| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Molecular Oncology, Markers, Clinical Correlates |
Department of Obstetrics and Gynecology, The University of Hong Kong, Hong Kong, S.A.R., China
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Genomic imprinting, the differential expression of parental alleles of a gene in somatic cells, is considered to play a role in human disease and cancer (11 , 12) . Thus far, a cluster of imprinting genes has been identified in chromosome 11p15.5, including IGF-II, H19, IPW, and p57kip. LOI of the IGF-II and H19 genes has been found in some embryonal and adult human cancers (11, 12, 13, 14, 15, 16, 17, 18) . Constitutional relaxation of IGF-II genomic imprinting has also been observed in normal kidney and peripheral blood leukocytes of a patient with Wilms’ tumor (13) . LOI of IGF-II was found in 40–60% of Wilms’ tumors (11 , 13) . Also, LOI of both IGF-II and H19 has been identified in 50% (5 of 10) and 17% (2 of 12) of cervical cancers (18) . LOI of IGF-II has been proposed to be one of the factors responsible for the abnormal overexpression of IGF-II in both human tumors and experimental animal models (13 , 19) .
It has been demonstrated that IGF-II gene imprinting is promoter-specific, in that expression from P1 promoter is biallelic, whereas that from the P2-P4 promoter is monoallelic, implying that both transcriptional repression (imprinting) and activation (LOI) can simultaneously occur within a single gene (20) . Hypermethylation of promoter 3 in some tumor cell lines shows that activation of IGF-II promoter 1 is linked to LOI, which suggested that activation of the silenced alleles in cancer (LOI) may be due to the activation of promoter 1, which is expressed only in liver with biallelic expression (21) .
To further understand the possible role of genomic imprinting in epithelial ovarian cancer, we studied the allelic gene expression of both IGF-II and H19 in ovarian cancer and LMP ovarian tumor. LOI of both IGF-II and H19 was involved in ovarian cancer with overexpression of IGF-II but not of H19. Activation of the P1 promoter may account for the mechanism of LOI of IGF-II in some of the LOI ovarian cancer.
|
PATIENTS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
DNA and RNA Extraction and Purification.
DNA from cancer samples and normal tissues or lymphocytes were
extracted using proteinase K/phenol chloroform protocol. RNA was
extracted by Tripure isolation reagent (Roche Molecular
Biochemicals) according to the manufacturer’s protocol. One
µg of RNA was treated with RNase free-DNase I (Life Technologies,
Inc.) for 15 min at room temperature to eliminate DNA contamination,
followed by heating for 10 min at 65°C to inactivate the DNase I.
Identification of Genomic Polymorphism of IGF-II and H19.
IGF-II exon 9 ApaI polymorphism site and the last two
exons of H19 RsaI and AluI polymorphism sites
were studied using PCR with 2.5 units of Taq, 10 pmol of primers, and
50 µl of total reaction volume. Ten µl of PCR products were
digested with ApaI, RsaI, or AluI at
37°C for 3 h (10 units of enzyme with 10 µl of PCR product and
2 µl of buffer at a final volume of 20 µl); run through a
3% agarose gel; and visualized with ethidium bromide. The
primers used were as follows: for IGF-II ApaI, P1F,
5'-CTTGGACTTTGAGTCAAATTGG-3', and P1R, 5'-GGTCGTGCCAATTACATTTCA -3';
for H19 RsaI, P2F, 5'- TGCTGCACTTTACAACCACTG-3', and P2R,
5'-GGTCGGAGCTTCCAGACTAG -3'; for H19 AluI: P3R,
5'-GTGGCCATGAAGATGGAGTC-3'. The PCR conditions for IGF-II were as
follows: DNA was denatured at 94°C for 4 min, amplified by 35 cycles
(94°C for 1 min, 55°C for 1 min, and 72°C for 1 min) and
extension at 72°C for 4 min. Conditions for H19 both
RsaI and AluI sites were as follows: 95°C for 2
min 30 s and 35 cycles of 94°C for 1 min, 58°C for 1 min, and
72°C 1 min; extension at 72°C for 7 min.
Allele-specific Gene Expression.
One µg of RNA was reverse-transcribed by 200 units of Moloney murine
leukemia virus teverse transcriptase with 200 ng of random hexamer, 0.5
mM dNTP, 40 units of RNase inhibitor (Promega), 50
mM Tris-HCl, 75 mMKCl, 3 mM MgCl2,
10 mM DTT in a total volume of 20 µl for 1 h at
37°C. RT was inactivated by heating for 10 min at 70°C. cDNA (50
ng) was amplified by PCR using primers for IGF-II and H19
RsaI and H19 AluI sites, respectively,
under the same conditions used for genomic DNA, except that the
number of cycles was increased to 40. The amplified cDNA was also
digested with ApaI for IGF-II (yielding a 292-bp fragment or
231- and 61-bp fragments) and with RsaI or AluI
for H19. Because the amplified area of IGF-II gene did not
include an intron, RT-PCR was performed both with and without teverse
transcriptase. No PCR products were observed when reverse
transcriptase was not added. We used P2F and P2R for the H19
RsaI site and P2F and P3R for the H19 AluI
site, both of which included an 80-bp intron. Contamination by genomic
DNA can be identified by the size of the DNA bands as well as by
carrying out the experiment without reverse transcriptase. For H19
RsaI site, the size of the DNA band is 635 bp for allele a
or 497 and 138 bp for allele b. However, RNA is 555 bp for the allele
A, or 417 bp and 138 bp for the allele B. For the H19
AluI site, the size of the DNA band is 239 bp for allele a
and 138 and 101 bp for allele b. RNA is 159 bp for the allele A and 101
bp and 58 bp for the allele B.
Semiquantitative RT-PCR in LOI Samples and Matched Normal Samples.
To determine whether imprinting status affects IGF-II or H19 mRNA
levels, semiquantitative RT-PCR was performed. One µg of total RNA
was reverse transcribed, and the amount of cDNA was compared with
GAPDH. Primers of GAPDH were as follows: P4F,
5'-CACCATCTTCCAGGAGCGAG-3'; P4R, 5'-TCACGCCACAGT-TTCCCGG A-3'. The
cycles were determined by the standard curve amplified from 16, 20, 24,
28, 32, 36, or 40 cycles. Conditions were chosen to give a
linear relationship between the amount of amplified product and the
input RNA (data not shown). For IGF-II and GAPDH, only 25
cycles were selected, and 30 cycles were performed for H19. Ten µl of
PCR product were electrophoresis on a 2% agarose gel and analyzed by
UVP Gel Work 1D for Windows. All data were compared with GAPDH.
P1 and P3 Promoter Expression of IGF-II in LOI Samples.
Activation of the P1 promoter concurrent with silencing of
the P3 promoter through hypermethylation has been observed in several
epithelial cancer cell lines, suggesting the promoter-specific
regulation of the IGF-II gene (21)
. To study
the role of promoter-specific regulation in the LOI mechanism, we
determined promoter-specific expression in the five LOI samples and
their matched normal tissues. For IGF-II LOI samples, cDNA were
amplified by PCR using standard buffer conditions. For IGF-II P1
promoter-specific PCR, the primers used were as follows: P5F,
5'-GGCCAGAGAGCCCAGTCCTGAGGTGA-3' (located in IGF-II exon 2, which is
exclusively transcribed from P1 promoter); P5R,
5'-TGGAAGAACTTGCCCACGGGGTATCT-3' (located in IGF-II exon 9). For IGF-II
P3 promoter, the primers used were P6F
(5'-TCCTCCCTGGACAATCAGACGAATTC-3'; located in IGF-II exon 5, which is
exclusively transcribed from P3) and the same exon 9 lower primer as
for P1. To confirm integrity of the RNA, the same amount of cDNA was
amplified by PCR using P4F and P4R for GAPDH as an internal control.
All samples were performed twice with or without reverse transcriptase
(21)
.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and 2
.
|
|
2
3
and
Figs. 1
and 2
. No LOI was found in all tumors with
LOH.
|
|
|
. The
normal and tumor samples showed approximately the same amounts of the
RT-PCR products for GAPDH. All five tumors with LOI of IGF-II
demonstrated significantly elevated levels of IGF-II mRNA when compared
with the normal matched specimens, whereas all five of these tumors
showed uniformly low levels of H19 mRNA expression regardless of
the imprinting status. The other six tumors with LOI of H19 showed same
expression of H19 and IGF-II except the two specimens with both LOI of
IGF-II. The results are shown in Fig. 3
.
|
. In
contrast, other samples all showed transcription from P3 promoter and
lack of expression from P1 promoter. Thus, expression of IGF-II from
the nonimprinted P1 promoter may account for some of the biallelic
expression the gene observed in ovarian cancer (Fig. 4)
.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In the present study, LOI of IGF-II gene was found in 25% (5 of 20) of ovarian cancer with no LOH. The frequency is lower than the report of Kim et al. (24) , which showed 54% (6 of 11) LOI. In that study, LOI was also involved in three benign ovarian cysts, suggesting that IGF-II LOI is one of the early event of ovarian carcinogenesis. In our study, LOI occurred in four early stage and one late stage ovarian cancer, including two mucinous, one serous, one endometrioid, and one clear cell carcinoma. This study was in contrary to the report of Yun et al. (16) that only monoallelic expression was found in 11 informative ovarian cancer cases. The frequency of LOI of IGF-II in ovarian cancer is lower than that of other malignant gynecological tumors, such as cervical cancer (Ref. 18 ; 50%, 5 of 10), and it is similar to that of endometrium carcinoma (Ref. 25 ; 20%, 1 of 5). In our study, LOI of the H19 gene was 30.2% on the RsaI and AluI sites. Two ovarian cancer samples showed LOI in both IGF-II and H19 sites. LOI was identified in one IA1 mucinous cystadenocarcinoma in tumor and both normal ovary and normal cervix of the same patient at the H19 RsaI site. LOI also occurred in one LMP ovarian tumor in the H19 AluI site. Similar findings have been reported in lung cancer (15) , brain gliomas (26) , and renal cell carcinomas (27) . The incidence of H19 LOI in our study is lower than that (62%) in the report of Kim et al. (24) . The authors of that study also indicated that LOI was more frequent in malignant epithelial ovarian carcinomas and was associated with advanced stages. The discrepancy between their study and ours is probably due to the differences in tumor cell types and total number in the study. This is the first report that LOI occurred in the LMP ovarian tumor and normal ovary in the H19 gene. From our results, LOI was detected not only in ovarian cancer but also in LMP ovarian tumor and normal ovary. LOI of IGF-II and H19 is probably an early event in ovarian carcinogenesis.
We observed that the expression of IGF-II in LOI samples was higher than that of the matched normal tissues, whereas there was no difference in the H19 expression in both the IGF-II LOI and H19 LOI samples. The findings of the present study are unlike that in Wilms’ tumor, in which LOI of IGF-II is associated with down-regulation of H19 (28 , 29) . The mechanism by which the genomic imprint is first established and then maintained is not well understood. However, in the case of IGF-II, the expression of a neighboring gene, H19, has been suggested to influence its transcription by competition for a common enhancer, thereby generating a mutually exclusive and allele-specific pattern of gene expression. Associated changes in CpG methylation in discrete areas of both genes have been implicated in maintenance of the imprint (30) . Joyce et al. (30) also reported in sporadic Beckwith-Wiedemann syndrome that IGF-II showed biallelic expression although H19 expression and methylation status were normal. This indicates that there must be an alternative H19-independent pathway by which allele-specific IGF-II expression is established or maintained. Thus, it is possible that the mechanisms of involvement in ovarian cancer may be different from Wilms’ tumor.
It has been demonstrated that IGF-II gene imprinting is promoter-specific, in that expression from P1 promoter is biallelic, whereas that from the P2-P4 promoter is monoallelic, which implies that both transcriptional repression (imprinting) and activation (LOI) can simultaneously occur within a single gene. In the present study, the expression of the IGF-II P1 promoter not P3 was identified in three of the five LOI tumors, and others were all transcribed from P3 promoter. This suggests that activation of the P1 promoter may attribute to the biallelic expression of IGF-II. Issa et al. (21) demonstrated that hypermethylation of the IGF-II gene was associated with low or absent activity of P3 promoter but maintained expression from P1, suggesting that it may contribute to the biallelic expression in cancer cells showing LOI (21) . However, not all LOI samples behave in the same way, suggesting that a different mechanism of LOI of IGF-II may exist. Yun et al. (16) suggest that IGF-II gene expression from the maternal P1 promoter can occur without expression from the maternal P3 promoter within the same IGF-II gene. They demonstrated independent allele activation of the promoter-specific transcription from a single cell. Biallelic P1 transcription can occur with monoallelic P2-P4 transcription.
Our present studies revealed that: (a) LOH of both IGF-II and H19 genes, although not frequent, tend to be found in advanced clinical stages of ovarian cancer; (b) LOI of IGF-II and H19 genes may be involved in ovarian carcinogenesis and were found in both LMP and invasive ovarian cancer; and (c) LOI of IGF-II associated with transcription from the P1 promoter may account for the biallelic expression of the IGF-II gene. However, reciprocal regulation of the IGF-II gene and the H19 gene was not found.
|
FOOTNOTES |
|---|
1 This study was supported by Committee on
Research and Conference Grant 10202161 from the University of
Hong Kong. 
2 To whom requests for reprints should be
addressed, at Department of Obstetrics and Gynecology, Queen Mary
Hospital, The University of Hong Kong, Pokfulam Road, Hong Kong,
S.A.R., China. Phone: 00852-28554684; Fax: 00852-28550947; E-mail: hysngan{at}hkucc.hku.hk
3 The abbreviations used are: LOH, loss of
heterozygosity; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
IGF-II, insulin-like growth factor II; LMP, low malignant potential;
LOI, loss of imprinting; RT, reverse transcription.
Received 8/ 9/99; revised 11/ 1/99; accepted 11/ 1/99.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  Q.-s. Ji, M. J. Mulvihill, M. Rosenfeld-Franklin, A. Cooke, L. Feng, G. Mak, M. O'Connor, Y. Yao, C. Pirritt, E. Buck, et al. A novel, potent, and selective insulin-like growth factor-I receptor kinase inhibitor blocks insulin-like growth factor-I receptor signaling in vitro and inhibits insulin-like growth factor-I receptor dependent tumor growth in vivo Mol. Cancer Ther., August 1, 2007; 6(8): 2158 - 2167. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Lu, D. Katsaros, A. Wiley, I. A. Rigault de la Longrais, H. A. Risch, M. Puopolo, and H. Yu The Relationship of Insulin-Like Growth Factor-II, Insulin-Like Growth Factor Binding Protein-3, and Estrogen Receptor-{alpha} Expression to Disease Progression in Epithelial Ovarian Cancer Clin. Cancer Res., February 15, 2006; 12(4): 1208 - 1214. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Lottin, E. Adriaenssens, T. Dupressoir, N. Berteaux, C. Montpellier, J. Coll, T. Dugimont, and J. J. Curgy Overexpression of an ectopic H19 gene enhances the tumorigenic properties of breast cancer cells Carcinogenesis, November 1, 2002; 23(11): 1885 - 1895. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. P. Sawiris, C. A. Sherman-Baust, K. G. Becker, C. Cheadle, D. Teichberg, and P. J. Morin Development of a Highly Specialized cDNA Array for the Study and Diagnosis of Epithelial Ovarian Cancer Cancer Res., May 1, 2002; 62(10): 2923 - 2928. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-L. Chen, S.-M. Ip, D. Cheng, L.-C. Wong, and H. Y. S. Ngan p73 Gene Expression in Ovarian Cancer Tissues and Cell Lines Clin. Cancer Res., October 1, 2000; 6(10): 3910 - 3915. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
