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The rat glial progenitor cell line CG-4 can be induced to differentiate into either oligodendrocytes or type-2 astrocytes. In order to identify genes whose expression varies coincident with such phenotypic differentiation, we employed representational difference analysis (RDA) of mRNA. Here, we report 38 cDNAs induced in type-2 astrocytes, oligodendrocytes, or both differentiated states. Among these were known transcription factors, membrane receptors, extracellular matrix proteins, secreted signaling modulators, chromatin regulators and myelin sheath components. In addition several novel genes were identified; among these was a gene induced during the very early stages of astrocyte differentiation that we have named Ieda (induced early in differentiating astrocytes). Several Ieda transcripts were detected by RT-PCR, and appeared to be produced by alternative splicing and promoter usage. The protein deduced from the longest Ieda mRNA exhibited sequence features characteristic of G-protein coupled receptors, including seven putative transmembrane domains, while the shorter Ieda transcripts encoded proteins that lacked several transmembrane segments. In the adult rat, Ieda transcripts were found exclusively in brain and testis. In the developing rat brain, Ieda expression was first detected at embryonic day 16, that is two days before the first appearance of mature astrocytes. Thus, this approach has yielded a potential source of markers for differentiation states of these two cellular types as well as genes predicted to be functionally involved in the differentiation process itself.

Glioblastoma multiforme (GBM) is the most aggressive type of glioma and GBMs frequently contain amplifications or mutations of the EGFR gene. The most common mutation results in a truncated receptor tyrosine kinase known as Delta EGFR that signals constitutively and promotes GBM growth. Here, we report that the 45-kDa variant of the protein tyrosine phosphatase TCPTP (TC45) can recognize Delta EGFR as a cellular substrate. TC45 dephosphorylated Delta EGFR in U87MG glioblastoma cells and inhibited mitogen-activated protein kinase ERK2 and phosphatidylinositol 3-kinase signaling. In contrast, the substrate-trapping TC45-D182A mutant, which is capable of forming stable complexes with TC45 substrates, suppressed the activation of ERK2 but not phosphatidylinositol 3-kinase. TC45 inhibited the proliferation and anchorage-independent growth of Delta EGFR cells but TC45-D182A only inhibited cellular proliferation. Notably, neither TC45 nor TC45-D182A inhibited the proliferation of U87MG cells that did not express Delta EGFR. Delta EGFR activity was necessary for the activation of ERK2, and pharmacological inhibition of ERK2 inhibited the proliferation of Delta EGFR-expressing U87MG cells. Expression of either TC45 or TC45-D182A also suppressed the growth of Delta EGFR-expressing U87MG cells in vivo and prolonged the survival of mice implanted intracerebrally with these tumor cells. These results indicate that TC45 can inhibit the Delta EGFR-mediated activation of ERK2 and suppress the tumorigenicity of Delta EGFR-expressing glioblastoma cells in vivo.

Innate and acquired resistance to chemotherapy and radiation therapy has been a major obstacle for clinical oncology. One potential adjunct to such conventional treatments is direct induction of cell death by activation of death receptor-mediated apoptosis. TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand), a recently identified member of the growing TNF superfamily, binds to its cognate "death" receptors DR4 and DR5 as well as "decoy" receptors DcR1 and DcR2. Upon binding, rapid apoptosis is enacted in a variety of human cancer cell lines independent of p53 status, but not in normal cell lines. TRAIL treatment results in significant growth suppression of TRAIL-sensitive human cancer xenografts in mice. Furthermore, combination treatment of TRAIL with genotoxic chemotherapeutic agents synergistically suppresses growth of tumor xenografts which are otherwise resistant to treatment with TRAIL or chemotherapy alone. Unlike the other death ligands TNF-alpha or FasL, systemic administration of soluble human TRAIL does not cause toxicity in mice and non-human primates. While further studies are needed to evaluate the possible cytotoxicity of TRAIL especially for human hepatocytes, indications are increasing that TRAIL may be a novel therapeutic agent for human cancer.

The urokinase plasminogen activator system is involved in angiogenesis and tumor growth of malignant gliomas, which are highly neovascularized and so may be amenable to antiangiogenic therapy. In this paper, we describe the activity of Å6, an octamer capped peptide derived from the non-receptor-binding region of urokinase plasminogen activator. Å6 inhibited human microvascular endothelial cell migration but had no effect on the proliferation of human microvascular endothelial cells or U87MG glioma cells in vitro . In contrast, Å6 or cisplatin (CDDP) alone suppressed subcutaneous tumor growth in vivo by 48% and 53%, respectively, and, more strikingly, the combination of Å6 plus CDDP inhibited tumor growth by 92%. Such combination treatment also greatly reduced the volume of intracranial tumor xenografts and increased survival of tumor-bearing animals when compared with CDDP or Å6 alone. Tumors from the combination treatment group had significantly reduced neovascularization, suggesting a mechanism involving Å6-mediated inhibition of endothelial cell motility, thereby eliciting vascular sensitivity to CDDP-mediated toxicity. These data suggest that the combination of an angiogenesis inhibitor that targets endothelial cells with a cytotoxic agent may be a useful therapeutic approach.

Differential display polymerase chain reaction analysis was used to compare five differentiation states of the O-2A progenitor-like cell line CG4: progenitor cells and cells at 12 h or 4 days after the induction of differentiation into oligodendrocytes or astrocytes. This led to the identification of 52 sequence tags that were expressed differentially with cellular phenotype. One sequence was upregulated during differentiation of CG4 cells and represented a novel gene that we named SETA (SH3 domain-containing gene expressed in tumorigenic astrocytes). This gene encodes an SH3 domain-containing adapter protein with sequence similarity to the CD2AP (CD2 adapter protein) and CMS (Cas ligand with multiple Src homology) genes. SETA mRNA was expressed at high levels in the developing rat brain but was barely detectable in the normal adult rat or human brain. However, SETA mRNA was found in approximately one half of the human gliomas tested, including astrocytomas grades II, III, and IV, as well as oligodendrogliomas, mixed oligoastrocytomas, and human glioma-derived cell lines. A rat glioma generated by treatment with the alkylating carcinogen ethylnitrosourea on postnatal day 1 and a derived cell line also expressed SETA mRNA. Furthermore, in an in vitro model of astrocytoma progression based on p53-/- astrocytes, expression of SETA was restricted to cells that are tumorigenic.

The PCAF gene encodes the p300/CBP-Associated Factor (PCAF), a histone acetyltransferase, which regulates p53 by acetylation of Lys320 in the C-terminal portion of p53. While the p53 gene is one of the most frequently mutated tumor suppressor genes in human tumors, such mutations occur in only 30% of astrocytic tumors. Since PCAF can regulate p53 activity, abrogation of PCAF function by PCAF gene mutation could be an alternate mechanism to inactivate the p53 pathway in tumors lacking p53 mutations. To test this hypothesis, we determined the nucleotide sequence of the entire PCAF coding region in 37 astrocytic tumors (17 glioblastomas, 10 anaplastic astrocytomas, 7 low-grade astrocytomas, and 3 pilocytic astrocytomas). We detected two single-nucleotide alterations that represented non-deleterious polymorphisms (GAG > GAA Glu103Glu, AAT > AGT Asn386Ser) but no obvious functional mutations. Moreover, the frequency of the Asn386Ser allele that contained Ser386 in glioma patients was not statistically different from its frequency in individuals without disease, and no significant association was observed between the PCAF polymorphisms and the presence or absence of p53 mutations in the tumors. We conclude that the PCAF gene is not mutated during the development of the astrocytic tumors studied here.

Malignant gliomas are among the most lethal and intractable of human tumors and drug resistance is one of the major obstacles to their successful treatment. Recent advances in the molecular biology and genetics of human cancers provide a detailed understanding of cellular and molecular responses to chemotherapy and how drug resistance may develop. Several oncogenes and tumor suppressor genes have been shown to confer resistance to tumor cells and should, therefore, provide novel and defined targets for cancer treatment. In addition to overcoming cellular resistance, special efforts to increase drug delivery to glial tumors need to be pursued because of the relatively unique problem of the blood-brain barrier. Treatments aimed at these targets will likely benefit from combined therapies including surgery, traditional chemotherapy and targeted disruption of other physiological processes such as angiogenesis. Copyright 1999 Harcourt Publishers Ltd.

Gross genetic lesions of chromosome 10 occur in 30–50% of sporadic human melanomas. To test the functional significance of this observation, we have developed an in vitro loss of heterozygosity approach in which a wild-type chromosome 10 was transferred into melanoma cells, where there was selection for its breakage and regional deletion to relieve its growth suppressive effects. The overlap of these events was at band 10q23, the site of the recently isolated PTEN/MMAC1 tumor suppressor gene, suggesting it as a potential target. Although the gene was expressed in the parental cells, both of its chromosomal alleles contained truncating mutations. In vitro loss of heterozygosity resulted in loss of the chromosomally introduced wild-type PTEN/MMAC1 , and ectopic expression of the gene caused cell growth suppression. Thus, this approach identified PTEN/MMAC1 as a target in malignant melanoma and may provide an alternative means to localizing tumor suppressor genes.

Deletions of all or part of chromosome 10 are the most common genetic alterations in high-grade gliomas. The PTEN gene (also called MMAC1 and TEP1 ) maps to chromosome region 10q23 and has been implicated as a target of alteration in gliomas and also in other cancers such as those of the breast, prostate, and kidney. Here we sought to provide a functional test of its candidacy as a growth suppressor in glioma cells. We used a combination of Northern blot analysis, protein truncation assays, and sequence analysis to determine the types and frequency of PTEN mutations in glioma cell lines so that we could define appropriate recipients to assess the growth suppressive function of PTEN by gene transfer. Introduction of wild-type PTEN into glioma cells containing endogenous mutant alleles caused growth suppression, but was without effect in cells containing endogenous wild-type PTEN . The ectopic expression of PTEN alleles, which carried mutations found in primary tumors and have been shown or are expected to inactivate its phosphatase activity, caused little growth suppression. These data strongly suggest that PTEN is a protein phosphatase that exhibits functional and specific growth-suppressing activity.

Deletions of chromosomal region 9p21 are among the most common genetic alterations observed during the clonal evolution of high grade malignant gliomas. Structural and functional evidence has suggested that homozygous deletion involving CDKN2A (the genetic locus encoding the cyclin-dependent kinase inhibitor p16(NK4a)) is a mechanism of inactivation of this gene and that it can be a growth suppressor in human gliomas. However, the presence of other potential suppressor genes in the 9p21 region and the relatively large sizes of the deletions has made it difficult to be certain that the CDKN2A gene is their actual target. Here, we tested this hypothesis by determining the growth suppressive effects, cell cycle inhibitions, and the activities of seven naturally occurring glioma-derived CDKN2A alleles carrying point mutations and found that two of them were functionally compromised. To resolve discrepancies among the different existing functional assays, we developed an assay for p16(INK4a) function that allowed us to demonstrate that the expression of wild-type CDKN2A, but not alleles with inactivating mutations, prevents pRB phosphorylation in vivo in human glioma cells. These data suggest that CDKN2A is a critical target for mutational inactivation in human malignant gliomas.

Deregulation of signaling by the epidermal growth factor receptor (EGFR) is common in human malignancy progression. One mutant EGFR (variously named DeltaEGFR, de2-7 EGFR, or EGFRvIII), which occurs frequently in human cancers, lacks a portion of the extracellular ligand-binding domain due to genomic deletions that eliminate exons 2 to 7 and confers a dramatic enhancement of brain tumor cell tumorigenicity in vivo. In order to dissect the molecular mechanisms of this activity, we analyzed location, autophosphorylation, and attenuation of the mutant receptors. The mutant receptors were expressed on the cell surface and constitutively autophosphorylated at a significantly decreased level compared with wild-type EGFR activated by ligand treatment. Unlike wild-type EGFR, the constitutively active DeltaEGFR were not down-regulated, suggesting that the altered conformation of the mutant did not result in exposure of receptor sequence motifs required for endocytosis and lysosomal sorting. Mutational analysis showed that the enhanced tumorigenicity was dependent on intrinsic tyrosine kinase activity and was mediated through the carboxyl terminus. In contrast with wild-type receptor, mutation of any major tyrosine autophosphorylation site abolished these activities suggesting that the biological functions of DeltaEGFR are due to low constitutive activation with mitogenic effects amplified by failure to attenuate signaling by receptor down-regulation.

A mutant epidermal growth factor receptor (DeltaEGFR) containing a deletion of 267 amino acids from the extracellular domain is common in human glioblastomas. We have previously shown that the mutant receptor fails to bind EGF, is constitutively phosphorylated, and confers upon U87MG glioblastoma cells expressing it (U87MG. DeltaEGFR), an increased ability to form tumors in mice. Here we demonstrate that the constitutively phosphorylated DeltaEGFR enhances growth of glioblastoma cells through increased activity of Ras: 1) there was an increase in the proportion of Ras present in the GTP-bound form, and 2) introduction of neutralizing anti-Ras 259 antibodies into U87MG and U87MG.DeltaEGFR cells by microinjection inhibited DNA synthesis to the same low level in both cell populations. We also show that the truncated EGF receptor constitutively associates with the adapter proteins Shc and Grb2 which are involved in the recruitment of Ras to activated receptors. Several derivatives of DeltaEGFR containing single, or multiple mutations at critical autophosphorylation sites were constructed and used to demonstrate that the major Shc binding site is Tyr-1148, and that Grb2 association occurs primarily through Tyr-1068. We conclude that the increased tumorigenic potential of glioblastoma cells expressing the truncated EGF receptor is due at least in part to Ras activation presumably involving the Shc and Grb2 adapter proteins.

The earliest genetic alteration in human astrocytoma progression is mutation of the p53 tumour suppressor gene, while one of the earliest phenotypic changes is the stimulation of neovascularization. Here, we tested the role of p53 in the angiogenic process by introducing a tetracycline-regulated wild type p53 gene into null glioblastoma cells. The parental cells expressed strong angiogenic activity while upon induction of wild type, but not mutant, p53 expression, the cells secreted a factor able to neutralize the angiogenicity of the factors produced by the parental cells as well as of basic fibroblast growth factor.

Alterations of the epidermal growth factor receptor (EGFR) gene are common in some forms of cancer and the most frequent is a deletion of exons 2-7. We have previously shown that this mutant receptor, called DeltaEGFR, confers enhanced tumorigenicity to glioblastoma cells through elevated proliferation and reduced apoptotic rates of the tumor cells in vivo. To understand the molecular mechanisms that underlie DeltaEGFR-enhanced proliferation, we examined the gene products that control cell cycle progression. We found that levels of the cyclin-dependent kinase (CDK) inhibitor, p27, were lower in U87MG.DeltaEGFR tumors than in parental U87MG or control U87MG.DK tumors. Consequently, CDK2-cyclin A activity was also elevated, concomitant with the RB protein hyperphosphorylation. In addition, activated phosphatidylinositol 3-kinase (PI3-K) and phosphorylated Akt levels were also elevated in the U87MG.DeltaEGFR tumors. U87MG.DeltaEGFR cells failed to arrest in G(1) in response to serum starvation in vitro and while maintaining high levels of PI3-K activity and hyperphosphorylated RB. Treatment of U87MG.DeltaEGFR cells with LY294002, a PI3-K inhibitor, caused reduced levels of phosphorylated Akt and concomitantly up-regulated levels of p27. Expression of a kinase dead dominant-negative Akt mutant in the U87MG.DeltaEGFR cells similarly resulted in up-regulation of p27 and down-regulation of tumorigenicity in vivo. These results suggest that the constitutively active DeltaEGFR can enhance cell proliferation in part by down-regulation of p27 through activation of the PI3-K/Akt pathway. This pathway may represent another therapeutic target for treatment of those aggressive glioblastomas expressing DeltaEGFR.

The rat glial progenitor cell line CG-4 can be induced to differentiate into either oligodendrocytes or type-2 astrocytes. In order to identify genes whose expression varies coincident with such phenotypic differentiation, we employed representational difference analysis (RDA) of mRNA. Here, we report 38 cDNAs induced in type-2 astrocytes, oligodendrocytes, or both differentiated states. Among these were known transcription factors, membrane receptors, extracellular matrix proteins, secreted signaling modulators, chromatin regulators and myelin sheath components. In addition several novel genes were identified; among these was a gene induced during the very early stages of astrocyte differentiation that we have named Ieda (induced early in differentiating astrocytes). Several Ieda transcripts were detected by RT-PCR, and appeared to be produced by alternative splicing and promoter usage. The protein deduced from the longest Ieda mRNA exhibited sequence features characteristic of G-protein coupled receptors, including seven putative transmembrane domains, while the shorter Ieda transcripts encoded proteins that lacked several transmembrane segments. In the adult rat, Ieda transcripts were found exclusively in brain and testis. In the developing rat brain, Ieda expression was first detected at embryonic day 16, that is two days before the first appearance of mature astrocytes. Thus, this approach has yielded a potential source of markers for differentiation states of these two cellular types as well as genes predicted to be functionally involved in the differentiation process itself.

In some respects, the EGFR appears to be an attractive target for tumor-targeted antibody therapy: it is overexpressed in many types of epithelial tumor and inhibition of signaling often induces an anti-tumor effect. The use of EGFR specific antibodies, however, may be limited by uptake in organs that have high endogenous levels of the wild type EGFR such as the liver. The de2-7 EGFR (or EGFRvIII) is a naturally occurring extracellular truncation of the EGFR found in a number of tumor types including glioma, breast, lung and prostate. Antibodies directed to this tumor specific variant of the EGFR provide an alternative targeting strategy, although the lower proportion of tumors that express the de2-7 EGFR restricts this approach. We describe a novel monoclonal antibody (MAb 806) that potentially overcomes the difficulties associated with targeting the EGFR expressed on the surface of tumor cells. MAb 806 bound to de2-7 EGFR transfected U87MG glioma cells (U87MG.Delta 2-7) with high affinity (approximately 1 x 10(9) M(-1)), but did not bind parental cells that express the wild type EGFR. Consistent with this observation, MAb 806 was unable to bind a soluble version of the wild type EGFR containing the extracellular domain. In contrast, immobilization of this extracellular domain to ELISA plates induced saturating and dose response binding of MAb 806, suggesting that MAb 806 can bind the wild type EGFR under certain conditions. MAb 806 also bound to the surface of A431 cells, which due to an amplification of the EGFR gene express large amounts of the EGFR. Interestingly, MAb 806 only recognized 10% of the total EGFR molecules expressed by A431 cells and the binding affinity was lower than that determined for the de2-7 EGFR. MAb 806 specifically targeted U87MG.Delta 2-7 and A431 xenografts grown in nude mice with peak levels in U87MG.Delta 2-7 xenografts detected 8 h after injection. No specific targeting of parental U87MG xenografts was observed. Following binding to U87MG.Delta 2-7 cells, MAb 806 was rapidly internalized by macropinocytosis and subsequently transported to lysosomes, a process that probably contributes to the early targeting peak observed in the xenografts. Thus, MAb 806 can be used to target tumor cells containing amplification of the EGFR gene or de2-7 EGFR but does not bind to the wild type EGFR when expressed on the cell surface.

The death ligands CD95L and Apo2L/TRAIL are promising investigational agents for the treatment of malignant glioma. EGFR is overexpressed in a significant proportion of malignant gliomas in vivo. Here, we report that CD95L-induced cell death is enhanced by EGFR inhibition using tyrphostine AG1478 in 7 of 12 human malignant glioma cell lines. Conversely, CD95-mediated and Apo2L-induced cell death are both inhibited by overexpression of EGFR in LN-229 cells. CD95L-induced cell death augmented by AG1478 is accompanied by enhanced processing of caspase 8. LN-229 cells overexpressing the viral caspase inhibitor, crm-A, are not sensitized to CD95L-induced cell death by AG1478, indicating that EGFR exerts its antiapoptotic properties through a caspase 8-dependent pathway. These data define a modulatory effect of EGFR-activity on death ligand-induced apoptosis and indicate that EGFR inhibition is likely to improve the efficacy of death ligand-based cancer therapies. Furthermore, it is tempting to speculate that EGFR amplification protects tumor cells from death ligand-mediated host immune responses in vivo and that EGFR's effects on death receptor-mediated apoptosis may explain the anti-tumor effects of non-cytotoxic, unarmed anti-EGFR family antibodies.

The gene for the major angiogenic factor, vascular endothelial growth factor (VEGF), encodes several spliced isoforms. We reported previously that overexpression of two VEGF isoforms, VEGF(121) and VEGF(165), by human glioma U87 MG cells induced tumor-associated intracerebral hemorrhage, whereas expression of a third form, VEGF(189), did not cause vessel rupture. Here, we test whether these VEGF isoforms have distinct activities for enhancing vascularization and growth of gliomas in mice. U87 MG cells that overexpressed VEGF(165) or VEGF(189) grew more rapidly than the parental cells in both s.c. and intracranial (i.c.) locations. However, cells that overexpressed VEGF(121) only showed enhancement of i.c. tumor growth but had a minimal effect on s.c. glioma progression. At both anatomical sties, VEGF(165) and VEGF(189) strongly augmented neovascularization, whereas VEGF(121) only increased vessel density in brain tumors. In each type of glioma, expression of VEGF receptors -1 and -2 largely phenocopied the tumor vasculature, because increased VEGF/VEGF receptor-activated microvessel densities were strongly correlated with the angiogenicity and tumorigenicity elicited by the VEGF isoforms at both anatomical sites. One notable difference between the sites was the expression of vitronectin, a prototypic ligand of alpha(v)beta(3) and alpha(v)beta(5) integrins, detected in i.c. but not in s.c., gliomas. Endothelial cell migration stimulated by VEGF(121) was potentiated by vitronectin to a greater extent than that stimulated by VEGF(165). This data demonstrates that VEGF isoforms have distinct activities at different anatomical sites and suggest that the microenvironment of different tissues affects the function of VEGF isoforms.

Object. Activation of signaling by the epidermal growth factor receptor (EGFR) through gene amplification or rearrangement is common in human malignancy, especially in a large fraction of de novo glioblastomas multiforme (GBMs). The most common mutant EGFR, (ΔEGFR, also known as de2–7 EGFR and EGFRvIII) lacks a portion of the extracellular domain, enhances tumorigenicity in vivo, and causes resistance to the chemotherapeutic drug cisplatin (CDDP). This resistance is due to the suppression of CDDP-induced apoptosis by the constitutively active tyrosine kinase activity of the receptor. The authors have investigated whether inhibition of AEGFR signaling by the tyrosine kinase inhibitor, tyrphostin AG1478, could sensitize tumor xenografts to CDDP and, thereby, enhance its therapeutic efficacy in animals. Methods. Nude mice were inoculated either subcutaneously or intracerebrally with human GBM cells expressing ΔEGFR and were then systemically treated with CDDP and/or AG1478. Tumor volumes were monitored and tumor sections were analyzed by using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assays or MIB-1 staining. Expression of ΔEGFR, but not wild-type EGFR, conferred CDDP resistance to the cells in vivo. Inhibition of receptor signaling by the EGFR-specific tyrosine kinase inhibitor, AG1478, sensitized the xenografts to the cytotoxic effects of CDDP. This combined CDDP/AG1478 treatment significantly suppressed growth of subcutaneous xenografts in nude mice in a synergistic manner (p < 0.01 compared with vehicle control) without causing generalized toxicity, whereas treatments with CDDP or AG1478 alone were ineffective. The synergistic growth suppression by the CDDP/AG1478 combination was not observed in xenografts overexpressing wild-type EGFR or kinase-deficient ΔEGFR. The combined CDDP/AG1478 treatment induced tumor growth suppression, which correlated with increased apoptosis and reduced proliferation. This treatment also extended the life span of mice bearing intracerebral xenografts (p < 0.01 compared with controls). Conclusions. The results of this study may provide the basis for the development of a novel and safe therapeutic strategy for the very aggressive ΔEGFR-expressing GBM.

Alterations of the epidermal growth factor receptor (EGFR) occur frequently in malignant gliomas through gene amplification or rearrangement, especially in a large fraction of de novo type glioblastomas. The most common of these mutant EGFRs (variously named de2-7 EGFR, deltaEGFR or EGFRvIII) lacks a portion of the extracellular ligand-binding domain. Here, we review the evidence that shows that expression of deltaEGFR bestows in vivo growth advantages to human glioma cells through its constitutively active tyrosine kinase activity. Thus, deltaEGFR may provide a novel therapeutic target for the most aggressive type of glioblastoma.

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Cancers are associated with frequent deletions of genetic material that select for the loss of genes regulating normal cellular physiology. Although several cancer suppressor genes have been identified from these areas of deletion, the identities of the vast majority remain unknown, making approaches leading to their localization, identification, and validation an important continuing endeavor. Those currently characterized cancer suppressors include regulators of aspects of the cell cycle, growth and transcriptional regulators, DNA repair enzymes, differentiation factors, cell motility elements, and regulators of signal transduction. Several inherited cancer predisposition genes have been mapped and cloned using meiotic genetic linkage mapping but less success has been achieved identifying those genes involved in non-familial cancer. The future localization, identification, and validation of these genes are likely to involve a combination of complementary position-oriented and function-driven approaches, some of which are detailed in this article.

Alterations of the epidermal growth factor receptor ( EGFR ) gene occur frequently in human malignant gliomas. The most common of these is deletion of exons 2–7, resulting in truncation of the extracellular domain (ΔEGFR or EGFRvIII), which occurs in a large fraction of de novo malignant gliomas (but not in progressive tumors or those lacking p53 function) and enhances tumorigenicity, in part by decreasing apoptosis through up-regulation of Bcl-X L . Here, we demonstrate that the ΔEGFR concomitantly confers resistance to the chemotherapeutic drug cisplatin (CDDP) by suppression of CDDP-induced apoptosis. Expression of Bcl-X L was elevated in U87MG.ΔEGFR cells prior to and during CDDP treatment, whereas it decreased considerably in CDDP-treated parental cells. CDDP-induced activation of caspase-3-like proteases was suppressed significantly in U87MG.ΔEGFR cells. These responses were highly specific to constitutively kinase-active ΔEGFR, because overexpression of kinase-deficient ΔEGFR (DK) or wild-type EGFR had no such effects. Correspondingly, ΔEGFR specific tyrosine kinase inhibitors reduced Bcl-X L expression and potentiated CDDP-induced apoptosis in U87MG.ΔEGFR cells. Ectopic overexpression of Bcl-X L in parental U87MG cells also resulted in suppression of both caspase activation and apoptosis induced by CDDP. These results may have important clinical implications for the use of CDDP in the treatment of those malignant gliomas expressing ΔEGFR.

The vascular endothelial growth factor (VEGF) has been shown to be a significant mediator of angiogenesis during a variety of normal and pathological processes, including tumor development. Human U87MG glioblastoma cells express the three VEGF isoforms: VEGF 121 , VEGF 165 , and VEGF 189 . Here, we have investigated whether these three isoforms have distinct roles in glioblastoma angiogenesis. Clones that overexpressed each isoform were derived and inoculated into mouse brains. Mice that received VEGF 121 - and VEGF 165 -overexpressing cells developed intracerebral hemorrhages after 60–90 hr. In contrast, mice implanted with VEGF 189 -overexpressing cells had only slightly larger tumors than those caused by parental cells and little evidence of hemorrhage at these early times after implantation, whereas, after longer periods of growth, enhanced angiogenicity and tumorigenicity were apparent. There was rapid blood vessel growth and breakdown around the tumors caused by cells overexpressing VEGF 121 and VEGF 165 , whereas there was similar vascularization but no eruption in the vicinity of those tumors caused by cells overexpressing VEGF 189 , and none on the border of the tumors caused by the parental cells. Thus, by introducing VEGF-overexpressing glioblastoma cells into the brain, we have established a reproducible and predictable in vivo model of tumor-associated intracerebral hemorrhage caused by the enhanced expression of single molecular species. Such a model should be useful for uncovering the role of VEGF isoforms in the mechanisms of angiogenesis and for investigating intracerebral hemorrhage due to ischemic stroke or congenital malformations.

DNA amplification is a common mechanism invoked by many human tumors to elicit overexpression of genes whose products are involved in drug resistance or cell proliferation. Although amplified regions in tumor DNA may exceed several megabases in size, segments of amplicons with a high probability of containing gene sequences may be amenable to detection by restriction landmark genomic scanning (RLGS), a high-resolution DNA analysis that separates labeled NotI fragments in two dimensions. Here, we tested this by applying RLGS to matched samples of glioma and normal brain DNA and found tumor-specific amplification of the gene encoding cyclin-dependent kinase 6 (CDK6), an observation not previously reported in human tumors. The CDK6 gene has been localized to chromosome 7q21-22, but in the gliomas studied here, it was not coamplified with either the syntenic MET (7q31) or epidermal growth factor receptor (7p11-p12) genes, suggesting that this may be part of a novel amplicon in gliomas. We then corroborated this finding by identifying both amplification-associated and amplification-independent increases in CDK6 protein levels in gliomas relative to matched normal brain samples. These data implicate the CDK6 gene in genomic amplification and illustrate the potential of RLGS for the more general identification and cloning of novel genes that are amplified in human cancer.

Rhabdomyosarcoma is a tumor of skeletal muscle origin affecting children and young adults. Although relatively undifferentiated, cell lines derived from this tumor express myogenic regulatory factors and so may be useful models of abortive myogenic differentiation. In the present studies, we have determined the effect of increased intracellular cAMP on proliferation, morphologic differentiation, and expression of myogenic genes in the prototypic embryonal rhabdomyosarcoma cell line, RD. Whereas growth in dibutyryl cAMP (dbcAMP), forskolin, or butyrate led to morphologic differentiation, growth in dbcAMP inhibited proliferation, while growth in butyrate slowed but did not stop cell division. Expression of the genes for myogenin and myosin light chain was inhibited by dbcAMP, while butyrate decreased myogenin and increased myosin light chain transcription. MyoD and MRF4 expression was not altered under either condition and no myf5 expression was detected. We also determined the effects of dbcAMP and butyrate on total protein expression, as well as on a panel of muscle- and neural-specific proteins using functional assays, immunohistochemistry, and immunoprecipitation. The total protein levels of cells treated with either agent were double those of untreated cells. DbcAMP increased the activity of acetylcholinesterase (AChE) up to 10-fold compared to untreated cells, while butyrate had a substantially lesser effect. These increases were due to increased AChE protein synthesis and stability in dbcAMP treated cells, compared to butyrate or untreated cells. Finally, cells under all conditions expressed MAP2, a neural-specific microtubule associated protein. Together, these data suggest that intracellular cAMP levels modulate distinct subsets of the myogenic differentiation pathway in rhabdomyosarcoma cells. Moreover, they also indicate that RD cells are able to express markers of different cell lineages, which may help explain some of the paradoxical features of these tumors.

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.

P16INK4 is a cell cycle regulator that specifically binds to and inactivates cyclin-dependent kinase 4 (CDK4). Its encoding gene (p16/CDKN2) maps to chromosome 9p21, a region that undergoes frequent loss of heterozygosity in a variety of human tumors. We have analyzed the p16/CDKN2 gene and its expression in a series of primary glioma samples. Although homozygous deletion or mutation of the p16/CDKN2 gene was uncommon in this series and P16INK4 protein was detectable in all grade II tumors, it was present in only 50% of grade III and grade IV samples. Conversely, in some grade IV tumors that level of P16INK4 protein was elevated; in these cases, its target, CDK4, was amplified and overexpressed. These results suggest: (a) the involvement of P16INK4 in glioma progression; (b) that mechanisms other than mutation or deletion can down-regulate expression of the p16/CDKN2 gene; and (c) that the balance between CDK4 and its cognate inhibitor, P16INK4, may confer a cell growth advantage and facilitate tumor progression.