Article

Isolation and characterization of a novel gene from human glioblastoma multiforme tumor tissue

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Abstract

Using the technique of DD-PCR (differential display-polymerase chain reaction) we isolated a novel gene (D2-2) that is overexpressed in glioblastoma multiforme tissue (GMT) as compared to normal brain tissue (NBT). D2-2 is also highly expressed in recurrent glioma, colon tumor metastatic to brain, breast tumors, prostate tumors and a prostate tumor cell line (LNCaP). Northern blot analysis showed that D2-2 is highly expressed in several tumor cell lines (MOLT lymphoblastic leukemia, SW480 colorectal adrenocarcinoma, A549 lung carcinoma, HL-60 promyelocytic leukemia, S3 HeLa cells, K-562 chronic myelogeneous leukemia and G361 melanoma) as compared to NBT. Additionally, D2-2 is very highly expressed in cell lines derived from glioblastomas, grade IV astrocytomas, normal human fetal astrocytes (NHFA) and glioma. D2-2 is moderately expressed in neuroblastoma, neuroectodermal and medulloblastoma tumor cell lines. D2-2 expression is localized to the frontal lobe, occipital lobe and the cerebellum in the normal brain. Normal tissues such as thyroid, stomach, adrenal cortex, small intestine and pancreas show high expression of D2-2. We also show that D2-2 is expressed 28-fold higher in fetal brain (20 weeks) than in adult brain. Sequence analysis of a 2.0-kb fragment for D2-2 shows no homology to known sequences in the data base.

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Nucleotide Sequence
August 1997
... For gene-specific Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and labeling of gene-specific probes, PCR primers (Genset Oligos, La Jolla, CA) were used to amplify the randomly selected 6 genes and human ß-actin (Clontech) as a control amplifier set. Table II shows the base pair length of amplified cDNA of 6 genes under study and the sequence of sense and antisense primers to amplify those cDNAs (37). ...
... Gene-specific RT-PCR analysis. To confirm differential expression of the 6 genes under study, gene-specific probes were generated by gene-specific RT-PCR technique (37). Different amounts of cDNAs and several PCR cycles were used to generate gene-specific probes. ...
... Differentially expressed gene-specific DNA bands were then eluted from the gel and purified with the help of the QIAquick Gel Extraction kit (Qiagen, Inc., Valencia, CA). These genespecific DNA bands were used as a probe in Northern blot (37). ...
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... RAB3D is a member of the ras oncogene family with a role in vesicular traffic control in eukaryotic cells. (Sehgal, et al., 1997) identified this as a putative target in glioblastoma multiforme, and found increased expression in several different tumor types, including lung, with potential oncogenic function. ...
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... Differential display allows the mRNA profile of all genes expressed from two different samples to be compared side by side, on the same gel, making the simultaneous detection of both up-and downregulated gene expression possible (Liang et al, 1992). This technique has been widely used in studying up-or downregulated genes in many tumour tissues, including prostate, thyroid (Musholt et al, 1997), pancreatic (Ozaki et al, 1998), and breast cancers (Watson and Fleming, 1994), colorectal and oesophageal carcinomas (Graber et al, 1996; Chan et al, 1998), melanoma (Duncan et al, 1998), and glioblastoma (Chuaqui et al, 1997; Sehgal et al, 1997). However, to our knowledge, no study using this technique in primary SCCHN tissues has been performed, although a few studies have reported using mouse or tumour cell lines of SCCHN (Gorogh et al, 1997; Patel et al, 1997; Gottschlich et al, 1999; Dong et al, 2001). ...
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... Total RNA from tissues were isolated according to the method devised by Chomczynski and Sacchi [18]. Reverse transcription (RT), PCR and competitive PCR were followed with the previous studies [19][20][21]. The forward primer was nested hNIS-5' primer containing the sequences: ACCTGGAAATGCGCTTCAGC. ...
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... To confirm the differential expression of clones isolated by DD-PCR, we used a modified RT-PCR technique described by Sehgal et al. (1997b). Gene-specific RT-PCR was done using hNr-CAM-specific primers (5Ј-AACATATGGGTAGAGA GTATATTT-3Ј and 5Ј-CTTTGCATTCCAGTTCATATTAA-3Ј). ...
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... By generating reproducible cDNA expression data, it is possible to compare gene expression in two or more cell types, or developmental stages or tissues associated with diseases, and a technique to isolate unknown differentially expressed genes. For example, this method has been quite successful in identifying differentially expressed genes in normal versus tumor-derived human mammary epithelial cells [33], isolating the gene D2-2 that was over expressed in glioblastoma multiforne tissue as compared to normal brain tissue [34], as well as identifying and characterizing differentially expressed genes in various stages of prostate cancer development [35] and isolating light activated genes differentially expressed in Coprinus congregatus [36]. The differential display technique allows side-by-side comparison of two different cell populations and therefore helps to identify known genes as well as unidentified new genes. ...
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... To con®rm differential expression of the six genes under study, gene-speci®c probes were generated by a gene-speci®c RT-PCR technique [34]. Different amounts of cDNAs and various numbers of PCR cycles were used to generate gene-speci®c probes. ...
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Using the technique of differential hybridization of a human fetal brain library, we have identified a novel gene, brain 2 (BR-2). This gene is expressed in normal brain but has low or no expression in human oligodendrogliomas and other brain tumor samples. We have cloned and sequenced the full-length cDNA corresponding to this gene. A data base search for the nucleotide sequence homology was performed for BR-2. BR-2 sequence showed strong homolog to a human genomic clone from chromosome 2. Moderate sequence homology was observed between BR-2 and an EST from a human placenta library. Multiple tissue dot blot analysis indicated that the BR-2 gene is expressed in a number of tissues including brain, heart, lung, placenta, lymph node, trachea and kidney. The BR-2 gene is also expressed in fetal heart, spleen and lung tissue. An extremely high level of BR-2 expression is observed in the left atrium of the heart. Low or no expression of BR-2 expression is observed in sixteen human cancer cell lines. RT-PCR analysis indicated that the BR-2 gene is expressed at high levels in two of the five normal brain tissue samples analyzed. Except for low expression in one oligodendroglioma, no expression of BR-1 gene was observed in eight anaplastic astrocytomas and glioblastoma multiforme tissue samples. Four of nine glioblastoma tumor cell lines did show a low level of BR-2 expression. On the basis of its expression and sequence, we conclude that BR-2 is a novel gene with unique expression properties in human brain tumors.
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Using the technique of differential hybridization of a human fetal brain library, we identified a novel gene, brain 1 (BR-1). This gene is expressed in normal brain but has low or no expression in human gliomas. We have cloned and sequenced the full-length cDNA corresponding to this gene. A data base search for the nucleotide sequence homology was performed for BR-1. The BR-1 sequence showed strong homology to a human genomic clone from chromosome 2. Moderate sequence homology was observed between BR-1 and an expressed sequence tag (EST) from a human placenta library. Three different regions of BR-1 also showed homology to a mouse EST that is similar to EL-10 gene. Sequence analysis indicated that the protein sequence for BR-1 has one tyrosine kinase phosphorylation site and two N-myristoylation sites. Northern blot analysis indicated that the BR-1 gene is expressed in heart, placenta, lung, liver, skeletal muscle, kidney and pancreas. A low level of expression of BR-1 is observed in the cerebellum, cerebral cortex, spinal cord, occipital lobe and putamen. The BR-1 gene is also expressed in fetal brain, liver and kidney. Low expression of BR-1 gene was observed in a number of non-brain tumor cell lines. RT-PCR analysis indicated that the BR-1 gene was expressed in non-neoplastic (epilepsy specimens) but not in six oligodendrogliomas and three oligoastrocytoma tumor samples analyzed. BR-1 was not expressed in either seven low grade gliomas or eight grade IV glioblastoma tumor tissue samples analyzed. Three glioblastoma cell lines did show low expression of the BR-1 gene. On the basis of its expression properties, we conclude that BR-1 is a potential novel tumor suppressor gene.
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The Differential Display Reverse Transcriptase (DDRT) technique was adopted to isolate genetic markers specific for the two main grades of the Glial tumor, the Astrocytoma and the Glioblastoma. A total of 16 brain biopsies (4 Astrocytoma and 12 Glioblastoma) were analysed. The technique was modified in order to reduce the false-positive ratio by means of more stringent amplification conditions. Electrophoretic patterns with previously selected arbitrarily primers revealed differences between the grades, four of them were investigated through sequencing. These sequences did not show significant nucleotide and aminoacid similarity to any known sequences in the DataBase. Sensitivity of the method was documented by the evidence that only one of the selected markers was an artefact, while the others represented genetic markers of the human Glial neoplasm.
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A two-step strategy was developed consisting of differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) with cultured normal human fetal astrocytes and U-373MG glioma cells followed by reverse Northern analysis of normal brain and primary tumor tissues. hu-dek, alpha-NAC, ribosomal proteins L7a and L35a, and five novel genes were identified. Since none of these genes has been previously shown to be associated with malignant brain tumor formation, this approach may be useful to identify novel targets for the diagnosis and treatment of brain tumors.
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We have previously reported the isolation of a G protein-coupled receptor, CXCR-4, that is overexpressed in glioblastoma multiforme tumor tissue (GMTT), as compared to normal brain tissue (NBT). Gene-specific RT-PCR, Northern blotting, and in situ hybridization techniques were used to study its expression in a variety of normal tissues, tumor tissues, and cell lines, as well as during development. Antisense CXCR-4 was overexpressed in glioblastoma cells to study its effect on cell proliferation. Gene-specific RT-PCR analysis indicated that the CXCR-4 gene is overexpressed in several malignant glioma tissues, breast tumor tissues and cell lines. Northern blot analysis indicated that CXCR-4 is expressed at high levels in certain leukemias, uterine cancer, and Burkitt's lymphoma cell line. The occipital and temporal lobe showed high levels of CXCR4 in normal human brain. The CXCR-4 gene was expressed in all organs in the early stages of development (days 8-10). In adult mouse, CXCR-4 is expressed only in brain, spinal cord, bone marrow, and pituitary gland. Antisense CXCR-4 overexpression in glioblastoma cells caused inhibition of cell proliferation and induction of cellular differentiation in vitro. This suggests that CXCR-4 expression may play an important role during embryonic development and also in the genesis of human gliomas. On the basis of CXCR4 expression data and antisense overexpression data, we conclude that CXCR-4 plays an important role in the tumorigenic properties of brain, breast, and other tumor types. On the basis of its unique expression during mouse development, we conclude that it may play an important role in the normal functioning of brain, spinal cord, and bone marrow during development.
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Glioma constitutes the most frequent brain tumor in man with glioblastoma as the most prevalent and malignant type. Despite surgery, radiation and chemotherapy, patients with glioblastoma have only a median survival time of less than one year. Although numerous chromosomal deviations have been described in glioblastoma, only few genes have been associated with these changes. It is especially necessary to identify genes involved in early glioma development and in progression to glioblastoma. Here, we describe the identification of a novel glioma expressed antigen both in the benign form of pilocytic astrocytoma and the malignant glioblastoma. We established two lambda zap expression libraries from a glioblastoma WHO grade IV and from a pilocytic astrocytoma WHO grade I and screened the libraries with the corresponding autologous patient sera. Both screenings revealed several serum positive clones, but interestingly only one clone termed glioma expressed antigen 1 (GLEA1) was found immunoreactive in the glioblastoma serum and the pilocytic astrocytoma serum. None of the control sera including sera from patients with lung carcinoma, meningioma or control persons showed an immune response to GLEA 1. GLEA appears to be a glioma specific immunogenic antigen which lends itself as a potential diagnostic marker.
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Using the technique of differential hybridization of Atlas Human cDNA expression arrays, we previously reported the isolation of a G protein coupled receptor, CXCR-4, which is overexpressed in glioblastoma multiforme tumor tissue (GMTT) compared to normal brain tissue (NBT). Using gene specific reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization, we studied its expression in a variety of brain and breast tumor tissue samples. To demonstrate the requirement of CXCR-4 in glioblastoma cell proliferation an antisense construct was overexpressed. Glioblastoma cells were also treated with antibodies against CXCR-4 and its ligand, SDFbeta-1. Expression analysis indicated that CXCR-4 is overexpressed in 57% of the primary glioblastoma tissues and in 88% of the glioblastoma cell lines analyzed. Overexpression of CXCR-4 in glioblastoma cell lines enhanced their soft agar colony-forming capability. Expression of anti-sense CXCR-4 in glioblastoma cell lines caused neurite outgrowth and cellular differentiation. Treatment of glioblastoma cell lines with CXCR-4 and SDFbeta-1 specific antibodies caused inhibition of glioblastoma cell proliferation. On the basis of these results, we conclude that CXCR-4 gene is required for the proliferation of human glioblastoma tumors.
Article
Background and Objectives Using the technique of differential hybridization of AtlasTM Human cDNA expression arrays, we previously reported the isolation of a G protein coupled receptor, CXCR-4, which is overexpressed in glioblastoma multiforme tumor tissue (GMTT) compared to normal brain tissue (NBT).Methods Using gene specific reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization, we studied its expression in a variety of brain and breast tumor tissue samples. To demonstrate the requirement of CXCR-4 in glioblastoma cell proliferation an antisense construct was overexpressed. Glioblastoma cells were also treated with antibodies against CXCR-4 and its ligand, SDFβ-1.ResultsExpression analysis indicated that CXCR-4 is overexpressed in 57% of the primary glioblastoma tissues and in 88% of the glioblastoma cell lines analyzed. Overexpression of CXCR-4 in glioblastoma cell lines enhanced their soft agar colony-forming capability. Expression of antisense CXCR-4 in glioblastoma cell lines caused neurite outgrowth and cellular differentiation. Treatment of glioblastoma cell lines with CXCR-4 and SDFβ-1 specific antibodies caused inhibition of glioblastoma cell proliferation.Conclusions On the basis of these results, we conclude that CXCR-4 gene is required for the proliferation of human glioblastoma tumors. J. Surg. Oncol. 1998;69:99–104. © 1998 Wiley-Liss, Inc.
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Previously, we reported the isolation of C4-2 as a potential tumor suppressor gene in human brain tumors. To understand the function of this gene, we investigated its molecular characterization and expression during development. Human fetal brain library screening and 5'RACE-PCR method was used to isolate the full-length cDNA. The coding region of C4-2 was used for in situ hybridization to study its expression during development. We report here the complete sequence of this gene. Sequence analysis indicated that C4-2 has a 94% sequence identity to a family of cAMP-regulated phosphoproteins (ARPP-16/19) in the coding region. C4-2 has a 3.1 Kb long 3'UTR with variable identity to ARPP-16 and ARPP-19. Northern blot analysis indicated that C4-2 is expressed at high levels in normal brain compared to other tissues. Zoo blot analysis demonstrated that the coding region of C4-2 is highly conserved among different animals. In situ hybridization using C4-2 coding region demonstrated that it follows a unique expression pattern during mouse brain development. High level of C4-2 expression was also observed in the spinal cord and somites of the developing embryo. Expression analysis during brain development strongly suggests that this family of proteins may play an important role not only in normal functioning of the brain, but also during brain development.
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Expression of CD44 and of specific splice-variants of CD44 has been causally related to metastatic behaviour in a variety of carcinomas and lymphomas. To elucidate whether, in principle, similar splice-variants could be involved in glioma cell invasion we examined the expression of CD44 and its splice-variants in a series of 38 primary human brain tumors (28 astrocytomas, WHO grade I-III and 10 glioblastomas, WHO grade IV) and in cell lines derived from 9 glioblastomas. All brain tumors examined showed strong immunoreactivity for an N-terminal epitope present on all CD44 isoforms known. Using a polyclonal antiserum raised against the complete sequence encoded by variant exons v3 to v10, CD44 splice-variants could be detected irrespective of the grade of malingnancy in many of the tumor samples at a low level and often restricted to only a few clustered tumor cells. Thus, the N-terminal epitope probably indicates the presence of the smallest and most ubiquitous isoform CD44s. Interestingly, all glioblastomas expressed CD44 variants whereas expression in astrocytomas WHO grade I, II, and III could only be detected in about half of the tumor samples. Using reverse transcriptase-PCR we were able to detect different CD44 splice-variants in the glioblastoma cell lines and in cultured primary astrocytic cells. Glioblastoma cells analyzed by flow cytometry showed the expected binding capacity for hyaluronic acid which could be increased twofold after pretreatment with hyaluronidase. The results presented show that there is low expression of CD44 variants in human tumors of astrocytic origin. Expression of CD44 and its splice-variants could contribute to the migration capacity of neoplastic astrocytes, and may be considered as a target for new diagnostic and therapeutic approaches in the clinical management of brain tumors.
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Identification of the genes that are specifically expressed in tumor cells but not in normal cells (oncogenes), or vice versa (tumor suppressor genes), is important for understanding the molecular basis of cancer. The differential display technique was applied to compare mRNAs from normal and tumor-derived human mammary epithelial cells, cultured under the same conditions. Complementary DNA fragments corresponding to several apparently differentially expressed mRNAs were recovered and sequenced. They exhibit characteristics of the 3' end of eukaryotic mRNA, as predicted by the method. A complementary DNA fragment seen only in the normal cell was used as a probe to isolate its corresponding complementary DNA clone from a library. Northern analysis confirmed its differential expression. Thus, this method can be used for detecting, cloning, and sequencing of genes that are unique to a host of biological and disease processes.
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In this study, we investigated the expression of activated gelatinase A and membrane-type metalloproteinase (MT-MMP) induced by concanavalin A (ConA) in four highly invasive glioma cell lines (UWR2, UWR3, U251MG, and SNB-19). We also examined gelatinase A and MT-MMP expression in human brain tumor tissues in vivo. Gelatin zymography showed that all four cell lines expressed latent progelatinase A (M(r) 66,000). Activated gelatinase A (M(r) 62,000) was induced by ConA in only UWR2 or UWR3 cells. MT-MMP mRNA was present in all four cell lines prior to ConA treatment, and the relative hybridization signals were 1, 0.80, 0.25, and 0.15 in UWR2, UWR3, U251MG, and SNB-19 cells, respectively. These mRNA signals were dramatically increased (2,8-, 5.4-, and 2.2-fold in UWR2, UWR3, and U251MG cells, respectively) following ConA treatment; however, MT-MMP mRNA expression was unchanged in SNB-19 cells. MT-MMP protein was detected in various amounts in the four cell lines, but only after ConA pretreatment. The amount of MT-MMP mRNA was unchanged in SNB-19 after ConA treatment, and the MT-MMP mRNA level in ConA-treated U251MG was lower than in UWR2 and UWR3 without ConA treatment. MT-MMP protein was detected in SNB-19 and U251 cell lines only after ConA treatment. Gelatin zymography of human brain tumor tissues revealed that almost all samples examined contained a latent form of gelatinase A, whereas the activated form of gelatinase A was only seen in metastatic lung adenocarcinomas and malignant astrocytomas, and especially in glioblastomas. MT-MMP mRNA levels were significantly higher in malignant astrocytomas than in low-grade gliomas and normal brain tissues. These results were confirmed by PCR analysis, which showed that MT-MMP mRNA was absent or barely detectable in normal brain white matter but was easily detectable in malignant astrocytomas. Immunohistochemistry of MT-MMP in frozen sections showed that MT-MMP was localized in neoplastic astrocytes of malignant astrocytomas but was undetectable in normal white brain matter. The data indicate that MT-MMP is present in malignant human glial tumors and that MT-MMP expression correlates with expression and activation of gelatinase A during malignant progression in vivo. A direct correlation between the levels of MT-MMP protein and its transcripts was not found in vitro, suggesting that MT-MMP expression in glioma cell lines might be regulated either at the level of transcription message stability or at posttranscription. Altered MT-MMP expression might contribute, in part, to gelatinase A activation, which in turn facilitates invasion of these tumors.
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A modified reverse transcription polymerase chain reaction (RT-PCR)-based differential display procedure with selected primers (SPR) was developed to increase the bias toward isolating moderate- to low-abundance transcripts that are differentially expressed during synapse formation in a microscopic neuronal system, the embryonic chicken ciliary ganglion. Major modifications, in comparison with available arbitrarily primed RT-PCR protocols, include the use of (i) experimentally selected primer pairs (50% GC-rich 15-21-mers) that avoid the amplification of highly abundant ribosomal and mitochondrial transcripts; (ii) a higher PCR annealing temperature (50 degrees C instead of 40 degrees C); (iii) selection of sequencing gel bands that are dependent on the two primers for amplification; (iv) tests for reproducibility by SPR amplification of independent sets of RNA extractions and Southern blot analysis of the products with an isolated radiolabeled clone; and (v) quantitative RT-PCR, instead of Northern blot analysis, to confirm the differential expression of individual cDNAs. Thirty-six cDNAs were isolated and sequenced using SPR. None showed significant homology to highly abundant transcripts. In contrast, when no criterion for primer or band selection was applied, 22% of 55 cDNAs were identical to ribosomal and mitochondrial transcripts. Reproducible amplification of 9 out of 10 SPR-isolated cDNAs was established by Southern blot analysis. Differential expression was then confirmed for 4 selected sequences by quantitative RT-PCR. Thus, SPR is a reproducible and efficient procedure for identifying differentially regulated transcripts of moderate- to low-abundance in microscopic biological systems.
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Vascular endothelial growth factor (VEGF) has been investigated as a potent mediator of Drain tumor angiogenesis, vascular permeability, and glioma growth. Using a VEGF ELISA, we determined the concentration of VEGF in the sera and tumor extracts of 19 brain tumor patients including glioblastoma, anaplastic astrocytoma, low grade astrocytoma, meningi- oiiiii. malignant lymphoma, and metastatic brain tumor as well as normal brain. VEGF concentration in the tissue of glioblastomas was significantly higher than that in other types of tumors as well as normal brain. Although VEGF concentration of the serum was not correlated with that of the tissue, VEGF concentrations of glioblastoma cyst fluid were 200- 300-fold higher than those of serum in the patients. VEGF concentration in the tumors was significantly correlated with the vascularity measured by counting vessels stained with von Willebrand factor antibody. VEGF protein localized to the cytoplasm of tumor cells and vasculature in gliomas, predominantly in the peripheral microvessel "hot spots" as well as around the necrosis in glioblastomas. VEGF immunopositivities were well reflected with VEGF concentration determined by ELISA. VEGF ELISA demonstrated time-dependent increase of the VEGF concentration in the serum-free conditioned medium of various glioma cell lines. The conditioned medium with high VEGF concentration induced endothelial cell migration. These observations suggest that VEGF represents a useful marker and measurable element of glioblastoma angiogenesis. The meas urement of VEGF concentration by ELISA in tumor and tumor cyst fluid may allow for the assessment of vascularity in gliomas.
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BACKGROUND Alterations of the suppressor genes, such as the retinoblastoma (RB), p53, p16(CDKN2), and p15 genes, have been reported in human gliomas. These genes have been suggested as the cell cycle regulatory genes at the G1-S checkpoint.METHODS Alterations of the RB, p53, p16(CDKN2), and p15 genes in human astrocytomas were screened by single strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP analysis) and then confirmed by dideoxy sequencing. In addition, the expression of RB and p16 protein was examined by Western blot analysis.RESULTSAberrations of the RB gene were found in 3 of 23 surgical astrocytoma specimens (13%). Mutations were found at codon 754 in exon 22 (Val→Gly), codon 519 in exon 17 (Thr→Pro), and one base deletion at codon 903 resulting in stop codon at codon 905 in exon 26. These mutational locations were all near the regions associated with the functional domains of the RB gene. Aberrations of the p53 gene were found in 4 cases (17.4%). These mutations were found at codons 146 (Trp→Gly) and 165 (Gln→His) in exon 5, codon 73 (Val→Glu) in exon 4, and codon 313 (Ser→Asn) in exon 9. In addition, alterations of the p16(CDKN2) gene were found, with 5 cases (21.7%) having homozygous deletions, and 2 cases (8.7%) harboring point mutations. No p15 gene alteration was detected. The expression of p16 protein was undetectable in 10 cases (43.5%) by Western blot analysis, demonstrating an inverse correlation with the expression of RB protein.CONCLUSIONSA few cases had overlapping alterations, and the incidence of one or more RB, p53, or p16(CDKN2) changes appeared to be relatively high in human astrocytomas. These results suggest that cell cycle regulatory gene alterations may play an important role in the development of gliomas. Cancer 1996;78:287-93.
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Effective methods are needed to identify and isolate those genes that are differentially expressed in various cells or under altered conditions. This report describes a method to separate and clone individual messenger RNAs (mRNAs) by means of the polymerase chain reaction. The key element is to use a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer. The mRNA subpopulations defined by these primer pairs were amplified after reverse transcription and resolved on a DNA sequencing gel. When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.
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The cdk inhibitor p21WAF1/Cip1 (p21), which can be transcriptionally activated by p53, functions to block cell cycle progression. In this study, we analysed the expression of p21 in normal and reactive brain and in gliomas of various malignancy grades. Southern blotting showed no p21 gene deletion. Western blotting and immunohistochemical assay showed that the levels of p21 protein in normal and reactive brain tissue were very low; however, p21 was elevated in a majority of gliomas tested, regardless of their malignancy grades. In glioblastoma multiforme, marked elevation of p21 was observed in samples harboring either wild-type or mutant p53. But, in anaplastic astrocytomas, the level of p21 was not elevated in samples harboring mutant-type p53. Immunohistochemical staining of paraffin-embedded astrocytomas and glioblastomas showed that tumor cells and not contaminating normal cells were positive for p21. Therefore, overexpression of p21 appears to be an early event in the development of glial neoplasms and p53-dependent p21 expression appears to be tumor grade specific.
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To investigate how overexpression of MAD, an antagonist of MYC oncogenes influences the malignant phenotype of human cancer cells, an adenovirus vector system was used to transfer the human MAD gene (AdMAD) into human astrocytoma cells. Decreased growth potential of AdMAD-infected cells was evidenced by a decrease in [3H]thymidine incorporation, an increase in cell doubling time and alteration of cell-cycle distribution. Diminished malignant potential of AdMAD-infected cells was manifested by their loss of anchorage-independent growth in soft agar and by their inability, in general, to induce tumorigenesis in a xenograft animal model. These studies indicate that adenovirus constructs encoding MAD dramatically inhibit the proliferation and tumorigenicity of human astrocytoma cells and support the use of MAD for gene therapy of human tumours.
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Hyperglycemia is postulated to cause chronic changes in the vasculature of diabetic patients, suggesting structural or genetic alterations. We have characterized the glucose induced alterations of gene expression in cultured bovine aortic smooth muscle cells using the recently developed mRNA differential display method. After five days of incubation with either 5.5 or 22 mM glucose, RNA preparations were isolated from confluent cells and probed with 10 candidate clones identified after screening up to 3000 mRNA species. Among these, three clones (2A, 2C, 3) showed significant changes in expression by Northern blot analysis. Elevated glucose levels decreased the mRNA expression of clones 2A and 3 to 51 +/- 7% (P < .01) and 59 +/- 10% (P < .05) (mean% of control +/- SEM), respectively. Expression of clone 2C was increased in 22 mM glucose condition to 221 +/- 23% (P < .05). Nucleotide sequence analysis showed that clone 3 had 77% homology to the 3'-noncoding region of human elongation factor 2, a member of the GTPase family which is essential for polypeptide synthesis. Clones 2A and 2C do show no homology to known nucleotide sequences. These results indicate that physiologically attainable high glucose conditions can significantly effect gene expression in aortic smooth muscle cells. Furthermore, mRNA differential display can be used in metabolic studies to identify new genes regulated by nutrients such as glucose.
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Brain tumors are a relatively common cancer in all age groups, and the incidence of both primary and metastatic brain tumors is increasing. Fortunately, dramatic advances in methods of diagnosis, surgical technique, and adjuvant therapy have all contributed to an improved outlook for patients with brain tumors. This article reviews the classification, symptoms, diagnosis, and treatment of brain tumors.
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We have significantly Improved a method originally developed by Liang and Pardee [Science 257 (1992) 967–971] to display a broad spectrum of expressed genes and to detect differences In expression between different cell types. We have analysed various aspects of the technique and have modified It for both, the application to fast and efficient Identification of genes and the use with automatic analysis systems. Based on the mathematical background we have devised the appropriate number of optimal PCR primers. We have also Introduced nondenaturatlng gels for separating double stranded fragments as single bands. By applying the method to regenerating mouse liver, we have identified, out of a total of 38,000 bands, about 70 fragments where the expression of the corresponding genes seems to be differentially regulated at different time points. Application of the method to an automatic DNA sequencer was successfully done. Thus, we have confirmed the usefulness and Increased the power of the RNA display technique, which we named differential display reverse transcription PCR (DDRT-PCR), and have extended the range of Its application.
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The American Cancer Society's Department of Epidemiology and Statistics reports its 30th annual compilation of cancer incidence, survival, and mortality data for the United States and around the world.
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Mutation of the p53 gene is among the most common lesions in a variety of human tumors, including those of the central nervous system. In most instances, mutation of one p53 allele is followed by loss of the remaining wild-type allele, resulting in cells with a complete absence of functional wild-type p53 protein. However, in some situations, such as at initiation of spontaneously arising gliomas or as the germline configuration of patients with the Li-Fraumeni syndrome, cells clearly carry both wild-type and mutant p53 alleles. These observations lead to the hypothesis that p53 mutations can give rise to loss of tumor suppressor functions as well as to gain of oncogenic transformation capabilities. In this review, we define the types of mutations that occur in the p53 gene in various glial tumors, contrast that with the spectra described in other human tumor types, and discuss the biochemistry and physiology of the p53 protein and its ability to regulate and be regulated by other gene products. We use this information to propose roles for p53 in the initiation and progression of human gliomas.
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Progression of gliomas to more malignant phenotypes involves numerous molecular genetic alterations. The genes affected by these alterations, the steps in malignant progression for which they are responsible, their normal function in controlling diverse cellular functions such as differentiation, signal transduction, cell cycle progression and angiogenesis and how they may act in concert with other tumour suppressor genes or oncogenes are some of the questions finally coming into focus and being studied. As other genes are discovered, their association with tumour progression can be assessed, coupled with current histopathology and used to determine more accurately patient prognosis and strategies for intervention. With the generation of specific reagents, such as monoclonal antibodies directed to glioma derived antigens or emerging gene therapy techniques designed to deliver toxic, antisense or reconstituting genes specifically to tumour tissue, new approaches will be devised that may finally be used to treat these tumours effectively.
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Laminin may alter the biological behavior of gliomas. Therefore, we investigated the expression of two laminin receptors, alpha6 beta1 and alpha6 beta4 integrins in normal brain, astrogliotic brain, and astrocytomas as compared to other central nervous system (CNS) tumors. In most CNS tumors, the expression of these integrins was unchanged in neoplastic as compared to normal counterpart cells. In contrast, increased numbers of reactive and neoplastic astrocytes expressed beta4 integrin as compared to normal astrocytes, whereas alpha6 and beta1 integrin expression did not change. Conversely, lower numbers of astrocytoma blood vessels expressed beta4, whereas all blood vessels in normal brain expressed beta4. These data suggest that the profile of laminin receptors changes in neoplastic astrocytes and in astrocytoma blood vessels; this change may play an important role in astrocytoma pathogenesis.
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The technique of differential display polymerase chain reaction (DD-PCR) was used to identify cDNA sequences, which are temporally expressed during ferret tracheal airway development. Such differentially expressed cDNAs may ultimately prove to be useful markers in elucidating mechanisms of epithelial differentiation and submucosal gland development in the airway. Using two sets of oligonucleotide primers 15 differentially amplified cDNAs were isolated by comparative reverse transcriptase (RT) PCR of 6-h and 3-day postnatal tracheal poly-A mRNA. In situ hybridization was used to assess the reliability of this method and confirm the differential mRNA expression patterns of cloned cDNAs. Results of in situ hybridization analysis demonstrated that 10 of the 15 cDNA sequences gave a temporally regulated pattern of expression, which was concordant with that of the differential display. Furthermore, sequence analysis of the 15 isolated cDNAs revealed that the majority of clones were amplified from two inverted decamer primers. These findings demonstrate the lack of poly-T priming in the differential display reaction, which suggests that this method may yield substantially more information regarding the coding sequence of cloned genes. In support of this observation, 6 of the 15 cDNA sequences contained one complete open reading frame. Although the majority of cDNAs demonstrated no homology to sequence data bases at the DNA or amino acid level, clone FT-4, which demonstrated a differential expression pattern limited to 3-day tracheal time points, was composed of a 10-amino acid repeat domain that was structurally similar to neuropeptide anthoRFamide and barley D hordein seed protein. A second interesting clone, FT-3, demonstrated an infrequent pattern of expression within a subset of epithelial cells limited to early developmental time points (6 h) and was dramatically reduced by 3 days postnatally. Several additional clones with no homologies to previously cloned genes demonstrated expression patterns that were also temporally regulated throughout tracheal development. Although the function of these temporally regulated genes has not been determined, these genes may ultimately prove to be useful markers of cellular differentiation during tracheal development.
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Epidermal growth factor receptor (EGFR) is an operationally specific antigen in malignant gliomas; it is overexpressed in > 60% of these tumors, whereas its expression is very low in normal brain. This study aimed to evaluate whether an adequate amount of an anti-EGFR monoclonal antibody (MAb) could reach a tumor after a single intravenous administration. This study was open, nonrandomized, and uncontrolled. Single doses (20, 40, 100, 200, or 400 mg) of the murine MAb EMD55900 (MAb 425) were administered intravenously before surgery to 30 patients with malignant brain tumors. Serum samples were taken at defined time intervals during infusion, to determine EMD55900 concentrations, and 10, 21, and/or 42 days after infusion, to evaluate the development of human anti-mouse antibodies. Tumor samples were investigated for EGFR and EMD55900 contents. Tolerance to EMD55900 was good. Increased liver transaminase levels were noted for three patients with Grade 1 toxicity. Twenty patients developed significant human anti-mouse antibody titers, without correlation with the administered dose. The median half-life of EMD55900 in serum ranged from 6 hours for 20 mg to 24 hours for 400 mg. In the membrane fractions of the tumors, EGFR saturation by EMD55900 varied with the injected dose of MAb. No binding was detected after a 20-mg dose. After doses of 40, 100, 200, and 400 mg, the mean saturation levels were 33, 73, 89, and 71%, respectively. This study indicates that a single intravenous administration of EMD55900 is well tolerated and produces substantial in vivo tumor binding with doses > 100 mg.