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Carcinogenesis |
Department of Urology, University of California, San Francisco and Veterans Affairs Medical Center, San Francisco, California 94121 [M. S., A. D., Y. T., R. D.]; Department of Urology, Chonnam University Medical School, Kwangju, Korea 005 [B. R. O.]; and Department of Obstetrics and Gynecology, School of Medicine, Hokkaido University, Kitaku Sapporo, 060 Japan [S-i. F.]
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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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Endometrial cancer induced by hyperestrogenism is suppressed by progesterone (2) . This conclusion is based on studies of an association between endometrial cancer and polycystic ovarian disease as well as other hyperestrogenic states. In these diseases, the tumorigenic effect of estrogen is completely reversed by the addition of a progestational agent (3) . Progesterone counteracts the growth-stimulatory effects of estrogen by inducing glandular and stromal differentiation of endometrium. Progesterone treatment is effective in decreasing the growth of endometrial tumors, which express PRs. The activity of PR strongly correlates with successful endocrine treatment and patient’s survival (3) .
Progesterone effects are mediated via PR. However, several studies have shown that PR regulation may be more complex than suspected previously. The expression of human PR is controlled by two promoters (4) . Human PR has two isoforms, A and B, which originate from translational initiation at AUG2 and AUG1, respectively. Both PR isoforms have a hormone-binding domain at the COOH terminus, a DNA-binding domain through which the receptors contact DNA, and two variable-length NH2 termini. Several investigators have suggested that the unique NH2-terminal sequence of the B receptor contain a strong transcriptional activating function (5 , 6) . There are several reports that observed that the functional activities of PRA and PRB are different in a cell type-, promoter-, or ligand-specific manner (7, 8, 9) . It is possible that the different mechanisms for the regulation of PR isoforms reflect their expressions in the endometrium.
It has been speculated that PRA and PRB are expressed in the endometrium with equal frequency and have similar function (10) . However, recent studies uncovered a difference between PRA and PRB expression and their function in endometrial and breast cancers (11, 12, 13, 14, 15) . In the endometrium, cyclical effects of estrogen and progesterone are mediated by PR regulation. However, progesterone regulation of PR is not a uniform effect in the uterus (10, 11, 12, 13, 14, 15) . In glands, the persistence of PRB during the midsecretory phase suggests its significance in glandular secretion. In stroma, the predominance of PRA throughout the cycle suggests its significance in postovulatory events. These results support the view that PRA and PRB mediate distinct pathways of progesterone action in the glandular epithelium and stroma of the human uterus throughout the menstrual cycle (10) . Several researchers reported that the PRA:PRB ratio is abnormal in endometrial cancer, leading to a lack of normal progesterone protection against the growth-promoting effects of E2 (16 , 17) .
The PR gene contains CpG islands in the 5' upstream region (4) . CpG islands are areas rich in CG dinucleotides that are found within the promoter region of various genes (17 , 18) . Abnormal CpG island methylation seems to be a frequent event in most malignancies (19 , 20) . Hypermethylation of promoter-associated CpG islands has been associated with gene silencing. De novo methylation of promoter-associated CpG islands has been associated with the transcriptional inactivation of genes. Thus, such methylation may be functionally equivalent to an inactivating mutation for the silencing of these genes (20, 21, 22) . However, no previous evaluation of the CG-enriched region 5' to exon 1 of PR has been reported in normal endometrium or UEC cells.
It is not known whether the effects of methylation on the two different PR promoters lead to equivalent silencing in the levels of PRA and PRB expressions. This question is critical in understanding the complexity of PR expression and regulation in the UEC. In this report, we studied methylation status and the expression of the two isoforms of this gene using several UEC cell lines and cancerous and normal tissues from UEC patients. MSP was chosen as a sensitive method to detect methylation status for the two forms of PRs (23) . The effect of methylation on PR expression was also studied by RT-PCR, the 5'RACE method, and immunohistochemistry (23 , 24) . Also, we investigated whether de novo methylation has occurred in these isoforms using pairs of cancerous and normal samples from the UEC patients.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DNA Extraction and Sodium Bisulfite Treatment.
DNA was isolated from the samples scraped from paraffin-embedded
sections. Microdissections were done from these samples as described
previously (23)
. DNA (
100 ng) was denatured using NaOH
and treated with sodium bisulfite for 16 h (Introgen, Purchase,
NY) as described previously (23)
. Modified DNA was
resuspended in 50 µl of TE (0.1 M Tris-HCl, 1
mM EDTA) and stored immediately at -20°C.
MSP Conditions.
The primers and PCR conditions are summarized in Table 1
and Fig. 1
. These primer sequences were chosen for regions containing frequent
cytosines (to distinguish unmodified from modified DNA) and CpG pairs
near the 3' end of primers (23)
. These primer sets are
located on the 5' upper region of each promoter for distinguishing PRA
and PRB separately (22)
. A fragment of DNA to be amplified
was intentionally small, because larger fragments are not possible from
paraffin blocks. In this examination, we made three sets of primers (U,
M, and W). Primer set (U) will anneal to unmethylated DNA that has
undergone a chemical modification. Primer set (M) will anneal to
methylated DNA that has undergone a chemical modification. Unmodified
DNA was amplified with the W primer set, which serves as a positive
control for PCR. The unmethylated or methylated sequence of promoter A
of this gene was detected with PRA-Uf and PRA-Ur or PRA-Mf and PRA-Mr,
respectively. The unmethylated or methylated sequence of promoter B of
this gene was detected with PRB-Uf and PRB-Ur or PRB-Mf and PRB-Mr,
respectively. PCR was performed with 10 ng of DNA solution
containing 1.5 mM MgCl2, 0.8
mM deoxynucleotide triphosphate, and 0.5 unit of Taq
polymerase (Applied Biosystems, Inc., Foster City, CA) in a total
volume of 20 µl. An 8-µl aliquot of each PCR product was mixed with
1 µl of 10x loading dye and then run on 3% agarose gel.
Electrophoresis was carried out at 180 V at ambient temperature. The
bands on the gels were visualized by ethidium bromide staining. Each
sample was examined at least three times. Only the cases that showed
clear bands were incorporated in the study. We used a breast cancer
cell line, MDA-MB-231, as a control for methylated bands, and this cell
line was treated with 5-azaC as a control for unmethylated bands.
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. Primers
for ß-actin were chosen specifically to cross two exons in the
ß-actin gene. In the presence of contaminating
genomic DNA, additional larger bands would be amplified. The lack of
amplification of any larger bands would indicate that there was no
contamination with any genomic DNA. Negative controls without RNA and
without reverse transcriptase were also performed. PRA mRNA cannot be distinguished from PRB by conventional RT-PCR because PRA has no specific sequence to distinguish it from PRB mRNA. A modified 5'RACE method was used for accurate evaluation of each mRNA expression (24) . RNA (5 µg) was reverse-transcribed using a custom-designed 24-mer oligonucleotide, RACE-REV, 5'-AACCTTGCACCCGGACCGGCTCAT-3. First- and second-strand cDNA synthesis were carried out using RT-PCR kits (Perkin-Elmer Corp.). The cDNA was circularized at 16°C overnight using T4 DNA ligase (Life Technologies, Inc., Grand Island, NY). The circularized cDNA was subjected to PCR using primer sets RACE-s and RACE-as. These primers were complementary to PR cDNA and numbered at the 5' base according to Kastner et al. (4) .
Sequencing.
For confirmation of MSP and 5'RACE, the PCR products were purified by
QIAquick PCR Purification kit (Qiagen, Valencia, CA) and applied to
second-PCR. Thirty ng of PCR products were used as a template for
sequencing (23)
. Double-strand sequence analysis was
performed using each primer set, an ABI 377 Sequencer, and a Dye
Terminator Cycle sequencing kit (Applied Biosystems, Inc., Foster City,
CA).
Immunohistochemistry.
Two anti-PR antibodies, one specific to PRB (B-30; Santa Cruz
Biotechnology, Santa Cruz, CA) and the other to PRA+B (C-19; Santa Cruz
Biotechnology), were used to identify PRB and PRA protein expression in
cancerous and normal endometrium by standard immunohistochemical
techniques (23)
. Anti-PRA+B recognizes both isoforms of
the receptor, whereas anti-PRB is specific to the B isoform. Because it
is not possible to raise an antibody specific to PRA, all
immunohistochemical analysis of the PRA subtype is by subtractive
inference. Paraffin-embedded endometrial cancer blocks were cut into
4-µm sections and dried at room temperature. After deparaffinization
and rehydration, sections were treated with 2% hydrogen peroxidase in
methanol for 20 min to inactivate endogenous peroxidase. Antigen
retrieval was done by autoclaving (121°C, 18 psi pressure) for 5 min
in an antigen unmasking solution (Vector, Burlingame, CA). After
blocking with 3% normal goat serum for 15 min, sections were incubated
with the two primary antibodies at a 1:100 dilution with 5% goat serum
in PBS overnight at 4°C under a humid chamber. Sections were washed
with in a solution containing 20 mM Tris, 150
mM NaCl, and 0.025% Tween (pH 7.8) and then incubated with
the secondary antibodies for 20 min. Immunostaining was done by using
an avidin-biotin-peroxidase method (Lab Vision, Fremont, CA) with
diaminobenzidine as the chromogen, followed by counterstaining with
hematoxylin.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows the schematic presentation of upstream regions of the
PR genes. The positions and orientation of MSP and RT-PCR
primers are indicated. Fig. 1
also shows a modified 5'RACE method. We
used a modified 5'RACE method for distinguishing each mRNA expression
because PRA has no unique sequences compared with PRB mRNA. mRNA was
reverse-transcribed using an oligonucleotide, RACE-REV. First- and
second-strand cDNA synthesis were carried out. The cDNA was
circularized using T4 DNA ligase. The circularized cDNA was subjected
to PCR using primer sets RACE-s and RACE-as. By this method, the
product from PRB had longer bands than that of PRA.
MSP and RT-PCR were performed using these cell lines treated with and
without the demethylating reagent 5-azaC (Table 2
; Fig. 2
). By MSP without 5-azaC treatment, only PRB was methylated in all of
the cell lines, although PRA was unmethylated (Table 2
; Fig. 2A
). By RT-PCR, no PRB expression was found, although PRA
expression was found in all UEC cell lines (Table 2
; Fig. 2B
). Treatment of cell lines with 5-azaC restored PRB
expression in all cancer cell lines (Table 2
; Fig. 2
). Fig. 2C
shows expression of PRA and PRB in Ishikawa cells using
the modified 5'RACE. A shorter band derived from PRA was observed after
5-azaC treatment because of inactivation of PRB. However, after 5-azaC
treatment, both longer and shorter bands were observed because of
re-expression of PRB. We also investigated PR genes using
the W primer sets, and no mutation or loss of heterozygosity was found
in the results (results not shown).
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. Sixty-two of 83 cancer samples (74.7%) had only methylated bands of
PRB, although all normal samples (100%) had only unmethylated bands.
All cancer and normal samples (100%) had only unmethylated bands of
PRA. There was a significant difference in methylation status of PRB in
cancerous and normal tissues (P < 0.001),
although there was no difference in methylation status of PRA.
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. To determine PR methylation and expression in normal endometrial
cells, we studied 33 normal endometrial samples. Twenty-six (78.8%)
and 24 of 33 normal samples (72.7%) were positive for PRA+B and PRB
expression, respectively (Table 4
; Fig. 3
). A significant difference was found in PRB positivity in cancerous
versus normal tissues (P < 0.001), although there was no such difference in PRA+B.
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shows the correlation of the methylation and immunohistochemical
positivity between cancer and normal samples. All of the 62 cancer
samples (100%) that had only methylated alleles were negative in
immunohistochemical analysis of PRB, whereas 11 of 12 cancer samples
(91.7%) that had only unmethylated alleles were positive
(P < 0.001; Table 5
).
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. Twenty of 32 cancer samples (62.5%) had only methylated alleles,
although all 32 normal samples (100%) had only unmethylated alleles
(Table 6)
. Seven of 8 cancer samples (87.5%) that had only
unmethylated PRB alleles were PRB positive. All cancer
samples that had only methylated alleles were negative for PRB
expression. The loss of unmethylated allele correlated well with
negativity in immunohistochemical staining for PRB
(P < 0.001).
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shows typical results of the MSP assay of the PR gene in a
pair of cancer and normal tissue from the same patient. Only
unmethylated bands of the promoter A were observed in both normal
(Lanes 1 and 2) and cancer tissues (Lanes
3 and 4). Only methylated alleles of PRB were observed
in cancer samples (Lanes 9 and 10), although only
unmethylated alleles of PRB were observed in normal tissue (Lanes
7 and 8). Fig. 5
shows direct sequencing of the PRB bands. CpG sites are underlined, and
all cytosines are deaminated and converted to thymines in the
unmethylated allele, whereas 5-methylcytosines remain unaltered in the
methylated allele.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The specific roles of these two PR subtypes are unclear. However, the relative levels of PRA and PRB in cells are critical for appropriate cellular response to progesterone (10 , 11) . Kumar et al. (16) revealed selective down-regulation of PRB in human UEC cells. They hypothesized that the PRA:PRB ratio may be abnormal in endometrial cancer, which may lead to a lack of normal progesterone protection against the growth-promoting effects of E2.
In this study, we investigated the methylation status of PRA and PRB in UEC cell lines using MSP. PRB is methylated in all human UEC cells, whereas PRA is unmethylated. We also investigated the expression of two isoforms of PR in UEC cell lines and found that PRB is inactivated. The data by the 5'RACE method confirmed only that PRB is inactivated in all cell lines and distinguished PRA and PRB independently.
To understand whether PRB mRNA expression is inactivated by methylation, we treated cells with 5-azaC. The treatment of 5-azaC restored PRB expression in all PRB-negative cell lines. Our results showed clearly that there is a close relationship between the selective inactivation of the PRB gene and abnormal methylation of PRB.
The molecular mechanisms of PRB inactivation in endometrial cancer are unknown. Several investigators have shown that PRB is protective against the hyperplastic effects of E2 (5 , 6) . It is well known that induction of endometrial cancer is related to hyperestrogenism (2) . The tumorigenic effect of estrogen is completely reversed by the addition of progestational agents (3) . Progesterone treatment is effective in decreasing the growth of endometrial tumors that express PRs. The activity of PR strongly correlates with successful endocrine treatment and patient’s survival (3) . It is possible that the PRB inactivation in endometrial cancer cannot protect proliferation of endometrial cells induced by E2 and thus cannot prevent carcinogenesis of the uterine endometrial cells.
De novo methylation in the islands of several genes in vitro can block transcription of downstream sequences (18, 19, 20, 21, 22) . Abnormal methylation has been observed for several genes in cancer cells (25, 26, 27) . Such methylation has been identified recently as an alternate mechanism of inactivating tumor suppressor genes during the development of cancer (19 , 20 , 22 , 27) .
CpG islands are found in other members of the hormone receptor superfamily including androgen, progesterone, and estrogen receptor genes (4 , 20, 21, 22) . We have recently observed methylation of the estrogen receptor promoter in prostate cancer during carcinogenesis (28) . In that report, we describe how selective PRB hypermethylation is associated with the loss of PRB expression in the endometrial cancer cell lines and endometrial cancer tissues (28) .
MSP is a new technology for the detection of gene methylation using small amounts of DNA (29, 30, 31) . This technique offers a highly sensitive approach. MSP also has the potential to define tumor suppressor gene functions and provides a new strategy for tumor detection research. Lapidus et al. (22) revealed that CpG islands in exon 1 of the PR gene is methylated in a significant fraction of primary human breast cancer. Thus, methylation-sensitive restriction sites in the PR gene CpG island are not methylated in normal breast specimens but methylated in PR-negative human breast tumors. Their data demonstrate that methylation of the PR gene CpG islands is associated with the lack of PR gene expression in a significant fraction of human breast cancers. However, they investigated only methylation status of exon 1 for both isoforms, because the promoter regions of PRA and PRB lack restriction enzyme recognition sites (22) .
In this study, we investigated the methylation status of PRA and PRB separately using 83 cancerous and 33 normal endometrial tissues from UEC patients. 74.7% of cancer samples had only methylated PRB alleles, although all normal samples had unmethylated PRB alleles. No significant changes were observed in the methylation status of PRA in normal and cancer samples. This is the first report that the selective methylation and silencing of PRB is observed in UEC.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Urology (112F), University of California
San Francisco and Veterans Affairs Medical Center, 4150 Clement Street,
San Francisco, CA 94121. Phone: (415) 750-6964; Fax: (415) 750-6639;
E-mail: Urologylab{at}aol.com 
2 The abbreviations used are: UEC, uterine
endometrial carcinoma; E2, estradiol; PR, progesterone
receptor; MSP, methylation-specific PCR; RT-PCR, reverse
transcription-PCR; 5'RACE, 5' rapid amplification of cDNA ends; 5-azaC,
5-aza-2'-deoxycytidine.
Received 1/10/00. Accepted 10/30/00.
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