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Tumor Biology |
4 Chain-containing Laminins in Human Glial Tumors Identified by Gene Microarray Analysis1
Maxine Dunitz Neurosurgical Institute [J. Y. L., A. J. L., A. L., M. S. R., K. L. B.], Department of Pathology [W. H. Y.], and Ophthalmology Research Laboratories [A. V. L.], Cedars-Sinai Medical Center, Los Angeles, California 90048; Renal Division, Washington University School of Medicine, St. Louis, Missouri 63110 [J. H. M.]; and Interdisciplinary Center for Clinical Research, University of Erlangen-Nuremberg, Erlangen 91054, Germany [L. M. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4 chain of laminin, a major blood vessel basement membrane component. Changes in expression of this laminin chain have not been previously associated with malignant tumors. Overexpression of laminin 4 chain in GBM and astrocytoma grade II by gene microarray analysis was confirmed by semiquantitive reverse transcription-PCR and immunohistochemistry. Importantly, an 4 chain-containing laminin isoform, laminin-8 (4ß1
1), was expressed mainly in blood vessel walls of GBMs and histologically normal tissues adjacent to GBMs, whereas another 4 chain-containing laminin isoform, laminin-9 (4ß21), was expressed mainly in blood vessel walls of low-grade tumors and normal brain. GBMs that overexpressed laminin-8 had a shorter mean time to tumor recurrence (4.3 months) than GBMs with overexpression of laminin-9 (9.7 months, P = 0.0007). Up-regulation of 4 chain-containing laminins could be important for the development of glioma-induced neovascularization and glial tumor progression. Overexpression of laminin-8 may be predictive of glioma recurrence. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key molecular genetic markers of tumor development include cellular proto-oncogenes, tumor suppressor genes (antioncogenes), and DNA mismatch repair mutations (2 , 3) . A few genes have been identified that are specific to particular neoplastic tissues (4 , 5) . However, for most tumors, including brain gliomas, the known genomic, chromosomal, and biochemical changes are largely nonspecific and are of limited use for tumor diagnostics or the design of effective treatment regimens (6) . Immunophenotyping of brain tumors is generally helpful. However, differential expression of protein markers (GFAP,4 vimentin, synaptophysin, and nestin) in gliomas has not altered existing therapeutic approaches, treatment success rate, and disease outcome prediction (7 , 8) .
Recent studies have found overexpression of certain genes and proteins in gliomas, including c-myc and c-met oncogenes, transcription factor AP-2 CD44, intercellular adhesion molecule 1, CD58 (LFA-3), smooth muscle actin, vimentin, matrix metalloproteinases, nitric oxide synthase, EGFR, TGF- and -ß, VEGF, IGF-II, and basic fibroblast growth factor. At the same time, decreased expression of connexin 43 and some tissue inhibitors of metalloproteinases has also been observed in glial tumors and their recurrences (6
, 9, 10, 11, 12, 13)
.
The ECM and its specialized structures, BMs, play important roles in tumor progression as barriers for invasion, migration substrata for invasive tumor cells, and components of newly formed tumor blood vessels. Proteolytic degradation of ECM mediated by the plasminogen activator system may facilitate cell migration and tumor invasion (14) . Along with ECM proteolysis necessary for invasion, human gliomas have increased expression of several ECM components associated with tumor neovasculature, including fibronectin, laminin, types III and IV collagen, vitronectin, and tenascin-C (6 , 9, 10, 11 , 15, 16, 17, 18, 19) .
Most previous studies of glioma markers have been conducted using either one or a few genes/proteins at a time, although families or cascades of genes are usually involved in biological events. Recently, the novel technology of gene array analysis allowed one to identify genes that were differentially expressed in tumors and analyze the interactions between multiple genes (18) . Multigene studies are difficult, however, because in addition to individual genome alterations, each tumor gene expression profile is unique. Another complication is presented by tumor progression, which can alter gene expression patterns differently in individual tumors.
Using the gene array method combined with RT-PCR and immunohistochemical evaluation of glial tumors, we detected 14 genes that were consistently overexpressed in GBMs. A novel finding was the overexpression of laminin 4 chain in both low and high-grade gliomas and in histologically normal tissues adjacent to GBM, compared with normal brain or benign meningiomas. The majority of GBMs had increased expression of laminin-8 (4ß11) chains in blood vessel walls, whereas low-grade tumors overexpressed laminin-9 (4ß21) chains. GBMs that predominantly expressed laminin-8 had a shorter time to recurrence than did GBMs that predominantly expressed laminin-9.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A total of 27 tissue samples were used for gene array, RT-PCR, and immunohistochemistry, including 15 primary gliomas, histologically normal tissues adjacent to the gliomas from five of the same patients, 3 meningiomas (benign extra-axial brain tumors), 3 normal brain tissues from trauma patients, and 1 sample of normal corpus callosum.
For gene array analysis, 12 tissues were used including 5 GBMs, 2 histologically normal brain tissues adjacent to 2 of the 5 GBMs, 2 grade II astrocytomas, 1 meningioma, and 2 normal brain tissues from trauma patients (Table 1)
. All of the samples were compared with normal human corpus callosum as an internal control tissue. RNA from corpus callosum (pooled RNA from tissues of 70 trauma patients) was purchased from Clontech (Palo Alto, CA). Corpus callosum contains mostly glial cells (22)
and was therefore chosen as a normal control for glial tumors.
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PCR was used to generate sequences for the microarray fabrication. PCR products were purified by gel filtration with Sephacryl-400 (Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated in 0.2x SSC. The filtrate was dried down and rehydrated in 0.1 volume of dH2O for arraying. The DNA solutions were arrayed by robotics on modified glass slides. After arraying, slides were processed to fix the DNA to the prepared glass surface and washed three times in dH2O at room temperature. Slides were then treated with 0.2% I-Block (Tropix, Bedford, MA), dissolved in 1x Dulbecco’s PBS (Life Technologies, Inc., Gaithersburg, MD) at 60°C for 30 min. GEM microarrays were then rinsed in 0.2% SDS for 2 min, followed by three 1-min washes in dH2O.
Fluorescent Labeling of Probe.
Poly(A)+ RNA (mRNA) was isolated from tissue samples as described previously (23)
. Isolated mRNA was reverse transcribed with 5' Cy3- or Cy5-labeled random 9-mers (Operon Technologies, Inc., Alameda, CA). Reactions were incubated for 2 h at 37°C with 200 ng of poly(A)+ RNA, 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), 4 mM DTT, 1 unit of RNase inhibitor (Ambion, Austin, TX), 0.5 mM deoxynucleotide triphosphates, and 2 µg of labeled 9-mers in a 25-µl volume with the enzyme buffer supplied by the manufacturer. The reaction was terminated by incubation at 85°C for 5 min. The paired reactions were combined and purified with a TE-30 column (Clontech), brought to 90 µl with dH2O, and precipitated with 2 µl of 1 mg/ml glycogen, 60 µl of 5 M NH4OAc, and 300 µl of ethanol. After centrifugation, the supernatant was decanted, and the pellet was resuspended in 24 µl of hybridization buffer containing 5x SSC, 0.2% SDS, and 1 mM DTT.
Hybridization.
Probe solutions were thoroughly resuspended by incubation at 65°C for 5 min with mixing. The probe was applied to the array, covered with a 22-mm2 glass coverslip, and placed in a sealed chamber to prevent evaporation. After hybridization at 60°C for 6.5 h, the slides went through three consecutive washes of decreasing ionic strength.
Scanning.
Microarrays were scanned in both Cy3 and Cy5 channels with Axon GenePix scanners (Axon Instruments, Inc., Foster City, CA) with a 10 µm resolution. The signal was converted into 16 bits/pixel resolution, yielding a 65,536 count dynamic range.
Normalization and Ratio Determination.
GEM Tools computer software program (Incyte Genomics) was used for image analysis. The elements were determined by a gridding and region detection algorithm. The area surrounding each element image was used to calculate a local background and subtracted from the total element signal. Background-subtracted element signals were used to calculate Cy3:Cy5 ratios. The average of the resulting total Cy3 and Cy5 signal gave a ratio that was used to balance or normalize the signals. According to the Incyte Genomics protocol, all balanced differential expression ratios between two samples equal to or higher than 2 were considered significant.
Semiquantitative RT-PCR.
This was conducted as described previously (24, 25, 26)
. cDNA was synthesized from total RNA (26)
and subjected to PCR using specific primers for the gene array-selected 4 laminin chain gene and for ß2-MG gene, which served as a standard for sample normalization. Primers listed below were designed using the Primer3 Internet software program (The Whitehead Institute, Boston, MA), and their specificity was confirmed by a BLAST Internet software-assisted search of a nonredundant nucleotide sequence database (National Library of Medicine, Bethesda, MD).
Primers were as follows: (a) laminin 4 chain, forward primer 5'-CTCCATCTCACTGGATAATGGTACTG-3' and reverse primer 5'-GACACTCATAAAGAGAAGTGTGGACC-3'; and (b) ß2-MG (25)
, forward primer 5'-CTCGCGCTACTCTCTCTTTCTG-3' and reverse primer 5'-GCTTACATGTCTCGATCCCACTT-3'.
PCR was conducted exactly as described previously (26)
. Each cycle consisted of 30 s of denaturation at 94°C, 30 s of annealing, and 45 s of elongation at 72°C, and 35 cycles were performed for the laminin 4 chain. Samples normalized using ß2-MG amplification were amplified in a linear range, which was established using serial cDNA dilutions and by varying the number of cycles. Negative controls without reverse transcriptase, water control, and a positive kit control were included in each reaction. Amplified products were separated electrophoretically in 3% agarose gels, visualized, and photographed under UV light after ethidium bromide staining.
To confirm the specificity of PCR products, selected bands were excised from the gels, purified using Wizard PCR Prep (Promega Biotech, Madison, WI), reamplified, cloned into Plasmid PCR II using a TA cloning kit (Invitrogen, Carlsbad, CA), and sequenced in an automatic DNA sequencer 373 (Applied Biosystems, Foster City, CA) at the Cedars-Sinai Sequencing Core Facility.
Immunofluorescent Analysis.
Nineteen tissue samples were used: 12 primary glial tumors; 3 GBM-adjacent tissues; 2 meningiomas; and 2 normal brains. There were 9 GBMs and 3 grade II astrocytomas among the glial tumors. Nine tissue samples were from the same cases in which laminin 4 chain gene expression was also analyzed by gene array and semiquantitative RT-PCR. Tissue samples were snap-frozen in liquid nitrogen by a pathologist immediately after surgery and embedded in OCT compound, and 8-µm sections were cut on a cryostat. Indirect immunofluorescence, photography, and routine negative controls were as described previously (27)
. Well-characterized polyclonal and monoclonal antibodies were used to laminin chains 2 (clone 1F9), 3 (BM165), ß1 (clone LT3), ß2 (clone C4), ß3 (clone 6F12), and 1 (clones A5 and 2E8) (27)
. Antibodies to 2 and 1 (clone 2E8) chains were a gift from Dr. Eva Engvall (The Burnham Institute, La Jolla, CA), and those to 3 and ß3 chains were donated by Dr. Robert E. Burgeson (Massachusetts General Hospital/Harvard Cutaneous Biology Research Center, Massachusetts General Hospital East, Charlestown, MA). Rabbit polyclonal antibodies to laminin 1 chain [donated by Dr. Donald Gullberg, University of Uppsala, Uppsala, Sweden (28)
] and laminin 4 chain [antibody 377 (29)
and antibody C0877 (30)
] were also used. Two polyclonal antibodies to laminin 4 chain gave very similar results, as did two monoclonal antibodies to laminin 1 chain. The monoclonal antibody to laminin 5 chain (clone 4C7) and secondary cross-species absorbed fluorescein- and rhodamine-conjugated donkey antimouse, antirat, and antirabbit antibodies were from Chemicon International (Temecula, CA). Monoclonal antibodies were used as straight hybridoma supernatants or at 10–20 µg/ml when purified, and polyclonal antibodies were used at 20–30 µg/ml. At least two independent experiments were performed for each marker, with identical results.
Statistical Analysis.
Immunostaining data were analyzed by the two-sided Fisher’s exact test using the InStat software program (GraphPad Software, San Diego, CA). To this end, the number of cases with an abnormal staining pattern in one experimental group (e.g., normal) was compared with the number of cases with an abnormal staining pattern in another experimental group (e.g., GBM). Intervals between recurrences for individual patients were compared by the two-tailed unpaired Student t test using the InStat program. With both methods, P < 0.05 was considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When normal brain was compared with corpus callosum, no major differences in gene expression were found. This is illustrated on a summary plot of gene expression (Fig. 1A)
. The overwhelming majority of gene expression level differences between normal brain and corpus callosum were within the error range (less than a 2-fold difference). Some genes, such as ectodermal-neural cortex protein (with a ratio of 4.8) and synapsin II (with a ratio of 3.2), may reflect differences in the percentage of neurons in normal brain versus corpus callosum (Fig. 1A)
.
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. There were pronounced differences in the gene expression profile between the two tissues. Gene expression analysis of 5 primary GBMs by GEM array detected a total of 2345 genes with increased expression and 719 genes with decreased expression compared with corpus callosum. Of these genes, 14 were significantly (with ratios of 
2) up-regulated in all 5 GBMs studied (Tables 2
and 3
), and 12 genes were down-regulated in all 5 GBMs studied (Table 4)
. The majority of down-regulated genes play a role in metabolic processes (Table 4)
. Among the overexpressed genes (Tables 2
and 3
), some genes have been previously associated with glial tumors, whereas other genes have not been described in gliomas before. Because members of this group were implicated in glioma development, this report concentrated on the study of overexpressed genes.
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. Elevated expression of all these genes apparently reflects the active cell growth in GBMs.
The second gene group is represented by genes coding for structural and ECM-related proteins, including vimentin, fibronectin, tenascin- C, type IV collagen 1 chain, laminin 4 chain, desmoplakin, and tropomodulin. Table 3
summarizes the results for these genes, which were overexpressed in all human gliomas studied.
Comparison of Gene Expression Patterns in Different Brain Tumors.
Next, we determined by gene array method the expression levels of these 14 GBM-overexpressed genes in low-grade astrocytomas. Two grade II astrocytomas from patients 34 and 53, one benign meningioma from patient 38, and two normal brain tissues from patients 44 and 46 were used (Table 1
; Fig. 2, A and B
; gene profiles for patients 38 and 44 are not shown). Simultaneous quantitative comparison of gene array data on select genes in GBM and low-grade astrocytoma is shown in Fig. 2, A and B
. For growth factor-related genes, the mean expression ratio was higher in GBMs (grade IV) than in grade II astrocytomas (Fig. 2A)
. In grade II astrocytomas from patients 34 and 53, only two genes from this group were significantly overexpressed, transcription factor AP-2 and EGFR (Tables 2
and 5
). From the structural and ECM-related gene group, only laminin 4 chain was significantly overexpressed in both low-grade astrocytoma and GBM compared with normal brain (Fig. 2B
; Tables 3
and 5
).
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. Both GBM-adjacent tissues were judged by a board-certified pathologist to be histologically normal (Fig. 4)
. The gene array analysis yielded different gene profiles for adjacent tissues from patients 16 and 39. GBM 16 and its adjacent tissue had, on average, lower levels of most GBM-related genes than GBM 39 and its adjacent tissue. The levels of gene expression for histologically normal adjacent tissues from patients 16 and 39 were between those for GBM and low-grade astrocytoma but were more similar to those of GBM than to those of normal tissue. Some genes that were up-regulated in a number of glial tumors, including VEGF, desmoplakin, and laminin 4 chain (Tables 2
and 3
), were also elevated in histologically normal tissues adjacent to GBM. At the same time, some genes had higher expression levels in tissues adjacent to GBM than in the respective GBMs. These genes included connective tissue growth factor, keratin 18, desmoplakin, and phospholipase A2 receptor (Tables 2
and 3
).
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and 3
) were more similar to normal tissue than GBMs and tissues adjacent to GBM. The gene expression ratios for grade II astrocytomas were generally significantly lower than those for GBMs.
Overexpression of Laminin 4 Chain in Gliomas.
Of many genes overexpressed in gliomas or GBM-adjacent tissues by GEM analysis, only EGFR and laminin 4 chain genes showed, on average, increased expression levels in all types of tumors and adjacent tissues studied. The EGFR gene is well known to be up-regulated in glial tumors (6)
. Such data on laminin 4 chain were absent. Therefore, the expression of this laminin chain in normal brain and brain tumors was characterized in more detail, using semiquantitative RT-PCR and tissue immunostaining. We also investigated which 4-containing laminin isoforms might be up-regulated in different brain tumors and whether other laminin chains not present on the gene array might have altered expression in gliomas.
The expression of laminin 4 chain gene was analyzed further to confirm the gene array data. Semiquantitative RT-PCR was performed using seven primary GBMs, histologically verified GBM-adjacent tissues from three patients, one grade II astrocytoma, one meningioma, two normal brain tissues from trauma patients, and one sample from corpus callosum. All GBMs that had been analyzed by gene microarray were included in semiquantitative RT-PCR. The results confirmed the gene array analysis data; all GBMs and their adjacent tissues highly expressed laminin 4 chain gene. Meningioma from patient 38 and normal brain from patient 46 had lower levels of 4 chain gene expression than did glial tumors, and even lower levels were seen in normal brain from patient 44 and corpus callosum (Fig. 5)
. RT-PCR data were thus in agreement with gene array results.
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4 chain staining in normal brain and benign meningioma was weak in blood vessel walls (Fig. 6)
, but in grade II astrocytoma, the staining was stronger (Fig. 6
; Table 6
). All three low-grade gliomas showed increased staining intensity compared with normal brain tissues and benign meningiomas (P < 0.03). In all GBMs and GBM-adjacent tissues, blood vessels generally showed much stronger staining for 4 chain (P < 0.002). These data were in complete accord with the gene array and RT-PCR analysis (Table 3
; Fig. 5
).
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4 Chain-containing Isoforms in Gliomas.4 chain can be part of laminin-8 (4ß11), laminin-9 (4ß21), and laminin-14 (4ß23; Refs. 15
, 30
, and 31
). Therefore, the staining patterns of all other chains of 4-containing laminins were investigated. Laminin-14 could not be found in brain or brain tumors because 4 and 3 chains were never found in the same locations.5
In contrast, laminin-8 and laminin-9 chains, ß1, ß2, and 1, codistributed with 4 chain. The 1 chain antibodies brightly stained blood vessel walls in all samples of normal brain and benign and malignant brain tumors (Fig. 6
; Table 6
). The ß1 chain was weak in normal brain, two of three low-grade astrocytomas, and three of nine GBMs (Table 6)
. In all of these cases, distinct to strong staining for ß2 chain was observed (Fig. 6
; Table 6
), compatible with predominant expression of laminin-9. However, in six GBMs and in two of three tissues adjacent to GBM, ß1 chain staining was significantly stronger than that in normal brain (Fig. 6)
. At the same time, ß2 chain staining was definitely weaker than ß1 staining, which suggested up-regulation of laminin-8 in two-thirds of GBMs and tissues adjacent to GBM (Fig. 6)
. Again, in terms of laminin expression, GBM-adjacent tissues tended to resemble their respective tumors more than normal brain tissue. Thus, normal brain and low-grade gliomas expressed relatively low levels of mostly laminin-9, whereas in the majority of GBMs, strong staining was seen for chains of laminin-8. Clinically, all six patients with high laminin-8 expression (patients 22, 39, 42, 45, 49, and 54) developed recurrences at a mean of 4.25 ± 0.51 months after surgery (mean ± SEM), whereas all patients with low laminin-8 expression (patients 47, 50, and 51) were diagnosed with recurrent tumors at a mean of 9.70 ± 0.91 months after surgery. This difference was highly significant (P = 0.0007).
Distribution Patterns of Laminin Chains in Gliomas.
Immunofluorescent staining was also performed with antibodies to all other known laminin chains. The 1, 2, and 5 chains were expressed in blood vessel walls, and their staining intensity did not show any significant difference between normal and malignant brain tissues (Table 7)
. The only exception was a lack of 2 chain in meningiomas (Table 7)
. 3 chain staining was negative in all tissue samples studied.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, gene array technology has further demonstrated the importance of knowing the molecular parameters of individual tumors. Novel molecular characteristics of different morphological forms of ovarian (36) and cervical cancer (37) and of metastatic versus primary breast cancer (38) were recently obtained using gene profiles. New subclasses of leukemia have also been identified using gene arrays (39) , which have become critical for the successful treatment of patients.
Using gene array analysis, we found a number of genes that were overexpressed (2345 genes; Tables 1
2
3
and 5
) or underexpressed (719 genes; Tables 1
and 4
) in glial tumors compared with normal brain. Most of these genes reflected individual tumor heterogeneity. Only 14 genes were significantly overexpressed in all 5 GBMs studied, and 3 of these genes were significantly overexpressed in both GBMs and low-grade astrocytomas.
A follow-up of GBM patients showed that the clinical course correlated with the tumor gene profiles. GBMs from patients 22, 39, and 45 overexpressed genes for transcription factor AP-2, EGFR, IGF-binding protein precursor 3, and IGF-II, which are known to promote tumor growth (6
, 9, 10, 11, 12, 13)
. These genes were expressed at lower levels in GBMs from patients 16 and 50. The same difference was seen in the expression of structural and ECM-related genes such as vimentin, fibronectin (see also Refs. 9, 10, 11)
, and 4 laminin chain. The tumor gene profile of patients 22, 39, and 45 may thus be regarded as "more aggressive/malignant" compared with the tumor gene profile of patients 16 and 50. Notably, the former three patients developed recurrence within 4 months. Patient 50, however, did not develop a recurrence until nearly 9 months after surgery (patient 16 died of other reasons).
Overexpression of growth-related genes, such as transcription factor AP-2, EGFR, IGF-II, VEGF, and IGF-binding proteins 3 and 5, has been described previously in glial tumors, although these genes were usually studied one gene at a time. The same is true for structural and ECM-related genes [vimentin, fibronectin, tenascin-C, and type IV collagen (6
, 9, 10, 11, 12, 13
, 40)
]. When we studied gene expression in human gliomas using the gene array method, coordinate overexpression of these genes was observed in all five GBMs (Tables 2
and 3
). In addition, overexpression in gliomas of certain other genes, such as laminin 4 chain and tropomodulin, was shown here for the first time.
Eighty percent of GBM recurrences occur within 2 cm of the resected primary tumor margin (1)
. We show that histologically normal tissues adjacent to GBM had high expression levels of 10 genes, including 7 genes that were overexpressed in GBMs (Tables 2
and 3
). Therefore, despite their normal histological appearance, these tissues may have had microinvasive foci that could have contributed to a tumor-like gene expression pattern. Alternatively, tumor-derived factors could increase the expression of specific genes in normal cells of tissues adjacent to GBM. This latter possibility may primarily concern genes with higher expression levels in normal adjacent tissues than in corresponding GBMs, such as connective tissue growth factor, phospholipase A2 receptor, and epithelial markers keratin 18 and desmoplakin (Tables 2
and 3
). The expression of some tumor-related genes in adjacent tissues before the appearance of morphological changes would mean that these genes might be important for tumor development and progression (41)
.
Interestingly, low-grade astrocytomas were more similar to normal brain tissue by gene profiles than to GBMs or tissue adjacent to GBM. Some genes that were overexpressed in low-grade tumors compared with normal brain were not overexpressed in GBMs or tissues adjacent to GBM (Table 5)
. These genes comprising 17 known genes and 2 ESTs may be initial regulators of the glial tumor development. Their role in this process should be explored further.
Of the many genes overexpressed in various glial tumors and tissues adjacent to GBM, only two genes were up-regulated in all groups compared with normal brain by GEM array analysis. These genes included EGFR and laminin 4 chain (Tables 2
and 3
). Because EGFR overexpression in gliomas has been well established (6)
, we focused on laminin 4 chain, which is associated with blood vessel walls.
Overexpression of laminin 4 chain in all glial tumors studied and tissues adjacent to GBM compared with normal brain or benign meningiomas is described here for the first time. This finding was first noted on gene arrays and confirmed by semiquantitative RT-PCR and immunostaining with two different antibodies. In agreement with previous results (29
, 30
, 42)
, this chain was associated with blood vessels (Fig. 6)
, with occasional extravascular staining in some cases.
Laminins are a large family of ECM glycoproteins containing , ß, and chain subunits, which take part in the maintenance of tissue architecture and the regulation of cell migration, differentiation, and proliferation (15
, 43)
. Genetic defects in laminins are associated with diseases, notably with muscular dystrophies (15
, 29)
.
Laminin 4 chain is found both in adult tissues and during development; in cardiac, skeletal, and smooth muscle fibers; and in vascular endothelium, lungs, synapses, peripheral nerves, and blood cells including monocytes, erythromegakaryocytes, and platelets (29
, 30 , 44, 45, 46, 47, 48, 49)
. It is strongly expressed in small and large intestine, muscle, placenta, liver, heart, lung, and ovary. However, only weak expression has been observed in pancreas, testis, prostate, spleen, kidney, and brain (29)
. 4 chain-containing laminins use integrin 6ß1 as their major cell surface receptor (46
, 48
, 49)
.
The laminin 4 chain is part of laminin-8, laminin-9, and laminin-14, which differ from each other by ß and chain composition (15
, 31
, 47
, 48)
. We found all chains of laminin-8 and laminin-9 including 4 chain in blood vessels of normal brain and brain tumors. Laminin-14 (4ß23) was not found because the 3 chain was not associated with vascular BM.6
In normal brain and meningiomas, only weak staining was seen for 4 chain and for laminin-8 chain ß1. However, the staining for laminin-9 chain ß2 in normal vessel walls was considerably stronger. Therefore, laminin-9 may be predominant in normal brain and meningiomas. In low-grade astrocytomas, 4 staining increased, but stronger staining for ß2 chain than for ß1 chain (more laminin-9) persisted. In GBMs, 4 staining was much stronger. In two-thirds of GBMs, ß1 staining was strong, but ß2 chain staining was comparable or weaker than that seen in normal brain (Fig. 6
; Table 6
). Thus, many GBMs predominantly express laminin-8. Importantly, our follow-up data suggest that laminin-8 may be associated with a more rapid recurrence of tumor.
The expression of other laminin chains, 1, 2, and 5 (3 was always absent), did not differ significantly between the tissues studied. Similar results were obtained previously for 1-containing laminin-1 and 2-containing laminin-2 in brain tumors (16)
. Our immunostaining results cannot completely rule out the possibility that several ß1 chain-containing laminins are increased in GBMs, with a concomitant decrease of ß2-containing isoforms. Based on all our data, however, it seems unlikely that laminin isoforms other than laminin-8 become overexpressed in GBM blood vessels.
Laminins are the major constituents of blood vessel BMs. Therefore, in GBMs, laminin-8 may be associated with neovascularization and may thus contribute to the tumor aggressiveness. Laminin-8 overexpressed in GBMs, together with factors promoting tumor growth, might be an important prognostic factor to predict the time to recurrence for GBMs and a potential target of glioma therapy.
In our study, GBMs were identical in major morphological parameters (multicellular tumors with mitoses, palisade necroses, and neovascularization) but differed somewhat in the expression of particular genes, such as IGF-II, IGF-binding proteins 3 and 5, fibronectin, and laminin-8. We can hypothesize that (a) gene profiles of human GBMs and adjacent tissues may correlate clinically with tumor growth and progression, and (b) within GBMs, there may be subgroups that differ in growth parameters and therefore differ in time of recurrence development. For low-grade astrocytomas, the situation may be the same. Future studies with a greater number of samples are needed to conclusively determine whether a patient prognosis can be made using a molecular gene profile of the respective tumor.
In summary, three genes were consistently overexpressed in both grade II astrocytoma and GBM by gene microarray analysis. These genes coding for transcription factor AP-2, EGFR, and laminin 4 chain may be related to glioma progression. Overexpression of laminin 4 chain in blood vessel walls of gliomas and tissues adjacent to GBM was confirmed by RT-PCR and immunohistochemistry. The majority of GBMs overexpressed a predominant 4 chain-containing laminin isoform, laminin-8 (4ß11), in contrast to normal brain, benign, and low-grade tumors that expressed mostly another 4 chain-containing laminin isoform, laminin-9 (4ß21). The laminin-8-containing GBMs had a significantly shorter time between surgical removal and tumor recurrence than did those with a predominant expression of laminin-9. Thus, overexpression of laminin-8 in tumor blood vessel walls may be an indicator of time to recurrence for patients with GBM.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center. 
2 To whom requests for reprints should be addressed, at Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, 8631 West Third Street, Suite 800-E, Los Angeles, CA 90048. Phone: (310) 423-0834; Fax: (310) 423-0810; E-mail: ljubimovaj@cshs.org.
3 American Cancer Society. Brain and Spinal Cord Cancers of Adults. http://www3.cancer.org/cancerinfo/load_cont.asp?st=wi&ct=3.
4 The abbreviations used are: AP-2, activator protein 2; GBM, glioblastoma multiforme; RT-PCR, reverse transcription-PCR; EGFR, epidermal growth factor receptor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; IGF, insulin-like growth factor; ECM, extracellular matrix; BM, basement membrane; EST, expressed sequence tag; poly(A)+ RNA, polyadenylated RNA; dH2O, distilled H2O; ß2-MG, ß2-microglobulin; GFAP, glial fibrillary acidic protein.
5 A. Ljubimov and M.-F. Steiner-Champliaud, unpublished data.
6 A. Ljubimov and M-F. Steiner-Champliaud, unpublished data.
Received 1/15/01. Accepted 5/ 7/01.
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