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Molecular Biology and Genetics |
Department of Internal Medicine II, Kumamoto University School of Medicine, Kumamoto 860-8556 [K. N., S. T., T. S., H. M., M. Mat.], Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507 [M. Mae.], Japan
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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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B, serum response factor (SRF), and cyclic
AMP-responsive element binding protein, Tax activates transcription of
both viral and cellular genes; on the other hand, Tax can also
trans-repress the transcription of certain genes,
such as lck and DNA polymerase ß (9
, 10) .
Moreover, Tax binds to IB and p16, resulting in their functional
suppression (11
, 12)
. These multiple functions of Tax are
believed to contribute to the immortalization of HTLV-I-infected cells,
especially CD4-positive T lymphocytes. Indeed, the proliferation of
HTLV-I-infected cells in vivo is generally clonal, as
detected by analysis of integration sites, and persistent proliferation
has been observed in HTLV-I carriers (13
, 14)
. After
initial infection with HTLV-I, a long-term latent period (
50 years
in Japan) precedes the onset of ATL (15)
. Such a long
latent period suggests that multistep tumorigenesis is involved in the
development of ATL. During the latent period, mutations are thought to
accumulate in infected cells. Among subtypes of ATL, chronic and
smoldering ATL are considered to be low-grade malignancies because of
their insidious clinical courses, although they frequently progress to
acute or lymphoma-type ATL after several years (16)
. The
presence of the HTLV-I provirus in ATL cells enables us to analyze each
of these steps of leukemogenesis (carrier, smoldering, chronic,
lymphoma-type, and acute ATL).
The CDKN2A gene was isolated as a tumor suppressor gene that
undergoes deletions and/or mutations in certain human tumors, including
acute lymphoblastic leukemia, bladder cancer, and melanoma
(17, 18, 19)
. The p16 protein encoded by CDKN2A is
an inhibitor of CDK4 and CDK6, competing with cyclin D to block CDK
activity, resulting in G1 arrest
(20)
. Deletions or rearrangements of the CDKN2A
gene have also been reported in 20% of ATL cases, mostly acute or
lymphoma-type ATL (21
, 22)
. However, the loss of
expression of the CDKN2A mRNA is seen in certain cancer
cells without evidence of deletion or recombination, suggesting that
epigenetic changes affect the expression of the CDKN2A gene
(23)
. Methylation of CpG sites in the promoter region of
the CDKN2A gene was recently shown to be associated with its
reduced expression, because demethylation by 5-aza-2'-deoxycytidine
restored its expression (24)
. These data suggest that
epigenetic changes, such as methylation, may be involved in the
inactivation of the CDKN2A gene.
In this study, we analyzed the methylation of CpG sites in the CDKN2A genes in ATL cells obtained from patients at various clinical stages and detected a high incidence of methylation in all stages of ATL. We also observed that the methylation of CpG sites in the CDKN2A gene increased as the disease progressed and caused the suppression of CDKN2A gene expression.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
HTLV-I transformed cell lines and ATL-derived cell lines were used in
this study (26
, 27)
. ED, ATL-43T, SKT1B, ATL-55T, TL-Om1,
and ATL-48T were cell lines derived from leukemic clones, as confirmed
by Southern blot analysis with an HTLV-I probe or a T-cell receptor
gene probe. MT-2, MT-4, ATL-2, ATL-14, ATL-16, ATL-17–2, ATL-18,
ATL-22, ATL-35T, ATL-KI, ATL-40T, ATL-41T, and Sez627 were derived from
nonleukemic clones. The human embryonic kidney cell line, 293, was
studied as a control. To demethylate the CDKN2A gene, cell
lines were grown in media supplemented with 10
µM 5-Aza-CdR (Sigma) for 3 days, and then RNAs
were isolated using Trizol (Life Technologies, Inc., Paisley, United
Kingdom). Reverse transcription-PCR using primers specific for the
CDKN2A gene amplified transcripts of the CDKN2A
gene.
Southern Blot Analysis.
Genomic DNA isolated from PBMCs or lymph node cells was first digested
with a methylation-sensitive enzyme, SacII, then digested
with EcoRI, and electrophoresed in a 1.0% agarose gel,
transferred to nylon membrane, and subjected to hybridization with a
probe corresponding to CDKN2A exon 1.
MSPCR.
MSPCR was performed as described previously by Herman et al.
(28)
. In brief, 2 µg of genomic DNA (10 µl) were
denatured by the addition of an equal volume of 0.6
N NaOH for 10 min, and then 208 µl of 3.6
M sodium bisulfite and 12 µl of 10
mM hydroxyquinone were added. This mixture was
incubated at 55°C for 16 h to convert cytosine to uracil.
Treated genomic DNA was subsequently purified using the Wizard clean up
system (Promega), precipitated with ethanol, and resuspended in 100
µl of distilled H2O. The genomic DNA obtained
was used in MSPCR using primers as follows: CDKN2A M
(methylated): sense primer 5'-TTATTAGAGGGTGGGGCGGATCGC-3', antisense
primer 5'-CCACCTAAATCGACCTCCGACCG-3'; CDKN2A U
(unmethylated): sense primer 5'- TTATTAGAGGGTGGGGTGGATTGT-3', antisense
primer 5'-CCACCTAAATCAACCTCCAACCA-3'; p15 M (methylated):
sense primer 5'-GCGTTCCTATTTTGCGGTT-3', antisense primer
5'-CGTACAATAACCGAACGACCGA-3'; and p15 U (unmethylated):
sense primer 5'-TGTGATGTGTTTGTATTTTGTGGTT-3', antisense primer
5'-CCATACAATAACCAAACAACCAA-3'. The PCR mixture contained 1x buffer
with 1.5 mM MgCl2, 0.2
µM of each primer, 0.1 mM
deoxynucleotide triphosphates, 2 units of Taq DNA polymerase (Promega),
and 100 ng of sodium bisulfite-treated genomic DNAs. Conditions of
amplification were as follows: 95°C for 3 min and 35 cycles of 95°C
for 30 s, 65°C for 30 s (60°C for CDKN2A U,
p15 M, and p15 U primers), 72°C for 30 s,
and finally 4 min at 72°C.
Direct Sequencing after Sodium Bisulfite Treatment.
Sodium bisulfite-treated genomic DNAs (100 ng) were amplified with the
CDKN2A gene-specific primers as follows (29)
.
Primers for amplification of the promoter region of the
CDKN2A gene (from -378 to -89) were
5'-TTTTTAGAGGATTTGAGGGATAGG-3' (sense) and 5'-
CTACCTAATTCCAATTCCCCTACA-3' (antisense). To obtain products for
sequencing, a second round of PCR was performed with 20 pmol of nested
primers: the sense primer, 5'-AGTATTAGGAGGAAGAAAGAGGAG-3', and the
antisense primer 5'-TCCAATTCCCCTACAAACTTC-3'. Amplification was carried
out for 35 cycles (30 s at 95°C, 1 min at 58°C, and 1 min at
72°C) and followed by a final 4-min extension at 72°C. Primers used
for amplification of the promoter and exon 1 (from -115 to 119) were
as follows: for the first PCR round, 5'-GAGGGGGTAGGGGATAT-3' (sense)
and 5'-ACCAATCAACCRAAAACTCCATACTA-3' (antisense), and for the nested
PCR, 5'-GTAGGTGGGGAGGAGTTTAGTT-3' (sense) and
5'-CCCACCCTATAATAACCAACCAA-3' (antisense). Conditions of amplification
were as follows: 35 cycles (30 s at 95°C, 1 min at 52°C, and 1 min
at 72°C) and then 4 min at 72°C. Sequencing was performed using Big
Dye Terminator (Applied BioSystems) with an ABI 377 autosequencer. Six
HTLV-I-transformed cell lines were analyzed [ED (CL-1), 43T (CL-2),
SKT-1B (CL-3), ATL-55T (CL-5), and Tl-Om1 (CL-6) were derived from
leukemic clones, whereas MT-2 (CL-4) was derived from nonleukemic
cells].
cDNA Synthesis and Semiquantitative PCR.
Total RNA was prepared from cell lines or PBMCs from ATL patients using
Trizol reagent (Life Technologies), and cDNAs were made from 5 µg of
total RNAs using the Superscript Preamplification System (Life
Technologies) according to the manufacturer’s protocol. Oligo dT
primers were used to prime first-strand synthesis for all of the
reactions. For PCR, 2 µl of the reverse transcriptase reaction
mixture were diluted with 50 µl of PCR buffer containing 0.2
mM each of deoxynucleotide triphosphates, 1.5
mM MgCl2, 1.25 units of Taq DNA
polymerase (Life Technologies), and 20 pmol of each primer. Primers
specific for the CDKN2A gene were 5'-TTCGGCTGACTGGCTGGCCA-3'
(sense, exon 1) and 5'-AGCTCCTCAGCCAGGTCCAC-3' (antisense, exon 2), and
primers for the GAPDH gene used as a control were
5'-ATGGGGAAGGTGAAGGTCGGAGTC-3' (sense) and
5'-CCATGCCAGTGAGCTTCCCGTTC-3' (antisense). PCR was performed in a Robo
Cycler (Stratagene, La Jolla, CA) for 30 cycles for the
CDKN2A gene and 22 cycles for the GAPDH gene under the
following conditions: 3 min at 94°C, 30 s at 94°C, 1 min at
60°C, 1 min at 72°C, and 4 min at 72°C. The PCR products were
visualized by ethidium bromide staining in 2% agarose gels. The
intensity of amplified bands was quantified by ATTO densitograph 4.0
(ATTO, Tokyo, Japan).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. MSPCR relies on the fact that methylated cytosines are resistant to
sodium bisulfite treatment, and thus primers can be made to distinguish
between unmethylated and methylated templates. In samples with highly
methylated CpG sites, only the methylated band was detected (Fig. 1
,
no. 3). When the CDKN2A gene was methylated
partially, both methylated and unmethylated bands were found (Fig. 1
,
no. 6). A total of 71 samples of primary cells (38 cases
with acute ATL, 8 cases with lymphoma-type ATL, 16 cases with chronic
ATL, 6 cases with smoldering ATL, and 3 asymptomatic carriers) and 19
HTLV-I-associated cell lines were analyzed with Southern blot and
MSPCR. This experiment detected a high incidence of methylation in both
cell lines and fresh samples, and methylation was more prevalent in the
more malignant forms of ATL, although MSPCR can detect methylation of
CpG sites only in the exon. In contrast, no methylation was detected in
asymptomatic HTLV-I carriers and uninfected healthy individuals. In
acute ATL, methylation and deletion of CDKN2A was found in
47 and 24% of cases, respectively, resulting in either genetic or
epigenetic changes of CDKN2A in 71% of acute ATL patients.
In lymphoma-type ATL, methylation of the CDKN2A gene was
found in 73% of cases without deletion. Methylation was less frequent
in chronic (17%) and smoldering ATL (17%), as assessed by MSPCR and
Southern blot analysis. Moreover, MSPCR with primers specific to the
p15 gene failed to detect the methylation in 36 ATL samples
(20 cases with acute ATL, 11 cases with chronic ATL, 3 cases with
lymphoma-type ATL, and 2 cases with smoldering ATL). Because the
CDKN2A gene is adjacent to the p15 gene, these
data support the idea that the CDKN2A gene is preferentially
methylated in lymphoid cells in contrast to the p15 gene.
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Direct Sequencing of Sodium Bisulfite-modified Genomic DNA.
A major limitation of Southern blot analysis and MSPCR is that these
methods detect methylation in only a few CpG sites in the
CDKN2A gene. In contrast, direct sequencing after treatment
with sodium bisulfite enables us to identify the methylation state of
all of the CpG sites in the amplified sequences. Therefore, we
amplified and sequenced the promoter region and exon 1 of the
CDKN2A gene using sodium bisulfite-treated genomic DNAs
obtained from PBMCs of patients at various clinical stages (13 cases
with acute ATL, 4 cases with lymphoma-type ATL, 11 cases with chronic
ATL, and 4 cases with smoldering ATL) and three carriers. The level of
methylation of the CpG sites (42 sites) was judged from the pattern of
the electropherogram. When >20% methylation of a CpG site was seen,
that site was determined to be a methylated one. Representative data
from this analysis are shown in Fig. 2
. Most of the CpG sites were heavily methylated in a case of acute ATL,
as shown in Fig. 2A
. Only partial methylation was observed
in a case of chronic ATL and another case with lymphoma type ATL (Fig. 2, B and C)
. A summary of this analysis is shown
in Fig. 3
, in which each column represents one CpG site of the CDKN2A
gene and the extent of its methylation. We frequently observed complete
methylation in HTLV-I-transformed cell lines, although the extent of
methylation differed among cell lines. Cell lines derived from leukemic
clones, like ATL-43T (CL-2), had heavily methylated CpG sites; however,
MT-2 cells (CL-4), which were derived from a nonleukemic cell
(30)
, had partially methylated CpG sites (Fig. 3)
. The 5'
region of the promoter was particularly less methylated.
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Although none of methylation-negative cases (C-10, C-11, S-3, and S-4) among chronic and smoldering type ATL showed progression to acute ATL, the clinical progression was frequently observed in methylation-positive ATL cases (8 of 11 cases), which showed that methylation-negative ATL cases had more indolent clinical courses than methylation-positive cases. Two different subgroups among patients with chronic ATL have been reported; patients in one group progress to acute crisis within a few years, and patients in the other group have indolent clinical courses (31) . Chronic ATL patients with risk factors identified previously (high WBC counts and high lactate dehydrogenase) had more methylation of the CDKN2A gene (C-1–6) when compared with those without risk factors (C-7–11).
In one case, partial methylation (50%) of the CDKN2A
gene was found in PBMCs, whereas no methylation was identified in lymph
node cells (Fig. 2D)
. It was confirmed that both ATL cell
samples had the same HTLV-I provirus integration site, showing that
multiple subclones with different methylation patterns were present in
the patient.
Semiquantitative Reverse Transcription-PCR of the
CDKN2A Gene.
Methylation of the CDKN2A gene may result in the suppression
of its transcription, leading to decreased expression. Therefore, we
analyzed the expression of the CDKN2A gene using
semiquantitative PCR (30 cycles) using primers specific for the
CDKN2A gene. As shown in Fig. 4A
,293 cells (Lane 1) and normal T lymphocytes
stimulated in the presence of interleukin 2 (Lane 6) contain
CDKN2A gene transcripts (330 bp) and were used as positive
controls. The relative levels of expression of the CDKN2A
gene were shown by the histogram after normalization with the GAPDH
transcript. Transcripts of the CDKN2A gene could not be
detected in any cell line, such as ED (Lane 3) or ATL 43T
(Lane 4), which had a heavily methylated CDKN2A
gene (Fig. 3)
, and the MT-2 cell line, which contains a partially
methylated gene, showed a markedly suppressed expression of the
CDKN2A gene (Lane 5). Thus, expression of
CDKN2A gene was inversely correlated with its methylation.
For example, ATL cells with extensive methylation, which ranged to the
promoter region, were found to express low levels of CDKN2A
gene transcripts (Lanes 7; A-5 in Fig. 3
and Lane
8; C-1 in Fig. 3
) when compared with controls. On the other hand,
ATL cells with no methylation had equivalent amounts of
CDKN2A gene transcripts (Lane 9, A-13; Lane
12, C-4). These results suggest that the decreased expression of
the CDKN2A gene is caused by the methylation of the gene. To
confirm this possibility, we tested whether a demethylating agent,
5-Aza-CdR, reversed the suppressive effects of methylation. As shown in
Fig. 4B
, an exposure of ATL cell lines (ED, ATL-43T, and
MT-2) to 5-Aza-CdR for 3 days reactivated or potentiated the expression
of CDKN2A mRNA, corroborating our observation that the
methylation of the CDKN2A gene was associated with
CDKN2A gene silencing in ATL cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Deletion of the CDKN2A gene was reported in 27% (21) and 11.4% (22) of ATL cases, whereas this study revealed that methylation of this gene was a more common phenomenon in ATL, suggesting that methylation is the predominant mechanism of suppressing the expression of the CDKN2A gene in ATL cells. In this work, we reported an increasing methylation as the disease progressed. Increasing methylation is also associated with progressive suppression of CDKN2A expression, as shown by analysis of CDKN2A transcripts. Examination of ATL cells at each clinical stage showed that methylation first occurs in the exon region and progresses to the promoter region with clinical progression of ATL. It has been reported that methylation in the exon region does not have any effect on the expression of the CDKN2A gene (29 , 35) . This study supports that finding because the ED and ATL-43T cell lines, which had completely methylated CDKN2A genes, did not express CDKN2A gene transcripts, whereas the MT-2 cell line with a less methylated promoter region expressed low levels of CDKN2A gene transcripts. Methylation of the promoter region is thought to be related to the silencing of CDKN2A gene transcription because 5-Aza-CdR could restore the transcription of the CDKN2A gene.
Analysis of bladder cancer cell lines with reactivated CDKN2A gene expression after 5-Aza-CdR treatment revealed that a small number of CpG sites can significantly down-regulate CDKN2A promoter activity (29) . In this study, methylation of a specific CpG site (-17) was observed frequently in ATL samples; this site may influence the expression of the CDKN2A gene. Transcriptional repression by DNA methylation has also been reported for other tumor suppressor genes such as E-cadherin (36) and the VHL tumor suppressor gene (37) . These reports suggest that methylation is a common mechanism of inhibiting tumor suppressor genes.
Among chronic ATL cases, there are variations in clinical course; some patients progress to acute or lymphoma-type ATL shortly, and other patients show no progression for a long time. Thus, the identification of the subgroup of chronic ATL cases who will progress to acute crisis is clinically important. Chronic ATL cases without CDKN2A gene methylation had more indolent clinical courses than those with methylation. Thus, analysis of the methylation status of the CDKN2A gene may be useful in predicting when the patients will progress to acute ATL.
In this study, we found different methylation patterns of the CDKN2A gene within a single patient, demonstrating the presence of subclones within leukemic cells carrying a single integration site of HTLV-I provirus. Cells with different methylation pattern of the CDKN2A gene in the same patient may have different tissue affinities, perhaps reflecting different patterns of expression of adhesion molecules.
Of various proteins associated with Tax, p16 has been shown to be functionally inactivated, resulting in cell cycle perturbation (12) . However, at the late stage of leukemogenesis, such as acute or lymphoma-type ATL, the expression of tax is significantly lower than in asymptomatic carriers (38) . Fresh leukemic cells in some acute ATL patients do not produce Tax in vitro because of deletion of the long terminal repeat and other mechanisms (39 , 40) . Thus, whether Tax expression is required in ATL cells remains unclear. On the other hand, such deletions in the HTLV-I provirus are rare in low-grade malignant ATL cases (smoldering or chronic ATL), suggesting that Tax may play an important role at such stages but may not be essential for late stages of leukemia. The increasing methylation of the CDKN2A gene shown in this study suggests that at an early stage of leukemogenesis, Tax is responsible for inactivating the p16 protein, whereas at a late stage, methylation or even deletion of the CDKN2A gene abolishes the expression of p16. Methylation might be a way to transform HTLV-I infected T cells in the absence of Tax expression. ATL cells express activation antigens like CD25 and secrete various cytokines; their phenotype resembles that of cells expressing Tax in vitro. Modification of methylation in ATL cells might fix a phenotype initially induced by Tax and replace the functions of Tax.
The progressive methylation of the CDKN2A gene in ATL cells observed in this study suggests that methylation suppresses the expression of the CDKN2A gene, which initially is functionally inactivated by Tax. Therefore, cells with methylated CDKN2A gene may not depend on the expression of viral proteins for their growth and thus may escape host immune surveillance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant-in-aid for Scientific
Research from the Ministry of Education, Science, Sports and Culture of
Japan. 
2 To whom requests for reprints should be
addressed, at Department of Internal Medicine II, Kumamoto University
School of Medicine, 1-1-1 Honjo, Kumamoto 860-8556 Japan. Phone:
81-96-373-5156; Fax: 81-96-363-5265; E-mail: matsuoka{at}kaiju.medic.kumamoto-u.ac.jp
3 Present address: Laboratory of Virus Immunology,
Research Center for AIDS, Institute for Virus Research, Kyoto
University, 53 Shogoin Kawaramachi, Sakyo-ku, Kyoto 606-8507, Japan.
Phone: 81-75-751-4048; Fax: 81-75-751-4049; E-mail: mmatsuok{at}virus1.virus.kyoto-u.ac.jp
4 The abbreviations used are: ATL, adult T-cell
leukemia; HTLV-I, human T-cell leukemia virus type I; CDK,
cyclin-dependent kinase; PBMC, peripheral blood mononuclear cell;
5-Aza-CdR, 5-aza-2'-deoxycytidine; MSPCR, methylation-specific PCR;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received 5/ 3/99. Accepted 12/15/99.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B and IB-
and induces nuclear translocation of NF-B for transcriptional activation. Oncogene, 10: 1199-1207, 1995.[Medline]
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