
[Cancer Research 61, 249-255, January 1, 2001]
© 2001 American Association for Cancer Research
Aberrant Promoter Methylation of Multiple Genes in Non-Small Cell Lung Cancers1
Sabine Zöchbauer-Müller,
Kwun M. Fong,
Arvind K. Virmani,
Joseph Geradts,
Adi F. Gazdar and
John D. Minna2
Hamon Center for Therapeutic Oncology Research [S. Z-M., A. K. V., A. F. G., J. D. M.], Departments of Pathology [A. F. G.], Internal Medicine [J. D. M.], and Pharmacology [J. D. M.], The University of Texas, Southwestern Medical Center at Dallas, Dallas, Texas 75390-8593; Department of Thoracic Medicine, The Prince Charles Hospital, Chermside, Brisbane 4032, Australia [K. M. F.]; and Nuffield Department of Pathology & Bacteriology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom [J. G.]
 |
ABSTRACT
|
|---|
Aberrant methylation of CpG islands acquired in tumor cells in promoter
regions is one method for loss of gene function. We determined the
frequency of aberrant promoter methylation (referred to as methylation)
of the genes retinoic acid receptor ß-2
(RARß), tissue inhibitor of metalloproteinase 3
(TIMP-3), p16INK4a,
O6-methylguanine-DNA-methyltransferase
(MGMT), death-associated protein kinase
(DAPK), E-cadherin (ECAD),
p14ARF, and glutathione
S-transferase P1 (GSTP1) in 107 resected
primary non-small cell lung cancers (NSCLCs) and in 104 corresponding
nonmalignant lung tissues by methylation-specific PCR. Methylation in
the tumor samples was detected in 40% for RARß, 26%
for TIMP-3, 25% for p16INK4a,
21% for MGMT, 19% for DAPK, 18% for
ECAD, 8% for p14ARF, and 7% for
GSTP1, whereas it was not seen in the vast majority of
the corresponding nonmalignant tissues. Moreover,
p16INK4a methylation was correlated with loss of
p16INK4a expression by immunohistochemistry. A total of
82% of the NSCLCs had methylation of at least one of these genes; 37%
of the NSCLCs had one gene methylated, 22% of the NSCLCs had two genes
methylated, 13% of the NSCLCs had three genes methylated, 8% of the
NSCLCs had four genes methylated, and 2% of the NSCLCs had five genes
methylated. Methylation of these genes was correlated with some
clinicopathological characteristics of the patients. In comparing the
methylation patterns of tumors and nonmalignant lung tissues from the
same patients, there were many discordancies where the genes methylated
in nonmalignant tissues were not methylated in the corresponding
tumors. This suggests that the methylation was occurring as a
preneoplastic change. We conclude that these findings confirm in a
large sample that methylation is a frequent event in NSCLC, can also
occur in smoking-damaged nonmalignant lung tissues, and may be the most
common mechanism to inactivate cancer-related genes in NSCLC.
 |
INTRODUCTION
|
|---|
Alterations of the pattern of DNA methylation have been recognized
as common changes in human cancers (1)
. These are thought
to have important implications for abnormalities of gene expression,
chromosome structure, timing of DNA replication, and chromatin
organization (1
, 2)
. Aberrant methylation of normally
unmethylated CpG-rich areas, also known as CpG islands, which are
located in or near the promoter region of many genes, has been
associated with transcriptional inactivation of defined
TSGs3
in human cancer (3)
. Thus, aberrant methylation serves as
an alternative to the genetic loss of a TSG function by deletion or
mutation (4)
.
Aberrant promoter methylation (referred to as methylation) has been
described for several genes in various malignant diseases including
lung cancer (5, 6, 7, 8, 9, 10, 11, 12, 13)
. In lung cancer, methylation of the
TSG p16INK4, the DNA repair gene
MGMT, and the detoxification gene GSTP1 has been
found in primary tumors (3
, 7
, 12
, 14)
. Moreover,
methylation of these genes and the apoptosis-associated gene
DAPK has been described in serum DNA of NSCLC patients
(15)
. Interestingly, p16INK4a
methylation has also been observed in precursor lesions of lung
carcinomas, which makes it a reasonable candidate biomarker for the
early diagnosis of lung cancer (16)
. Methylation of
TIMP-3 has been described in 4 of 21 (19%) NSCLCs
(11)
, and TIMP-3 is thought to suppress primary
tumor growth (17
, 18)
. The RARß-2 gene,
RARß, which may function as a TSG (19
, 20)
,
has been observed to be silenced by methylation in colon cancer and
breast cancer (6
, 9)
. ECAD, which plays a role
in invasion suppression, has been found methylated in breast and
prostate carcinomas (5)
. Esteller et al.
(13)
recently reported methylation of
p14ARF in 28% of primary colorectal carcinomas and
suggested that methylation-associated inactivation of
p14ARF is independent of p16INK4a
methylation and p53 mutational status.
Although several reports about methylation of various genes in lung
cancer have been published, in most cases, the methylation status has
been investigated for just a single gene or in a small number of
samples (3
, 7
, 11
, 12
, 15
, 21)
. Therefore, we decided to
investigate methylation of multiple genes in a large sample collection
of primary resected NSCLCs and their associated nonmalignant lung
tissues, for which we also had clinical data and results about certain
other molecular abnormalities. We determined the frequency of
methylation of the eight genes RARß, TIMP-3,
p16INK4a, MGMT, DAPK,
ECAD, p14ARF, and GSTP1 in 107
primary NSCLCs and 104 corresponding nonmalignant lung tissues by MSP.
Methylation of these genes was shown to occur in confirmed promoter
regions or in 5' CpG islands in or near putative promoter regions. This
analysis also provided us with the opportunity to determine whether the
methylation status of the individual genes occurred independently of
one another or with other molecular abnormalities. Finally, we wanted
to know whether methylation of these genes was correlated with clinical
features such as sex, age, smoking history, tumor stage, histology, and
overall survival of the patients.
 |
MATERIALS AND METHODS
|
|---|
Tumor Samples.
Primary tumor samples (n = 107) and
corresponding nonmalignant lung tissues (n = 104) were obtained from NSCLC patients who had been treated with
curative resectional surgery in The Prince Charles Hospital (Brisbane,
Australia) between June 1990 and March 1993. This cohort of patients
had been investigated previously for various genetic abnormalities
(22, 23, 24, 25, 26, 27, 28)
. There were 76 males and 31 females (age, 2881
years; mean age at diagnosis, 61 years). Sixty-one patients had stage I
disease, 21 patients had stage II disease, 24 patients had stage IIIA
disease, and 1 patient had stage IIIB disease. Histological subtypes
included 45 adenocarcinomas, 43 squamous cell carcinomas, 11
adenosquamous carcinomas, 4 large cell carcinomas, 3 atypical
carcinoids, and 1 typical carcinoid. Ninety-eight patients were smokers
(mean pack-years, 31), and the rest of patients were never smokers or
nonsmokers. Survival data of 5 or more years were available on most
patients.
MSP.
DNA was extracted as described previously (22)
, and
bisulfite modification of genomic DNA was performed as reported by
Herman et al. (29)
. Briefly, 1 µg of genomic
DNA was denatured with NaOH (final concentration, 0.2
M), and 10 mM hydroquinone
(Sigma) and 3 M sodium-bisulfite (Sigma) were
added and incubated at 50°C for 16 h. Afterward, modified DNA
was purified using Wizard DNA purification resin (Promega) followed by
ethanol precipitation. Treatment of genomic DNA with sodium bisulfite
converts unmethylated cytosines (but not methylated cytosines) to
uracil, which are then converted to thymidine during the subsequent PCR
step, giving sequence differences between methylated and unmethylated
DNA. PCR primers that distinguish between these methylated and
unmethylated DNA sequences were then used. Primer sequences of all
genes for both the methylated and the unmethylated form, annealing
temperatures, and the expected PCR product sizes are summarized in
Table 1
. The PCR mixture contained 10x PCR buffer (Qiagen), deoxynucleotide
triphosphates (1.25 mM), primers (final
concentration, 0.6 µM each per reaction), 1
unit of HotStarTaq (Qiagen), and bisulfite-modified DNA (
150 ng).
Amplification was carried out in a 9700 Perkin-Elmer Thermal Cycler.
DNA from peripheral blood lymphocytes of healthy individuals and water
blanks were used as a negative control for methylated genes. DNA from
peripheral blood lymphocytes treated with SssI methyltransferase (New
England Biolabs) was used as a positive control for methylated alleles.
Fifteen µl of each PCR reaction were loaded onto a 2% agarose gel
and visualized under UV illumination. The PCR for all samples
demonstrating methylation for the individual genes was repeated to
confirm these results.
Other Molecular Markers.
Data on immunohistochemistry of p16INK4a,
RB, and p53 in 102 samples have been described by
Geradts et al. (28)
. LOH analysis at 1p
(MYCL), 3p21 (D3S1029), 3p25.326.2 (D3S1038), 5q
(APC and MCC), 8p (LPL), 9p
(IFNA and D9S126), 11p (H-ras, INS,
RRM1, FSHB, and CAT), 13q
(RB and D13S260), 17q (NF1, NM23-H1,
D17S40, D17S21, and D17S4), and 18q (DCC) has also been
described previously by Fong et al. (22, 23, 24, 25, 26)
.
Other available molecular markers from previous studies included
K-ras codon 12 mutations and p53 exon 58
mutations (25)
.
Statistics.
Statistical analysis was performed using the
2
and Fishers exact test for differences between groups and
t tests between means. Overall survival was calculated using
Kaplan-Meier log-rank testing. To determine the overall rate of
methylation in individual samples, we used the MI. The MI is defined as
a fraction representing the number of genes methylated/the number of
genes tested. A previously designed Microsoft Visual Basic Program was
used for color formatting and visualization of our data
(30)
.
 |
RESULTS
|
|---|
Frequency of Methylation in Primary NSCLCs and Their Corresponding
Nonmalignant Lung Tissues.
We determined the frequency of methylation of RARß,
TIMP-3, p16INK4a, MGMT,
DAPK, ECAD, p14ARF, and
GSTP1 in 107 resected NSCLCs and in 104 corresponding
nonmalignant lung tissues by MSP (Fig. 1
; Table 2
). In the corresponding nonmalignant lung tissues, methylation of
p16INK4a, MGMT, ECAD, and
GSTP1 was not detected, but it was seen at low frequencies
for RARß (14%), TIMP-3 (8%), DAPK
(6%), and p14ARF (5%; Table 2
). The bands that
were seen for the methylated form in nonmalignant tissues, especially
for RARß, were faint. The detailed results of
methylation for each gene in all tumors compared with the corresponding
nonmalignant lung tissues are shown in Fig. 2
. The unmethylated form of all genes was detected in 100% of samples in
both tumors and nonmalignant tissues. Because the tumor specimens
represented macroscopically isolated samples that contained both tumor
and nonmalignant tissue, this was expected.

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 1. Methylation analysis of eight genes in primary NSCLCs and
their corresponding nonmalignant lung tissues by MSP. The gene studied
is given at the left of each panel. TU,
tumor; NL, nonmalignant lung tissue; Lane
U, amplified product with primers recognizing unmethylated
sequence; Lane M, amplified product with primers
recognizing methylated sequence. Peripheral blood lymphocytes
(L) were used as a negative control. In
vitro methylated DNA (IVD) was used as a
positive control for methylation. H2O, water
blanks. The PCR product sizes of all of the genes are summarized in
Table 1
.
|
|

View larger version (103K):
[in this window]
[in a new window]
|
Fig. 2. Summary of methylation of RARß,
TIMP-3, p16INK4a,
MGMT, DAPK, ECAD,
p14ARF, and GSTP1 in resected
primary NSCLCs (left) and corresponding nonmalignant
lung tissues (right). Green boxes
represent samples that are not methylated; blue boxes
represent samples that are methylated.
|
|
We found that at least one of these eight genes had methylation in 82%
of the tumors; 37% of the tumors had only one gene methylated, 22% of
the tumors had two genes methylated, 13% of the tumors had three genes
methylated, 8% of the tumors had four genes methylated, and 2% of the
tumors had five genes methylated, giving MIs of 0 in 19 tumors, 0.1 in
39 tumors, 0.3 in 24 tumors, 0.4 in 14 tumors, 0.5 in 9 tumors, and 0.6
in 2 tumors, respectively. A statistically significant
correlation was found for the methylation status between
RARß and MGMT (P = 0.0005), whereas the methylation status of the other genes was
independent when compared with each other.
Clinicopathological Correlations.
We analyzed the methylation changes in the tumors and the clinical data
obtained from these patients (Table 3)
. Overall, we found no correlation between the MI (overall
fraction of genes methylated) and any of the clinicopathological
characteristics of the patients. A significantly longer overall
survival was found for patients whose tumors showed methylation of
ECAD (P = 0.005, Kaplan-Meier
log-rank test). This result was seen particularly in stage I disease.
We found no association between methylation of RARß,
TIMP-3, p16INK4a, MGMT,
DAPK, p14ARF, or GSTP1 and
survival, regardless of whether we analyzed all stages combined or
performed a separate analysis for stage I, II, and III disease.
Lymph node involvement is a well-established prognostic indicator for
resected NSCLC, and we found lymph nodes were involved with the tumor
in 41% of samples with any gene methylated, but in only 11% of
samples in which no genes showed methylation (P = 0.012). To summarize our findings regarding other
clinical parameters, methylation of TIMP-3 was detected more
frequently in women than in men, DAPK methylation and
p16INK4a methylation were more frequent in men than
in women, and p16INK4a methylation was more frequent
in squamous carcinomas than in adenocarcinomas and was seen only in
smokers. When making multiple comparisons of clinical data with
multiple biomarkers like the methylated genes, such as was done in this
study, caution must be used with conservative statistical corrections
(such as the Bonferroni, Tukey, and Newman-Kauls post tests)
before deciding that significant correlations exist. These also need to
be confirmed in other data sets and larger series. Thus, we feel the
most conservative approach is to present the data in tabular form for
future reference without drawing any statistical conclusions of
significance (Table 3)
.
Immunohistochemistry and Molecular Correlations.
These tumor samples had been scored previously for immunohistochemical
staining for p16INK4a, RB, and
p53 (28)
. As expected, we found a significant
correlation between p16INK4a methylation and loss of
p16INK4a tumor staining (P = 0.009), whereas an inverse correlation (which was also expected)
was found between loss of RB protein expression and
p16INK4a methylation (P = 0.009). No correlation was seen between methylation of any gene and
p53 immunohistochemical or mutational (exons 58) status.
Data about LOH on different chromosomal regions (1p, 3p21,
3p25.326.2, 5q, 8p, 9p, 11p, 13q, 17q, and 18q) were compared with
the results about methylation of the various genes. Given the large
number of comparisons, no significant association was seen for LOH on
other chromosomal regions and methylation of the investigated genes.
Specifically, no association was detected between LOH on 9p21 and
p16INK4a methylation.
 |
DISCUSSION
|
|---|
This report describes the frequency of methylation of the genes
RARß, TIMP-3, p16INK4a,
MGMT, DAPK, ECAD,
p14ARF, and GSTP1 as well as the
correlation of these methylation changes with clinicopathological
characteristics and molecular abnormalities in a large number of
patients. Methylation of RARß, TIMP-3,
p16INK4a, MGMT, DAPK, and
GSTP1 had been described previously in lung cancer cell
lines or small numbers of primary lung tumors, and our results are
similar to the previously reported data (3
, 7
, 11
, 12
, 14
, 15
, 21)
. However, methylation of ECAD and
p14ARF had not been reported in lung cancer. We also
investigated methylation in the corresponding nonmalignant lung
tissues. Several studies reported the lack of methylation in
nonmalignant tissues and described methylation as a tumor-restricted
event. However, we found methylation of RARß,
TIMP-3, DAPK, and p14ARF in
some of the matched nonmalignant lung tissues. We cannot exclude that
this may be due in some cases to contamination with adjacent malignant
cells. However, from many previous studies, we have learned that
preneoplastic/preinvasive and even histologically normal
smoking-damaged epithelium has suffered genetic changes
(31, 32, 33)
. Thus, a possible explanation for detecting
methylated alleles in the nonmalignant lung samples is that they
represent premalignant changes. To begin to address this issue, we
compared the specific genes methylated in the tumors and nonmalignant
tissues from the same patient (Fig. 2
; Table 4
). In the majority of cases, the genes methylated in the nonmalignant
tissues were not those methylated in the corresponding tumors from the
same patients. This would indicate that the methylation changes in
these cases do not represent tumor contamination but are more likely
premalignant changes. This was especially true in the case of
RARß methylation, which we found relatively frequently but
with weak signals in the corresponding nonmalignant lung tissues.
RARß mRNA and protein expression have been lost in some
smoking-damaged lungs (34, 35, 36)
. These findings are also in
agreement with the results reported by Cote et al.
(6)
and Bovenzi et al. (9)
, who
described RARß methylation in a few samples of
corresponding normal colon and breast tissues associated with cancers.
The studies by Belinsky et al. (16)
, who
reported the appearance of p16INK4a methylation in
hyperplasias associated with squamous lung cancers, and Esteller
et al. (13)
, who reported a similar rate of
p14ARF methylation in colorectal adenomas and
colorectal carcinomas, are other examples where methylation may be an
early event in cancer development. However, we found no examples of
p16INK4a methylation and only a few examples of
p14ARF methylation in nonmalignant lung samples from
lung cancer patients. It has been described that aging is associated
with methylation of certain genes (4)
. Therefore, the
aging mechanism could also be a possible explanation for detecting
methylation in nonmalignant lung tissues.
View this table:
[in this window]
[in a new window]
|
Table 4 Comparison of methylation of specific genes between tumors and
nonmalignant lung tissues from the same patients
Data from Fig. 2
comparing the methylation status of each gene in the
tumor and corresponding nonmalignant lung tissues from the same
patient. The +/+ group could represent contamination of the
nonmalignant tissue with tumor DNA. However, the (+) nonmalignant
tissue/(-) tumor group (n = 25) must
represent distinct methylation events occurring only in the
nonmalignant tissue of that patient.
|
|
Overall, at least one gene was methylated in 82% of the NSCLCs. To
identify a subset of tumors that have concordant methylation of
multiple loci and may therefore lead to the simultaneous inactivation
of multiple genes, we determined the MI of the individual samples. In
our study, 2 tumors had a MI of 0.6, 9 tumors had a MI of 0.5, 14
tumors had a MI of 0.4, 24 tumors had a MI of 0.3, 39 tumors had a MI
of 0.1, and 19 tumors had a MI of 0, whereas in the corresponding
nonmalignant lung tissues, only 4 had a MI of 0.3, 26 had a MI of 0.1,
and 74 had a MI of 0. These results indicate there is a subset
of lung cancers with widespread acquired methylation. Tumors with a
high MI may have a distinct pathogenesis from tumors with a low MI.
Patients whose tumors have several genes inactivated by methylation are
appropriate candidates for clinical trials of drugs blocking
methylation. In this case, if preclinical data showed that reactivation
of expression of genes extinguished by methylation led to inhibition of
tumor cell growth or induction of apoptosis, there would appear to be a
strong rationale for testing such drugs in patients.
Another aim of this study was to investigate whether methylation of
RARß, TIMP-3, p16INK4a,
MGMT, DAPK, ECAD,
p14ARF, and GSTP1 in NSCLC is associated
with clinicopathological parameters, particularly survival, of these
patients. With the exception of ECAD, the presence of
methylation of these genes or a group of genes was not associated with
different survival. The longer survival associated with ECAD
methylation will need to be confirmed by other series. However, a
possible explanation for this surprising finding might be the fact that
methylation of ECAD seems to be dynamic and heterogenous as
described by Graff et al. (37)
. In the Graff
et al. (37)
study, breast cancers under
conditions favoring invasion (with loss of adhesion) exhibited densely
methylated ECAD promoter with reduced ECAD expression,
whereas their survival and growth under conditions similar to
metastatic deposits (spheroids) requiring cell adhesion actually showed
loss of ECAD methylation with reestablished ECAD expression.
It will be of interest to see whether similar findings occur in lung
cancer primary lesions and metastatic deposits. Although it was not
correlated with survival, the presence of finding any gene methylated
was correlated with lymph node positivity. Methylation of
TIMP-3 was seen more frequently in women, whereas
methylation of DAPK and p16INK4a was more
common in men. The reason for this gender difference is unknown.
Methylation of p16INK4a was more frequent in
squamous cell carcinomas than in adenocarcinomas. These results
demonstrate that methylation of certain genes may be associated with
some clinicopathological characteristics of these patients. These
clinical correlations need to be confirmed in other independent
studies.
For p16INK4a and RB, which are involved
in the p16INK4a/cyclin D1/cyclin-dependent
protein kinase 4/RB pathway, data about protein expression from
immunohistochemistry studies were available. We found a statistically
significant correlation between loss of p16INK4a
expression and methylation of p16INK4a and, as
expected, an inverse correlation between loss of RB
expression and p16INK4a methylation. These findings
confirm previous data showing that methylation is a mechanism for gene
silencing of p16INK4a. This finding is also in
agreement with finding either p16INK4a or
RB mutations or loss of protein expression, but not both, in
the same tumor (28
, 38)
. Esteller et al.
(13)
reported that methylation-associated inactivation of
p14ARF is independent of p16INK4a
methylation and p53 mutational status in colon carcinomas.
In agreement with their study, we also found methylation-associated
inactivation of p14ARF to be independent of
p16INK4a methylation and p53 mutational
status.
In conclusion, our study about methylation of RARß,
TIMP-3, p16INK4a, MGMT,
DAPK, ECAD, p14ARF, and
GSTP1 in primary resected NSCLCs stresses the high frequency
of methylation in a large collection of samples and demonstrates that
methylation may be the most common mechanism to inactivate
cancer-related genes in NSCLC. Why certain genes are targeted for
methylation and the enzymes involved in this methylation merit special
attention, and the answers should be of translational value. In the
meantime, the detection of methylated genes is an attractive biomarker
for testing the early detection of lung cancer and for monitoring
chemoprevention efforts as proposed by Belinsky et al.
(16)
. In these studies, it will be necessary to show in
prospective clinical trials that persons at high risk for developing
lung cancer (such as smokers with a heavy smoking history who go on to
develop lung cancer) have certain key genes methylated in the
nonmalignant tissues (e.g., sputum or bronchial brushes and
washings) before the cancer becomes clinically evident.
 |
ACKNOWLEDGMENTS
|
|---|
We thank Luc Girard for analysis with the Visual Basic Program.
 |
FOOTNOTES
|
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by Grants J1658-MED and J1860-MED from
the Austrian Science Foundation, Lung Cancer SPORE (Special Program of
Research Excellence) P50 CA70907, and The G. Harold and Leila Y.
Mathers Charitable Foundation. 
2 To whom requests for reprints should be
addressed, at Hamon Center for Therapeutic Oncology Research, The
University of Texas, Southwestern Medical Center at Dallas, 6000 Harry
Hines Boulevard, Dallas, TX 75390-8593. Phone: (214) 648-4900; Fax:
(214) 648-4940; E-mail: John.Minna{at}UTSouthwestern.edu 
3 The abbreviations used are: TSG, tumor
suppressor gene; NSCLC, non-small lung cancer; RAR, retinoic acid
receptor; TIMP, tissue inhibitor of metalloproteinase; MGMT,
O6-methylguanine-DNA-methyltransferase;
DAPK, death-associated protein kinase; ECAD, E-cadherin; GSTP1,
glutathione S-transferase P1; RB, retinoblastoma; LOH, loss of
heterozygosity; MI, methylation index; MSP, methylation-specific PCR. 
Received 6/ 5/00.
Accepted 10/30/00.
 |
REFERENCES
|
|---|
-
Antequera F., Boyes J., Bird A. High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell, 62: 503-514, 1990.[Medline]
-
Keshet I., Lieman-Hurwitz J., Cedar H. DNA methylation affects the formation of active chromatin. Cell, 44: 535-543, 1986.[Medline]
-
Merlo A., Herman J. G., Mao L., Lee D. J., Gabrielson E., Burger P. C., Baylin S. B., Sidransky D. 5' CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat. Med., 1: 686-692, 1995.[Medline]
-
Baylin S. B., Herman J. G., Graff J. R., Vertino P. M., Issa J. P. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv. Cancer Res., 72: 141-196, 1998.[Medline]
-
Graff J. R., Herman J. G., Lapidus R. G., Chopra H., Xu R., Jarrard D. F., Isaacs W. B., Pitha P. M., Davidson N. E., Baylin S. B. E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. Cancer Res., 55: 5195-5199, 1995.[Abstract/Free Full Text]
-
Cote S., Sinnett D., Momparler R. L. Demethylation by 5-aza-2'-deoxycytidine of specific 5-methylcytosine sites in the promoter region of the retinoic acid receptor ß gene in human colon carcinoma cells. Anticancer Drugs, 9: 743-750, 1998.[Medline]
-
Esteller M., Corn P. G., Urena J. M., Gabrielson E., Baylin S. B., Herman J. G. Inactivation of glutathione S-transferase P1 gene by promoter hypermethylation in human neoplasia. Cancer Res., 58: 4515-4518, 1998.[Abstract/Free Full Text]
-
Katzenellenbogen R. A., Baylin S. B., Herman J. G. Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies. Blood, 93: 4347-4353, 1999.[Abstract/Free Full Text]
-
Bovenzi V., Le N. L., Cote S., Sinnett D., Momparler L. F., Momparler R. L. DNA methylation of retinoic acid receptor ß in breast cancer and possible therapeutic role of 5-aza-2'-deoxycytidine. Anticancer Drugs, 10: 471-476, 1999.[Medline]
-
Melki J. R., Vincent P. C., Clark S. J. Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia. Cancer Res., 59: 3730-3740, 1999.[Abstract/Free Full Text]
-
Bachman K. E., Herman J. G., Corn P. G., Merlo A., Costello J. F., Cavenee W. K., Baylin S. B., Graff J. R. Methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene suggests a suppressor role in kidney, brain, and other human cancers. Cancer Res., 59: 798-802, 1999.[Abstract/Free Full Text]
-
Esteller M., Hamilton S. R., Burger P. C., Baylin S. B., Herman J. G. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res., 59: 793-797, 1999.[Abstract/Free Full Text]
-
Esteller M., Tortola S., Toyota M., Capella G., Peinado M. A., Baylin S. B., Herman J. G. Hypermethylation-associated inactivation of p14ARF is independent of p16INK4a methylation and p53 mutational status. Cancer Res., 60: 129-133, 2000.[Abstract/Free Full Text]
-
Kashiwabara K., Oyama T., Sano T., Fukuda T., Nakajima T. Correlation between methylation status of the p16/CDKN2 gene and the expression of p16 and Rb proteins in primary non-small cell lung cancers. Int. J. Cancer, 79: 215-220, 1998.[Medline]
-
Esteller M., Sanchez-Cespedes M., Rosell R., Sidransky D., Baylin S. B., Herman J. G. Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. Cancer Res., 59: 67-70, 1999.[Abstract/Free Full Text]
-
Belinsky S. A., Nikula K. J., Palmisano W. A., Michels R., Saccomanno G., Gabrielson E., Baylin S. B., Herman J. G. Aberrant methylation of p16INK4a is an early event in lung cancer and a potential biomarker for early diagnosis. Proc. Natl. Acad. Sci. USA, 95: 11891-11896, 1998.[Abstract/Free Full Text]
-
Bian J., Wang Y., Smith M. R., Kim H., Jacobs C., Jackman J., Kung H. F., Colburn N. H., Sun Y. Suppression of in vivo tumor growth and induction of suspension cell death by tissue inhibitor of metalloproteinases (TIMP)-3. Carcinogenesis (Lond.), 17: 1805-1811, 1996.[Abstract/Free Full Text]
-
Anand-Apte B., Bao L., Smith R., Iwata K., Olsen B. R., Zetter B., Apte S. S. A review of tissue inhibitor of metalloproteinases-3 (TIMP-3) and experimental analysis of its effect on primary tumor growth. Biochem. Cell Biol., 74: 853-862, 1996.[Medline]
-
Houle B., Rochette-Egly C., Bradley W. E. Tumor-suppressive effect of the retinoic acid receptor ß in human epidermoid lung cancer cells. Proc. Natl. Acad. Sci. USA, 90: 985-989, 1993.[Abstract/Free Full Text]
-
Berard J., Laboune F., Mukuna M., Masse S., Kothary R., Bradley W. E. Lung tumors in mice expressing an antisense RARß2 transgene. FASEB J., 10: 1091-1097, 1996.[Abstract]
-
Virmani A. K., Rathi A., Zöchbauer-Müller S., Sacchi N., Fukuyama Y., Bryant D., Maitra A., Heda S., Fong K. M., Thunnissen F., Minna J. D., Gazdar A. F. Promoter methylation and silencing of the retinoic acid receptor-ß gene in lung carcinomas. J. Natl. Cancer Inst. (Bethesda), 92: 1303-1307, 2000.[Abstract/Free Full Text]
-
Fong K. M., Zimmerman P. V., Smith P. J. Correlation of loss of heterozygosity at 11p with tumour progression and survival in non-small cell lung cancer. Genes Chromosomes Cancer, 10: 183-189, 1994.[Medline]
-
Fong K. M., Kida Y., Zimmerman P. V., Ikenaga M., Smith P. J. Loss of heterozygosity frequently affects chromosome 17q in non-small cell lung cancer. Cancer Res., 55: 4268-4272, 1995.[Abstract/Free Full Text]
-
Fong K. M., Zimmerman P. V., Smith P. J. Tumor progression and loss of heterozygosity at 5q and 18q in non-small cell lung cancer. Cancer Res., 55: 220-223, 1995.[Abstract/Free Full Text]
-
Fong K. M., Zimmerman P. V., Smith P. J. Microsatellite instability and other molecular abnormalities in non-small cell lung cancer. Cancer Res., 55: 28-30, 1995.[Abstract/Free Full Text]
-
Fong K. M., Kida Y., Zimmerman P. V., Smith P. J. MYCL genotypes and loss of heterozygosity in non-small-cell lung cancer. Br. J. Cancer, 74: 1975-1978, 1996.[Medline]
-
Fong K. M., Biesterveld E. J., Virmani A., Wistuba I., Sekido Y., Bader S. A., Ahmadian M., Ong S. T., Rassool F. V., Zimmerman P. V., Giaccone G., Gazdar A. F., Minna J. D. FHIT and FRA3B 3p14. 2 allele loss are common in lung cancer and preneoplastic bronchial lesions and are associated with cancer-related FHIT cDNA splicing aberrations. Cancer Res., 57: 2256-2267, 1997.[Abstract/Free Full Text]
-
Geradts J., Fong K. M., Zimmerman P. V., Maynard R., Minna J. D. Correlation of abnormal RB, p16ink4a, and p53 expression with 3p loss of heterozygosity, other genetic abnormalities, and clinical features in 103 primary non-small cell lung cancers. Clin. Cancer Res., 5: 791-800, 1999.[Abstract/Free Full Text]
-
Herman J. G., Graff J. R., Myohanen S., Nelkin B. D., Baylin S. B. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. USA, 93: 9821-9826, 1996.[Abstract/Free Full Text]
-
Girard L., Zöchbauer-Müller S., Virmani A. K., Gazdar A. F., Minna J. D. Genome-wide allelotyping of lung cancer identifies new regions of allelic loss, differences between small cell lung cancer and non-small cell lung cancer, and loci clustering. Cancer Res., 60: 4894-4906, 2000.[Abstract/Free Full Text]
-
Bennett W. P., Colby T. V., Travis W. D., Borkowski A., Jones R. T., Lane D. P., Metcalf R. A., Samet J. M., Takeshima Y., Gu J. R., et al p53 protein accumulates frequently in early bronchial neoplasia. Cancer Res., 53: 4817-4822, 1993.[Abstract/Free Full Text]
-
Westra W. H., Baas I. O., Hruban R. H., Askin F. B., Wilson K., Offerhaus G. J., Slebos R. J. K-ras oncogene activation in atypical alveolar hyperplasias of the human lung. Cancer Res., 56: 2224-2228, 1996.[Abstract/Free Full Text]
-
Wistuba I. I., Lam S., Behrens C., Virmani A. K., Fong K. M., LeRiche J., Samet J. M., Srivastava S., Minna J. D., Gazdar A. F. Molecular damage in the bronchial epithelium of current and former smokers. J. Natl. Cancer Inst. (Bethesda), 89: 1366-1373, 1997.[Abstract/Free Full Text]
-
Gebert J. F., Moghal N., Frangioni J. V., Sugarbaker D. J., Neel B. G. High frequency of retinoic acid receptor ß abnormalities in human lung cancer. Oncogene, 6: 1859-1868, 1991.[Medline]
-
Geradts J., Chen J. Y., Russell E. K., Yankaskas J. R., Nieves L., Minna J. D. Human lung cancer cell lines exhibit resistance to retinoic acid treatment. Cell Growth Differ., 4: 799-809, 1993.[Abstract]
-
Xu X. C., Sozzi G., Lee J. S., Lee J. J., Pastorino U., Pilotti S., Kurie J. M., Hong W. K., Lotan R. Suppression of retinoic acid receptor ß in non-small-cell lung cancer in vivo: implications for lung cancer development. J. Natl. Cancer Inst. (Bethesda), 89: 624-629, 1997.[Abstract/Free Full Text]
-
Graff J. R., Gabrielson E., Fujii H., Baylin S. B., Herman J. G. Methylation patterns of the E-cadherin 5' CpG island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression. J. Biol. Chem., 275: 2727-2732, 2000.[Abstract/Free Full Text]
-
Sekido Y., Fong K. M., Minna J. D. Progress in understanding the molecular pathogenesis of human lung cancer. Biochim. Biophys. Acta, 1378: F21-F59, 1998.[Medline]
This article has been cited by other articles:

|
 |

|
 |
 
D. M. Euhus, D. Bu, S. Milchgrub, X.-J. Xie, A. Bian, A. M. Leitch, and C. M. Lewis
DNA Methylation in Benign Breast Epithelium in Relation to Age and Breast Cancer Risk
Cancer Epidemiol. Biomarkers Prev.,
May 1, 2008;
17(5):
1051 - 1059.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
J. D.F. Licchesi, W. H. Westra, C. M. Hooker, and J. G. Herman
Promoter Hypermethylation of Hallmark Cancer Genes in Atypical Adenomatous Hyperplasia of the Lung
Clin. Cancer Res.,
May 1, 2008;
14(9):
2570 - 2578.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
P. Gade, S. K. Roy, H. Li, S. C. Nallar, and D. V. Kalvakolanu
Critical Role for Transcription Factor C/EBP-{beta} in Regulating the Expression of Death-Associated Protein Kinase 1
Mol. Cell. Biol.,
April 15, 2008;
28(8):
2528 - 2548.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
M.-R. Pan, H.-C. Chang, L.-Y. Chuang, and W.-C. Hung
The Nonsteroidal Anti-Inflammatory Drug NS398 Reactivates SPARC Expression via Promoter Demethylation to Attenuate Invasiveness of Lung Cancer Cells
Experimental Biology and Medicine,
April 1, 2008;
233(4):
456 - 462.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
Q. Feng, S. E. Hawes, J. E. Stern, L. Wiens, H. Lu, Z. M. Dong, C. D. Jordan, N. B. Kiviat, and H. Vesselle
DNA Methylation in Tumor and Matched Normal Tissues from Non-Small Cell Lung Cancer Patients
Cancer Epidemiol. Biomarkers Prev.,
March 1, 2008;
17(3):
645 - 654.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
T. A. Rauch, X. Zhong, X. Wu, M. Wang, K. H. Kernstine, Z. Wang, A. D. Riggs, and G. P. Pfeifer
High-resolution mapping of DNA hypermethylation and hypomethylation in lung cancer
PNAS,
January 8, 2008;
105(1):
252 - 257.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
D. M. Euhus, D. Bu, R. Ashfaq, X.-J. Xie, A. Bian, A. M. Leitch, and C. M. Lewis
Atypia and DNA Methylation in Nipple Duct Lavage in Relation to Predicted Breast Cancer Risk
Cancer Epidemiol. Biomarkers Prev.,
September 1, 2007;
16(9):
1812 - 1821.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
W. A. Zagorski, E. S. Knudsen, and M. F. Reed
Retinoblastoma Deficiency Increases Chemosensitivity in Lung Cancer
Cancer Res.,
September 1, 2007;
67(17):
8264 - 8273.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
L. Xu and R. K. Jain
Down-Regulation of Placenta Growth Factor by Promoter Hypermethylation in Human Lung and Colon Carcinoma
Mol. Cancer Res.,
September 1, 2007;
5(9):
873 - 880.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
C. J. Marsit, E. A. Houseman, A. R. Schned, M. R. Karagas, and K. T. Kelsey
Promoter hypermethylation is associated with current smoking, age, gender and survival in bladder cancer
Carcinogenesis,
August 1, 2007;
28(8):
1745 - 1751.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
W. Yue, S. Dacic, Q. Sun, R. Landreneau, M. Guo, W. Zhou, J. M. Siegfried, J. Yu, and L. Zhang
Frequent Inactivation of RAMP2, EFEMP1 and Dutt1 in Lung Cancer by Promoter Hypermethylation
Clin. Cancer Res.,
August 1, 2007;
13(15):
4336 - 4344.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
C. A. Righini, F. de Fraipont, J.-F. Timsit, C. Faure, E. Brambilla, E. Reyt, and M.-C. Favrot
Tumor-Specific Methylation in Saliva: A Promising Biomarker for Early Detection of Head and Neck Cancer Recurrence
Clin. Cancer Res.,
February 15, 2007;
13(4):
1179 - 1185.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
J. Subramanian and R. Govindan
Lung Cancer in Never Smokers: A Review
J. Clin. Oncol.,
February 10, 2007;
25(5):
561 - 570.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
J. Gu, D. Berman, C. Lu, I. I. Wistuba, J. A. Roth, M. Frazier, M. R. Spitz, and X. Wu
Aberrant Promoter Methylation Profile and Association with Survival in Patients with Non-Small Cell Lung Cancer
Clin. Cancer Res.,
December 15, 2006;
12(24):
7329 - 7338.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
P. Modena, E. Lualdi, F. Facchinetti, J. Veltman, J. F. Reid, S. Minardi, I. Janssen, F. Giangaspero, M. Forni, G. Finocchiaro, et al.
Identification of Tumor-Specific Molecular Signatures in Intracranial Ependymoma and Association With Clinical Characteristics
J. Clin. Oncol.,
November 20, 2006;
24(33):
5223 - 5233.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
Y. Yuan, J. Wang, J. Li, L. Wang, M. Li, Z. Yang, C. Zhang, and J. L. Dai
Frequent Epigenetic Inactivation of Spleen Tyrosine Kinase Gene in Human Hepatocellular Carcinoma.
Clin. Cancer Res.,
November 15, 2006;
12(22):
6687 - 6695.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
C. J. Marsit, E. A. Houseman, B. C. Christensen, K. Eddy, R. Bueno, D. J. Sugarbaker, H. H. Nelson, M. R. Karagas, and K. T. Kelsey
Examination of a CpG Island Methylator Phenotype and Implications of Methylation Profiles in Solid Tumors
Cancer Res.,
November 1, 2006;
66(21):
10621 - 10629.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
J. Wang, G. Walsh, D. D. Liu, J. J. Lee, and L. Mao
Expression of {Delta}DNMT3B Variants and Its Association with Promoter Methylation of p16 and RASSF1A in Primary Non-Small Cell Lung Cancer.
Cancer Res.,
September 1, 2006;
66(17):
8361 - 8366.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
T. Rauch, H. Li, X. Wu, and G. P. Pfeifer
MIRA-Assisted Microarray Analysis, a New Technology for the Determination of DNA Methylation Patterns, Identifies Frequent Methylation of Homeodomain-Containing Genes in Lung Cancer Cells
Cancer Res.,
August 15, 2006;
66(16):
7939 - 7947.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
M. C. Shin, S. J. Lee, J. E. Choi, S. I. Cha, C. H. Kim, W. K. Lee, S. Kam, Y. M. Kang, T. H. Jung, and J. Y. Park
Glu346Lys Polymorphism in the Methyl-CpG Binding Domain 4 Gene and the Risk of Primary Lung Cancer
Jpn. J. Clin. Oncol.,
August 1, 2006;
36(8):
483 - 488.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
M. F. Reed, W. A. Zagorski, J. A. Howington, J. T. Zilfou, and E. S. Knudsen
Inhibition of retinoblastoma tumor suppressor activity by RNA interference in lung cancer lines.
Ann. Thorac. Surg.,
July 1, 2006;
82(1):
249 - 253.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
J. S. Kim, J. W. Kim, J. Han, Y. M. Shim, J. Park, and D.-H. Kim
Cohypermethylation of p16 and FHIT Promoters as a Prognostic Factor of Recurrence in Surgically Resected Stage I Non-Small Cell Lung Cancer.
Cancer Res.,
April 15, 2006;
66(8):
4049 - 4054.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
S. Toyooka, M. Tokumo, H. Shigematsu, K. Matsuo, H. Asano, K. Tomii, S. Ichihara, M. Suzuki, M. Aoe, H. Date, et al.
Mutational and Epigenetic Evidence for Independent Pathways for Lung Adenocarcinomas Arising in Smokers and Never Smokers
Cancer Res.,
February 1, 2006;
66(3):
1371 - 1375.
[Abstract]
[Full Text]
[PDF]
|