I. Introduction
Primary brain tumors are neoplasms that originate from
various intracranial tissues. About 17,000 new cases occur annually and primary
cancer of the central nervous system (CNS) is the cause of death of
approximately 13,000 individuals per year (Surawicz et al, 1998). More than 60%
of all brain tumors have a glial origin, including pilocytic astrocytoma,
low-grade astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma,
anaplastic oligodendroglioma, mixed oligoastrocytoma and low-grade and anaplastic
ependymomas (Kleihues and Cavenee, 2000).
Pilocytic astrocytoma (a slow-growing tumor with a
World Health Organization (WHO) grade I) is considered to be the most common
glioma in children, accounting for 10% of cerebral and 85% of cerebellar
astrocytomas. It constitutes the principal CNS neoplasm in neurofibromatosis 1
(NF1) (Burger et al, 2000). Cytogenetic analysis of pilocytic astrocytoma has
revealed normal karyotypes or a variety of aberrations, primarily involving
gains of chromosomes 7 and 8 (Rey et al, 1987; Karnes et al, 1992; White et al,
1995). Allelic losses at 1p36 or at 17q have been identified in a few cases
(von Deimling et al, 1993; Bello et al, 1995), and comparative genomic
hybridization analysis identified gains of chromosomes 19, 22 and 9q34.1-qter,
and losses of chromosome 19 (Sanoudpu et al, 2000). Regarding TP53 gene mutations discordant data are
available; early studies identified sequence changes in a few tumors (von
Deimling et al, 1993), whereas 35% of samples (7 of 20) analyzed by Hayes et
al. (1999) displayed mutations of this gene. The only consistent gene
alteration described in this astrocytoma subtype is a loss of NF1 alleles that occurs in up to 90% of
informative NF1-associated cases, in contrast to only 4% of sporadic tumors
(Burger et al, 2000). LOH analysis at 1p, 10, 17 and 19q, and mutation
detection at TP53, p16INK4a and EGFR has been performed on 12 samples,
including three NF1-associated tumors (Tada et al, 2003). None of the genetic
abnormalities commonly detected in higher-grade astrocytomas were found in the
sporadic cases. In contrast, LOH 10 and 17q (including the PTEN and NF1 regions,
respectively) and homozygous deletion of p16INK4a
were identified in the NF1-associated samples. These data support the
hypothesis that some NF1-associated pilocytic astrocytomas would differ
genetically from sporadic cases.
Diffuse astrocytic gliomas are the most common primary
neoplasm occurring in the CNS and are histologically classified as WHO grade II
astrocytomas, WHO grade III anaplastic forms, and WHO grade IV glioblastoma
(Kleihues and Cavenee, 2000). Low grade (WHO grade II) tumors and anaplastic
grade III astrocytomas usually occur in adults and show a strong tendency
toward progression. Glioblastoma, the most malignant subtype of glioma, may
develop either from diffuse or anaplastic tumors (secondary glioblastoma) or de novo (primary glioblastoma) without a
defined prior tumor lesion. Multiple genetic alterations have been identified
in these astrocytic neoplasms; these alterations primarily involve inactivation
or amplification/over-expression of TP53,
p16INK4a, RB1, PTEN, MDM2, and EGFR genes (Kleihues and Cavenee, 2000). Several other non-random
anomalies are also characteristic features of these gliomas, including loss of
heterozygosity at 1p, 10p, 10q, 11p, 19q and 22q, although the putative tumor
suppressing genes remain unidentified. A distinct pattern of involvement of
these genes and chromosomal regions characterizes both forms of glioblastoma.
The main differences consist of EGFR
gene amplification and TP53
mutations, which respectively characterize primary and secondary glioblastomas
(Kleihues and Cavenee, 2000).
Tumors with a major oligodendroglial component account
for 4% of all primary brain tumors and represent between 5% and 18% of all
intracranial gliomas, including oligodendroglioma (WHO grade II), anaplastic
oligodendroglioma (WHO grade III) and mixed oligoastrocytoma (Kleihues and
Cavenee, 2000). They arise preferentially in the cerebral hemispheres of adult
patients with a mean age at diagnosis of ~40 years. Low-grade
oligodendrogliomas are characterized by a high incidence of loss of chromosome
arms 1p and 19q, and anaplastic forms accumulate allelic losses on the short
arm of chromosome 9 and on chromosome 10 (for review see Kleihues and Cavenee,
2000).
Ependymomas represent 3-9% of all intracranial brain
tumors and about 60% of spinal tumors, and commonly arise in children
(Kleihhues and Cavenee, 2000). Cytogenetic and molecular biology studies have
demonstrated a preferential involvement of chromosome 22 (by losses), parallel
to the inactivation of the NF2 gene
(located at 22q12), primarily in sporadic cord tumors. Additional genomic
abnormalities include chromosome 7 gains and overrepresentation of chromosomes
2, 5, 9, 12, 15, 18, 20q and X, and proportional losses of 13q. Losses of 6q
and 9p, with gains of 1q, have primarily been found in intracranial ependymomas
(Weremowicz et al, 1992; Rubio et al, 1994; Ebert et al, 1999; Hulsebos et al,
1999; Rousseau-Merk et al, 2000; Kraus et al, 2001; Alonso et al, 2002). These
findings, thus, suggest that intracranial and spinal cord ependymomas progress
along different genetic pathways that may influence differences in the clinical
behavior of these gliomas.
Tumorogenesis of gliomas seems to be a multi-step
process composed of genetic and epigenetic alterations involving tumor
suppressor genes, cell cycle regulatory genes, oncogenes, and as yet
unidentified genes located at specific chromosomal regions (Kleihues and
Cavenee, 2000). Transcriptional silencing by hypermethylation of CpG islands
located in the promoter regions is considered a common epigenetic mechanism for
inactivation of tumor-related genes (Esteller, 2003). CpG islands are 05-2 Kb
regions rich in cytosine-guanine dinucleotides, present in the 5 region of
about half of all human genes (Baylin et al, 1998). Little information is
available on the CpG island methylation status of neurogenic neoplasms.
Isolated previous studies focus on high-grade astrocytomas, primarily the
anaplastic forms and glioblastoma multiforme (Costello et al, 1996; Park et al,
2000; Nakamura et al, 2001a; 2001b; Yin et al, 2002; Gonzalez-Gomez et al,
2003a, 2003b; Uhlmann et al, 2003) and less frequently on low-grade
astrocytomas (Costello et al, 2000; Gonzalez-Gomez et al, 2003a; 2003b; 2003c;
Uhlmann et al, 2003), oligodendrogliomas (Watanabe et al,
2001a;Wolter et al, 2001; Yin et al, 2002; Alonso et al, 2003; Hong et al,
2003; Uhlmann et al, 2003), pilocytic astrocytoma (Gonzalez-Gomez et
al, 2003c; Uhlmann et al, 2003) and ependymomas (Rousseau et al, 2003; Alonso
et al, 2004).
In the present study we
determined the frequency of methylation of three genes: RB1, p14ARF and p16INK4a
in a series of 198 gliomas, including astrocytic, oligodendroglial and
ependymal tumors, and in two normal brain tissue samples, using polymerase
chain reaction (PCR)-based techniques involving sodium bisulfite modification
of DNA (MSP) and sequencing of the PCR products.
II.
Materials and methods
A. Sample collection and DNA preparation
Fresh tumor tissues were
obtained from 198 patients with malignant gliomas, including 16 WHO grade I
pilocytic astrocytomas; 26 WHO grade II diffuse astrocytomas; 23 WHO grade III
anaplastic astrocytomas; 53 WHO grade IV glioblastomas multiformes (43 primary
and 10 secondary); one giant cell astrocytoma; 24 WHO grade II
oligodendrogliomas; 16 WHO grade III anaplastic oligodendrogliomas, six WHO
grade II-III mixed oligo-astrocytomas; two WHO grade I ependymomas, 24 WHO
grade II ependymomas, five anaplastic (WHO grade III) ependymomas, and two
ependymoblastomas (WHO grade IV) as well as two nonneoplastic brain samples.
Tumors were diagnosed according to the WHO guidelines (Kleihues and Cavenee,
2000), and the tumor cell content was estimated by histologic examination to be
approximately 75-90%. DNA was prepared from frozen tissues using standard
methods, as described (Rey et al, 1992).
B.
Bisulfite treatment of DNA, methylation-specific
polymerase chain reaction (MSP) and sequencing
Bisulfite modification of genomic DNA was performed as reported (Herman
et al, 1996). Briefly, 2mg of genomic DNA
was denatured with 2mol/L NaOH (37C for 10 min), followed by incubation with 3mol/L sodium bisulfite (pH
5.0) at 55-56C for 16 hours in
the dark. After treatment, DNA was purified using the DNA clean-up Kit
(Promega, Madison, WI) as recommended by the manufacturer, incubated with
3mol/L NaOH (room temperature for 5 min), precipitated with 10mol/L ammonium
acetate and 100% ethanol, washed with 70% ethanol and re-suspended in 30 ml distilled water. The primer sequences of
these genes for the methylated and unmethylated reactions were as reported
(Xing et al, 1999; Simpson et al, 2000). PCR was performed for the methylated and
unmethylated alleles using a thermal cycler in standard conditions with
variable (55-66C) annealing
temperatures. Each PCR reaction (20ml) was loaded directly onto non-denaturing 6% polyacrylamide gels or
2-3% agarose gels, stained with ethidium bromide, and visualized under UV
illumination. Samples giving signals approximately equivalent to the positive
control were designated as methylated. As positive control for methylated
alleles, we used DNA (from lymphocytes of healthy volunteers) treated with SssI
methyl-transferase (New England Biolabs), then subjected to bisulfite
treatment. To verify the identity of PCR products, they were purified and
sequenced (after PCR re-amplification with the same primer set) using the ABI
PRISM Byg-Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer
Applied Biosystems) on the Applied Biosystem model 3100 or 377 DNA sequencers.
Each amplicon was sequenced bidirectionally.
III. Results
Examples of methylation of all three genes, sequences
from methylated alleles aligned against the corresponding wild type sequence,
and bar diagrams that illustrate the methylation frequency of each gene in the
different histological glioma subtypes are shown in Figure 1.
All but 92 study samples displayed CpG island
hypermethylation in at least one gene (54%). The methylation frequency of the RB1, p14ARF
and p16INK4a genes was
13%, 21%, and 37%, respectively. In contrast, methylation of these genes did
not occur in either of the two normal brain samples. The methylation
frequencies for the three genes in every tumor type studied are shown in Table 1.
RB1 was primarily methylated in
astrocytic and mixed tumors, with frequencies ranging from 13% (in anaplastic
astrocytoma) to 40% in secondary glioblastoma. On the other hand, pure
oligodendrogliomas and ependymomas presented 0-4% aberrant RB1 methylation. In contrast, p14ARF
hypermethylation rates were higher in oligodendroglial tumors (46% and 50%
in low-grade and anaplastic oligodendrogliomas, respectively), and values of
about 20-30% characterized p14ARF
in anaplastic astrocytomas and ependymomas. On the other hand pilocytic
astrocytomas and secondary glioblastoma displayed a 0-6% methylation rate for
this gene. The p16INK4a gene
was hypermethylated by >35% of each subtype of astrocytic tumor, including
pilocytic astrocytomas. However, the methylation rate for this gene was lower
than 25% in pure oligodendrogliomas and ependymal tumors.
As shown in Table
2, simultaneous promoter methylation of two or three genes was demonstrated
in some tumors as follows: RB1 and p16INK4a in 7% (14/198) of
cases, p16INK4a together
with p14ARF in 8% (15/198)
of cases, and RB1 plus p14ARF in 0.5% (1/198) of
cases. Three gliomas showed hypermethylation of all three genes (1.5%).
Promoter methylation of either RB1 or
p16INK4a was present in
41% of samples (82/198).
In most tumors, gene hypermethylation was always
accompanied by amplification of the unmethylated reaction (Figure 1). This finding was expected, since the tumor specimens
were macroscopically isolated samples that contained both tumor and a small
fraction of non-malignant tissues. Alternatively, cytosine hemimethylation,
that is the cytosine on one chromosome is methylated but its homologue on the
other chromosome is not, might also explain the finding. Sequencing of the
corresponding methylated PCR products demonstrated the presence of invariable
CpG as was expected (Figure 1).
IV. Discussion
The results presented here clearly demonstrate that
CpG island hypermethylation of cell-cycle control genes is a frequent event in
gliomas, as methylation of one or more genes was observed in 54% of the
analyzed samples (106 of 198). Our study shows that hypermethylation rates vary
by gene, and that it occurs in most instances at early stages of gliomagenesis,
because it is already present in the low-grade forms. This was primarily
evident for p16INK4a,
which presented methylation rates >40% in low-grade astrocytic tumors such
as pilocytic astrocytoma and WHO grade II tumors. For its part p14ARF was hypermethylated in
<15% of these low-grade astrocytomas.
RB1 is located on the long arm of
chromosome 13 (13q14) and is the classical example of a tumor suppressor gene.
It encodes a nucleoprotein (pRB) that plays a key role in the cell cycle
regulation complexes that govern the G1-S transition of cells, thus allowing
mitosis and cell division (Friend et al, 1986; Lee et al, 1987).
Table
2:
Isolated and concurrent methylation frequencies of cell-cycle control genes in
gliomas
|
Gene
|
Frequency
|
|
RB1
|
13%
|
(26/198)
|
|
p14ARF
|
21%
|
(42/198)
|
|
p16INK4a
|
37%
|
(74/198)
|
|
|
|
|
|
RB1 + p14ARF
|
0.5%
|
(1/198)
|
|
RB1 + p16INK4A
|
7%
|
(14/198)
|
|
p14ARF + p16INK4a
|
8%
|
(15/198)
|
|
RB1 + p14ARF +
p16INK4a
|
1.5%
|
(3/198)
|
|
|
|
|
|
RB1 or p16INK4a
|
41%
|
(82/198)
|
Loss of RB1 function has been described in a variety of tumor types, and
hypermethylation of the promoter is recognized as an important RB1 silencing mechanism (Ohtani-Fujita
et al, 1993). RB1 methylation was
most frequent in astrocytic neoplasms in our tumor series and, in agreement
with previous reports (Nakamura et al, 2001a; Gonzalez-Gomez et al, 2003a), we
found RB1 promoter methylation more
frequently in secondary (40%) than in primary (12%) glioblastomas. The
non-random frequencies (13-35%) of aberrant RB1
methylation that we detected in the low-grade and anaplastic astrocytomas might
be indicative of a subgroup of astrocytic tumors that further develop towards
secondary glioblastoma, thus displaying a more aggressive biological behavior.
In fact, loss of RB1 expression has
been associated with a higher grade of malignancy in several human neoplasms
(Cryns et al, 1994; Tsuda et al, 2000). In oligodendroglial and ependymal
tumors we found very low rates or no methylation of this gene. Since RB1 inactivating mutations are also
infrequent in these tumors (Gonzalez-Gomez et al, 2003a), the inactivation of
the RB1 cell cycle control pathways
should occur through the silencing of another alternative genes. However,
oligo-astrocytomas presented a 17% rate of aberrant RB1 methylation, probably corresponding to the astrocytic component
in these mixed tumors.
Low rates of p14ARF
hypermethylation were found in the pilocytic astrocytoma group (6%) and
low-grade astrocytomas (15%) and this frequency increased slightly in
anaplastic astrocytomas (22%). Loss of p14ARF
expression has been shown to be an important event in the genetic pathways for the
development of both primary and secondary glioblastoma (Nakamura et al, 2001b;
Ghimenti et al, 2003). Although promoter hypermethylation of this gene has been
reported as more frequent in secondary tumors (Nakamura et al, 2001b), we found
21% p14ARF methylation in
primary glioblastoma and no secondary glioblastoma in our series displayed this
anomaly. This finding may be due to the small number of secondary tumors
included in our study; p14ARF
promoter methylation has been proposed as an early event in a subset of
low-grade astrocytomas (as occurs in our tumor series) that may undergo
malignant progression to secondary glioblastoma. Some of these highly malignant
(secondary) tumors display homozygous deletion of p14ARF (Nakamura et al, 2001b). With regard to
oligodendroglial tumors, previous studies on p14ARF methylation have shown variable results: Dong et
al (2001) found 2%, whereas Wolter et al (2001) found 41%, and Watanabe et al
(2001b) detected p14ARF
methylation in 21% of grade II oligodendrogliomas and 15% of the anaplastic
forms. Our data show 46% and 50% in grade II and grade III forms, respectively,
whereas 17% of our mixed tumors showed epigenetic alteration in this gene;
these figures are closer to those we detected in astrocytomas. The epigenetic
inactivation of p14ARF is
a frequent alteration in ependymal tumors, since about 30% of the samples
(low-grade and anaplastic forms) displayed this alteration in our series. These
findings agree with the data recently provided by Rousseau et al (2003), who
detected 21% epigenetic change in this gene in this glioma subtype and, in
agreement with their data, aberrant p14ARF
hypermethylation was more frequently identified in the intracranial ependymal
tumors. An inverse correlation has been reported for p14ARF and TP53 mutations
in glioblastomas (Ichimura et al, 2000). Since oligodendrogliomas and
ependymomas rarely present TP53
inactivating mutations (for review see Kleihues and Cavenee, 2000), silencing
of the p14ARF gene through
aberrant promoter methylation may be a mechanism to inactivate the p14ARF/MDM2/TP53 cell-cycle
signaling pathway in these glioma subtypes. Recent data have demonstrated a
high percentage of p14ARF
inactivation in glioblastomas with classical, astrocytic or oligodendroglial
differentiation areas (Ghimenti et al, 2003), suggesting again that silencing
this gene is an important step in tumorogenesis and/or progression of distinct
glioma subtypes Aberrant methylation was the molecular gene silencing mechanism
in some of those cases.
Oligodendrogliomas and ependymomas showed a lower rate
of p16INK4a promoter
hypermethylation than astrocytic tumors. We found a 25% rate of p16INK4a aberrant
hypermethylation in oligodendrogliomas and a 12.5% rate in the anaplastic
forms, as well as a 25% rate in the low-grade ependymomas. Dong et al, (2001),
Walter et al (2001) and Watanabe et al, (2001) also showed variable results in
methylation studies of this gene. They respectively report rates of 12%, 32%
and 0%. Rates of about 21% aberrant methylation in the p16INK4a gene were identified in ependymomas (Rousseau
et al, 2003; Alonso et al, 2004). Variable frequencies of p16INK4a hypermethylation have also been reported in
secondary glioblastoma, and range from 70% to <5% (Costello et al, 1996; Fueyo et al, 1996;
Hegi et al, 1997; Burns et al, 1998; Nakamura et al, 1998; Schmidt et al, 1998;
Park et al, 2000; Yin et al, 2002; Gonzlaez-Gomez et al, 2003). Our results corroborate the
proposal that a high rate of p16INK4a
CpG island hypermethylation is characteristic of both primary and secondary
glioblastoma subtypes and also of the lower grade astrocytic forms. A 44% rate
for p16INK4a CpG island
hypermethylation was found in the pilocytic astrocytomas included in our
series. The mixed oligo-astrocytoma group was different from the pure
oligodendrogliomas, perhaps due to their astrocytic component. Interestingly,
an association between a worse prognosis and p16INK4a inactivation either by deletion/mutation or
aberrant promoter methylation has been recently reported in oligodendrogliomas
(Bortolotto et al, 2000).
Concurrent aberrant hypermethylation was identified in
several cases; for instance methylation occurred in both p16INK4a
and either RB1 or p14ARF in 7-8% of cases,
whereas only one sample (0.5%) displayed concurrent RB1 plus p14ARF
methylation. Accordingly, promoter hypermethylation of the p14ARF gene seems to be independent of the
methylation status of p16INK4a
even though their promoters are very close to each other on 9p21 and they
share two exons, albeit in different reading frames (Sherr et al, 1996; Kamijo
et al, 1998), and they are frequently co-deleted in glioblastoma (Newcomb et
al, 2000). The transcriptional activity of p14ARF
is regulated independently of p16INK4a
and participates in a regulatory feedback loop with p53 and MDM2 (Kamijo et
al, 1998). The p16INK4a
protein function is closely related to RB1
in cell cycle regulation; it regulates G1-S phase transition by inhibiting the
activity of cyclin-dependent kinases CDK4 and CDK6. This cell-cycle control
pathway was inactivated in 41% of the gliomas we studied, since methylation of
both or either the RB1 or p16INK4a gene occurred. Some
cases in our series displayed a concurrent aberrant hypermethylation of both RB1 and p16INK4a that might represent a redundant epigenetic
alteration of this cell-cycle control pathway.
In conclusion, this study demonstrates that CpG
island methylation of cell-cycle control genes is a common event in gliomas. It
generally occurs at early stages of carcinogenesis, since it is already present
in the low-grade forms. Some differences in the pattern of gene methylation
were observed: hypermethylation of p14ARF
occurred more frequently in oligodendrogliomas and ependymomas than in
astrocytic tumors. On the other hand p16INK4a
and RB1 were frequently methylated in
astrocytic gliomas. In agreement with previous data (Gonzalez-Gomez et al,
2003c; Uhlmann et al, 2003) our findings suggest an important role for
epigenetic changes in the development of pilocytic astrocytoma, a glial tumor
in which no consistent genetic alteration has been identified previously.
Finally, epigenetic inactivation of the cell-cycle control genes in some glioma
subtypes might be indicative or predictive of an aggressive behavior: RB1 or p14ARF in astrocytic tumors (Nakamura et al, 2001a;
2001b), or p16INK4a in
oligodendrogliomas (Bortolotto et al, 2000). Accordingly, therapies addressed
to promoting re-expression of these genes (Swanton, 2004) might be useful in
the management of glioma patients. An accurate glioma-subtype histological
diagnosis together with an inequivocal identification of samples displaying
promoter hyperemethylation, in combination with gene mutational/expression
analyses, will contribute to optimizing the clinical application of molecular
characterization of gliomas; this should lead to a firm establishment of
predictive prognostic factors and specific therapies.
Acknowledgements
This study was supported by Grants 02/0669, 03/0235
from Fondo de Investigaciones Sanitarias (FIS), Ministerio de Sanidad, and
Grant 08.1/0040/2003.1 from Consjeria de Educacion, Comunidad de Madrid, and
grant from Fundaci½n MAPFRE-Medicina. M. Eva Alonso is supported by a
predoctoral felowship from Consejeria de Educacion, Comunidad de Madrid. C.
Casartelli is supported by Fundaao de Amparo a Pesquisa de estado do Sao Paolo
(FAPESP) and Coordenaao de Aperfeioamento de Pesoal de Nivel Superior
(CAPES). We are indebted to Dr J.L. Sarasa and Dr. M. Gutierrez for
histological diagnosis of tumor samples.
References
Alonso ME,
Bello MJ, Arjona D, Gonzalez-Gomez P, Lomas J, de Campos JM, Kusak ME, Isla A,
Rey JA (2002) Analysis of the NF2
gene in oligodendrogliomas and ependymomas. Cancer Genet Cytogenet 134, 1-5.
Alonso ME,
Bello MJ, Gonzalez-Gomez P, Arjona D, de Campos JM, Gutierrez M, Rey JA (2004) Aberrant CpG island methylation of multiple genes in ependymal
tumors. J Neuro-Oncol 67, 159-165.
Alonso ME,
Bello MJ, Gonzalez-Gomez P, Arjona D, Lomas J, De Campos JM, Isla A, Sarasa JL,
Rey JA (2003) Aberrant promoter
methylation of multiple genes in oligodendrogliomas and ependymomas. Cancer Genet Cytogenet 144,134-142.
Baylin SB,
Herman JF, Graff JR, Vertino PM, Issa JP (1998)
Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res 72, 141-196.
Bello MJ,
Leone PE, Nebreda P, de Campos JM, Kusak ME, Vaquero J, Sarasa JL,
Garcia-Miguel P, Queizan A, Hernandez-Moneo JL, Pestaa A, Rey JA (1995) Allelic status of chromosome 1 in
neoplasms of the nervous system. Cancer
Genet Cytogenet 83, 160-164.
Bortolotto S,
Chado.Piat L, Cavalla P, Bosone I, Chio A, Mauro A, Schiffer D (2000) CDKN2A/p16 inactivation in the prognosis of oligodendrogliomas. Int J Cancer 88, 554-557.
Burger PC,
Scheithauer BW, Paulus W, Szymas J, Giannini C, Kleihues P (2000) Pilocytic astrocytoma. In:
Pathology and genetics of tumors of the nervous system. World health
organization classification of tumors. Kleihues P and Cavenee WK (eds). IARC
Press, Lyon, pp 45-51.
Burns KL, Ueki
K, Jhung SL, Koh J, Louis DL (1998)
Molecular genetic correlates of
p16, cdk4, and pRb immunohistochemistry in glioblastomas. J Neuropathol Exp Neurol 57, 122-130.
Costello JF,
Berger MS, Huang HS, Cavenee WK (1996)
Silencing of p16/CDKN2 expression in
human glomas by methylation and chromatin condensation. Cancer Res 56, 2405-2410.
Costello JF,
Plass C, Cavenee WK (2000) Aberrant
methylation of genes in low-grade gliomas. Brain
Tumor Pathol 17, 49-56.
Cryns VL, Thor
A, Xu HJ, Hu SX, Wierman ME, Vickery AL, Benedict WF, Arnold A (1994) Loss of the retinoblastoma
tumor-suppressor gene in parathyroid carcinoma. N Engl J Med 330, 757-761.
Dong S-M, Pang
JC-S, Poon W-S, Hu J, To K-F, Chang AR, Ng H-K (2001) Concurrent hypermethylation of multiple genes is associated
with grade of oligodendroglial tumors. J
Neuropathol Exp Neurol 60, 808-816.
Ebert C, von
Haken M, Meyer-Pullitz B, Wiestler OD, Reifenberger G, Pietsch T, von Deimling
A (1999) Molecular genetic analysis
of ependymal tumors. NF2 mutations and chromosome 22q loss occur preferentially
in intramedullary spinal ependymomas. Am
J Pathol 155, 627-632.
Esteller M (2003) Relevance of DNA Methylation in
the Management of Cancer. Lancet Oncol
4, 351-358.
Friend SH,
Bernards R, Rogelji S, Weinberg RA, Rapaport JM, Alberts DM, Dryja TP (1986) A human DNA segment with
properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 323, 643-646.
Fueyo J,
Gomez-Manzano C, Bruner JM, Saito Y, Zhang B, Zhang W, Levin VA, Yung WK,
Kyritsis AP (1996) Hypermethylation
of the CpG island of p16/CDKN2 correlates with gene inactivation in gliomas. Oncogene 13, 1615-1619.
Ghimenti C,
Fiano V, Chiado-Piat L, Chio A, Cavalla P, Schiffer D (2003) Deregulation of the p14ARF/Mdm2/p53 pathway and G1/S
transition in two glioblastoma sets. J
Neuro-Oncol 61, 95-102.
Gonzalez-Gomez
P, Bello MJ, Alonso ME, Arjona D, Lomas J, De Campos JM, Isla A, Rey JA (2003a) CpG island methylation status
and mutation analysis of the RB1 gene essential promoter region and
protein-binding pocket domain in nervous system tumours. Br J Cancer 88, 109-114.
Gonzalez-Gomez
P, Bello MJ, Arjona D, Lomas J, Alonso ME, de Campos JM, Vaquero J, Isla A,
Gutierrez M, Rey JA (2003b) Promoter
hypermethylation of multiple genes in astrocytic gliomas. Int J Oncol 22, 601-608.
Gonzalez-Gomez
P, Bello MJ, Lomas J, Arjona D, Alonso ME, Aminoso C, De Campos JM, Vaquero J,
Sarasa JL, Casartelli C, Rey JA (2003c)
Epigenetic Changes in Pilocytic Astrocytomas and Medulloblastomas. Int J Mol Med 11, 655-660.
Hayes VM,
Dirven CM, Dam A, Verlind E, Molenaar WM, Mooij JJ, Hofstra RM, Buys CH (1999) High freqeuncy of TP53 mutations
in juvenile pilocytic astrocytomas indicates role of TP53 in the development of
this tumors. Brain Pathol 9,
463-467.
Hegi ME, zur
Hausen A, Ruedi D, Malin G, Kleihues P (1997)
Hemizygous or homozygous deletion of the chromosomal region containing the p16INK4A
gene is associated with amplification of the EGF receptor gene in
glioblastomas. Int J Cancer 73,
57-63.
Herman JG,
Graff JR, Myohanen S, Nelkin BD, Baylin SB (1996) Methylation-specific PCR: a novel PCR assay for methylation
status of CpG islands. Proc Natl Acad
Sci USA 93, 9821-9826.
Hong C, Bollen
AW, Costello JF (2003) The contribution
of genetic and epigenetic mechanisms to gene silencing in olgodendrogliomas. Cancer Res 63, 7600-7605.
Hulsebos TJM,
Oskam NT, Bijleveld EH, Westerveld A, Hermsen MA, van den Oweland AMW, Hamel
BC, Tijssen CC (1999) Evidence for
an ependymoma tumor suppressor gene in chromosome region 22pter-22q11.2. Br J Cancer 81, 1150-1154.
Ichimura K,
Bolin MB, Goike HM, Schmidt EE, Moshref A, Collins VP (2000) Deregulation of the p14ARF/MDM2/p53 pathway is a prerequisite
for human astrocytic gliomas with G1-S transition control gene abnormalities. Cancer Res 60, 417-424.
Kamijo T,
Weber JD, Zambetti G, Zindy F, Roussel MF, Sher CJ (1998) Functional and phisical interactions of the ARF tumor
suppressor with p53 and Mdm2. Proc Natl
Acad Sci USA 95, 8292-8297.
Karnes PS,
Tran TN, Cui MY, Raffel C, Gilles FH, Barranger JA, Ying KL (1992) Cytogenetic analysis of 39
pediatric central nervous system tumors. Cancer
Genet Cytogenet 92, 169-174.
Kleihues P,
Kavenee WK (2000) Pathology and
genetics of tumors of the nervous system. World health organization
classification of tumors. IARC Press, Lyon.
Kraus JA, de
Millas W, Srensen N, Herbold C, Schichor C, Tonn JC, Wiestler OD, von Deimling
A, Pietsch T (2001) Indications for
a tumor suppressor gene at 22q11 involved in the pathogenesis of ependymal
tumors and distinct from hSNF5/INI1. Acta Neuropathol 102, 69-74.
Lee WH, Shew
JY, Hong F, Sery T, Donoso LA, Young LJ, Bookstein R, Lee EYHP (1987) The retinoblastoma susceptibility
gene product is a nuclear phosphoprotein associated with DNA binding activity. Nature 329, 642-645.
Nakamura M,
Konishi N, Hiasa Y, Tsunoda, Nakase H, Tsuzuki T, Aoki H, Sakitani H, Inui T,
Sasaki T (1998) Frequent alterations
of cell-cycle regulators in astrocytic tumors as detected by molecular genetic
and immunohistochemical analyses. Brain
Tumor Pathol 15, 83-88.
Nakamura M,
Watanabe T, Klangby U, Asker C, Wiman K, Yonekawa Y, Kleihues P, Ohgaki H (2001b) p14ARF deletion and
methylation in genetic pathways to glioblastomas. Brain Pathol 11,159-168.
Nakamura M,
Yonekawa Y, Kleihues P, Ohgaki H (2001a)
Promoter hypermethylation of the RB1 gene in glioblastomas. Lab Invest 81,77-82.
Newcomb EW,
Alonso M, Sung T, Miller DC (2000)
Incidence of p14ARF gene
deletion in high-grade adult and pediatric astrocytomas. Hum Pathol 31, 115-119.
Ohtani-Fujita
N, Fujita T, Aoike A, Osifchin NE, Robbins PD, Sakai T (1993) CpG island methylation inactivates the promoter activity of
the human retinoblastoma tumor suppressor gene. Oncogene 8, 1063.1067.
Park S-H, Jung
KC, Ro JY, Kang GH, Khang SK (2000)
CpG island methylation of p16 is associated with absence of p16 expression in
glioblastomas. J Korean Med Sci 15,
555-559.
Rey JA, Bello
MJ, de Campos JM, Kusak ME, Moreno S (1987)
Chromosomal composition of a series of 22 human low-grade gliomas. Cancer Genet Cytogenet 29, 223-237.
Rey JA, Bello
MJ, Jimeze-Lara A, Vaquero J, Kusak ME, de Campos JM, Sarasa JL, Pestaa A (1992) Loss of heterozygosity for distal
markers on 22q in human gliomas. Int J
Cancer 51, 703-706.
Rousseau E,
Ruchoux MM, Scaravilli F, Chapon F, Vinchon M, De Smet C, Godfrain C, Vikkula M
(2003) CDKN2A, CDKN2B and p14ARF are frequently and
differently methylated in ependymal tumours. Neuropathol Applied Neurobiol 29, 574-583.
Rousseau-Merk
M, Versteege I, Zattara-Cannoni H, Figarella D, Lena G, Aurias A, Vagner-
Capodano A (2000) Fluorescence in
situ hybridization determination of 22q12-q13 deletion in two intracerebral
ependymomas. Cancer Genet Cytogenet
121, 223-227.
Rubio MP,
Correa KM, Ramesh V, MacCollin MM, Jacoby LB, von Deimling A, Gusella JF, Louis
DN (1994) Analysis of the
neurofibromatosis 2 gene in human ependymomas and astrocytomas. Cancer Res 54, 45-47.
Sanoudpu D,
Tingby O, Ferguson-Smith MA, Collins VP, Coleman N (2000) Analysis of pilocytic astrocytoma by comparative genomic
hybridization. Br J Cancer 82,
1218-1222.
Schmidt EE,
Ichimura K, Messerle KR, Goike HM, Collins VP (1997) Infrequent methylation of CDKN2A(MTS1/p16)
and rare mutation of both CDKN2A and CDKN2B(MTS2/p15) in primary astrocytic
tumors. Br J Cancer 75, 2-8.
Sher CJ (1996) Cancer cell cycles. Science 274, 1672-1677.
Simpson DJ,
Hibberts NA, McNicol AM, Clayton RN, Farrell WE (2000) Loss of pRb expression in pituitary adenomas is associated
with methylation of the RB1 CpG island. Cancer
Res 60, 1211-1216.
Surawicz TS,
Davis F, Freels S, Laws ER, Menk HR
(1998) Brain tumor survival:
Results from the National Cancer Data base. J Neuro-Oncol 40, 151-160.
Swanton C (2004) Cell-cycle targeted therapies. The Lancet Oncology 5, 27-36.
Tada K, Kochi
M, Saya H, Kuratsu J, Shiraishi S, Kamiryo T, Shinohima N, Ushio Y (2003) Preliminary observations on
genetic alterations in pilocytic astrocytomas associated with neurofibromatosis
1. Neuro-Oncol 5, 228-2234.
Tsuda H,
Yamamoto K, Inoue T, Uchiyama I, Umesaki N (2000) The role of p16-cyclin D/CDK-pRb pathway in the tumorigenesis
of endometroid-type endometrial carcinoma. Br
J Cancer 82, 675-682.
Uhlmann K,
Rohde K, Zeller C, Szymas J, Vogel S, Marczinek K, Thiel G, Nrnberg P, Laird
PW (2003) Distinct methylation
profiles of glioma subtypes. Int J
Cancer 106, 52-59.
Von Deimling
A, Louis DN, Menon AG, von Ammon K, Ellison D, Wiestler OD, Seizinger BR (1993) Deletions on the long arm of
chromosome 17 in pilocytic astrocytoma. Acta
Neuropathol 86, 81-85.
Watanabe T,
Nakamura M, Yonekawa Y, Kleihues P, Ohgaki H (2001a) Promoter hypermethylation and homozygous deletion of the p14ARF and p16INK4a genes in
oligodendrogliomas. Acta Neuropathol 101,
185-189.
Watanabe T,
Yokoo H, Yokoo M, Yonekawa Y, Kleihues P, Ohgaki H (2001b) Concurrent inactivation of RB1 and TP53 pathways in
anaplastic oligodendrogliomas. J
Neuropathol Exp Neurol 60, 1181-1189.
Weremowicz S,
Kupsky WJ, Morton CC, Fletcher JA (1992)
Cytogenetic evidence for a chromosome 22 tumor suppressor gene in ependymoma. Cancer Genet Cytogenet 61, 193-196.
White FV,
Anthony DC, Yunis EJ, Tarbel NJ, Scott RM, Schofield DE (1995) Nonrandom chromosomal gains in pilocytic astrocytomas of
childhood. Hum Pathol 26, 979-986.
Wolter M,
Reifenberger J, Blaschke B, Ichimura K, Schmidt EE, Collins VP, Reifenberger G
(2001) Oligodendroglial tumors
frequently demonstrate hypermethylation of the CDKN2A (MTS1, p16INK4a), p14ARF, and CDKN2B (MTS2, p15INK4b)
tumor suppressor genes. J Neuropathol
Exp Neurol 60, 1170-1180.
Xing EP, Nie
Y, Song Y, Yang G-Y, Cai YC, Wang L-D, Yang CS (1999) Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a
genes in human esophageal squamous cell carcinoma. Clin Cancer Res 5, 2704-2713.
Yin D, Xie D,
Hofman W-K, Miller CW, Black KL, Koeffler HP (2002) Methylation, expression, and mutation analysis of the cell
cycle control genes in human brain tumors. Oncogene
21, 8372-8373.
