![](metilacao_cancer_3_files/shim.gif) Science, Vol 302, Issue 5648,
1153 , 14 November 2003
[DOI: 10.1126/science.1089523]
Comment on "Chromosomal Instability and Tumors Promoted by DNA
Hypomethylation" and "Induction of Tumors in Mice by Genomic
Hypomethylation"
Eden et al. (1)
and Gaudet et al. (2)
reported increased tumorigenesis in mice with DNA
hypomethylation and attributed these results to increased
chromosomal instability. Both Gaudet et al. (2)
and Lengauer (3)
caution that these results could have negative
implications for the use of hypomethylating agents in the
treatment of cancer. However, we would like to caution
against overinterpretation of these papers.
It is clear that the data collected in (1,
2)
are the result of rather extreme modeling in mice. The
authors present data showing large decreases in
repetitive element DNA methylation as a result of genetic
manipulation, but these decreases may have limited
relevance to human neoplasia and therapy. To address this
issue, we first wished to ascertain whether the high degree
of hypomethylation observed in the genetically manipulated
mice is ever observed in human tumors. We examined the
methylation of LINE-1 transposable elements and Alu
elements using a quantitative COBRA (4)
assay. PCR primers were designed based on the conserved
Alu and LINE-1 sequences. Previous cloning and sequencing
experiments have suggested that, in practice, the assay
amplifies about 15,000 distinct Alu elements. Using this
assay to quantitate global methylation, we found that in
19 pairs of colon cancer, the mean Alu methylation was
identical to adjacent normal colon mucosa (normal: 82.5%
methylation, SEM = 1.3%; cancer: 82.3% methylation, SEM =
1.0%; Fig.
1). The extent of Alu element methylation did vary in
individual cases from normal to tumor, but 12 tumors
showed an increase in Alu methylation (range = 1.2 to
7.4%), compared to only 7 tumors that showed a decrease
(range = 2.1 to 12.0%). We did find hypomethylation of
LINE-1 in colon cancer, but the mean decrease in
methylation was only 7.0% (n = 23; range = 5.2 to
27.8%) compared to uninvolved adjacent mucosa. Normal
colon mucosa had a mean LINE-1 methylation of 69.7% (SEM
= 2.2%) while colon tumors showed 62.7% (SEM = 1.0%)
LINE-1 methylation (Fig.
1).
Fig. 1. Alu and
LINE-1 methylation in primary colon tumors and cancer cell
lines. Bisulfite-treated DNA was amplified using PCRprimers
located in the conserved regions of Alu or LINE-1 elements.
Amplified DNA was digested with restriction enzymes that
distinguish methylated from unmethylated alleles, and the
alleles were separated by polyacrylamide gel electrophoresis
or capillary electrophoresis. Methylation was quantitated
based on the density of the digested band versus the
undigested band. For Alu methylation, the % methylation value
was calculated by comparison to DNA that was fully methylated
with SssI methylase. [View
Larger Version of this Image (16K GIF file)]
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Next, we investigated the relationship between
hypomethylation and chromosomal instability. Lengauer
et al. (5,
6)
previously reported an inverse relationship between
mismatch repair (MMR) deficiency and chromosomal
instability (CIN) in colon cancer cell lines. Cells that
were MMR deficient used genetic instability as a method
to drive tumorigenesis, and had chromosomal stability.
Conversely, cells that were MMR proficient had chromosomal
instability as the mechanism driving tumorigenesis. We
therefore analyzed a similar cell line panel for
differences in Alu element and LINE-1 methylation. Alu
element methylation averaged 83.1% (SEM = 1.4%) in stable
cell lines (Hct116, LoVo, SW48, RKO, DLD1) and 78.9% (SEM
= 5.2%) in unstable cell lines (HT29, SW480, SW837,
Colo205) (Fig.
1). LINE-1 methylation in stable versus unstable cell
lines was 56.6% (SEM = 6.77%) and 50.6% (SEM = 8.76%),
respectively. Thus, there was only a small, nonsignificant
decrease in methylation in CIN-versus CIN+ cell lines
(P = 0.48, two-tailed t-test).
Gaudet et al. (2)
and Lengauer (3)
caution that the therapeutic use of hypomethylating
agents such as 5-aza-2'deoxycytidine (5-aza-dC) could be
associated with the development of secondary tumors. We
have used the Alu element assay to measure demethylation
induced by this drug in 41 patients with leukemia treated
at different doses of 5-aza-dC. As shown in Fig.
2, 5-aza-dC resulted in an average Alu demethylation
of 7.7% (SEM = 1.68, SD = 9.82%) 4 to 7 days after
treatment, with 31.5% being the maximum decrease seen in
a single patient. In addition, methylation returned to
baseline levels within as few as 15 days of finishing
treatment. Thus, 5-aza-dC-induced demethylation is at
least one order of magnitude lower than that reported by
Gaudet et al. (2)
and Eden et al. (1).
Furthermore, the pharmacologically induced demethylation
appears to be very transient, with methylation returning
to pretreatment levels within two weeks following
therapy.
Fig. 2. Demethylation
of Alu elements by 5-aza-dC in patients with leukemia.
Peripheral blood was collected on serial days from leukemia
patients being treated with 5-aza-dC. DNA methylation was
quantitated using the assay described for Fig.
1. The data represent the mean methylation for 41
patients. [View
Larger Version of this Image (21K GIF file)]
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Finally, we reviewed the clinical histories of 195 patients
with leukemia treated with 5-aza-dC at the University of
Texas M.D. Anderson Cancer Center. Due to the
investigational nature of the studies, we primarily
treated patients with a poor prognosis. Fifty-three
patients survived 6 months or longer after initiation of
therapy. Of these patients, 45 have died (24 patients at
6 months to 1 year, 13 at 1 to 2 years, and 8 at 2 years
after beginning therapy). Of the 8 surviving patients, 5
are alive at 2 years, 2 at 4 years, and 1 at 5 years after
therapy began. Excluding six patients who were lost to
follow-up, all deaths were the result of the initial
disease, and no patient has developed a secondary
malignancy to date. While the follow-up is short and the
number of patients limited, we note that other drugs can
cause malignancies within months of therapy, and we
suggest that 5-aza-dC is likely to be relatively safe in
this regard. Of course, longer periods of administration
of this drug could be associated with other problems and,
indeed, some studies have found it to be mutagenic (7).
Overall, however, our studies caution against extension
of the results observed by Gaudet et al. (2)
to physiologic conditions in human neoplasia and
therapy.
Allen S. Yang Marcos
R.H. Estecio Guillermo Garcia-Manero
Hagop M. Kantarjian Jean-Pierre J.
Issa Department of Leukemia M.D.
Anderson Cancer Center 1515 Holcombe Boulevard
Houston, TX 77030, USA E-mail: jpissa@mail.mdanderson.org
| References
1. |
A. Eden, F. Gaudet, A. Waghmare, R.
Jaenisch, Science 300, 455 (2003).[Free Full Text] |
2. |
F. Gaudet et al.,
Science 300, 489 (2003).[Abstract/Free Full Text] |
3. |
C. Lengauer, Science
300, 442
(2003).[Abstract/Free Full Text] |
4. |
Z. Xiong, P. W. Laird, Nucleic
Acids Res. 25, 2532 (1997).[Abstract/Free Full Text] |
5. |
C. Lengauer, K. W. Kinzler, B.
Vogelstein, Nature 386, 623 (1997).[CrossRef][ISI][Medline] |
6. |
C. Lengauer, K. W. Kinzler, B.
Vogelstein, Proc. Natl. Acad. Sci. U.S.A. 94,
2545 (1997).[Abstract/Free Full Text] |
7. |
L. Jackson-Grauby et al.,
Proc. Natl. Acad. Sci. U.S.A. 94, 4681 (1997).[Abstract/Free Full Text] | 20
June 2003; accepted 27 August
2003 10.1126/science.1089523 Include this information when
citing this paper.
Related articles in Science:
- Chromosomal Instability and Tumors Promoted by DNA
Hypomethylation
- Amir Eden, François Gaudet, Alpana Waghmare, and Rudolf
Jaenisch
Science 2003 300: 455. (in Brevia) [Full
Text]
- Induction of Tumors in Mice by Genomic
Hypomethylation
- François Gaudet, J. Graeme Hodgson, Amir Eden, Laurie
Jackson-Grusby, Jessica Dausman, Joe W. Gray, Heinrich Leonhardt,
and Rudolf Jaenisch
Science 2003 300: 489-492. (in Reports)
[Abstract]
[Full
Text]
- Response to Comment on "Chromosomal Instability and
Tumors Promoted by DNA Hypomethylation" and "Induction of Tumors
in Mice by Genomic Hypomethylation"
- Amir Eden, François Gaudet, and Rudolf Jaenisch
Science
2003 302: 1153. (in Technical Comments) [Full
Text]
Volume 302, Number 5648, Issue of 14 Nov 2003, p. 1153.
Copyright © 2003 by The American Association for the
Advancement of Science. All rights reserved.
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