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Molecular Biology and Genetics |
Division of Neurosurgery [L. M. L.], Departments of Neurology [A. B., J. M. B.], Pathology [J. P. G.], Pharmacology [H. I. K.], and Human Genetics [S. F. N.], and the Jonsson Comprehensive Cancer Center [L. M. L., S. F. N.], University of California at Los Angeles School of Medicine, Los Angeles, California 90095; Brookwood Biomedical, Birmingham, Alabama 35209 [R. L. L.]; and Research Genetics Inc., Huntsville, Alabama 35801 [R. S. S.]
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ABSTRACT |
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INTRODUCTION |
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A variety of methods are currently used to isolate genes associated with particular differential phenotypes. Subtractive hybridization (6) , differential display (7, 8, 9, 10) , representational difference analysis (11, 12, 13, 14) , serial analysis of gene expression (5 , 15) , and suppression subtractive hybridization (16 , 17) all allow for the cloning and identification of differentially expressed sequences. Although all these techniques identify tissue-enriched mRNAs, none select for tissue-specific proteins. Because we initially were interested in identifying glioma-associated antigens that may be potential targets for brain tumor immunotherapy, we set out to devise a differential screening technique that provided actual confirmation of the presence of a protein product, not just the capacity to synthesize a protein. Furthermore, we wanted to select for proteins with antigenic determinants that may be potentially recognized by the immune system.
Here we report an approach to identifying differentially expressed gene products that are actually translated from mRNA species, using antibody-based screening of a cDNA expression library. We further show that this method, which we termed DIA,3 can be coupled to cDNA microarray hybridization and allowed the identification of a putative growth factor gene, granulin, which may play a role in the malignant progression of glioblastomas.
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MATERIALS AND METHODS |
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Antisera were raised against the GBM tumor homogenate by s.c. and i.m. immunization of New Zealand White rabbits, using complete and incomplete Freund’s adjuvants. Several bleeds were collected from two animals, pooled, and diluted 1:2 with 0.1 M BBS (pH 8.4). The diluted antiserum was passed over the GBM affinity column, and unbound material was washed off with BBS. Bound material was eluted off using glycine buffers adjusted to pH 3, pH 2, and then pH 1. The effluent and eluate were monitored at 280 nm (LKB), and the antiserum was passed repeatedly through the column until depleted. The eluate was then collected into BBS, checked for neutral pH, and cross-absorbed repeatedly (until depleted of cross-reactive antibodies) against the column of non-tumor brain to select out antibodies that may bind normal brain antigens. The unbound material was further cross-absorbed against normal human plasma to select out nonspecific antibodies. The final product was concentrated using YM30 columns (Amicon) and dialyzed into carbonate buffer (pH 9.5). The antibodies were biotinylated at a molar ratio of 15:1 using NHS long-chain biotin (Sigma) and repurified using a column of G-25 (Pharmacia). These biotinylated antibodies were then used to screen a glioblastoma phagemid cDNA expression library.
Construction and Screening of cDNA Expression Library.
For construction of the glioblastoma cDNA expression library, a human
GBM tumor was snap frozen in liquid nitrogen at the time of surgery and
stored at -80°C. Total RNA was extracted from 500 mg of fresh frozen
tumor tissue, using Trizol reagent according to the manufacturer’s
protocol (Life Technologies, Inc.). mRNA from a total of 30 µg
of RNA was isolated using double chromatography on oligo-dT cellulose
columns (Life Technologies, Inc.). Double-stranded cDNA was synthesized
from this mRNA, using a Superscript II cDNA synthesis kit (Life
Technologies, Inc.), and the cDNAs were ligated into a 
ZipLox
phagemid vector (Life Technologies, Inc.). We obtained a library titer
estimated at 5.0 x 106
plaque-forming units. Approximately 2.0 x 106 plaque-forming units were plated and grown in
the presence of isopropyl-1-thio-ß-D-galactoside, lifted
onto nitrocellulose membranes, and incubated with biotinylated anti-GBM
antibodies (1:1000 dilution). The membranes were then incubated with
streptavidin-horseradish peroxidase and diaminobenzidine
tetrahydrochloride (Pierce). Positive clones were isolated,
re-screened, and subcloned into the pZL1 plasmid vector (Life
Technologies, Inc.) by in vivo excision. Inserts were
verified by agarose gel electrophoresis and partially sequenced using a
dsDNA cycle sequencing kit (Life Technologies, Inc.) according to the
manufacturer’s protocol.
Microarraying of Cloned DIA Products.
After the subtractive products were cloned into the pZL1 vector,
plasmid inserts were PCR-amplified using vector-specific primers. PCR
was performed in 50-µl reactions containing 10 mM Tris
(pH 9.0), 50 mM KCl, 0.1% gelatin, 2.5 units of Taq DNA
polymerase, and 150 µM deoxynucleotide triphosphates.
Thermal cycling conditions consisted of an initial denaturation at
94°C for 2 min, followed by 35 cycles of 94°C for 1 min, 68°C for
1 min, and 72°C for 1.5 min, with a final 72°C extension for 10
min, in a PTC100 thermal cycler (MJ Research). Five microliters of each
PCR amplification product were examined by agarose gel electrophoresis
with ethidium bromide staining. A single band was detected in 26 of the
28 PCR reactions performed. Each of the 26 successfully amplified PCR
products (1–2 µg) was recovered from the remaining 45 µl of each
PCR reaction by ethanol precipitation.
The PCR products were arrayed onto glass slides, following a protocol
similar to that described previously (18)
. Briefly, the
PCR products were resuspended in 15 µl of 1x SSC. A
custom-built arraying robot picked up 
600 nl of DNA solution and
deposited 1–4 nl of DNA solution in triplicate onto a silanized glass
slide surface (Sigma). After printing, the slide was hydrated for
10 s over a 37°C water bath, snap dried for 2 s on a
100°C heating block, and then UV cross-linked with 4000 mJ short-wave
irradiation (Stratagene Stratalinker). The slide was then washed for 2
min sequentially in 0.2% SDS and distilled water. The bound DNA was
denatured in distilled water at 100°C, desiccated in an ice-cold bath
of 95% ethanol, and air-dried.
Probe labeling, microarray hybridization, and washes were performed as described previously (19) . mRNA from a large batch of pooled tumor and non-tumor brain specimens was used to make cDNA labeled with Cy5. The Cy5-labeled cDNA from this collective batch served as the common reference probe in all hybridizations. mRNA samples (2 µg) from 10 individual tumor and non-tumor brain specimens (e.g., 8 gliomas and 2 normal brain tissues) were used to make cDNA labeled with Cy3.
After hybridization with the arrayed subtractive clones, Cy3 and Cy5
intensities were scanned using a custom-built two-color laser scanning
fluorometer. The image files were analyzed with custom-written software
that performed quantification similar to that published previously
(20
, 21)
. The relative abundance of each of our 26
subtractive clones (L1–L26) in tumor versus normal brain
was calculated using the equation:
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Northern Blot Analysis of Granulin mRNA Expression.
Tissue total RNA was extracted using Trizol reagent (Life Technologies,
Inc.) according to the manufacturer’s instructions, and 10 µg/lane
were separated on 1.2% denaturing agarose gels, transferred overnight
to Hybond membranes (Amersham) using 10x SSC, and irreversibly fixed
by UV cross-linking. Prehybridization and hybridization were performed
at 65°C in ExpressHyb solution (Clontech).
32P-labeled cDNA probes were generated from our
plasmid DNA containing granulin cDNA using random
primers according to the manufacturer’s protocol (NEB). After
hybridization, membranes were washed (2x SSC containing 0.1% SDS at
37°C for 20 min, followed by 0.2x SSC containing 0.1% SDS at 61°C
for 20 min), and exposed to X-ray film (Kodak) at 80°C. Blots were
then stripped with 0.1% SDS at 100°C for 15 min and reprobed with
32P-labeled ribosomal 18S cDNA to control for gel
loading and RNA integrity.
In Situ Hybridization of Granulin mRNA Expression.
In situ hybridization was performed using
35S-labeled riboprobes following previously
published protocols (22)
. Briefly, surgically resected
human brain tissues (tumor and non-tumor) were rapidly frozen in
isopentane directly from the operating room. Frozen tissues were
sectioned on a cryostat at 20-µm thickness, post-fixed in 4%
paraformaldehyde, washed, and stored at -75°C. Sections were washed,
acetylated, defatted, and incubated with
35S-labeled sense or antisense
granulin cRNA probe (107 cpm/ml) at
60°C overnight (18–24 h). Following RNase A (20 µg/ml) treatment
at 45°C, sections were washed in descending concentrations of SSC,
air dried, and dipped for emulsion autoradiography in Kodak NTB2 (1:1
dilution). Following exposure to emulsion for 5 weeks, the slides were
developed and counterstained with H&E.
Hybridization densities were measured from the in situ slides by counting silver grains within representative cells, using an image analysis computer (Olympus microscope and MCID Imaging software; Imaging Research, Inc.; Ref. 23 ). Sections through several different tumor and non-tumor human brain specimens that had been hybridized with granulin cRNA were chosen for counts. Briefly, two independent observers outlined labeled regions within each slide, and the computer determined the absorbance and quantity of silver grains within each outlined area. Ten measurements were performed for each slide and averaged into single values per mm2 per specimen. These values were then divided by the estimated number of cells per mm2 for each specimen to get the average units of silver grains per cell. The average quantity of silver grains per cell for each tumor was compared to that of non-tumor brain specimens using the Student’s t-test.
Cell Cultures.
Primary cultures of rat astrocytes from the brains of adult Fischer 344
rats were isolated following a protocol described previously
(24)
. Cultures were maintained in DMEM (Life Technologies,
Inc.) supplemented with 10% FBS, L-glutamine, and
antibiotic drugs (100 units/ml penicillin and 100 µg/ml streptomycin)
at 37°C in 5% CO2.
Primary human glioblastoma cell cultures were established in our laboratory using a protocol similar to that published previously (25) . Tumors were taken directly from the operating room at the time of surgery. Tissues were finely minced using sterile scissors, rinsed with PBS, and dispersed with trypsin-EDTA. Monolayer cells were plated in T75 flasks (Costar) and cultured in DMEM/Ham’s F12 (Irvine Scientific) supplemented with 10% FBS (Life Technologies, Inc.), L-glutamine, and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin).
Measurement of Cell Proliferation.
The effect of granulin D peptide and granulin antibody on the
proliferation of primary rat astrocytes and three early-passage human
glioblastoma cell lines were examined. Synthetic peptide, consisting of
the 55-amino acid sequence of granulin D (26)
, was
provided by Research Genetics. For the antibody studies, a polyclonal
antibody was raised against this 55-amino acid synthetic peptide
conjugated to keyhole limpet hemocyanin. The IgG fraction was isolated
from sera, using protein A-Sepharose (Zymed), concentrated using a
Centri-cell concentrator (Amicon), and stored in borate buffer
consisting of 25 mM sodium borate, 100 mM boric
acid, 75 mM NaCl, and 5 mM EDTA.
The biological effects of increasing concentrations of granulin D
peptide and antibody on in vitro cell growth rates were
assayed using [3H]thymidine incorporation.
Cells were grown to 60% confluence in T75 flasks (Costar) and then
plated in 12-well plates (Corning) at a density of
104 cells/well in 1 ml of DMEM supplemented with
10% FBS. One day after plating, the medium was removed and replaced
with medium containing increasing concentrations of either synthetic
granulin D peptide (0–1000 ng/ml) or granulin D antibody (1:1000 to
1:100) in triplicate. Three days later, the medium was again replaced
by fresh medium and supplemented with increasing amounts of peptide or
antibody. After 3 days, 0.5 µCi/well of
[3H]thymidine was added for overnight
incubation at 37°C. Wells were then washed twice with 1 ml of
ice-cold PBS and collected by treatment with trypsin-EDTA. Cell
suspensions were transferred to scintillation vials, and radioactivity
was counted with a scintillation counter. Six separate experiments were
performed on each cell line, using triplicate wells per experiment
(n = 18). The Student’s t test
was used to interpret the significance of differences between groups.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Using this approach, which we term DIA, positive reactions were found
in 28 plaques. Positive clones were isolated, subcloned into the pZL1
plasmid vector by in vivo excision, and partially sequenced.
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. Of the 19 novel sequences that were identified, Northern blot
analyses were performed on 9 of these cDNAs and differential expression
was confirmed in tumor tissues versus normal brain in all
nine of the clones tested (data not shown).
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. As seen in Fig. 2
, for the majority of the 26 genes tested, the three
glioblastoma samples analyzed appeared to have higher levels of
differential expression than the other tumor types. Furthermore,
analysis of the expression of these candidate tumor-specific genes
revealed that one of our isolated clones, L5, consistently had much
higher levels of expression in gliomas compared with normal brain
(3–30-fold).
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Granulin mRNA Expression in Human Gliomas.
The differential expression of granulin in human gliomas was confirmed
by Northern blot analysis, which showed a transcript of 2.1-kb
expressed in 86% (18 of 21) of human gliomas and 0% (0 of 3) of the
non-tumor brain tissues analyzed (Fig. 3A)
. Interestingly, of the three gliomas that had absence of
any granulin signal, one was from a patient who had received
previous radiation therapy and one was from a low-grade
oligodendroglioma. These data would suggest that granulin
expression may be mitigated by radiation and/or may be related to
higher malignancy and tumor progression. Further studies with greater
numbers of irradiated or low-grade glioma samples would be necessary to
confirm this hypothesis.
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.
Differential expression of granulin mRNA was also seen in
tumor versus non-tumor tissues, using in situ
hybridization. Granulin antisense riboprobe hybridized
predominantly to hypercellular areas of tumor tissue (Fig. 4, A and B)
. The identity of these cells labeled by
in situ hybridization was suggested by counterstaining the
tissue sections with H&E, which revealed that the majority of the RNA
was within tumor cells and not in the tissue stroma (Fig. 4, C and D)
. Sense strand riboprobe cDNA was used as
a control and showed no specific labeling (data not shown), indicating
that the cellular hybridization obtained with the antisense probe was
specific for the granulin mRNA. Quantitation of
granulin hybridization densities was measured from the
in situ slides, using image analysis software. This analysis
revealed significantly greater numbers of silver grains within cells of
the most malignant brain tumors (e.g., anaplastic
astrocytomas and GBM) compared with non-tumor glial cells
(P = 0.006), confirming that elevated levels
of granulin mRNA are expressed in high-grade primary brain
tumors (Table 2)
.
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6 kDa that is derived from a larger precursor of
593 amino acids, known as acrogranin (38, 39, 40)
.
The acrogranin cDNA (clone L5) that we isolated from human
glioblastomas contained the entire sequence for granulin D(bp 1254–2099; Ref. 26
).
The implication of granulin molecules in growth regulation with a
tertiary structure reminiscent of that of EGF suggested a potentially
important role for our L5/granulin D clone as a putative growth factor.
To determine whether granulin D may modulate glial cell proliferation,
we synthesized a 55-amino acid peptide corresponding to the known
sequence of granulin D (26)
. We then studied the effect of
this synthetic peptide on proliferation rates of rat astrocytes in
culture, using a standard [3H]thymidine
incorporation assay. As shown in Fig. 5
, addition of synthetic granulin D peptide stimulated DNA synthesis of
rat astrocytes in vitro up to 300% in a dose-dependent
manner (Fig. 5, A and B)
. Statistically
significant increases in cell proliferation (up to 150% of controls)
were seen with the addition of as little as 1 ng/ml (169
pM) of granulin D to cell culture
(P = 0.025). Interestingly, this synthetic
peptide had a much more modest effect on the proliferation of primary
human glioblastoma cells in culture, showing only a 120–150% increase
(P = 0.068) in growth with the addition of
>1000 ng/ml (169 nM) granulin D (Fig. 5C)
. This may be explained by the fact that these human
cells were tumorous and already expressed high levels of granulin (as
shown by Northern blot and in situ hybridization). Thus, the
putative receptors of this potential autocrine growth factor may be
saturated by endogenous granulin and thereby preclude further growth
stimulation by the addition of exogenous peptide. To further evaluate
the growth regulatory role of granulin D on human tumor cells in
vitro, a polyclonal antibody was raised against the 55-amino acid
granulin D peptide and assayed for its ability to inhibit cell
proliferation in three primary human glioblastoma cultures. As shown in
Fig. 5D
, the addition of increasing concentrations of
purified granulin D antibody to early-passage human brain tumor cell
cultures significantly inhibited cell growth in vitro.
[3H]Thymidine incorporation was suppressed down
to only 18.6% of controls with the highest concentration of antibody
tested (1:100 dilution; P = 0.035).
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DISCUSSION |
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With current microarray technology, it is feasible to screen relatively
large numbers of tumor samples for the expression of subtractive
products. This allows easy discrimination of redundant clones and rapid
confirmation of truly differentially expressed genes. Although we
isolated and screened only 26 clones from our DIA method, it is
conceivable that thousands of differentially expressed gene products
could be identified among the 15,000 individual mRNA species in a
pair of human cell populations (i.e., tumor
versus normal). This is based on the assumption that perhaps
15% of the estimated 100,000 genes in the human genome are expressed
in any individual cell type at a particular time (41)
.
Even if thousands of differential clones are generated from our
subtractive approach, current robotic microarray technology allows for
the fabrication of arrays containing up to 20,000 distinct cDNA targets
(18)
. The expression of these thousands of targets can be
monitored in multiple tissue samples, just as we measured the relative
expression of our 26 clones in various brain tumor tissues. Thus,
microarrays in concert with subtractive gene hunting methods could
serve as useful tools for the identification of biologically intriguing
and clinically relevant human gene sequences.
Using the combination of DIA and microarray hybridization, we have identified a potentially interesting candidate oncogene, granulin D, with a likely function in glial cell proliferation. This granulin peptide belongs to a family of putative growth factors that previously have been characterized by a unique structural motif and implicated in growth regulation (28 , 30 , 31 , 33, 34, 35) . Structurally, granulins consist of 12 cysteines with four cysteine pairs flanked by two single cysteines at both the NH2 and the COOH termini (26 , 42) . The predicted protein architecture consists of four stacked ß-hairpins, each connected to the next with two parallel disulfide bridges, and a peptide backbone arranged as two ladders in a left-handed superhelix (34) . Interestingly, this tertiary structure is partially homologous to that of EGF. Functionally, several independent investigators have found granulin peptides to be regulators of cellular proliferation, with biological activities reminiscent of the actions of other polypeptide growth factors such as EGF. Granulin proteins have been shown to have mitogenic activity in murine embryonic 3T3 cells, in the tumorigenic teratoma-derived PC cell line, in human epithelial and fibroblastic cells, and in murine keratinocytes (27 , 30 , 33 , 35) . We now report similar growth regulatory effects of this peptide in primary rat astrocytes and in early-passage human glioblastoma cell lines.
The surprising parallels between the granulin and EGF systems are of interest. Given that amplification of the EGFR gene is one of the most common findings in glioblastomas and malignant astrocytomas (1 , 43) , it is intriguing that one of the glioblastoma-associated clones identified via our novel DIA technique may be related to the EGF/EGFR system. Nevertheless, there are many molecules that have EGF-like domains, and other investigators have found that granulin does not bind to wild-type EGFR (also called erbB-1; Ref. 44 ). Furthermore, Western blot analysis of EGFR expression in the human glioblastomas used in our bioassay revealed EGFR overexpression in only one of the three tumors tested, with no direct correlation between EGFR overexpression and granulin-induced growth regulation. Interestingly, however, all three of the tumors we studied had overexpression of the closely related EGFR-like transmembrane receptor tyrosine kinase erbB-2 (also called HER-2 or neu; data not shown). The significance of this coincident overexpression of granulin and erbB-2 is unknown at present.
Also intriguing in this context is the fact that both granulin and erbB-2 are genes located on chromosome 17 (29 , 45) . Previous reports in the literature have found that high-grade gliomas have overrepresentation of chromosome 7 and gain of chromosome 17q at the cytogenetic level (46 , 47) , which presumably relates to amplification of EGFR and erbB-2 at the gene expression level (48, 49, 50) . Although overexpression of erbB-2 has been found in a subset of primary brain tumors, its putative ligand in brain cancers is not yet known. It would be interesting to determine whether tumors with amplification of chromosome 17q have coordinate overexpression of erbB-2 and granulin, which may support the idea of granulin being a ligand for the erbB-2 proto-oncogene autocrine/paracrine loop. Additional studies of granulin binding data and signal transduction pathways are warranted and may aid in our understanding of the oncogenesis of brain neoplasms. Given the tissue-specificity of granulin for tumor versus normal brain, its EGF-like domains, its location on chromosome 17, and its implicated role in glial cell growth regulation, it is conceivable that this gene product may be a useful target for the development of new therapeutics for malignant brain tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by grants from the National
Cancer Institute (Grant NIH CA 82666-01), the American Cancer Society
(Grant IN-131), the Howard Hughes Medical Institute (Grant
76296-549701), and the STOP Cancer Foundation (awarded to L. M. L.). We also thank the generous support of the Henry E.
Singleton Brain Cancer Research Fund. 
2 To whom requests for reprints should be
addressed, at Division of Neurosurgery, UCLA School of Medicine, Center
for Health Sciences, CHS 74-134, 10833 Le Conte Avenue, Box 956901, Los
Angeles, CA 90095-6901. Phone: (310) 794-5664; Fax: (310) 825-7245;
E-mail: lliau{at}mednet.ucla.edu
3 The abbreviations used are: DIA, differential
immuno-absorption; GBM, glioblastoma multiforme; BBS, borate-buffered
saline; FBS, fetal bovine serum; EGF, epidermal growth factor; EGFR,
EGF receptor.
Received 10/13/99. Accepted 1/ 5/00.
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 J. M. MARKERT, C. M. FULLER, G. Y. GILLESPIE, J. K. BUBIEN, L. A. MCLEAN, R. L. HONG, K. LEE, S. R. GULLANS, T. B. MAPSTONE, and D. J. BENOS Differential gene expression profiling in human brain tumors Physiol Genomics, February 7, 2001; 5(1): 21 - 33. [Abstract] [Full Text] [PDF]
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