Replication-defective Retroviral Vector Research Articles

General steps in the production and establishment of transgenic chicken lines carrying a bacterial lacZ gene expressing β-galactosidase

Replication-defective retroviral vectors are efficient vehicles for the delivery of exogenous genes, and they may be used in the generation of transgenic animals. The replication-defective retroviral SNTZ vector carrying the lacZ gene with a nuclear localized signal was injected into the subgerminal cavity of freshly laid eggs. Subsequently, the eggs were allowed to hatch, and the chickens were screened for the lacZ gene by using the polymerase chain reaction. Eight of 15 male chickens that survived to sexual maturity contained the lacZ gene in their semen. Subsequently, these males were mated with wild-type female chickens. In this study, performance of the SNTZ vector carrying the Eschrichia coli lacZ gene to express a nuclear-localized β-galactosidase was injected into the subgerminal cavity of embryos collected from newly laid eggs was reviewed.

Peroxisome Proliferator-activated Receptor γ (PPARγ) Mediates a Ski Oncogene-induced Shift from Glycolysis to Oxidative Energy Metabolism

Overexpression of the Ski oncogene induces oncogenic transformation of chicken embryo fibroblasts (CEFs). However, unlike most other oncogene-transformed cells, Ski-transformed CEFs (Ski-CEFs) do not display the classical Warburg effect. On the contrary, Ski transformation reduced lactate production and glucose utilization in CEFs. Compared with CEFs, Ski-CEFs exhibited enhanced TCA cycle activity, fatty acid catabolism through β-oxidation, glutamate oxidation, oxygen consumption, as well as increased numbers and mass of mitochondria. Interestingly, expression of PPARγ, a key transcription factor that regulates adipogenesis and lipid metabolism, was dramatically elevated at both the mRNA and protein levels in Ski-CEFs. Accordingly, PPARγ target genes that are involved in lipid uptake, transport, and oxidation were also markedly up-regulated by Ski. Knocking down PPARγ in Ski-CEFs by RNA interference reversed the elevated expression of these PPARγ target genes, as well as the shift to oxidative metabolism and the increased mitochondrial biogenesis. Moreover, we found that Ski co-immunoprecipitates with PPARγ and co-activates PPARγ-driven transcription.

Krüppel-like Factor 4 Inhibits Epithelial-to-Mesenchymal Transition through Regulation of E-cadherin Gene Expression

The Krüppel-like factor 4 (KLF4) is a transcriptional regulator of proliferation and differentiation in epithelial cells, both during development and tumorigenesis. Although KLF4 functions as a tumor suppressor in several tissues, including the colon, the role of KLF4 in breast cancer is less clear. Here, we show that KLF4 is necessary for maintenance of the epithelial phenotype in non-transformed MCF-10A mammary epithelial cells. KLF4 silencing led to alterations in epithelial cell morphology and migration, indicative of an epithelial-to-mesenchymal transition. Consistent with these changes, decreased levels of KLF4 also resulted in the loss of E-cadherin protein and mRNA. Promoter/reporter analyses revealed decreased E-cadherin promoter activity with KLF4 silencing, while chromatin immunoprecipitation identified endogenous KLF4 binding to the GC-rich/E-box region of this promoter. Furthermore, forced expression of KLF4 in the highly metastatic MDA-MB-231 breast tumor cell line was sufficient to restore E-cadherin expression and suppress migration and invasion. These findings identify E-cadherin as a novel transcriptional target of KLF4. The clear requirement for KLF4 to maintain E-cadherin expression and prevent epithelial-to-mesenchymal transition in mammary epithelial cells supports a metastasis suppressive role for KLF4 in breast cancer.

310 PRODUCTION OF TRANSGENIC CLONED PORCINE EMBRYOS EXPRESSING EGFP OR LacZ GENE THROUGH REPLICATION DEFECTIVE RETROVIRAL VECTOR

We have demonstrated previously that retroviral-mediated gene transfer is a promising method to produce transgenic avian, porcine, and bovine embryos. This study was designed to evaluate the development potential of transgenic porcine embryos produced by somatic cell nuclear transfer (SCNT) of fetal fibroblast (pFF) cells transfected by a robust replication-defective retroviral vector harboring enhanced green fluorescent protein (EGFP) or β-galactosidase (LacZ) gene. Moloney murine leukemia virus (MoMLV)-based retroviral vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein and harboring EGFP or LacZ under the control of β-actin promoter were produced and used to transfect primary pFF cells that were subsequently used for SCNT of enucleated porcine oocytes matured in vitro. Our results showed that all surviving cells after transfection and antibiotic selection expressed the genes without any evidence of replication-competent retrovirus. The fusion, cleavage, and blastocyst rates were 85.6 � 6.5, 53.6 � 6.4, and 12.0 � 5.7% for EGFP; 83.5 � 8.2, 57.5 � 6.3 and 10.1 � 4.1% for LacZ; and 80.5 � 4.2, 60.9 � 8.2 and 12.3 � 4.0% for controls, respectively. Mosaicism was not observed in any of the group as evidenced by the expression of LacZ or EGFP in individual blastomeres of all embryos upon staining with β-galactosidase (for LacZ) or when visualized under UV illumination of an epifluorescent microscope using the fluorescein isothiocyanate (FITC) filter set (for EGFP). Further recloning of EGFP-expressing blastomeres, obtained from 4-cell-stage cloned embryos produced by SCNT of pFF cells infected with EGFP harboring vector, into enucleated metaphase II (MII) oocytes resulted in consistent expression of EGFP in recloned blastocysts. Interspecies SCNT (iSCNT) of transfected pFF into enucleated bovine oocytes could also result in consistent gene expression without any adverse effect on blastocyst rate (5.5 v. 4.9%) compared with non-transfected pFF. These data indicate that the replication-defective retroviral vector used in the present study is robust and independent of the genes inserted. Furthermore, introduction of transgenes by this method does not influence the in vitro development rate of cloned embryos. This work was supported by a grant from Biogreen 21 Program, RDA, Republic of Korea.

Neu-Induced Retroviral Rat Mammary Carcinogenesis: A Novel Chemoprevention Model for Both Hormonally Responsive and Nonresponsive Mammary Carcinomas

Clinically relevant animal models of mammary carcinogenesis are crucial for the development and evaluation of new breast cancer chemopreventive agents. The neu-induced retroviral rat mammary carcinogenesis model is based on the direct in situ transfer of the activated neu oncogene into the mammary epithelium using a replication-defective retroviral vector. The resulting mammary carcinomas in intact Wistar-Furth rats exhibit a mixed hormonal response in the same proportion as has been observed in women. In intact rats, approximately 50% of mammary carcinomas can be prevented by tamoxifen treatment. In ovariectomized animals, the mammary carcinomas are hormonally nonresponsive and cannot be prevented by tamoxifen. We evaluated the efficacy of retinoic X receptor-selective retinoids (rexinoids) in this novel model of mammary carcinogenesis. The rexinoids LG100268 and bexarotene (LG1069, Targretin) were highly efficacious in the prevention of neu-induced mammary carcinomas. Dietary LG100268 at 100 mg/kg diet decreased tumor multiplicity by 32% (P = 0.0114) in intact rats and 50% (P < 0.0001) in ovariectomized rats. Bexarotene treatment at a dose of 250 mg/kg diet was associated with reductions in tumor multiplicity of 84% (P < 0.0001) and 86% (P < 0.0001) in intact and ovariectomized animals, respectively. In addition to tumor multiplicity, proliferation and apoptosis were modulated by bexarotene treatment independently of estrogen signaling. The neu-induced retroviral rat mammary carcinogenesis model represents a valuable addition to existing rodent chemoprevention models. The model is useful for assessing the efficacy of chemopreventive agents, specifically those compounds that target hormonally nonresponsive tumors.

Acute myeloid leukemia is associated with retroviral gene transfer to hematopoietic progenitor cells in a rhesus macaque

We report, for the first time, a replication-defective retroviral vector-associated neoplasia in a nonhuman primate. Five years after transplantation with CD34+ cells transduced with a retroviral vector expressing enhanced green fluorescent protein (eGFP) and a drug-resistant variant of the dihydrofolate reductase gene (L22Y), a rhesus macaque developed a fatal myeloid sarcoma, a type of acute myeloid leukemia. Tumor cells contained 2 clonal vector insertions. One insertion was found in BCL2-A1, an antiapoptotic gene. This event suggests that currently available retroviral vectors may have long-term side effects, particularly in hematopoietic stem and progenitor cells.

Transgenic birds for the production of recombinant proteins.

Transgenic birds were expected to be an excellent transgenic bioreactor for the production of recombinant pharmaceutical proteins. However, the only successful transgenic bioreactors have been based on mammals. We have developed two key techniques for obtaining transgenic birds. For bird embryo culture, we identified that the low rate of hatchability of cultured embryos is caused by limited oxygen and calcium availability. In quail embryo culture using a chicken eggshell as a culture vessel, hatchability increased to 80% by the supplement of calcium lactate in addition to oxygen aeration. A fully artificial vessel for quail embryo culture using a gas-permeable Teflon membrane was also designed. Although the hatchability was lower than that of cultures using a surrogate eggshell, we succeeded in hatching of bird embryos using a fully artificial vessel. For transgene introduction, a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus (MoMLV) pseudotyped with vesicular stomatitis virus G protein (VSV-G) was injected to laid embryos at the blastodermal stage, and the embryos were hatched in vitro to generate G0 birds. The viral vector sequence was detected in the tissues of all G0 birds. The germ-line transmission efficiency was more than 80%. Plural copies of the transgene were inserted into the genome of G1 transgenic progeny.

Validating the hen as a bioreactor for the production of exogenous proteins in egg white

Increased demand for the production of human biopharmaceuticals in transgenic organisms has led to an intensive effort to develop the hen as a bioreactor producing exogenous proteins in egg white via transgenesis. To date, however, robust methods for transgenic modification of the avian genome have been lacking. We have used a replication-defective retroviral vector derived from avian leukosis virus (ALV) to generate transgenic chickens expressing bacterial beta-lactamase secreted into serum and egg whites through several generations. Expression was driven by the ubiquitous cytomegalovirus (CMV) promoter. Here we describe results from a transgenic lineage (Harvey et al., 2002a, b) in which (1) the transgene was stably transmitted from a G1 founder male (5657) through several generations without silencing, (2) the protein was biologically active, and (3) the level of expression in egg whites was doubled in a G3 homozygote.

Development of transgenic chickens expressing bacterial beta-galactosidase.

Replication-defective retroviral vectors are efficient vehicles for the delivery of exogenous genes, and they may be used in the generation of transgenic animals. The replication-defective retroviral SNTZ vector carrying the lacZ gene with a nuclear localized signal was injected into the subgerminal cavity of freshly laid eggs. Subsequently, the eggs were allowed to hatch, and the chickens were screened for the lacZ gene by using the polymerase chain reaction. Eight of 15 male chickens that survived to sexual maturity contained the lacZ gene in their semen. Subsequently, these males were mated with wild-type female chickens. From one of the eight lacZ-positive G(0) males, two lacZ-positive male chickens were produced from a total of 224 G(1) progeny for a germline transmission rate of 0.89%. Both G(1) male chickens carrying the lacZ gene were mated with wild-type female chickens and 46.5% of the G(2) progeny contained the lacZ gene, which is consistent with the expected Mendelian 50% ratio for a heterozygous dominant allele. The product of the lacZ gene, nuclear localized beta-galactosidase, was expressed in primary myoblast cultures derived from G(2) chickens, and it was also expressed in whole G(2) chicken embryos.

Production of anti-prion scFv-Fc fusion proteins by recombinant animal cells

We constructed a replication-defective retroviral vector plasmid for the expression of a single-chain antibody fragment (scFv), derived from a chicken anti-human prion protein monoclonal antibody, fused with the Fc region of human IgG1. CHO-K1 and NS-1 cells were transformed with the viral vector pseudotyped with vesicular stomatitis virus G protein (VSV-G), and scFv-Fc producer clones were established. Among the established clones, CHO-2A9 cells produced a large amount of the product with an antibody-like dimerized structure in serum-free culture that facilitated the purification of scFv-Fc. The scFv-Fc specifically recognized the epitope sequence of prion protein in solid-phase enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The injection test into quails revealed that the scFv became more stable in vivo by fusion with the Fc region. The scFv-Fc will be a useful tool for the detection of mammalian prion proteins.

Production of Anti-Prion scFv-Fc Fusion Proteins by Recombinant Animal Cells

We constructed a replication-defective retroviral vector plasmid for the expression of a single-chain antibody fragment (scFv), derived from a chicken anti-human prion protein monoclonal antibody, fused with the Fc region of human IgG1. CHO-K1 and NS-1 cells were transformed with the viral vector pseudotyped with vesicular stomatitis virus G protein (VSV-G), and scFv-Fc producer clones were established. Among the established clones, CHO-2A9 cells produced a large amount of the product with an antibody-like dimerized structure in serum-free culture that facilitated the purification of scFv-Fc. The scFv-Fc specifically recognized the epitope sequence of prion protein in solid-phase enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The injection test into quails revealed that the scFv became more stable in vivo by fusion with the Fc region. The scFv-Fc will be a useful tool for the detection of mammalian prion proteins.

Liver as an ideal target for gene therapy: expression of CTLA4Ig by retroviral gene transfer.

Liver has been a target organ for gene therapy as it plays a central role in metabolism and production of serum proteins. Many metabolic disorders result from a deficiency of liver-derived protein products. In transplantation settings, modulation of the immune responses caused by CTLA4Ig protein has been shown to be an attractive direction. In this study, we investigated the efficacy of hepatocyte transduction via introduction of the exogenous CTLA4Ig gene to the rat liver graft by retrovirus vector, and examined the presence of target serum protein after gene transfers. We constructed a replication defective retroviral vector that contained the CTLA4Ig gene. The liver graft regeneration index was first examined by 5-bromo-2-deoxyuridine, Ki-67 and proliferating cell nuclear antigen antibodies to determine the optimal time of gene transduction. The liver graft was then perfused with the retroviral vector, and animals were killed at constant time points to examine for the presence of CTLA4 protein in the graft and peripheral blood. CTLA4 protein was detected on postoperative days 5, 9 and 14, with liver graft tissue transduction indexes of 7.2, 10.9 and 1.8, respectively. Blood protein levels were at 151.6, 26.5 and 21.4 rhog/mL, respectively. A transduction index reaching 22.1 was observed in the graft with the most rapid liver regeneration. We had established the gene delivery model in rat with auxiliary partial liver transplantation. Expression of the exogenous gene delivered by retrovirus was demonstrated in the liver with secretion of diffusible protein in the bloodstream. The present study provides important information for gene transfer using the liver to produce the target protein in situ and as serum protein. This will also be applicable to the treatment of other metabolic diseases.

Retrovirus-mediated transfer of prothymosin gene inhibits tumor growth and prolongs survival in murine bladder cancer.

To explore the potential use of prothymosin alpha(ProT), a putative thymic hormone, in gene therapy for bladder cancer, we generated a replication-defective recombinant retroviral vector encoding ProT and tested its antitumor effect on the MBT-2 murine bladder cancer. C3H/HeN mice injected with MBT-2 cells in conjunction with retroviruses encoding ProT exhibited smaller tumor mass, lower tumor incidence and higher survival rate, as well as higher antitumor cytotoxic activities compared with those injected with control viruses. However, such effects were not observed in severe combined immunodeficiency mice, suggesting that ProT exerts antitumor effects through its immunomodulatory activities. Cell growth in monolayer culture and colony formation in soft agar were enhanced in ProT gene-modified MBT-2 clones, and such growth-promoting activities of ProT could be reversed if its nuclear localization signal (NLS) was deleted. To circumvent the proliferation-promoting effect of ProT on tumor cells, a retroviral vector encoding ProT lacking NLS was constructed. Our results showed that retroviruses encoding NLS-deleted ProT was more efficacious than those encoding wild-type ProT in prolonging survival of tumor-bearing mice. This is the first report indicating that ProT, in particular NLS-deleted ProT, delivered by retroviral vectors may be further explored for the treatment of bladder cancer.

Production of Transgenic Quails with High Frequency of Germ-Line Transmission Using VSV-G Pseudotyped Retroviral Vector

We report here the production of transgenic quails using a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus (MoMLV) pseudotyped with vesicular stomatitis virus G protein (VSV-G). The retroviral vector was injected into laid quail embryos at the blastodermal stage, and the embryos were incubated to hatch to produce G0 transgenic quails. Among 134 embryos subjected to viral injection, 37 hatched. The viral vector sequence was detected in the tissues of all G0 quails. The germ-line transmission efficiency of G0 quails mated with nontransgenic quails was more than 80% on average. Southern blot analysis revealed that the G1 transgenic progeny had one to three copies of the transgene. The expression of vector-encoded neomycin-resistance gene under the control of the Rous sarcoma virus (RSV) promoter was observed in several tissues including heart and muscle of both G1 and G2 transgenic offspring. Due to the high frequency of germ-line transmission, this method may markedly facilitate the production of transgenic avian.

Generation of high-titer retroviral vector-producing macrophages as vehicles for in vivo gene transfer

The goal of this project was to develop a novel gene transfer system based on macrophages (Mphi) as shuttles of recombinant retroviral vectors carrying therapeutic or marker genes. The murine Mphi cell line WGL5 was used as a source of Mphi for this study. We generated retrovirus-producing Mphi by transducing the WGL5 cells with a replication-defective retroviral vector carrying the enhanced green fluorescent protein (EGFP) reporter gene and the Moloney murine leukemia virus (MoMLV) as helper virus. We demonstrated stable integration of the recombinant retrovirus in the Mphi genome, efficient recombinant retrovirus production, and EGFP gene delivery to different cell lines in vitro. To evaluate Mphi-mediated EGFP gene transfer in vivo, allogeneic mice were injected s.c. with the retrovirus-producing WGL5 Mphi, that gave rise to solid tumor masses at the injection site, highly infiltrated with host leukocytes. We observed EGFP fluorescence in tumor-infiltrating CD4(+) and CD8(+) host T lymphocytes, providing direct evidence of the ability of engineered Mphi to mediate EGFP gene delivery to host cells in vivo. Moreover, we showed that retrovirus-producing Mphi could home to different organs in vivo following i.v. injection into mice. These data demonstrate that Mphi can be engineered as cellular vehicles for recombinant retroviruses carrying heterologous genes and suggest potential applications of this novel vector system for gene therapy.

Transgenic monkeys produced by retroviral gene transfer into mature oocytes.

Transgenic rhesus monkeys carrying the green fluorescent protein (GFP) gene were produced by injecting pseudotyped replication-defective retroviral vector into the perivitelline space of 224 mature rhesus oocytes, later fertilized by intracytoplasmic sperm injection. Of the three males born from 20 embryo transfers, one was transgenic when accessible tissues were assayed for transgene DNA and messenger RNA. All tissues that were studied from a fraternal set of twins, miscarried at 73 days, carried the transgene, as confirmed by Southern analyses, and the GFP transgene reporter was detected by both direct and indirect fluorescence imaging.

Medial Edge Epithelial Cell Fate during Palatal Fusion

To explain the disappearance of medial edge epithelial (MEE) cells during palatal fusion, programmed cell death, epithelial–mesenchymal transformation, and migration of these cells to the oral and nasal epithelia have been proposed. However, MEE cell death has not always been accepted as a mechanism involved in midline epithelial seam disappearance. Similarly, labeling of MEE cells with vital lipophilic markers has not led to a clear conclusion as to whether MEE cells migrate, transform into mesenchyme, or both. To clarify these controversies, we first utilized TUNEL techniques to detect apoptosis in mouse palates at the fusion stage and concomitantly analyzed the presence of macrophages by immunochemistry and confocal microscopy. Second, we in vitro infected the MEE with the replication-defective helper-free retroviral vector CXL, which carries the Escherichia coli lacZ gene, and analyzed β-galactosidase activity in cells after fusion to follow their fate. Our results demonstrate that MEE cells die and transform into mesenchyme during palatal fusion and that dead cells are phagocytosed by macrophages. In addition, we have investigated the effects of the absence of transforming growth factor β3 (TGF-β3) during palatal fusion. Using environmental scanning electron microscopy and TUNEL labeling we compared the MEE of the clefted TGF-β3 null and wild-type mice. We show that MEE cell death in TGF-β3 null palates is greatly reduced at the time of fusion, revealing that TGF-β3 has an important role as an inducer of apoptosis during palatal fusion. Likewise, the bulging cells observed on the MEE surface of wild-type mice prior to palatal shelf contact are very rare in the TGF-β3 null mutants. We hypothesize that these protruding cells are critical for palatal adhesion, being morphological evidence of increased cell motility/migration.

DM-GRASP Is Necessary for Nonradial Cell Migration during Chick Diencephalic Development

Cell migration is fundamental to normal CNS development. Radial migration, along radial glial fibers, has been the principal pathway studied, however, nonradial or tangential cell migration has increasingly been identified at all levels of the CNS. Receptors, cell adhesion molecules, and extracellular matrix molecules have all been shown to participate in radial cell migration. In contrast, the molecular basis of nonradial cell migration has only recently begun to be elucidated. Using replication defective retroviral vectors we have determined the location and time when nonradial cell migration begins in the developing chick diencephalon. We have identified three molecules that are expressed in spatially and temporally restricted domains that are consistent with them playing a role in nonradial cell migration. One of these molecules, DM-GRASP, a transmembrane protein with five extracellular Ig domains, is expressed on the nonradially migrating cells in addition to axons. To test the hypothesis that DM-GRASP participates in guiding nonradial cell migration, we injected a replication-defective retroviral vector used for lineage tracing followed by a DM-GRASP blocking antibody. Embryos injected with the blocking antibody showed a near complete block in nonradial cell migration specifically where DM-GRASP is expressed. Furthermore, morphological analyses revealed disruption of the normal architecture of the diencephalon indicating nonradial cell migration is necessary for normal morphological development of the brain. Our data indicate that DM-GRASP is necessary for nonradial cell migration in the chick diencephalon and have provided a system to further explore the function of nonradial cell migration during CNS development.

Retroviral Labeling is an Appropriate Marker for Dividing Cells

5-Bromo-2′-deoxyuridne (BrdU) and 3H-thymidine label mitotically active cells, but they do not adequately mark the progeny of dividing cells for long term study. An alternative method is to label cells using the replication-defective CXL retroviral vector, which carries the lacZ gene encoding β-galactosidase; however, the ability of the CXL retroviral vector to pulse-label mitotically active cells selectively is not known. Cultures of proliferating muscle cells were simultaneously incubated with the CXL retrovirus and BrdU (10 μM) for 2 hr. After removing the retrovirus containing medium, the cells were maintained for an additional 24 hr in vitro before they were stained to detect β-galactosidase and BrdU simultaneously. More than 95% of β-galactosidase positive cells were also BrdU positive suggesting that the majority of β-galactosidase positive cells were in the S-phase of the cell cycle at the time of CXL retroviral administration. Therefore, the CXL retroviral vector is an appropriate pulse marker for dividing cells, and it is useful when it is desirable to know the fate of the progeny of a particular cell following a mitotic event.

Treatment of progressive or recurrent pediatric malignant supratentorial brain tumors with herpes simplex virus thymidine kinase gene vector--producer cells followed by intravenous ganciclovir administration

Object The outcome for children with recurrent malignant brain tumors is poor. The majority of patients die of progressive disease within months of relapse, and other therapeutic options are needed. The goal of this Phase I study was to evaluate the safety of in vivo suicide gene therapy in 12 children with recurrent, malignant, supratentorial brain tumors. Methods After optimal repeated tumor resection, multiple injections of murine vector--producing cells shedding murine replication--defective retroviral vectors coding the herpes simplex virus thymidine kinase type 1 (HSV-Tk1) gene were made into the rim of the resection cavity. Fourteen days after the vector-producing cells were injected, ganciclovir was administered for 14 days. The retroviral vector that was used only integrated and expressed HSV-Tk1 in proliferating cells, which are killed after a series of metabolic events lead to cell death. The median age of the patients was 11 years (range 2--15 years). Treated brain tumors included seven malignant gliomas, two ependymomas, and three primitive neuroectodermal tumors. The patients were treated with one of three escalating dose concentrations of vector-producer cells. Four transient central nervous system adverse effects were considered possibly related to the vector-producing cells. In no child did permanent neurological worsening or ventricular irritation develop, and tests for replication-competent retroviruses yielded negative findings. Conclusions This Phase I study demonstrates that in vivo gene therapy in which a replication-defective retroviral vector in murine vector--producing cells is delivered by brain injections can be performed with satisfactory safety in a select group of children with localized supratentorial brain tumors.