainier S, Feinberg AP 1997 Reversal of loss of imprinting in tumor cells by 5-aza-2’-deoxycytidine. Cancer Res 57:48-50
39. Jones PA, Taylor SM 1980 Cellular differentiation, cytidine and DNA meth- ylation. Cell. 20:85-93
40. Wolffe AP 2000 Transcriptional control: imprinting insulation. Curr Biol 10: 463-465
41. Logié A, Boulle N, Gaston V, Perin L, Boudou P, Le Bouc Y, Gicquel C 1999 Autocrine role of IGF-II in proliferation of human adrenocortical carcinoma NCI H295R cell line. J Mol Endocrinol 23:23-32
42. Eden S, Constanica M, Hashimshony T, Dean W, Goldstein B, Johnson AC, Keshet I, Reik W, Cedar H 2001 An upstream repressor element plays a role in IGF-II imprinting. EMBO J 20:3518-3525
43. Wilkin F, Gagne N, Paquette J, Oligny LL, Deal C 2000 Pediatric adreno- cortical tumors: molecular events leading to insulin-like growth factor II gene overexpression. J Clin Endocrinol Metab 85:2048-2056
44. Dao D, Walsh CP, Yuan L, Gorelov D, Feng L, Hensle T, Yamashiro DJ, Bestor TH, Tycko B 1999 Multipoint analysis of human chromosome 11p15/ mouse distal chromosome 7: inclusion of H19/IGF2 in the minimal WT2 region, gene specificity of H19 silencing in Wilms’ tumorigenesis and meth- ylation hyper-dependence of H19 imprinting. Hum Mol Genet 8:1337-1352
45. Jones BK, Levorse JM, Tilghman SM 1998 Igf2 imprinting does not require its own DNA methylation or H19 RNA. Genes Dev 12:2200-2207
46. Bell AC, Felsenfeld G 2000 Methylation of a CTCF-dependent boundary controls imprinted expression of the igf2 gene. Nature 405:482-485
47. Hark AT, Schoenherr CJ, Katz DJ, Ingram RS, Levorse JM, Tilghman SM 2000 CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/igf2 locus. Nature 405:486-489
48. Vu TH, Li T, Nguyen D, Nguyen BT, Yao XM, Hu JF, Hoffman AR 2000 Symmetric and asymmetric DNA methylation in the human IGF2-H19 im- printed region. Genomics 64:132-143