SSU 748

Seminars in Surgical Oncology 1998; 14:3–12

Molecular Changes During the Genesis of

Human Gliomas
ANIL SEHGAL, PhD*
Deke Slayton Center for Brain Cancer Studies and Pacific Northwest Cancer Foundation,
Northwest Hospital, Seattle, Washington

Neoplastic transformation in the normal human brain occurs as a result of the accumulation
of a series of genetic alterations. These genetic alterations include the loss, gain or amplifica-
tion of different chromosomes which lead to altered expression of proteins that play impor-
tant roles in the regulation of cell proliferation. Several common genetic alterations at the
chromosomal level (loss of 17p, 13q, 9p, 19, 10, 22q, 18q and amplification of 7 and 12q)
have been observed. These alterations lead to changes in the expression of several genes;
protein 53 (p53), retinoblastoma (RB), interferon (INF)α/β, cyclic AMP dependent kinase
number 2 (CDKN2), mutated in multiple advanced cancers 1 (MMAC1), deleted-in-colon
carcinoma (DCC), epidermal growth factor receptor (EGFR), platelet derived growth factor
(PDGF), platelet derived growth factor receptor (PDGFR), MDM2, GLI, CDK4 and SAS
during the genesis and progression of human gliomas. Recent studies suggest that altered
expression of several other genes [MET; MYC; transforming growth factor β (TGFβ); CD44;
vascular endothelial growth factor (VEGF); human neuroglial-related cell adhesion molecule
(hNr-CAM); neuroglial cell adhesion molecule (NCAM L1); p21waf1/Cip1; TRKA; mismatch
repair genes (MMR); C4-2; D2-2] and proteins [e.g., cathepsins, tenascin, matrix
metalloproteases, tissue inhibitors of metalloproteases, nitric oxide synthase, integrins,
interleukin-13 receptor (IL-13R), Connexin43, urokinase-type plasminogen activator recep-
tors (uPARs), extracellular matrix proteins and heat shock proteins] are associated with the
genesis of human gliomas. Taken together, these findings point to the accumulation of mul-
tiple genetic mutations coupled with extensive changes in gene expression in the etiology of
human gliomas. Semin. Surg. Oncol. 14:3–12, 1998. © 1998 Wiley-Liss, Inc.

KEY WORDS: brain neoplasms/etiology; glioma; human chromosomes; chromosome

deletion; genes; gene deletion; genome; mutation; gene expression;
astrocytoma

INTRODUCTION are the most frequent brain tumors of children between

Brain tumors are one of the leading causes of death the ages of 8 and 13 years. It is a slowly growing lesion
among young children and adults. In 1996, 13,300 people that rarely undergoes progression to neoplasia.
died of brain tumors [1] and recent statistics estimate that 2) Low-grade astrocytomas (WHO grade II), on the other
17,900 new cases of brain tumors will be diagnosed in 1997 hand, manifest themselves in adults between 30 and 40
[2]. This increased incidence of brain tumors is evident in years of age. The low-grade astrocytomas account for 25%
both young children and adults [3]. of all gliomas and are more infiltrative in nature.
Gliomas are the most common of the primary brain tu- Gemistocytic and fibrillary astrocytomas are the two ma-
mors and account for more than 40% of all central ner- jor forms of low-grade astrocytomas.
vous system neoplasms [4]. For practical purposes, various 3) Anaplastic malignant astrocytomas (WHO grade III)
histopathological characteristics of gliomas are condensed are highly malignant gliomas with increased cellularity,
into a grading scheme. According to the World Health
Organization (WHO), four major grades of gliomas are
defined [5].
*Correspondence to: Anil Sehgal, PhD, Pacific Northwest Cancer Foun-
1) Astrocytomas are predominantly composed of neo- dation, 120 Northgate Plaza, Room 230, Seattle, WA 98125-7001 USA.
plastic astrocytes. Pilocytic astrocytomas (WHO grade I) Fax: (206) 368-3009. E-mail: asehgal@nwhsea.org

© 1998 Wiley-Liss, Inc.

4 Sehgal

pleomorphism and nuclear atypia. Anaplastic astrocytomas esis can occur either at the chromosomal level or at the
show an increased tendency to progress to glioblastoma. gene expression level. Furthermore, changes at the genetic
4) Glioblastoma multiforme (GM)(WHO grade IV) is a level can be either due to the loss of a major portion of a
highly malignant brain tumor and typically affects adults chromosome or due to point mutations within a single gene.
between 45 and 60 years of age. GMs are composed of In this review, the role of several genes (at the chromo-
poorly differentiated, fusiform round or pleomorphic cells. somal or at the expression level) that are shown to be in-
Mitotic activity in these tumors is very high (5-25%). In- volved in the process of initiation and progression of human
creased necrosis and vascular endothelial proliferation are gliomas is discussed.
two major histological markers for GM diagnosis [4]. The
poor prognosis of patients with GMs is largely due to the MOLECULAR EVENTS DURING THE GENESIS
spread of tumor cells to other regions of the brain. Two OF GRADE I AND GRADE II ASTROCYTOMAS
major pathways for the occurrence of GMs have been pro- There are three major events associated with the devel-
posed [4,6,7]; first, GMs may develop rapidly without any opment of early grade astrocytomas.
precursor lesion in a de novo fashion, and second, GMs
may develop slowly by progression from a less malignant p53 Mutation and 17p Loss
precursor lesion. The p53 gene was discovered in 1979 [13]. Molecular
Although gliomas are thought to be derived from astro- cloning and functional analysis indicated that p53 could
cytomas, oligodendrocytes or ependymal cells, they dis- transform cells in culture, particularly in combination with
play a broad spectrum of histopathological features. The other oncoproteins such as RAS, and could elicit tumor
variation in the phenotype and biological behavior of formation in transgenic animals [14]; this indicated that
gliomas reflects the type of transformation genes operative p53 is a dominantly acting oncogene. Later experiments
in neoplastic development [4]. Thus, in order to understand demonstrated that it was the mutated form of p53 rather
the pathological changes during the genesis of human glio- than the normal p53 that accumulates in tumor cells [15].
mas, it is imperative to understand changes that take place Further experiments clearly demonstrated that over-
at the cellular and molecular levels. expressionof wild type p53 caused growth suppression of
It is estimated that the human genome has approximately tumors. The p53 gene has been localized to the p13 re-
60,000–70,000 genes, and approximately 30,000 of these gion of the chromosome 17 [16]. This gene is found only
are expressed in the brain [8]. This fact underscores the in vertebrates and the sequence is highly conserved. It has
complexity of cellular functions, particularly of cellular been demonstrated that the tumor suppressor function of
proliferation. The specific role of a number of genes and p53 requires its localization to the nucleus. The wild type
their protein products have been documented in normal p53 binds to DNA and regulates the transcription of sev-
and tumors cells [9]. In the brain, the most abundant genes eral genes [17].
are those involved in structural and regulatory function In human tumors, over 200 naturally occurring muta-
(transcription factors and signal transduction)[8]. In the con- tions have been described for p53, e.g., missense, frame-
text of tumorigenesis, genes are divided into two main cat- shift, premature termination, and deletion mutations [18].
egories, oncogenes and tumor suppressor genes. Oncogenes Sequence analysis indicates that there are no brain spe-
are those genes that when mutated and/or overexpressed in cific mutations in p53, but mutation at codon 273 is the
normal cells cause the cells to become tumorigenic [10]. most common [19]. The analysis of 75 families with p53
Tumor suppressor genes, on the other hand, are genes that germline mutations revealed that brain tumors accounted
play an important role in normal cell growth, differentia-
for 13% of the neoplasms. Germline mutation analysis also
tion and progression through the cell cycle. The loss of
indicated that sporadic brain tumors, in which p53 is mu-
tumor suppressor gene function can be responsible for ab-
tated, are largely restricted to astrocytic origin. On the ba-
errant cell proliferation. Insertion and expression of tumor
suppressor genes in tumor cells causes them to become sis of extensive mutation analysis, it was concluded that
non-tumorigenic. More than 50 oncogenes and tumor sup- p53 mutations in brain tumors are involved both in the
pressor genes have been isolated and characterized [11]. initiation and progression of astrocytic tumors [4]. The p53
Recent advances in cell and molecular biology techniques mutations in tumors of other origin (colon, pancreas and
have led to the identification not only of chromosomal aber- esophagus) are shown to play an important role in tumor
rations but also of oncogenes and tumor suppressor genes progression as opposed to tumor initiation. Several stud-
associated with these aberrations and the initiation or pro- ies have reported that one of the earliest detectable genetic
gression of brain tumorigenesis. events in the tumorigenesis of astrocytomas is the muta-
Brain tumors like other tumors types arise as a result of tion of p53. The allelic loss of chromosome 17p is ob-
gradual accumulation of several genetic aberrations in pre- served in approximately one-third of WHO grades II–IV
cursor cells [12]. Genetic changes during brain tumorigen- adult astrocytomas [12]; thus the conclusion that inactiva-
 Molecular Change During Glioma Genesis 5

tion of p53 is an important event in the genesis of low- of glioblastoma. In the majority of gliomas, the
grade astrocytomas. In a study of 31 non-glioblastoma as- overexpression of PDGFR was not due to gene amplifica-
trocytomas it was demonstrated that mutated p53 tion. In a few cases where amplification was observed, it
accumulated in 71% of juvenile pilocytic astrocytomas resulted in deletion of the extracellular domain, and po-
(WHO grade I), 63% of astrocytomas (WHO grade II) and tentially, a constitutively activated PDGFR.
63% of anaplastic astrocytomas (WHO grade III). These
data suggest that mechanisms other than p53 gene inacti- Loss of Chromosome 22q
vation by mutation result in the accumulation of p53 in Allelic loss of chromosome 22q has been observed in
non-glioblastoma astrocytomas. The p53 protein is in- 20–30% of astrocytomas and it is thought to be an impor-
volved not only in the early but also in the late events of tant event in the early stages of astrocytoma progression
brain tumorigenesis such as angiogenesis. In a recent [24]. The potential tumor suppressor gene responsible has
study, it was shown that adenovirus mediated transfer not been cloned, but the NF2 gene located on the same chro-
of the p53 gene produces rapid and generalized death mosome is not involved in genesis of astrocytomas [25].
of human glioma cells via apoptosis [20]. On the basis of
studies mentioned above it is clear that wild type p53 plays MOLECULAR EVENTS DURING GENESIS
an important role as a tumor suppressor gene and its muta- OF ANAPLASTIC ASTROCYTOMAS
tion is a frequent early initiation event in the genesis of (WHO GRADE III)
astrocytomas.
Anaplastic astrocytomas occur typically in individuals
Overexpression of Platelet Derived Growth Factor between 40 and 50 years of age. These tumors are highly
(PDGF) and PDGF Receptor (PDGFR) cellular and they exhibit extensive morphological hetero-
geneity. Several molecular events play an important role
PDGF was originally discovered as a platelet α-gran- in the progression of early grade astrocytomas to anaplas-
ule release product [21]. PDGF is a 30 KD protein con- tic astrocytomas. These include RB gene mutation and 13q
sisting of disulfide bonded A- or B-chains thus forming loss; loss of chromosome 9p; and loss of chromosome 19q.
AA or BB homodimers or AB heterodimers. The genes
for PDGF A- and B-chains are located on human chro- RB Gene Mutation and 13q Loss
mosomes 7p22 and 22q12.3-13.1, respectively. Two
The RB (retinoblastoma) locus was originally identified
types of PDGF receptors (PDGFRα and -β) have been
as a locus commonly mutated in children with retinoblas-
identified. Both belong to the protein-tyrosine kinase
toma tumors [26]. The gene product of RB, pRB, is an ubiq-
family of growth factor receptors. The α- receptor binds
uitously expressed nuclear protein that is phosphorylated
both PDGF AA and PDGF AB whereas the β-receptor
in synchrony with specific cell cycle transition [27]. Mi-
binds only to PDGF BB. PDGF and PDGFR are ex-
croinjection of the RB protein inhibits cell cycle progres-
pressed in a variety of tissues and are shown to play an
sion through the G1 phase; this clearly indicates that it is a
important role in embryonic development [21]. Several
tumor suppressor gene. The phosphorylation state of the
human malignant gliomas and astrocytomas coexpress
RB protein is very important not only for its association
high levels of both PDGF and PDGFR as compared to
with other proteins but also for its cell cycle controlling
normal brain [22]. activity [28]. Two cyclin dependent protein kinases (CDK4
In situ hybridization and immunohistochemical studies and CDK6) can phosphorylate RB to control its function.
have demonstrated a role for PDGF-A and PDGFRα in It has been suggested that inhibitors of CDKs (such as p15
tumor cell proliferation; however, PDGF B is thought to and p16) can indirectly regulate the RB function. Analysis
promote tumor angiogenesis. RNA analysis of a collec- of anaplastic astrocytomas indicates that 50% of these tu-
tion of astrocytoma and glioblastoma tumors and cell lines mors show allelic loss of 13q [29]. Molecular analysis of
have demonstrated that both PDGF AA and PDGF BB different regions of chromosome 13 revealed that it is the
are expressed at high levels. It has been demonstrated in RB1 locus (13q14) that is commonly affected in brain tu-
another study that several glioma cell lines express high mors. Approximately 30% of the high-grade astrocytomas
levels of both A- and B-chain mRNA. The most abundant showed loss of heterogeneity on the RB1 locus. Thus, loss
product in these tumor cell lines was PDGF AA as com- of RB function can lead to the generation of retinoblasto-
pared to PDGF AB or PDGF BB [23]. In situ analysis mas and astrocytomas [30].
of various grades of astrocytic tumors demonstrated that
expression of PDGF AA and PDGF BB is highest in Loss of Chromosome 9p
glioblastoma multiforme as compared to low-grade as- Analysis of primary anaplastic astrocytomas and glio-
trocytomas [23]. PDGFRα was also detected at high blastomas revealed a frequent loss of one or both type I
levels in GMs and PDGFRβ in capillary endothelium interferon complements (INFβ or INFα) in 50% of the
6 Sehgal

cases [31]. It has been suggested that loss of one allele for localized this putative tumor suppressor gene to the
9p is usually seen in anaplastic astrocytomas, whereas both 19q13.2-13.3 region [39].
the alleles are lost in glioblastomas [32]. Thus, the dosage
effect of IFNs may be important in controlling transition The 12q Amplicon
from early-grade to high-grade astrocytomas. This may in- The 12q region of human chromosome 12 (which has
dicate that these two genes can act as tumor suppressor several genes: D12S8, MDM2, CDK4, SAS, GADD, GLI
genes. In fact, transfer of the INFβ gene into cultured and A2MR) undergoes amplification during the genesis of
glioma cells results in marked growth suppression [33]. late stages of anaplastic astrocytomas and early stages of
Alternatively, deletions in 9p has been narrowed down to glioblastomas [40]. In a recent study, 234 tumors were
the 9p21-p22 region. This indicates that there could be analyzed and 19 were found to show amplification of
another candidate tumor suppressor gene in this region of 12q13-14 region. Approximately 15% of glioblastomas and
the chromosome 9. 17% anaplastic astrocytomas showed 12q13-14 region
Recently, a candidate tumor suppressor gene, CDKN2, amplification. The MDM2 gene is located on chromosome
has been localized to the 9p21 region of the chromosome 12q13-14 and it is found to be amplified in 10% of the
9. This gene plays an important role in cell cycle progres- anaplastic astrocytomas and glioblastomas. The MDM2
sion [34]. CDKN2 encodes a 156 amino acid, 16 KD cell gene product regulates p53 protein function by binding and
cycle inhibitor protein, which normally blocks abnormal inactivating its transcriptional activity. Tumors that show
cell growth and proliferation by binding to complexes of amplification of the MDM2 gene lack p53 mutations. The
cyclin-dependent kinases (CDK4) and cyclin D. This bind- degree of MDM2 amplification ranged between eight and
ing leads to inhibition of CDK and arrest of the cell cycle 70 times higher in astrocytomas and glioblastomas as com-
in the G1 phase. Mutant p16 does not form these com- pared to normal tissue [41]. In addition, the GLI gene has
plexes, thus leading to uncontrolled cell proliferation. The been mapped to this region and is shown to be amplified
importance of this gene product is demonstrated by the and overexpressed in glioblastoma [42]. Other genes in
fact that it is mutated in a number of different tumors types this region include CDK4, SAS and GADD153, all of which
[35]. Transfection of CDKN2 gene into glioma cell lines were overexpressed at the mRNA level, and their gene
resulted in the suppression of cell proliferation which amplification status is currently under investigation.
clearly supports the fact that CDKN2 can function as a
tumor suppressor gene. The mutated form of CDKN2 MOLECULAR EVENTS DURING GENESIS
is non-functional and thus leads to accumulation of RB OF GLIOBLASTOMA MULTIFORME
in its growth inhibitory dephosphorylated form. Ho- (WHO GRADE IV)
mozygous deletion of the CDKN2 gene has been ob- The glioblastoma multiforme (GM) tumor (WHO grade
served frequently in primary astrocytomas (14 of 46) IV) occurs typically between the ages of 45 and 60 years
and glioblastomas [36]. In addition to the CDKN2, the of age. Histologically, these tumors show marked cyto-
9p21 locus has another that codes for a p15 protein. logical diversity with poorly differentiated, fusiform, round,
The p15 protein can act as a potential inhibitor of CDK4 or pleomorphic cells and multinucleated giant cells. Poor
kinase that regulates G1 progression through the cell prognosis of patients with GMs is due essentially to rapid
cycle. One study demonstrated that 27 of 38 glioblas- tumor growth and spreading to other regions of the brain.
toma tumors had deletions in the p15 gene [37]. This GMs also show a high level of necrosis and vascular pro-
suggests that intact expression of p15, like p16, is im- liferation, and they may develop rapidly in de novo fash-
portant in the normal cell proliferation. On the basis of the ion or more slowly from a malignant precursor lesion.
above information, it appears that the loss of chromosome Extensive work has been carried out to identify the mo-
9 is an important step in the progression of low-grade as- lecular events involved in the progression of anaplastic
trocytomas to anaplastic astrocytomas. astrocytomas to GMs [4]. These include the loss of chro-
mosome 10 and overexpression and/or rearrangement of
Loss of Chromosome 19 the epidermal growth factor receptor (EGFR) gene.
Loss of chromosome 19 has been observed very fre-
quently (up to 40%) in human gliomas. In a study of 161 Loss of Chromosome 10
gliomas from 156 patients, it was demonstrated that loss Cytogenetic analysis of glioblastomas has shown that
of heterozygosity of 19q was observed in 3/19 (WHO grade monosomy of chromosome 10 is very frequent [43]. Al-
II), 12/27 anaplastic astrocytomas (WHO grade III) and lelic loss of chromosome 10 occurs in more than 90% of
16/76 cases of glioblastoma multiforme [38]. Results from glioblastomas but rarely in low-grade astrocytomas [44]
this and several other studies suggest the presence of a and this suggests the presence of a putative tumor sup-
tumor suppressor gene on chromosome 19. Deletion pressor gene on this chromosome. Identification of this
mapping analysis of the long arm of chromosome 9 has putative tumor suppressor gene has been difficult since, in
 Molecular Change During Glioma Genesis 7

most glioblastomas, the entire chromosome is lost. It has clearly indicate that EGFR amplification and rearrange-
been demonstrated that the introduction of a copy of chro- ment appears to be a frequent event in the genesis of glio-
mosome 10 by microcell-mediated transfer into brain tu- blastomas.
mor cell lines resulted in suppression of the transformed
and tumorigenic phenotypes in vivo and in vitro. To iden- Loss of Deleted-in-Colon Carcinoma (DCC)
tify the tumor suppressor gene involved in glioblastoma Expression
progression, several cytogenetic experiments were per- The DCC gene is a tumor suppressor gene that was iso-
formed. As a result, two suppressive regions on chromo- lated in colorectal cancers and is located on chromosome
some 10 (10pter-q11 and 10q24-q26) were identified [45]. 18q21 [56]. The DCC protein is a transmembrane cell ad-
Recently, a gene (MMAC1) was identified that transcribes hesion molecule of the neural cell adhesion molecule
into a 5.5-Kb mRNA [46]. The predicted MMAC1 protein (NCAM) family. Loss of the DCC locus has been found in
contains motifs that have significant homology to the cata- 50% of large adenomas, 70% of carcinomas and 100% of
lytic domain of protein phosphatases and to the cytoskeletal hepatic metastasis. In a recent study, a panel of 57 human
proteins, tenacin and auxilin. Sequence analysis of MMAC1 gliomas were analyzed for the DCC locus deletion. Re-
did show mutations in a number of gliomas and other tu- sults from this study showed that 53% of high-grade glio-
mor types [46]. On the basis of information described blastomas and 7% of low-grade gliomas showed deletion
above, it appears that loss of chromosome 10 is one of the of the DCC locus [57]. These findings suggested that DCC
most frequent events involved in progression of anaplas- expression is preferentially, but not exclusively, lost in the
tic astrocytomas to GMs. genetic pathway to glioblastomas.
EGFR Overexpression Overexpression of D2-2
EGFR is a class I tyrosine kinase transmembrane gly- Using the technique of differential display-polymerase
coprotein that binds epidermal growth factor and trans- chain reaction (DD-PCR), a novel gene was isolated (D2-
forming growth factor α. EGFR is thought to play an 2) that is overexpressed in glioblastoma multiforme tissue
important role in normal cell proliferation [47]; it is en- (GMT) as compared to normal brain tissue (NBT)[57]. D2-
coded by the c-erbB gene, and has a role in the genesis of 2 is also expressed at high levels in recurrent glioma, co-
certain cancers. The human EGFR gene has been local- lon tumor metastatic to brain, breast tumors, prostate
ized to chromosome 7p11-p12 [48]. In the past few years, tumors and a prostate tumor cell line (LNCAP). Northern
several studies have demonstrated that the EGFR is blot analysis showed that D2-2 is expressed at high levels
overexpressed in glioblastomas [49]. Cytogenetic analy- in several tumor cell lines (MOLT lymphoblastic leuke-
sis of these tumors demonstrated that as compared to nor- mia, SW480 colorectal adrenocarcinoma, A549 lung car-
mal cells, the EGFR gene is amplified several times in cinoma, HL-60 promyelocytic leukemia, S3 HeLa cells,
tumor cells. More than 300 cases of glioblastomas (WHO K-562 chronic myelogeneous leukemia, G361 melanoma)
grade IV) have been examined and the cumulative inci- as compared to NBT. Additionally, D2-2 is expressed at
dence of EGFR amplification is about 40% [50]. EGFR very high levels in cell lines derived from glioblastomas,
copy number is amplified 20 times in most of the glioblas- grade IV astrocytomas, normal human fetal astrocytes
tomas and can be detected in extrachromosomal double (NHFA) and glioma. D2-2 is expressed at moderate levels
minutes. Approximately 33% of glioblastomas with EGFR in neuroblastoma, neuroectodermal and medulloblastoma
amplification also have EGFR gene rearrangements, some tumor cell lines. D2-2 expression is localized to frontal
of which result in a protein that is very similar to the viral lobe, occipital lobe and the cerebellum of the normal brain.
erbB-1 oncogene. One of the most common rearrangements Normal tissues such as thyroid, stomach, adrenal cortex,
observed in the glioblastomas’ EGFR is the deletion of small intestine and pancreas show high expression of D2-
exons 2-7 [51]. This results in deletion of a majority of the 2. D2-2 is expressed 28 times higher in fetal brain (20
extracellular domain of the receptor and is observed in 30- weeks) than in adult brain [58]. Sequence analysis of a
50% of gliomas [52]. As a result of this deletion, the trun- 2.0-Kb fragment for D2-2 shows no homology to known
cated receptor is constitutively active, constantly sending sequences in the database. This indicates that D2-2 can
signals to the nucleus initiating cell proliferation of tumor serve as a novel marker for early detection and perhaps
cells. It has been shown that transfection of normal cells therapy of GMs.
with this truncated EGFR leads to ligand independent cell
transformation [53]. Molecular analysis of EGFR deletion Loss of C4-2(ARPP-16/19) Expression
has recently revealed creation of a new epitope that is highly Clone C4-2 was also isolated using the technique of DD-
tumor specific [54]. Monoclonal antibodies specific to this PCR. It is expressed in NBT but not in GMT [59]. The
epitope are currently being developed for diagnostic and coding region of C4-2 has 94% sequence identity to the
therapeutic applications [55]. The studies discussed above previously isolated ARPP-16 (cAMP-regulated phospho-
8 Sehgal

protein of Mr = 16,000)[60]. C4-2 is expressed at high Other Genes

levels in normal brain and is either not expressed or ex- In addition to genetic alterations described above, sev-
pressed at low levels in several brain tumor cell lines. Fur- eral other genes are shown to be overexpressed or
ther, C4-2 was either not expressed or expressed at low underexpressed in glioblastomas (Table I, Fig. 1). These
levels in meningioma, B-cell lymphoma, recurrent glioma, include MET [65], MYC [66], TGFβ [67], CD44 [68], vas-
LNCAP (prostate tumor cell line), breast tumor or pros- cular endothelial growth factor (VEGF)[69], neuroglial cell
tate tumor tissue. Expression analysis of C4-2 indicates
that it may function as a potential tumor suppressor gene.
TABLE I. Expression Pattern of Genes During the Genesis
Overexpression of hNr-CAM of Human Gliomas

Using the technique of DD-PCR, another cDNA frag- Genes/chromosome Status Reference
ment was isolated that is overexpressed in GMT as com- p53/17p Mutation [18]
PDGF/7p22 Overexpression [22]
pared to NBT. Sequence analysis indicated that this
PDGFR Overexpression [22]
sequence is identical to the previously isolated human Chromosome 22q Loss [24]
neuron-glial related cell adhesion molecule, hNr-CAM (un- RB/13q Loss [29]
published observations). Gene specific reverse transcrip- IFNα/9p21 Loss [31]
tion-polymerase chain reaction (RT-PCR) analysis CDKN2/9p21 Loss [36]
Chromosome 19 Loss [38]
indicated that hNr-CAM is overexpressed in high-grade
MDM2/12q Amplification [41]
astrocytomas, gliomas, and glioblastoma tumor tissues as GLI Amplification [42]
compared to normal brain tissue. High levels of hNr-CAM MMAC1/10q Loss [46]
expression were also observed in cell lines derived from EGFR/7p11 Amplification
astrocytomas, gliomas, and GM tumors. Low levels of hNr- Alteration
Overexpression [50]
CAM expression were observed in neuroblastoma, menin-
DCC/18q21 Loss [57]
giomas, melanoma, normal breast and prostate tumor D2-2 Overexpression [58]
tissues. Northern blot analysis showed an alternatively C4-2 Loss [59,60]
spliced mRNA of 1.4 Kb in several tumors as compared to Connexin43 Under expression (Unpublished
the 7.5-Kb transcript found inNBT. Genomic Southern blot observations)
MET Overexpression [65]
analysis of the DNA from three brain tumor cell lines
MYC Overexpression [66]
showed that overexpression of hNr-CAM in brain tumors TGFβ Overexpression [67]
was not due to gene amplification. In situ hybridization CD44 Overexpression [68]
analysis indicated that 11 of the 20 human brain tumor VEGF Overexpression [69]
samples studied showed hNr-CAM overexpression. These NCAM L1 Under expression [70]
hNr-CAM Overexpression (Unpublished
results suggest that hNr-CAM is overexpressed in malig-
observations)
nant brain tumors and can serve as a novel marker for brain p21WAF1/Cip1 Overexpression [71]
tumor detection and, perhaps, for therapy. Tenasin Overexpression [72]
NOS Overexpression [73]
Loss of Connexin43 Expression Cathepsin Overexpression [74]
MMPs Overexpression [75]
Gap junctions play an important role in tissue homeo-
TIMPs Overexpression [76]
stasis and cell proliferation. These junctions are comprised IL-13R Overexpression [77]
of a family of homologous proteins called connexins [61]. TRKA Under expression [78]
In the normal brain, astrocytes express Connexin43 uPARs Overexpression [79]
whereas oligodendrocytes and some neurons express MMRs Under expression [80]
ECMs Overexpression [81]
Connexin32. Transfection of Connexin43 into rat astrocy-
Heat Shock Proteins Overexpression [82]
toma cell lines and dog kidney cells leads to an increase in α6β4α and α6β1 Integrins Overexpression [83]
intercellular communication and growth suppression
[62,63]. A recent study clearly indicates decrease in the p53, protein 53; PDFG, platelet derived growth factor; PDFGR, platelet
derived growth factor receptor; RB, retinoblastoma; IFN, interferon;
Connexin43 expression in several brain tumor cell lines CDKN2, cyclic AMP dependent kinase number 2; MMAC1, mutated in
(unpublished observations). Overexpression of Con- multiple advanced cancers 1; EGFR, epidermal growth factor receptor;
nexin43 suppresses growth of these tumor cell lines, and DCC, deleted-in-colon carcinoma; VEGF, vascular endothelial growth
this strongly suggests that Connexin43 can act as a poten- factor; NCAM, neuroglial cell adhesion molecule; hNr-CAM, human
tial brain tumor suppressor gene and loss of its expression neuroglial-related cell adhesion molecule; NOS, nitric oxide synthase;
MMPs, matrix metalloproteases; TIMPs, tissue inhibitors of
could be an important step in the genesis of human glio- metalloproteases; IL-13R, interleukin-13 receptor; uPARS, urokinase-
mas. Connexin43 may have potential use in gene therapy type plasminogen activator receptors; MMRs, mismatch repair genes;
of human brain tumors [63,64]. ECMs, extracellular matrix.
 Molecular Change During Glioma Genesis 9

Fig. 1. Schematic of molecular changes during the genesis of human cers 1; DCC, deleted-in-colon carcinoma; NOS, nitric oxide synthase;
gliomas. PDGFα, platelet derived growth factor alpha; PDGFr, platelet VEGF, vascular endothelial growth factor; TIMPs, tissue inhibitors of
derived growth factor receptor; RB, retinoblastoma; INF, interferon; metalloproteases; Nr-CAM, neuroglial-related cell adhesion molecule;
CDKN2, cyclic AMP dependent kinase number 2; EGFR, epidermal NCAM L1, neuroglial cell adhesion molecule; TGF, transforming growth
growth factor receptor; MMAC1, mutated in multiple advanced can- factor; uPARs, urokinase-type plasminogen activator receptor.
10 Sehgal

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