Gene Therapy and Molecular Biology Vol 1, page 133
Gene Ther Mol Biol Vol 3, 133-148. August 1999.
Glioblastoma multiforme: molecular biology and new perspectives for therapy
Giorgio PalÃ¹, Luisa Barzon, and Roberta Bonaguro
Institute of Microbiology, University of Padova Medical School, Padova, Italy
Corresponding Author : Giorgio PalÃ¹, MD, Institute of Microbiology, Via A. Gabelli 63, 35121 Padova, Italy. Tel: +39-049-8272350; Fax: +39-049-8272355; E-mail: email@example.com
Key words : Therapy, gene therapy, brain tumors, gliomas, glioblastoma multiforme, molecular biology, pathogenesis, immunotherapy, neoangiogenesis, oncogenes
Abbreviation : GBM , glioblastoma multiforme
Received: 23 November 1998; accepted 30 November 1998
Pathogenic features of glioblastoma multiforme and of other gliomas are reviewed in the present article. Emphasis is given to those genetic alterations which are involved in oncogenesis, to the process of tumor neoangiogenesis and to the role played by the immune system in controlling neoplastic growth. Aspects which are relevant to therapeutic interventions are also dissected, and gene therapy in particular. A new gene therapy approach that combines tumor suicide, via enzyme- directed prodrug activation, and cytokine-promoted immune rejection is reported, together with results from the first application of this approach in humans.
The outcome of malignant gliomas remains extremely poor, in spite of aggressive use of currently available therapies. Recent advances in elucidating the molecular biology of gliomas have led to the development of innovative therapeutic strategies. The more promising approaches involve gene therapy, aiming at increasing tumor cell chemosensitivity and/or immunogenicity, by transfer of genes expressing cytokines and prodrug activating enzymes.
Glioblastoma multiforme (GBM) represents 15-20% of all intracranial tumors and 50% of gliomas (Russel and Rubistein, 1989). It affects 5,000 Americans and 1,000
Italians every year, and typically occurs in adults, with a peak incidence in the fifth and sixth decades of life. It is a very aggressive tumor, with a uniform and profound morbidity. Because of its morbidity it contributes to the cost of cancer on a pro capite basis more than any other tumor. Despite surgery, radiotherapy and/or chemotherapy, the prognosis is extremely poor and has not substantially changed over the last two decades, death resulting in 80%
of patients for tumor recurrence within 6-12 months from treatment.
Glioblastoma multiforme (grade IV astrocytoma) is usually located in the cerebral hemispheres, though it occasionally appears at other sites, such as the cerebellum, the brain stem and the spinal cord. Histology shows marked cytological diversity, ranging from tumors composed of small cells with scant cytoplasm to those composed of multinucleated giant cells. The World Health Organization (WHO) classification recognizes two distinct subvariants of the tumor: (i ) giant cell glioblastoma, characterized by a predominance of enormous, multinucleated giant cells and, on occasion, an abundant stromal reticulin network; and (ii ) gliosarcoma, in which hyperplastic vascular elements have undergone sarcomatous transformation.
Current therapies for malignant gliomas include surgical removal of the tumor mass, which is mandatory for precise diagnosis, and irradiation. Although surgery improves the prognosis (Levin et al, 1993), the infiltrative behavior of malignant gliomas precludes their complete
PalÃ¹ et al: Perspectives for therapy of glioblastoma multiforme
resection, and 90% of GBM recur within 2 cm of the primary site. Postoperative radiotherapy is therefore commonly administered, with a significant improvement in survival (Hochberg and Pruitt, 1980; Walker et al,
1980). Despite surgery and irradiation, however, only a few patients are alive two years after diagnosis. Results of chemotherapy trials are disappointing (Hosli et al, 1998). This is due both to the tumor intrinsic chemoresistance (Petersdorf and Berger, 1996) and to the tumor location within the central nervous system, which limits the penetration of drugs (Janzer and Raff, 1987; Mak et al,
1995). Among malignant gliomas, GBM is the least responsive to medical treatment. Available protocols include both monochemotherapy and polychemotherapy regimens. Nitrosoureas are the leading drugs in glioma chemotherapy, with response rates as single agents varying from 10% to 40% (Young et al, 1973; Fewer et al, 1972; Hoogstraten et al, 1972). Other drugs, evaluated in monochemotherapy (Forsyth and Cairncross, 1996), occasionally showed clinically and radiologically objective responses. Among these are vincristine (Smart et al,
1968), procarbazine (Rodriguez et al, 1989), paclitaxel (Chamberlain and Kormanik, 1995; Prados et al, 1996), and temozolomide (Newlands et al, 1996). However, methodological bias present in most studies raise doubts about the validity of these results. The most commonly used polychemotherapy regimens for gliomas are PVC (i.e. a combination of CCNU, procarbazine, and vincristine) and MOP (i.e. a combination of procarbazine, vincristine, and mechlorethamine). Response rates (complete or partial) of
17-37% have been reported for glioblastomas (Levin et al,
1980; Coyle et al, 1990). More recently, interesting results have been obtained in GBM patients with the ICE regimen (ifosfamide, carboplatin and etoposide), although in association with severe hematological toxicity (Sanson et al, 1996). The role of PVC as adjuvant chemotherapy is controversial (Fine et al, 1993), and, overall, there is no clear-cut evidence that survival of glioblastoma patients is improved by chemotherapy (Hosli et al, 1998).
II. Molecular biology of glioblastoma multiforme and corrective gene therapy
As for most cancers, brain tumors derive from a multi- step process of successive alterations, including loss of cell cycle control, neoangiogenesis and evasion of immune control. Figure 1 summarizes the genetic alterations associated with the malignant transformation of astrocytes. Most of these changes involve the loss of putative tumor suppressor genes or activation of proto-oncogenes.
Gene therapy of cancer, in its most direct form, should aim at replacing a mutated gene with its correct form, or at suppressing the abnormal oncogenic function. At present, however, such a corrective gene therapy, faces the
insurmountable task that gene replacement, or gene suppression, should simultaneously involve a number of different genes, and should be applied to all tumor cells to reverse the malignant phenotype. Hence, corrective gene therapy seems to be quite difficult to propose as a single therapeutic approach.
A. Genetic alterations
Several members of the protein-tyrosine kinase receptor family are over-expressed by gene amplification in malignant gliomas, including the epidermal growth factor receptor (EGFR), the platelet-derived growth factor receptor- (PDGFR ) and the c-met genes (Furnari et al,
1995). A high percentage of glioblastomas also have EGFR gene rearrangements that may lead to the expression of a truncated, constitutively activated receptor. The transfer of a mutant human EGFR gene into glioblastoma cells caused constitutive self phosphorylation and a pronounced enhancement tumorigenicity in nude mice
Figure 1. Simplified representation of oncogenes and tumor suppressor genes contributing to malignant progression of astrocytic tumors.
Gene Therapy and Molecular Biology Vol 1, page 135
(Ekstrand et al, 1994; Nishikawa et al, 1994). Numerous strategies are currently being investigated to specifically inhibit EGFR using antibodies, immunoconjugates or antisense technology. The selective inhibition of EGFR in human GBM cells with kinase-deficient mutants inhibited cell proliferation and transforming efficiency in athymic mice (O'Rourke et al, 1997), and converted radioresistant human glioblastoma cells to a more sensitive phenotype (O'Rourke et al, 1998), providing a rationale for gene therapy applications.
Other dominant oncogenes, such as N-myc, fos, src, H- ras or N-ras, and mdm2 are amplified and highly expressed in gliomas (Collins, 1993). GBM produce high levels of insulin-like growth factor I (IGF-I). When this alteration has been targeted by a vector expressing an antisense anti- IGF-I gene, rejection of genetically altered rat C6 glioma cells was observed. Injection, even at a site distal to the tumor, caused regression of established brain GBM. Destruction of the tumor was mediated by a glioma- specific T CD8+ (CTL) response (Trojan et al, 1993).
2. Genes associated to cell immortalization
A role in cell immortalization has been proposed for telomerase, the RNA-protein complex that elongates telomeric DNA. Telomerase is expressed almost exclusively in cancer cells, but not in normal cells, suggesting the possibility that gene therapy may be applied to inhibit this function. A successful example of treatment via antisense oligonucleotides directed against human telomerase suppressed glioma cells growth and survival, both in vitro and in vivo, through the induction of apoptosis (Kondo et al, 1998).
3. Tumor suppressor genes
Molecular and cytogenetic analyses of gliomas have shown frequent losses of genetic material, suggesting the inactivation of putative tumor suppressor genes. Loss of heterozygosity (LOH) has been described in chromosome
1p, 9p, 10p, 10q, 11p, 13q, 17p, 19q, and 22q, and in some cases the tumor suppressor gene involved in LOH has been identified. This is the case of the p53 tumor suppressor gene, which maps in 17p. Wild-type p53 protein is involved in G1 cell cycle arrest and apoptosis of DNA-damaged cells and is therefore crucial in preventing mutation or deletion of functional genes. Mutations of p53 seem to be an early event in glioma tumorigenesis, being frequently detected also in low grade astrocytomas. Along with p53 mutations, amplification of the mdm2 oncogene, whose product binds to and degrades p53, accounts for p53 inactivation in gliomas. Since p53 plays a key role in the pathogenesis of most cancers, it has raised great interest as a target for cancer gene therapy. Transduction of malignant
cells with wild type p53 can significantly inhibit growth and neoangiogenesis, or can induce apoptosis in p53 mutant cells in several tumor models in vitro, including gliomas (Badie et al, 1995; Van Meir et al, 1995; Gomez- Manzano et al, 1996). The presence of functional p53 has also been shown to modulate chemoresistance. Consequently, another possible advantage of the restoration of wild type p53 may be sensitization to chemotherapy and radiotherapy. Indeed, the combination of p53 gene transduction with radiation or chemotherapy (Lowe et al,
1994) has resulted in local tumor control superior to either therapy alone (Fujiwara T et al, 1994; Gjerset et al, 1995; Ngyuyen et al, 1996, Lang et al, 1998). This combined therapy is currently under investigation in clinical trials (Roth and Cristiano, 1997; Nielsen and Maneval, 1998).
The cell cycle regulator genes provide an additional target for corrective gene therapy. The p105Rb product of the retinoblastoma tumor suppressor gene (Rb) is one of the most critical regulators of cellular proliferation. The Rb protein (pRb), when unphosphorylated, is responsible for arrest of cell cycle by inhibition of the activity of the E2F family of transcription factors. Normal cell cycle progression requires inactivation of Rb through phosphorylation by cyclin-dependent kinases (CDK). This process, in turn, is regulated by CDK inhibitors. Among these, p21 protein is induced directly by p53; p16 protein, and its homologue p15, specifically bind to and inhibit CDK4, and may therefore regulate Rb phosphorylation, and cell cycle progression. Dysregulation of cell cycle control is a frequent finding in malignant gliomas, like deletion or loss of expression of p16 and p15 tumor suppressor genes (Jen et al, 1994; Nishikawa et al, 1995), amplification of CDK4 (He et al, 1994), and deletion or mutation of the Rb tumor suppressor gene (Henson et al,
1994). Interestingly, both of the latter events take place when the p16 gene is intact and correctly expressed (He et al, 1995). Restoration of wild-type p16 gene in glioma cells through an adenoviral vector arrested cells in G0-G1 phases of the cell cycle (Fueyo et al, 1996) and suppressed glioma cells invasion in vitro (Chintala et al, 1997). Overexpression of p21 increases the susceptibility of glioblastoma cells to cisplatin-induced apoptosis (Kondo et al, 1996), whereas adenovirus-mediated transfer of exogenous E2F-1 protein induced massive apoptosis and suppressed glioma growth in vivo and in vitro (Fueyo et al, 1998). The possibility that E2F-responsive promoters may be more active in tumor cells relative to normal cells, because of loss of pRb function, has been exploited to design adenoviral vectors containing transgenes driven by the E2F-1 promoter for gene therapy of gliomas (Parr et al,
1997). These vectors showed tumor-selective gene expression in vivo and reduced toxicity of the normal tissue with respect to standard adenoviral vectors.
PalÃ¹ et al: Perspectives for therapy of glioblastoma multiforme
Inhibition or inactivation of genes/factors involved in DNA repair and/or cellular SOS response could represent a gene therapy approach that potentiates radiation therapy. In fact, inhibition of the RAD51 gene by antisense oligonucleotides enhanced the radiosensitivity of mouse malignant gliomas, both in vitro and in vivo, improving survival (Ohnishi et al, 1998). This gene, a homologue of the yeast RAD51 and E. coli RecA genes, is involved in repair of DNA double-strand breaks, in recombination repair, and in various SOS responses to DNA damage caused by gamma-irradiation and alkylating agents.
Deletions of large regions or even of the entire copy of chromosome 10 are a genetic hallmark of GBM. At least two tumor suppressor genes located on chromosome 10 (one on each arm) have been demonstrated to participate to glial oncogenesis. A first candidate tumor suppressor gene, called PTEN (Phosphatase and tensin homologue deleted on chromosome TEN) was recently characterized (Li et al,
1997). The DNA region encoding PTEN is altered in glioblastoma multiforme, but not in lower grade astrocytic tumors (Tohma et al, 1998; Ichimura et al, 1998; Chiariello et al, 1998).
Tumors may remain in a state of dormancy until they establish a blood supply for receiving oxygen and nutrients. The complex process of neoangiogenesis is regulated by numerous factors, some with angiogenic properties, i.e. vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF ), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin-8, and by endogenous inhibitors of angiogenesis, i.e. thrombospondin-1, platelet factor 4 (PF4), angiostatin, endostatin. VEGF, which binds to two specific tyrosine-kinase receptors, called Flk-1 and Flt-1, has been demonstrated to play a key role in angiogenesis of gliomas. Indeed, VEGF and its receptors are downregulated in the normal adult brain, whereas, VEGF is highly produced by GBM cells. Since both flt-1 and flk-1 genes are expressed by proliferating endothelial cells of gliomas, this leads to the establishment of a paracrine loop. Moreover, VEGF expression is higher around necrotic areas and seems to be stimulated by hypoxia.
Glioblastoma multiforme is one of the most highly vascularized solid neoplasms; therefore, treatments that target neoangiogenesis would be of great interest in clinical practice. Co-injection of rat C6 glioma cells, either subcutaneously or intracerebrally in nude mice, together with cells producing retroviral vectors encoding a dominant-negative mutant of the Flk-1 receptor showed inhibition of neoangiogenesis, reduction of tumor growth, and survival improvement (Millauer et al, 1994; 1996). Antisense VEGF oligonucleotides (Saleh et al, 1996) and
ribozymes against VEGF mRNA have been successfully employed to reduce VEGF expression in glioma cells (Ke et al 1998), once more suggesting a potential role for antiangiogenic gene therapy. Similarly, bFGF antisense cDNA decreased C6 glioma cells proliferation (Redekop and Naus, 1995). Besides inhibiting the production of angiogenic factors, a therapeutic intervention could also consist of providing tumors with antiangiogenic factors. Indeed, retroviral and adeno-associated viral vectors expressing a modified PF4 were reported to inhibit endothelial cells proliferation in vitro and the growth of intracerebrally implanted gliomas (Tanaka et al, 1997). Retroviral and adenoviral vectors transducing angiostatin gene increased apoptotic death of glioma tumor cells (Tanaka et al, 1998). Additionally, the intratumoral delivery of angiostatin gene by an adenoviral vector produced inhibition of tumor growth in vivo, suppression of neovascularization, and a marked increase of tumor cells apoptosis (Griscelli et al, 1998). Damage of tumor microvasculature was reported also in human malignant glioma xenografts, after gene therapy followed by radiotherapy. The treatment consisted of intratumoral injection of adenoviral vectors expressing tumor necrosis factor- (TNF- ), under control of the Egr-1 promoter (Staba et al, 1998). The use of viral vectors containing radiation-inducible promoters, such as Egr-1, has the advantage of selectively, spatially, and temporally limiting the effects of the therapeutic gene in the radiation field. Recently, this strategy has yielded interesting results in rat
9L glioma cells (Manome et al, 1998).
III. Suicide gene therapy
Suicide gene therapy operates by tumor transduction of genes converting a prodrug into a toxic substance; independently, the gene product and the prodrug are non- toxic. The prototype of this approach exploits the selective intracellular phosphorylation of ganciclovir (GCV), driven by the herpes simplex virus thymidine kinase gene product (HSV-TK). This activation generates a toxic drug metabolite that inhibits DNA synthesis, inducing cell death. For in vivo gene transfer of the HSV-TK gene to malignant cells, packaging cells that produces retroviral vectors expressing HSV-TK, have been injected directly into the tumor to transduce replicating cells. An interesting feature of the HSV-TK/GCV system is the bystander killing of nontransduced cells.
The mechanisms that are responsible for this effect have not been fully defined, but are likely to include: (i ) transfer of non-diffusible, phosphorylated GCV to neighboring cells through gap junctions; (ii ) endocytosis by nontransduced cells of cellular debris containing toxic GCV; and (iii ) stimulation of host antitumor immune response. The therapeutic efficacy of the HSV-TK/GCV
Gene Therapy and Molecular Biology Vol 1, page 137
system may be further increased by the use of adenoviral vectors, since these vectors can also transduce resting tumor cells. However, adenoviral vectors will infect also normal cells; hence, the inclusion of sequences able to restrict gene expression only in tumor cells can circumvent this problem. Selective tumor toxicity was obtained positioning the suicide gene under control of the E2F- responsive promoter elements which are de-repressed in glioma cells (Parr et al, 1997).
The effectiveness of suicide gene therapy has been explored for a variety of neoplasms, especially for refractory and localized diseases such as GBM. The HSV- TK/GCV scheme has been demonstrated to be effective in animal models, by ex vivo and in vivo transduction with retroviral, adenoviral, or adenoviral-associated vectors expressing HSV-TK (Ezzeddine et al, 1991; Culver et al,
1992; Takamiya et al, 1992, 1993; Barba et al, 1993,
1994; Ram et al, 1993; Kim et al, 1994; Vincent et al,
1996; Maron et al, 1996; Mizuno et al, 1998). Novel gene delivery systems were also applied in a mouse model for gene therapy of meningeal gliomatosis. Liposomes coated with Sendai virus envelope protein were highly efficient in delivering the therapeutic gene in disseminated glioma cells (Mabuchi et al, 1997).
A factor that may limit the effectiveness of HSV- TK/GCV therapy is the GCV crossing through the blood- brain barrier. This can be circumvented by the use of the bradykinin analogue and potent blood-brain barrier permeabilizer RMP-7, which, administered intravenously, increase the delivery of GCV into rat brain tumors, enhancing the cytotoxic and bystander effects of HSV- TK/GCV (LeMay et al, 1998).
Several clinical trials exploiting the HSV-TK/GCV system have been initiated. It is too early to estimate the effectiveness of these therapeutic procedures. Notwithstanding the evidence for growth-suppressive activities of HSV-TK plus GCV, cure rates are low. Explanation for lack of complete response in humans may reside in the different biological behavior of GBM cells when injected into animals (Sturtz et al, 1997).
The antitumor effects elicited by HSV-TK/GCV prompted to explore other prodrug activating enzymes. A promising system exploits the ability of E. coli cytosine deaminase (CD) to convert the relatively non toxic 5- fluorocytosine (5-FC) to the chemotherapeutic agent 5- fluorouracil (5-FU). Significant antitumor effects of CD/5- FC were observed in nude mice bearing tumors derived from C6 glioma cells and transduced with CD. A "bystander" effect could also be demonstrated (Ge et al,
1997), suggesting a potential role for gene therapy of glioblastoma.
As already reported for p53, both the CD and HSV-TK
systems sensitize cancer cells to radiation. Animal models
have shown encouraging results both in vitro and in vivo (Khil et al, 1995; Kim et al, 1997). 9L glioma cells transduced with a retrovirus encoding a CD/HSV-TK fusion gene exhibited enhanced sensitivity to both GCV and 5-FC, as well as increased radiosensitivity (Rogulski et al, 1997). This experiment suggests the feasibility of a combined approach with two suicide genes associated with radiotherapy.
Another prodrug activation system is represented by cytochrome P450 2B1 (the liver enzyme catalyzing cyclophosphamide and ifosfamide activation) gene transfer followed by cyclophosphamide or ifosfamide administration. Metabolites of these drugs produce inter- strand DNA cross-linking in a cell cycle-independent fashion. C6 and 9L rat glioma cells, when stably transfected with the P450 2B1 gene, become highly sensitive to cyclophosphamide in in vitro and in vivo models (Wei et al, 1994; Manome et al, 1996). Rabbit cytochrome P450 isozyme CYP4B1, which converts the inert prodrugs 2-amonianthracene (2-AA) and 4-ipomeanol (4-IM) into highly toxic alkylating metabolites, also showed high antitumor effects, both in vitro and in vivo. The treatment had relatively low toxicity and was associated with a bystander effect, not requiring cell-to-cell contact (Rainov et al, 1998).
Another suicide gene system is based on E. coli purine nucleoside phosphorylase (PNP), which generates toxic purine nucleoside analogues intracellularly, either from 6- methylpurine-2â-deoxyriboside or arabinofuranosyl-2- fluoroadenine monophosphate. Significant antitumor activity and low systemic toxicity were reported in nude mice bearing human malignant D54MG glioma tumors expressing PNP (Parker et al, 1997).
Phosphorylation of the prodrug cytosine arabinoside (ara-C) by deoxycyticine kinase (dCK) is a limiting step for activation. Thus, ara-C, a potent antitumor agent for hematological malignancies, has only minimal activity against most solid tumors. Transduction of the dCK cDNA by retroviral and adenoviral vectors also resulted in marked sensitization of glioma cell lines to ara-C in vitro, and in significant antitumor activity in vivo (Manome et al,
Unlike other prodrug activating enzymes, E. coli gpt sensitizes cells to the prodrugs 6-thioxanthine (6TX) and 6- thioguanine (6-TG), and confers resistance to different regimens (mycophenolic acid, xantine, and hypoxanthine), providing a means to select for gpt-positive cells. Rat C6 glioma cells transduced with a retroviral vector expressing the gpt gene exhibited significant 6TX and 6GT susceptibility and a "bystander" effect in vitro. An antiproliferative effect was demonstrated in vitro and in vivo (Tamiya et al, 1996; Ono et al, 1997).
PalÃ¹ et al: Perspectives for therapy of glioblastoma multiforme
Tumor suicide can also be achieved by direct infection of tumor cells with a conditionally replicative virus, i.e. an infectious agent able to replicate and to kill only dividing cells. In the case of tumors highly proliferating in the context of a completely post-mitotic tissue, such as the brain, gene transfer can ideally be obtained by using neurotropic herpes viral vectors, which are rendered conditionally replicative after deletion of non-essential genes (Lachmann and Efstathiou, 1997).
Tumor specific cell death has already been provided in animal glioma models by HSV vectors, deleted in neurovirulence genes, such as 34.5, thymidine kinase and ribonucleotide reductase (Chambers et al, 1995; Mineta et al, 1995; Boviatsis et al, 1994; Miyatake et al, 1997; McKie et al, 1996; Andreansky et al, 1996). Their direct injection into gliomas produced tumor regression with minimal bystander effects on surrounding normal tissue. Enhancement of replication of defective HSV vectors lacking 34.5 gene and a significant reduction of tumor mass was observed combining ionizing radiation (Advani et al, 1998).
Another way to treat malignant gliomas emerged from the discovery that these tumors often express functional Fas (CD95) (Weller et al, 1994). Fas is a transmembrane glycoprotein belonging to the nerve growth factor/TNF receptor superfamily: when activated, Fas can transduce an apoptotic signal through its cytoplasmic domain. Apoptosis is triggered by the binding of Fas to its natural ligand (FasL) or by cross-linking with anti-Fas antibodies. A high proportion of human glioma cell lines are sensitive to apoptosis mediated by anti-Fas antibodies in vitro. Some other cell lines are resistant, but may be rendered sensitive after stable transfection with human Fas cDNA (Weller et al, 1995). These results offer new possibilities for treating gliomas with anti-Fas antibodies or soluble FasL. One possible drawback of such an approach is that other Fas-positive cells may be affected, like infiltrating leukocytes. Thus their activity may be reduced, restricting strategies relying upon simultaneous immune response enhancement.
IV. Immunotherapy of cancer
Glioblastoma multiforme has the propensity to microscopically infiltrate normal structures since its early stage of development. This characteristic makes therapeutic success rather difficult by any approach. Moreover, it becomes virtually impossible to obtain specific targeting of all tumor cells and sparing of normal ones. Therefore, inducing an efficient immune response against malignant cells becomes an attractive and essential treatment strategy. In this perspective, the natural circulatory properties of cells of the immune system offer also an important support for the recognition of secondary lesions.
Despite the location in the central nervous system (CNS), a long-believed âimmunologically privileged siteâ, glioblastoma cells may interact with immune cells. These interactions are mediated by receptor-ligand recognition during cell to cell contact and by a plethora of cytokines. An imbalance in the tumor-host relationship, resulting in deficit in some components of the response, may explain the aggressive growth of malignant gliomas. Before discussing the designed strategies to increase the immune response against glioblastoma cells, we review the more recent acquisitions in the âdialogueâ between these neoplastic cells and the immune system (Dietrich et al,
A. Antigenicity of glioblastoma cells
The presence of specific antigens at the surface of tumor cells to be recognized by cells of the immune system is essential for the generation of a specific antitumor immune response. At present, no tumor antigen able to elicit an immune response has been identified in glioblastoma in vivo. MAGE-1 (melanoma antigen) was the first tumor-specific antigen to be identified (Van der Bruggen et al, 1991), and MAGE family members are expressed by some glioblastoma cell lines (Rimoldi et al,
1993), but not in uncultured tumors (De Smet et al, 1994). This observation could be explained with different levels of DNA methylation induced by culture, where MAGE expression was regulated by methylation (De Smet et al,
Proteins that are structurally altered during malignant transformation, or that contribute to this process, are possible tumor antigen candidates. The frequent alterations of p53 in the early stages of carcinogenesis, for example, may provide new antigenic peptides that could trigger an immune response. Consistent with this possibility, specific cytotoxic T-cell clones were generated in vitro against mutated p53 protein (Houbiers et al, 1993). Additionally, in vivo immunization with mutated p53 peptide was shown to induce specific CTL clones able to lyse MHC-matched tumor cells expressing the mutated p53 gene (Noguchi et al, 1994).
The future identification of glioblastoma-specific antigens would aid new treatment strategies.
B. Tumor-induced immunosuppression
A high proportion of glioblastomas have shown to be infiltrated by lymphocytes, mostly T lymphocytes, but also B and NK cells. Differently from what reported for other tumors, the level of lymphocyte infiltration does not relate with a better prognosis. In fact, tumor-infiltrating lymphocytes (TIL) of gliomas appear to be functionally defective. Abnormalities range from abnormal
Gene Therapy and Molecular Biology Vol 1, page 139
hypersensitivity responses, depressed response to mitogens, decreased humoral responses (CD4+ T helper cells deficit?), and impaired T cell-mediated cytotoxicity. These functional alterations may be explained, at least in part, by a defective high-affinity IL-2 receptor (IL-2 R) (Roszman et al, 1991).
It has been demonstrated that glioblastoma cells produce and release soluble factors that are responsible for a depressed immune response. T lymphocytes from normal individuals exhibit immunologic abnormalities when grown in presence of supernatant obtained from glioblastoma cell line cultures (Roszman et al, 1991). The most important soluble suppressing factor seems to be TGF- 2. This cytokine acts as a growth-inhibitory factor (Sporn et al, 1986; Sporn et al, 1987), and has a defined variety of immunoregulatory properties, including: inhibition of: (i ) T cell proliferation; (ii ) IL-2R induction (Kehrl et al, 1986); (iii ) cytokine production (Espevik et al, 1987; Espevik et al, 1988); (iv ) natural killer cell activity (Rook et al, 1986); (v ) cytotoxic T cell development (Jin et al, 1989; Ranges et al, 1987); (vi ) LAK cell generation (Espevik et al, 1987; Jin et al, 1989); and (vii ) production of tumor-infiltrating lymphocytes (Kuppner et al, 1989). Most cells secrete TGF- in a latent form (Sporn et al, 1986), but glioblastoma cells have also the capacity to convert it to an active form, through proteolytic cleavage. This was demonstrated by an experimental work in which T-cell suppression mediated by TGF- 2 was inhibited when proteolytic enzymes were blocked by protease inhibitors (Huber et al, 1992).
Other soluble factors, namely prostaglandin E2 (PGE2), IL-1 receptor (IL-1 R) antagonist, and interleukin-10 (IL-
10) may be implicated in immunosuppression, although in vivo intervention has not been fully defined. A potential down regulation of some immune functions was shown for IL-10 cytokine, which was produced both by GBM cells and normal brain tissue (Nitta et al, 1994; Merlo et al,
1993). Furthermore, in different animal models, human and murine IL-10 was demonstrated to stimulate the acquisition of a specific and efficient antitumor immune response (Berman et al, 1996).
C. Costimulatory molecules
A complete T-cell effector function needs not only antigen presentation, but also the delivering of activation signals to the T cell, which is mediated by the so-called costimulatory molecules. Unresponsiveness of T cells (anergy) may be due to absence of the second signal, essentially given by the B7-CD28 interaction (June et al,
1994). Two other members of the B7 family have been cloned, B7.1 and B7.2; their counter-receptors on T cells are CD28 and CTLA-4, respectively. CD28 mediates stimulatory effects, while CTLA-4 appears to be a negative
regulator of T cell responses. Glioblastoma cells and monocytes that infiltrate the tumor are not expressing B7 costimulatory molecules, while monocytes in the normal tissue that surrounds the tumor are B7-positive (Tada et al,
1996). This suggests the possible intervention of local mechanisms able to down-regulate B7 expression in glioblastomas, impeding efficient T-cell priming and favoring T-cell anergy. Moreover, B7-CD28 interactions in the CNS have been shown to be essential to generate a valid CTL response towards viral antigens (Kuendig et al,
1996). Hence, restoring B7 expression by gene transfer might become an interesting task to elicit a proper immune recognition of glioblastoma cells, and an appropriate immune response.
V. Restoring a proper immune response
Many approaches have been tented to restore a proper immune response towards malignant gliomas. As previously stated, T lymphocytes play a major role in the antitumor response, and priming of T lymphocytes requires antigen recognition, with or without help from APC. Since no specific antigens have yet been identified for glioblastomas, a vaccination approach has been proposed by administration of genetically modified tumor cells. Moreover, tumor cells transfected to produce various cytokines have been used to enhance lymphocyte responsiveness in animal models.
The most interesting results were obtained with cells of murine glioma transfected with an expression vector containing the murine interleukin 7 cDNA (Aoki et al,
1992). IL-7 transfected glioma cells were vigorously rejected by a CD8+ T-cell-mediated immune response, that was proportional to the level of IL-7 production. Moreover, the response was tumor-specific, since no effect was observed against other syngeneic tumor cells (melanoma and fibrosarcoma cells). IL-7 is a very interesting cytokine being able to increase IL-2R chain expression on CD4+ T lymphocytes and to inhibit TGF- mRNA expression and production by murine macrophages (Dubinett et al, 1993). IL-12 can also be considered a promising agent to enhance the antitumor response, since it augments T-cell and natural killer-cell activities, induces IFN- production, and promotes the differentiation of uncommitted T cells to Th1 cells (Hendrzak and Brunda,
1995). Vector-mediated delivery of IL-12 into established tumors suppresses tumor growth (Caruso et al, 1996) and can induce immune responses against challenge tumors (Bramson et al, 1996). Moreover, IL-12 has other non- immune properties such as anti-angiogenic effects (Voest et al, 1995).
IL-4, another cytokine with pleiotropic functions, increases T cell proliferation and cytotoxicity, and enhances eosinophil and B cell proliferation and differentiation. It
PalÃ¹ et al: Perspectives for therapy of glioblastoma multiforme
also exerts direct anti-proliferative effects in vitro against many tumor cell lines (Tepper, 1993). Antitumor effects induced by IL-4-transfected cells were reported in nude mice, suggesting T cell-independent mechanisms (Tepper,
1993; Yu et al, 1993). A strong recruitment of eosinophils and subsequent inhibition of the tumor growth was noted. Eosinophil depletion was not performed as a control; hence a direct anti-proliferative effect mediated by IL-4 cannot be excluded. In any respect, a possible non-T-cell-dependent mechanism could be an advantage in the glioblastoma setting, considering the various abnormalities of T cell function.
In an attempt to overcome the local immunosuppression mediated by TGF- 2 Fakhrai et al conducted an experimental work on rats by antisense gene therapy. 9L gliosarcoma transfected cells inoculated sub- cutaneously became highly immunogenic and were able to induce eradication of an established wild-type tumor (Fakhrai et al, 1996).
The enhancement of antigen presentation and T cell co- stimulation has also been considered and may be achieved with genes coding for cytokines, like GM-CSF, co- stimulatory molecules, like B7, or CIITA, a transcription factor playing a critical role in the regulation of MHC class II molecules. Encouraging results have been reported in a murine melanoma model located in the CNS, whereby an efficient antitumor response was induced by sub- cutaneous vaccination with irradiated, GM-CSF-producing tumor cells (Sampson et al, 1996). Vaccination with cells co-transfected with B7 and IL-2 was able to mediate rejection of established tumors (Gaken et al, 1997), suggesting a possible application of such an intervention for the treatment of glioblastomas.
VI. Combined gene therapy approach in humans
The strict localization of glioblastomas in the CNS, with only exceptional metastases, makes these tumors candidates for approaches of direct intra-tumoral gene delivery. Retrovirus-mediated gene therapy of GBM is particularly attractive, since these viral vectors transduce only mitotically active cells, sparing the normal neuronal tissue composed of non-replicating cells.
Gene therapy of brain tumors by intra-tumoral injection of retroviral vector producing cells (RVPC) in human patients was initiated by Oldfield and colleagues in
1993 (Oldfield et al, 1993). The gene being transferred was that expressing the herpes simplex thymidine kinase (HSV-TK ), which conferred sensitivity to the anti-herpes drug ganciclovir. This treatment has proved free of toxicity and safe for there was no evidence of systemic spread of the retroviral vector (Long et al, 1998). However, a clinical benefit was limited to very small tumors (1.5 ml), probably because only malignant cells adjacent to the RVPC were transfected (Ram et al, 1997).
A new treatment strategy combining two different modalities, enzyme-directed prodrug activation (tumor suicide) along with cytokine-promoted tumor rejection, has been recently devised to amplify the antitumor response, and proved to be efficacious in animal models (Castleden et al, 1997) (Figure 2 ). A bicistronic retroviral vector co- expressing HSV-TK and human interleukin-2 genes has been designed to pursue this new approach of cancer gene therapy in humans (Pizzato et al, 1998).
Figure 2. Structure of a bicistronic retroviral vector for transduction of genes coding for a cytokine and a prodrug- activating enzyme, expressed via a cap-dependent and an internal ribosome entry site (IRES)-dependent mechanism, respectively. A selectable marker (neomycin phosphotransferase gene, neo) is expressed under the control of a SV40 promoter.
Gene Therapy and Molecular Biology Vol 1, page 141
Figure 3. Gene therapy approach for treatment of glioblastoma multiforme, via intratumoral stereotactic injection of cells producing a triple gene retroviral vector.
Figure 4. Contrast-enhanced MRI sagittal images of left parietal GBM lesion before (left ), and one month after completion of GCV treatment (to the right ).
Gene Therapy and Molecular Biology Vol 1, page 142
Figure 5. Histology of stereotactic biopsies from patients treated by HSV/TK-IL-2 combined gene therapy. A) Toluidine blue staining - Evidence of a large number of infiltrating inflammatory cells; Immunostaining with B ) monoclonal antibodies marking CD3+ cells; C) Mac 387 antibodies recognizing young/activated macrophages; D) monoclonal antibodies marking CD1+ infiltrating cells.
After in vitro characterization of efficacy and safety (Pizzato et al, 1998), the vector was employed in a pilot study to treat four patients with recurrent glioblastoma multiforme (Colombo et al, 1997; PalÃ¹ et al, 1998) (Figure 3 ). A significant and sustained reduction (>50% of the initial volume) of the tumor mass (80 ml) was demonstrated by magnetic resonance imaging (MRI) and computerized tomography (CT) in one patient (Figure 4 ). In this case, the objective response was associated with a dramatic clinical improvement. The other three patients showed areas of tumor necrosis (2 ml) around the site of stereotactic RVPC injection and stabilized disease for a long period of time (11-12 months).
In stereotactic biopsies taken before ganciclovir administration, large tumor infiltrates of immune- inflammatory cells (T lymphocytes, mostly CD4+ but also CD8+ granzyme B-positive cells, activated macrophages, NK cells, neutrophils) were present, notwithstanding the standard steroid therapy (Figure 5 ). The observed inflammatory response has never been reported in previous
trials with thymidine kinase (Ram et al, 1997; Ostertag and Chiocca, personal communications).
Interestingly, endothelial cells stained positive for TK by in situ hybridization, indicating that the vector had targeted the neo-vascular component, a highly replicative population in glioblastomas. This is consistent with an anti-angiogenic effect of this therapeutic approach, that, in addition to direct tumor suicide and immune activation, may be relevant to the bystander phenomenon and to the clinical response. It is noteworthy that IL-2 was measurable in the cerebral-spinal fluid, even after GCV treatment. This cytokine might have derived from an autocrine-paracrine secretion of recruited infiltrating immune-inflammatory cells, after primary expression in transduced cells.
Efforts to achieve more efficient gene transfer systems are being sought for. These include the development of new generation retroviral vectors, produced at higher titres and characterized by higher transduction efficiency. Strategies involving envelope pseudotyping, use of new
Gene Therapy and Molecular Biology Vol 1, page 143
packaging cell lines of human origin, and substitution of promoter elements will contribute to the improvement of current available vectors.
New therapeutic gene combinations should also be accomplished in order to promote a more generalized immune response. Genes for cytokines other than IL-2 (i.e., IL-4, IL-7, IL-12, GM-CSF) as well as genes targeting neoangiogenesis deserve further consideration for combined treatment approaches.
The authors wish to acknowledge Fondazione Cassa di Risparmio di Padova e Rovigo and Regione Veneto for financial support.
Advani SJ, Sibley GS, Song PY, Hallahan DE, Kataoka Y, Roizman B, Weichselbaum RR. (1998 ) Enhancement of replication of genetically engineered herpes simplex viruses by ionizing radiation: a new paradigm for destruction of therapeutically intractable tumors. Gene Ther 5, 160-165.
Andreansky SS, He B, Gillespie GY, Soroceanu L, Markert J, Chou J, Roizman B, Whitley RJ. (1996 ) The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors. Proc Natl Acad Sci USA 93, 11313-11318.
Aoki T, Tashiro K, Miyatake S, Kinashi T, Nakano T, Oda Y, Kikuchi H, Honjo T. (1992 ) Expression of murine interleukin 7 in a murine glioma cell line results in reduced tumorigenicity in vivo Proc Natl Acad Sci USA 89,
Badie B, Drazan KE, Kramar MH, Shaked A, Black KL. (1995 ) Adenovirus-mediated p53 gene delivery inhibits 9L glioma growth in rats. Neurol Res 17, 209-216.
Barba D, Hardin J, Ray J, Gage FH. (1993 ) Thymidine kinase-mediated killing of rat brain tumors. J Neurosurg
Barba D, Hardin J, Sadelain M, Gage FH. (1994 ) Development of antitumor immunity following thymidine kinase-mediated killing of experimental brain tumors. Proc Natl Acad Sci USA 91, 4348-4352.
Berman RM, Suzuki T, Tahara H, Robbins PD, Narula SK, Lotze MT. (1996 ) Systemic administration of cellular IL-
10 induces an effective, specific, and long-lived immune response against established tumors in mice. J Immunol
Boviatsis EJ, Scharf JM, Chase M, Harrington K, Kowall NY, Breakefield XO, Chiocca EA. (1994 ) Antitumor activity and reporter gene transfer into rat brain neoplasms inoculated with herpes simplex virus vectors defective in thymidine kinase or ribonucleotide reductase. Gene Ther
Bramson JL, Hitt M, Addison CL, Muller WJ, Gauldie J, Graham FL. (1996 ) Direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and long-lasting immunity that is associated with highly localized expression of interleukin-12. Hum Gene Ther
Caruso M, Pham-Nguyen K, Kwong Y-L, Xu B, Kosai K-I, Finegold M, Woo SLC, Chen SH. (1996 ) Adenovirus- mediated interleukin-12 gene therapy for metastatic colon cancer. Proc Natl Acad Sci USA 93, 11302-11306
Castleden SA, Chong H, Garcia-Ribas I, Melcher AA, Hutchinson G, Roberts B, Hart IR, Vile RG. (1997 ) A family of bicistronic vectors to enhance both local and systemic antitumor effects of HSVTK or cytokine expression in a murine melanoma model. Hum Gene Ther 8, 2087-102.
Chamberlain MC, Kormanik P. (1996 ) Salvage chemotherapy with paclitaxel for recurrent primary brain tumors. J Clin Oncol 13, 2316-21.
Chambers R, Gillespie GY, Soroceanu L, Andreansky S, Chatterjee S, Chou J, Roizman B, Whitley RJ. (1995 ) Comparison of genetically engineered herpes simplex viruses for the treatment of brain tumors in a scid mouse model of human malignant glioma. Proc Natl Acad Sci USA 92, 1411-1415.
Chen CY, Chang YN, Ryan P, Linscott M, McGarrity GJ, Chiang YL. (1995 ) Effect of herpes simplex virus thymidine kinase expression levels on ganciclovir- mediated cytotoxicity and the "bystander effect". Hum Gene Ther 6, 1467-1476.
Cheney IW, Johnson DE, Vaillancourt MT, Avanzini J, Morimoto A, Demers GW, Wills KN, Shabram PW, Bolen JB, Tavtigian SV, Bookstein R (1998 ) Suppression of tumorigenicity of glioblastoma cells by adenovirus- mediated MMAC1/PTEN gene transfer. Cancer Res 58,
Chiariello E, Roz L, Albarosa R, Magnani I, Finocchiaro G. (1998 ) PTEN/MMAC1 mutations in primary glioblastomas and short-term cultures of malignant gliomas. Oncogene 16, 541-545.
Chintala SK, Fueyo J, Gomez Manzano C, Venkaiah B, Bjerkvig R, Yung WK, Sawaya R, Kyritsis AP, Rao JS. (1997 ) Adenovirus-mediated p16/CDKN2 gene transfer suppresses glioma invasion in vitro. Oncogene 15, 17,
Chiocca EA. ( 1995 ) Brain tumor gene therapy in mice with a novel "suicide" gene, the cyclophosphamide-activating CYP2B1 gene. Clin Neurosurg 42, 370-82.
Collins VP. (1993 ) Amplified genes in human gliomas.
Semin Cancer Biol 4, 27-32.
Colombo F, Zanusso M, Casentini L, Cavaggioni A, Franchin E, Calvi P, PalÃ¹ G. (1997 ) Gene stereotactic neurosurgery for recurrent malignant gliomas. Stereotact Funct Neurosurg 68, 245-251.
PalÃ¹ et al: Perspectives for therapy of glioblastoma multiforme
Coyle T, Baptista J, Winfield J. ( 1990 ) Mechlorethamine, vincristine and procarbazine chemotherapy for recurrent high-grade glioma in adults, A phase II study. J Clin Oncol 8, 2014-2018.
Culver KW, Ram Z, Wallbridge S, Ishii I, Oldfield EH, Blaese RM. (1992 ) In vivo gene transfer with retroviral vector- producer cells for treatment of experimental brain tumors. Science 256, 1550-1552.
De Smet C, Courtois SJ, Faraoni I, Lurquin C, Szikora JP, De Backer O, Boon T. (1995 ) Involvement of two Ets binding sites in the transcriptional activation of the MAGE-1 gene. Immunonogenetics 42, 282-290.
De Smet C, Lurquin C, Van Der Bruggen P, De Plaen E, Brasseur F, Boon T. (1994 ) Sequence and expression pattern of the human MAGE 2 gene. Immunogenetics
Dietrich P-Y, Walker PR, Saas P, de Tribolet N. (1997 ) Immunobiology of Gliomas, New perspectives for therapy. Ann NY Acad Sci 824, 124-140.
Dubinett SM, Huang M, Dhanani S, Wang J, Beroiza T. (1993 ) Down-regulation of macrophage transforming growth factor-beta messenger RNA expression by IL-7. J Immunol 151, 6670-6680.
Ekstrand AJ, Longo N, Hamid ML, Olson JJ, Liu L, Collins VP, James CD. (1994 ) Functional characterization of an EGF receptor with a truncated extracellular domain expressed in glioblastomas with EGFR gene amplifacation. Oncogene 9, 2313-20.
Espevik T, Figari IS, Ranges GE, Palladino MA Jr. (1988 ) Transforming growth factor- 1 (TGF- 1) and recombinant human tumor necrosis factor reciprocally regulate the generation of lymphokine-activated killer cell activity. Comparison between natural porcine platelet-derived TGF-
1 and TGF- 2, and recombinant human TGF- 1. J Immunol 140, 2312-2316.
Espevik T, Figari IS, Shalaby MR, Lackides GA, Lewis GD, Shepard HM, Palladino MA Jr. (1987 ) Inhibition of cytokine production by cyclosporin A and transforming growth factor . J Exp Med 166, 571-576.
Ezzeddine ZD, Martuza RL, Platika D, Short MP, Malick A, Choi B, Breakefield XO. (1991 ) Selective killing of glioma cells in colture and in vivo by retrovirus transfer of the herpes simplex virus thymidine kinase gene. New Biol 3, 608-614.
Fakhrai H, Dorigo O, Shawler DL, Lin H, Mercola D, Black KL, Royston I, Sobol RE. (1996 ) Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy. Proc Natl Acad Sci USA 93, 2909-2914.
Fewer D, Wilson CB, Boldrey EB. (1972 ) Phase II study of CCNU in the treatment of brain tumors. Cancer Chem Rep , 56, 421-7.
Fine HA, Dear KBG, Loeffler JS. (1993 ) Meta-analysis of radiation therapy with an without adjuvant chemotherapy for malignant glionas in adults. Cancer 71, 2585-97.
Forsyth PA, Cairncross JG. (1996 ) Chemotherapy of malignant gliomas. In Cerebral Gliomas. Baillere's Clin Neurol 5, 371-93.
Fueyo J, Gomez Manzano C, Yung WK, Clayman GL, Liu TJ, Bruner J, Levin VA, Kyritsis AP. (1996 ) Adenovirus- mediated p16/CDKN2 gene transfer induces growth arrest and modifies the transformed phenotype of glioma cells. Oncogene 12, 103-110.
Fueyo J, Gomez Manzano C, Yung WK, Liu TJ, Alemany R, McDonnell TJ, Shi X, Rao JS, Levin VA, Kyritsis AP. (1998 ) Overexpression of E2F-1 in glioma triggers apoptosis and suppresses tumor growth in vitro and in vivo. Nat Med 4, 685-690.
Fujiwara T, Grimm EA, Mukhopadhayay T, Zang WW, Owen- Schaub LB, Roth JA. (1994 ) Induction of chemosensitivity in human lung cancer in vivo by adenoviral mediated tranfer of the wild type p53 gene. Cancer Res 54, 2287-2291.
Furnari FB, Su Huang HJ, Cavenee WK. (1995 ) Genetics and malignant progression of human brain tumors. Cancer Surv 25, 233-275.
Gaken JA, Hollingsworth SJ, Hirst WJR, Buggins AGS, Galea J, Peakman M, Kuiper M, Patel P, Towner P, Patel PM, Collins MK, Mufti GJ, Farzaneh F, Darling DC. (1997 ) Irradiated NC adenocarcinoma cells transduced with both B7.1 and interleukin-2 induce CD4+-mediated rejection of established tumors. Hum Gene Ther 8, 477-488.
Ge K, Xu L, Zheng Z, Xu D, Sun L, Liu X. (1997 ) Transduction of cytosine deaminase gene makes rat glioma cells highly sensitive to 5-fluorocytosine. Int J Cancer 71, 675-9.
Gjerset RA, Turla ST, Sobol RE, Scalise JJ, Mercola D, Collins H, Hopkins PJ. (1995 ) Use of wild type p53 to achieve complete treatment sensitization of tumor cells expressing endogenous mutant p53. Mol Carcinog 14,
Gomez-Manzano C, Fueyo J, Kyritsis AP, Steck PA, Roth JA, McDouwell TJ, Steck KD, Levin VA, Yung WK. (1996 ) Adenovirus-mediated tranfer of the p53 gene produces rapid and generalized death of human glioma cells via apoptosis. Cancer Res 56, 694-699.
Griscelli F, Li H, Bennaceur Griscelli A, Soria J, Opolon P, Soria C, Perricaudet M, Yeh P, Lu H. (1998 ), Angiostatin gene transfer, inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest. Proc Natl Acad Sci USA 95, 11, 6367-
Hamel W, Magnelli L, Chiarugi VP, Israel MA. (1996 ) Herpes simplex virus thymidine kinase/ganciclovir-mediated apoptotic death of bystander cells. Cancer Res 56,
He J, Allen JR, Collins VP, Allalunis-Turner MJ, Godbout R, Day RS 3rd, James CD. (1994 ) CDK4 amplification is an alternative mechanism to p16 gene homozygous deletion in glioma cell lines. Cancer Res 54, 5804-5807.
Gene Therapy and Molecular Biology Vol 1, page 145
He J, Olson JJ, James CD. (1995 ) Lack of p16ink4 or retinoblastoma protein (pRb) or amplification-associated overexpression of cdk4 is observed in dinstinct subsets of malignant glial tumors and cell lines. Cancer Res 55,
Hendrzak JA and Brunda MJ. (1996 ) Interleukin-12, biologic activity, therapeutic utility, and role in disease. Lab Invest 72, 619-637.
Henson JW, SchniTKer BL, Correa KM, von Dimling A, Fassbender F, Xu HJ, Benedict WF, Yandell DW, Louis DN. (1994 ) The retinoblastoma gene is involved in malignat progression of astrocytomas. Ann Neurol 36, 714-721.
Hochberg FH, Pruitt A. (1980 ) Assumptions in the radiotherapy of glioblastoma. Neurology 30, 907-911.
Hoostraten B, Gottlieb JA, Caoili A. 1973 CCNU in the treatment of cancer, A phase II study. Cancer 32, 38-43.
Hosli P, Sappino AP, de Tribolet N, Dietrich PY. (1998 ) Malignant glioma, Should chemotherapy be overthrown by experimental treatments? Ann Oncol 9, 589-600.
Houbiers JG, Nijman HW, Van Der Burg SH, Drijfhout JW, Kenemans P, Van Der Velde CJ, Brand A, Momburg F, Kast WM, Melief CJ. (1993 ) In vitro induction of human cytotoxic T lymphocyte responses against peptides of mutant and wild-type p53. Eur J Immunol 23, 2072-
Huber D, Philipp J, Fontana A. (1992 ) Protease inhibitors interfere with the transforming growth factor-(-indipendent pathway of tumor cell-mediated immunosuppression. J Immunol 148, 277-284.
Ichimura K, Schmidt EE, Miyakawa A, Goike HM, Collins VP. (1998 ) Distinct patterns of deletion on 10p and 10q suggest involvement of multiple tumor suppressor genes in the development of astrocytic gliomas of different malignancy grades. Genes Chromosomes Cancer 22,
Janzer RC, Raff MC. (1987 ) Astrocytes induce blood brain barrier properties in eÃ¿dotelial cells. Nature 325, 253-
Jen J, Harper JW, Bigner SH, Bigner DD, Papadopoulos N, Markowitz S, Willson JK, Kinzler KW, Vogelstein B. (1994 ) Deletion of p16 and p15 genes in brain tumors. Cancer Res 54, 6353-6358.
Jin B, Scott JL, Vadas MA, Burns GF. (1989 ) TGF- down regulates TLiSA1 expression and ihibits the differentiation of precursor lymphocytes into CTL and LAK cells. Immunology 66, 570-576
June CH, Bluestone JA, Nadler LM, Thompson CB. (1994 ) The B7 and CD28 receptor families. Immunol Today 15,
Ke LD, Fueyo J, Chen X, Steck PA, Shi YX, Im SA, Yung WK. (1998 ) A novel approach to glioma gene therapy, down- regulation of the vascular endothelial growth factor in glioma cells using ribozymes. Int J Oncol 12, 1391-6.
Kerhl JH, Wakefield LM, Roberts AB, Jakowlew S, Alvarez- Mon M, Derynck R, Sporn MB, Fauci AS. (1986 ) Production of transforming growth factor ( by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 163, 1037-1050.
Kim JH, Kim SH, Brown SL, Freitag SO. (1994 ) Selective enhancement by an antiviral agent of the radiation induced cell killing of human glioma cells transduced with HSV-TK gene. Cancer Res 54, 6053-6056.
Kim JH, Kim SH, Kolozsvary A,Brown SL, Kim OB, Freytag SO. ( 1995 ) Selective enhancement of radiation response of herpes simplex virus thymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo by antiviral agents. Int J Radiat Oncol Biol Phys 33, 861-868.
Kim SH, Kim JH, Kolozsvary A, Brown SL, Freytag SO. (1997 ) Preferential radiosensitization of 9L glioma cells transduced with HSV-TK gene by acyclovir. J Neurooncol 33, 189-194.
Kondo S, Barna BP, Kondo Y, Tanaka Y, Casey G, Liu J, Morimura T, Kaakaji R, Peterson JW, Werbel B, Barnett GH. (1996 ) WAF1/CIP1 increases the susceptibility of p53 non-functional malignant glioma cells to cisplatin- induced apoptosis. Oncogene 13, 1279-85.
Kondo S, Kondo Y, Li G, Silverman RH, Cowell JK. (1998 ) Targeted therapy of human malignant glioma in a mouse model by 2-5A antisense directed against telomerase RNA. Oncogene, 16, 25, 3323-30.
Kundig TM, Shahinian K, Kawai K, Mittruecker HW, Sebzda E, Bachmann MF, Mak TW, Ohashi PS. (1996 ) Duration of TCR stimulation determines costimulatory requirement of T cells. Immunity , 5, 41-52
Kuppner MC, Hamou M-F, Sawamura Y, Bodmer S, de Tribolet N. (1989 ) Inhibition of lymphocyte function by glioblastoma-derived transforming growth factor 2. J Neurosurg 71, 211-217
Lachmann RH, Efstathiou S. (1997 ) The use of herpes simplex virus-based vectors for gene delivery to the nervous system. Mol Med Today 3, 404-411.
Lang FF, Yung WK, Raju U, Libunao F, Terry NH, Tofilon PJ. (1998 ) Enhancement of radiosensitivity of wild-type p53 human glioma cells by adenovirus-mediated delivery of the p53 gene. J Neurosurg 89, 1, 125-132.
LeMay DR, Kittaka M, Gordon EM, Gray B, Stins MF, McComb JG, Jovanovic S, Tabrizi P, Weiss MH, Bartus R, Anderson WF, Zlokovic BV. (1998 ) Intravenous RMP-7 increases delivery of ganciclovir into rat brain tumors and enhances the effects of herpes simplex virus thymidine kinase gene therapy. Hum Gene Ther 9, 989-995.
Levin VA, Edwards MS, Wright DC. (1980 ) Modified procarbazine, CCNU and vincristine (PCV3) combination chomtherapy in the treatment of malignant brain tumors. Cancer Treat Rep 64, 237-241.
Levin VA, Gutin PH, Leibel S. Cancer, Principles and Practice of Oncology. In De Vita VT Jr, Hellman S, Rosemberg SA
PalÃ¹ et al: Perspectives for therapy of glioblastoma multiforme
(eds) (1993 ) , Neoplasms of the Central Nervous
System , Philadelphia, JB Lippincot, 1679-737.
Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Puc J, Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanella BC, Ittmann M, Tycko B, Hibshoosh H, Wigler MH, Parsons R. (1997 ) PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science, 275, 1943-1947.
Libermann TA, Nusbaum HR, Razon N, Kris R, Lax I, Soreq H, Whittle N, Waterfield MD, Ullrich A, Schlessinger J. (1985 ) Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumors of glial origin. Nature 313, 144-147.
Long Z, Li LP, Grooms T, Lockey C, Nader K, Mychkovsky I, Mueller S, Burimski I, Ryan P, Kikuchi G, Ennist D, Marcus S, Otto E, McGarrity G. (1998 ) Biosafety monitoring of patients receiving intracerebral injections of murine retroviral vector producer cells. Hum Gene Ther 9, 1165-1172.
Lowe SW, Bodis S, McClatchey A, Remington L, Ruley HE, Fisher DE, Housman DE, Jacks T. (1994 ) p53 status and the efficacy of cancer therapy in vivo. Science 266, 807-
Mabuchi E, Shimizu K, Miyao Y, Kaneda Y, Kishima H, Tamura M, Ikenaka K, Hayakawa T. (1997 ) Gene delivery by HVJ-liposome in the experimental gene therapy of murine glioma. Gene Ther 4, 768-72.
Mak M, Fung L, Strasser JF, Saltzman WM. (1995 ) Distribution of drugs following controlled delivery to the brain interstitium. J Neurooncol 26, 91-102.
Manome Y, Kunieda T, Wen PY, Koga T, Kufe DW, Ohno T. (1998 ) Transgene expression in malignant glioma using a replication-defective adenoviral vector containing the Egr-1 promoter, activation by ionizing radiation or uptake of radioactive iododeoxyuridine. Hum Gene Ther 9,
Manome Y, Wen PY, Chen L, Tanaka T, Dong Y, Yamazoe M, Hirshowitz A, Kufe DW, Fine HA. (1996 ) Gene therapy for malignant gliomas using replication incompetent retroviral and adenoviral vectors encoding the cytochrome P450 2B1 gene together with cyclophosphamide. Gene Ther 3, 513-520.
Manome Y, Wen PY, Dong Y, Tanaka T, Mitchell BS, Kufe DW, Fine HA. (1996 ) Viral vector transduction of the human deoxycytidine kinase cDNA sensitizes glioma cells to the cytotoxic effects of cytosine arabinoside in vitro and in vivo. Nat Med 2, 567-573.
McKie EA, MacLean AR, Lewis AD, Cruickshank G, Rampling R, Barnett SC, Kennedy PG, Brown SM. (1996 ) Selective in vitro replication of herpes simplex virus type 1 (HSV-1) ICP34.5 null mutants in primary human CNS tumours-- evaluation of a potentially effective clinical therapy. Br J Cancer 74, 745-752.
Merlo A, Juretic A, Zuber M, Filgueira L, Luscher U, Caetano V, Ulrich J, Gratzl O, Heberer M, Spagnoli G. (1993 ) Cytokine gene expression in primary brain tumors,
metastases and meningiomas suggests specific transcription patterns. Eur J Cancer 29A, 2118-2125.
Millauer B, Longhi MP, Plate KH, Shawer LK, Risau W, ulrich A, Strawn LM. (1996 ) Dominant-negative inhibition of Flk-1 suppress the growth of many tumor types in vivo. Cancer Res 56, 1615-1620.
Millauer B, Shawer LK, Plate KH, Risau W, Ulrich A. (1994 ) Glioblastoma growth inhibited in vivo by a dominant- negative Flk-1 mutant. Nature, 367, 576-579.
Mineta T, Rabkin SD, Yazaki T, Hunter WD, Martuza RL. (1995 ) Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas. Nat Med, 1, 9,
Miyatake S, Martuza RL, Rabkin SD. (1997 ) Defective herpes simplex virus vectors expressing thymidine kinase for the treatment of malignant glioma. Cancer Gene Ther 4, 222-228.
Mizuno M, Yoshida J, Colosi P, Kurtzman G. (1998 ) Adeno- associated virus vector containing the herpes simplex virus thymidine kinase gene causes complete regression of intracerebrally implanted human gliomas in mice, in conjunction with ganciclovir administration. Jpn J Cancer Res, 89, 1, 76-80.
Newlands ES, O'Reilly SM, Glaser MG. (1996 ) The Charing Cross hospital experience with Temozolomide in patients with gliomas. Eur J Cancer, 32A, 2236-41.
Nguyen DM, Spitz FR, Yen N, Cristiano RJ, Roth JA. (1996 ) Gene therapy for lung cancer, enhancement of tumor suppression by a combination of sequential systemic cisplatin and adenovirus-mediated p53 gene transfer. J Torac Cardiovasc Surg, 112, 1372-1377.
Nielsen LL, Maneval DC. (1998 ) p53 tumor suppressor gene therapy for cancer. Cancer Gene Ther 5, 52-63.
Nishikawa R, Furnari FB, Lin H, Arap W, Berger MS, Cavenee WK, Su Huang HJ. (1995 ) Loss of p16ink4 expression is frequent in high-grade gliomas. Cancer Res 55, 1941-
Nishikawa R, Ji XD, Harmon RC, Armon RC, Lazar CS, Gill GN, Cavenee WK, Huang HG. (1994 ) A mutant epidermal growth factor receptor common in human gliomas confers enhanced tumorigenicity. Proc Natl Acad Sci USA
Nitta T, Hishii M, Sato K, Okumura K. (1994 ) Selective expression of interleukin-10 gene within glioblastoma multiforme. Brain Res , 649, 122-128.
Noguchi Y, Chen YT, Old LJ. (1994 ) A mouse mutant p53 product recognized by CD4+ and CD8+ T cells. Proc Natl Acad Sci 91, 3171-3175.
Ohnishi T, Taki T, Hiraga S, Arita N, Morita T. ( 1998 ) In vitro and in vivo potentiation of radiosensitivity of malignant gliomas by antisense inhibition of the RAD51 gene. Biochem Biophys Res Commun 245, 319-
Oldfield EH, Ram Z, Culver KW, Blaese RM, De Vroom HL, Anderson WF. ( 1993 ) Clinical Protocol, Gene therapy for
Gene Therapy and Molecular Biology Vol 1, page 147
the treatment of brain tumors using intra-tumoral transduction with the thymidine kinase and intravenous ganciclovir. Hum Gene Ther 4, 39-69.
Ono Y, Ikeda K, Wei MX, Harsh GR 4th, Tamiya T, Chiocca
EA. (1997 ) Regression of experimental brain tumors with
6-thioxanthine and Escherichia coli gpt gene therapy.
Hum Gene Ther 8, 2043-2055.
O'Rourke DM, Kao GD, Singh N, Park BW, Muschel RJ, Wu CJ, Greene MI. (1998 ) Conversion of a radioresistant phenotype to a more sensitive one by disabling erbB receptor signaling in human cancer cells. Proc Natl Acad Sci USA 95, 10842-10847.
O'Rourke DM, Qian X, Zhang HT, Davis JG, Nute E, Meinkoth J, Greene MI. (1997 ) Trans receptor inhibition of human glioblastoma cells by erbB family ectodomains. Proc Natl Acad Sci USA 94, 3250-3255.
PalÃ¹ G, Cavaggioni A, Calvi P, Franchin E, Pizzato M, Boschetto R, Parolin C, Chilosi M, Ferrini S, Zanusso A, Colombo F. (1998 ) Gene therapy of glioblastoma multiforme via combined expression of suicide and cytokine genes, a pilot study in humans. Gene Ther (in press)
Parr MJ, Manome Y, Tanaka T, Wen P, Kufe D, Kaelin WG Jr, Fine HA. (1997 ) Tumor-selective transgene expression in vivo mediated by an E2F-responsive adenoviral vector. Nature Med, 3, 1145-9.
Petersdorf SH, Berger MS. (1996 ) Concepts in Neurosurgery, The molecular basis of neurosurgical disease. In Raffel C, Harsh IV GR (eds), Molecular Basis of Chemotherapy for Brain Tumors, Baltimore, Williams and Wilkins, chapter 12 (Vol 8), 198-210.
Pizzato M, Franchin E, Calvi P, Boschetto R, Ferrini S, Colombo M, PalÃ¹ G. (1998 ) Production and characterization of a bicistronic Moloney-based retroviral vector expressing human interleukin 2 and herpes simplex virus thymidine kinase for gene therapy of cancer. Gene Ther 5, 1003-1007.
Prados MD, Schold SC, Spence AM, Berger MS, Mc Allister LD, Mehta MP, Gilbert MR, Fulton D, Kuhn J, Lamborn K, Rector DJ, Chang SM. (1996 ) Phase II study of paclitaxel in patients with recurrent malignant glioma. J Clin Oncol 14, 2316-2321.
Rainov NG, Dobberstein KU, Bahn H, Holzhausen HJ, LautenschlÃ¤ger C, Heidecke V, Burkert W. (1997 ) Prognostic factors in malignant glioma, influence of the overexpression of oncogene and tumor-suppressor gene products on survival. J Neurooncol 35, 13-28.
Rainov NG, Dobberstein KU, Sena-Esteves M, Herrlinger U, Kramm CM, Philpot RM, Hilton J, Chiocca EA, Breakefield XO. (1998 ) New prodrug activation gene therapy for cancer using cytochrome P450 4B1 and 2- aminoanthracene/4-ipomeanol. Hum Gene Ther 9,
Ram Z, Culver KW, Oshiro EM, Viola JJ, De Vroom HL, Otto E, Long Z, Chiang Y, McGarrity GJ, Muul LM, Katz D, Blaese RM, Oldfield EH. (1997 ) Therapy of malignant
brain tumors by intratumoral implantation of retroviral vector-producing cells. Nature Med 3, 1354-1361
Ram Z, Culver KW, Wallbridge S, Blaese RM, Oldfield EH. (1993 ) In situ retroviral-mediated gene transfer for the treatment of brain tumors in rats. Cancer Res 53, 83-88.
Ranges GE, Figari IS, Espevik T, Palladino MA Jr. (1987 ) Inhibition of cytotoxic T cell development by transforming growth factor and reversal by recombinant tumor necrosis factor . J Exp Med 166, 991-998.
Redekop GJ, Naus CC. (1995 ) Transfection with bFGF sense and antisense cDNA resulting in modification of malignant glioma growth. J Neurosurg 82, 83-90.
Rimoldi D, Romero P, Carrel S. (1993 ) The human melanoma antigen/encoding gene, MAGE-1, is expressed by other tumor cells of neuroectodermal origin such as glioblastomas and neuroblastomas. Int J Cancer 54,
Rodriguez LA, Prados M, Silver P. (1989 ) Reevaluation of procarbazione for the treatmentof recurrent malignant central nervous system tumors. Cancer 64, 2420-2423.
Rogulski KR, Kim JH, Kim SH, Freytag SO. (1997 ) Glioma cells transduced with an Escherichia coli CD/HSV-1 TK fusion gene exhibit enhanced metabolic suicide and radiosensitivity. Hum Gene Ther 8, 73-85.
Rook AH, Kerhl JH, Wakefield LM, Roberts AB, Sporn MB, Burlington DB, Lane HC, Fauci AS. (1986 ) Effects of transforming growth factor ( on the functions of natural killer cells, depressed cytolytic activity and blunting of interferon responsiveness. J Immunol 136, 3916-3920.
Rosemberg SA, Lotze MT, Muul LM, Chang AE, Avis FP, Leitman S, Linehan WM, Robertson CN, Lee RE, Rubin JT, Seipp CA, Simpson CG, White DE. ( 1987 ) A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high dose of interleukin-2 alone. New Engl J Med , 316, 889-905.
Roszman T, Elliott L, Brooks W. (1991 ) Modulation of T- cell function by gliomas. Immunol Today 12, 370-374.
Roth JA, Cristiano R. (1997 ) Gene therapy for cancer, What we done and where we are going? J Natl Cancer Inst ,
Russell DS, Rubistein LJ. (1989 ) Pathology of Tumors of the Central Nervous System , 5th Edition, Edward Arnold, London.
Saleh M, Stacker SA, Wilks AF. (1996 ) Inhibition of growth of C6 glioma cells in vivo by expression of antisense vascular endothelial growth factor sequence. Cancer Res
Sampson JH, Archer GE, Ashley DM, Fuchs HE, Hale LP, Dranoff G, Bigner DD. (1996 ) Subcutaneous vaccination with irradiated, cytokine-producing tumor cells stimulates CD8+ cell mediated immunity against tumors located in the immunologically privileged central nervous system. Proc Natl Acad Sci USA 93, 10399-10404.
PalÃ¹ et al: Perspectives for therapy of glioblastoma multiforme
Sanson M, Ameri A, Monjour A, Sahmoud T, Ronchin P, Poisson M, Delattre JY. ( 1996 ) Treatment of recurrent malignant supratentorial gliomas with ifosfamide, carboplatin, and etoposide, A phase II study. Eur J Cancer 32A, 2229-2235.
Smart CR, Ottomoan RE, Rochlin DB. (1968 ) Clinical experience with vincristine with tumors of the central nervous system and other malignant diseases. Cancer Chem Rep 52, 733-741.
Sporn MB, Roberts AB, Wakefield LM, Assoian RK. (1986 ) Transforming growth factor-(, biological fusion and chemical structure. Science 233, 532-534
Sporn MB, Roberts AB, Wakefield LM, de Crombrugghe B. (1987 ) Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol
Staba MJ, Mauceri HJ, Kufe DW, Hallahan DE, Weichselbaum RR. (1998 ) Adenoviral TNF-alpha gene therapy and radiation damage tumor vasculature in a human malignant glioma xenograft. Gene Ther 5, 293-300.
Sturtz FG, Waddell K, Shulok J, Chen X, Caruso M, Sanson M, Snodgrass HR, Platika D. (1997 ) Variable efficiency of the thymidine kinase/ganciclovir system in human glioblastoma cell lines, implications for gene therapy. Hum Gene Ther 8, 1945-53.
Tada M, Diserens AC, Hamou MF, Jaufeerally R, Van Meir E, de Tribolet N. (1996 ) Brain Tumor Res Ther , 327-
Takamiya Y, Short MP, Ezzeddine ZD, Moolten FL, Breakefield XO, Martuza RL. (1992 ) Gene therapy of malignant brain tumors, a rat glioma line bearing the herpes simplex virus type 1-thymidine kinase gene and wild type retrovirus kills other tumor cells. J Neurosc Res 33, 493-503.
Takamiya Y, Short MP, Moolten FL, Fleet C, Mineta T, Breakefield XO, Martuza RL. (1993 ) An experimental model of retrovirus gene therapy for malignant brain tumors. J Neurosurg 79, 104-110.
Tamiya T, Ono Y, Wei MX, Mroz PJ, Moolten FL, Chiocca EA. (1996 ) Escherichia coli gpt gene sensitizes rat glioma cells to killing by 6-thioxanthine or 6- thioguanine. Cancer Gene Ther 3, 3, 155-162.
Tamura M, Shimizu K, Yamada M, Miyao Y, Hayakawa T, Ikenaka K. (1997 ) Targeted killing of migrating glioma cells by injection of HTK-modified glioma cells. Hum Gene Ther 8, 381-91.
Tanaka T, Cao Y, Folkman J, Fine HA. (1998 ) Viral vector- targeted antiangiogenic gene therapy utilizing an angiostatin complementary DNA. Cancer Res 58, 3362-
Tanaka T, Manome Y, Wen P, Kufe DW, Fine HA. ( 1997 ) Viral vector-mediated transduction of a modified platelet factor 4 cDNA inhibits angiogenesis and tumor growth. Nat Med 3, 437-442.
Tepper RI. (1993 ) The antitumor and proinflammatory actions of IL-4. Res Immunol 144, 633-637.
Tohma Y, Gratas C, Biernat W, Peraud A, Fukuda M, Yonekawa Y, Kleihues P, Ohgaki H. (1998 ) PTEN (MMAC1) mutations are frequent in primary glioblastomas (de novo) but not in secondary glioblastomas. J Neuropathol Exp Neurol 57, 684-689.
Trojan J, Johnson TR, Rudin SD, Ilan J, Tykocinski ML, Ilan J. (1993 ) Treatment and prevention of rat glioblastoma by immunogenic C6 cells expressing antisense insulin- like growth factor I RNA. Science 259, 94-97.
Van Der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van Den Eynde B, Knuth A, Boon T. (1991 ) A gene encoding an antigen recognized by cytolityc T lymphocytes on a human melanoma. Science 254, 1643-
Van Meir E, Roemer K, Diserens AC, Kikuchi T, Rempel SA, Haas M, Huang HG, Friedmann T, de Tribolet N, Cavenee WK. (1995 ) Single cell monitoring of growth arrest and morphological changes induced by transfer of wild-type p53 alleles to glioblastoma cells. Proc Natl Acad Sci USA 92, 1008-1012.
Vincent AJ, Vogels R, Someren GV, Esandi MC, Noteboom JL, Avezaat CJ, Vecht C, Bekkum DW, Valerio D, Bout A, Hoogerbrugge PM. (1996 ) Herpes simplex virus thymidine kinase gene therapy for rat malignant brain tumors. Hum Gene Ther 7, 197-205.
Voest EE, Kenyon BM, OâReilly MS, Truitt G, DâAmato RJ, Folkman J. (1995 ) Inhibition ofangiogenesis in vivo by interleukin-12. J Natl Cancer Inst 87, 581-586.
Walker MD, Green SB, Byar DP. (1980 ) Randomized comparisons of radiotherapy and nitrosoureas for the treatment of malignant glioma after surgery. N Engl J Med 303, 1323-1329.
Wei M, Tamiya T, Chase M, Bobiatsis EJ, Chang TK, Kowall NW, Hochberg FH, Waxman DJ, Breakefield XO, Chiocca EA. (1994 ) Experimental tumor therapy in mice using the cyclophosphamide-activating cytochrome P450 2B1 gene. Hum Gene Ther 5, 969-978.
Weller M, Malipiero U, Rensing Ehl A, Barr PJ, Fontana A. (1995 ) Fas/APO-1 gene transfer for human malignant glioma. Cancer Res 55, 2936-2944.
Young RC, Walker MD, Canellos GP. 1973 Initial clinical trials with methyl-CCNU 1-(2-chloroethyl)-3-(4-methyl cycloexyl)-I-nitrosourea (MeCCNU). Cancer 31, 1164-
Yu JS, Wei MX, Chiocca EA, Martuza RL, Tepper RI. (1993 ) Treatment of glioma by engineered interleukin 4-secreting cells. Cancer Res 53, 3125-3128.
Zerrouqi A, Rixe O, Ghoumari AM, Yarovoi SV, Mouawad R, Khayat D, Soubrane C. (1996 ) Liposomal delivery of the herpes simplex virus thymidine kinase gene in glioma, improvement of cell sensitization to ganciclovir. Cancer Gene Ther 3, 385-392.
Gene Therapy and Molecular Biology Vol 1, page 149
PalÃ¹ et al: Perspectives for therapy of glioblastoma multiforme
integrated viral vector
LTR IL-2 IRES TK SV NEO LTR
GCV GCV-P GCV-3P TK
producer cells inoculation
Gene Therapy and Molecular Biology Vol 1, page 151
TK SV NEO
regulatory elements therapeutic genes
gene for positive selection