Eiichiro Mabuchi

Usefulness of a Mouse Myelin Basic Protein Promoter for Gene Therapy of Malignant Glioma: Myelin Basic Protein Promoter Is Strongly Active in Human Malignant Glioma Cells

We have searched for suitable promoters to regulate the expression of suicide genes for use in gene therapy. We have shown that the 1.3‐kb fragment of the mouse myelin basic protein (MBP) promoter region initiates transcription in mouse glioma cells more efficiently than glial flbrillary acidic protein (GFAP) or myelin proteolipid protein (PLP) promoter. Among three different lengths of the MBP promoter, the shortest (256‐bp) core promoter region initiates transcription as efficiently as 650‐bp or 1.3‐kh MBP promoter lengths in RSV‐M glioma cells. To assess the suitability of the MBP promoter for use in clinical trials of malignant glioma gene therapy, we also had to show that it (the 1.3‐kb length in this case) Is effective in human glioma cells, as well as in murine glioma cells. The activity of the MBP promoter is much higher than that of GFAP or PLP promoter in most human glioma cells, suggesting that the MBP promoter would be best for directing toxic gene expression in gene therapy for patients with malignant glioma. Human glioma cells in which the MBP promoter was strongly active were sensitive to ganciclovir when they were transduced with MBP promoter/herpes simplex virus thymidine kinase gene‐bearing retroviruses. In conclusion, retrovirus‐targeted gene therapy for malignant glioma using this MBP promoter is a promising candidate for clinical trials.

Monoclonal antibody ONS-M21 recognizes integrin α3 in gliomas and medulloblastomas

SummaryThe monoclonal antibody ONS-M21 recognizes an antigen found on the surface of glioma and medulloblastoma cells but does not react with the antigens of normal brain tissue. We purified and identified the 140-kDa protein by means of an antibody-binding affinity column. This 140-kDa antigen has sequences homologous to the amino-terminal region and five parts of the internal domain of integrin α3. When the integrin α3-related sequences was amplified and used to analyse the mRNA of glioma and medulloblastoma surgical specimens, the transcription level of integrin α3 mRNA appeared to be quantitatively correlated with the grade of malignancy. These findings suggest that the ONS-M21 antibody, which reacts with integrin α3, might be useful in the diagnosis of gliomas and medulloblastomas.

Gene delivery by HVJ-liposome in the experimental gene therapy of murine glioma.

We investigated the delivery of foreign genes into mouse glioma cells in vivo using hemagglutinating virus of Japan (HVJ)-liposomes, which are coated by Sendai virus envelope protein. HVJ-liposomes, containing lacZ gene or herpes simplex virus thymidine kinase (HSVtk) gene-bearing plasmid DNA were applied in the meningeal gliomatosis (MG) mouse model system. Highly efficient delivery was observed in disseminated glioma cells, and 80% of MG mice expressing the HSVtk gene were cured by treatment with ganciclovir. These results suggest that this novel gene delivery system may be applicable for the in vivo gene therapy of human malignant glioma.

Systemic interleukin 12 displays anti-tumour activity in the mouse central nervous system

In various systemic cancers, interleukin 12 (IL-12) induces anti-tumour immunity mediated by T lymphocytes and natural killer cells. To determine whether IL-12 has anti-tumour activity against malignant gliomas in the central nervous system (CNS), which is considered to be an immunologically privileged site, we treated mice with meningeal gliomatosis by intraperitoneal (i.p.) or intrathecal (i.t.) administration of recombinant murine IL-12. Although untreated mice revealed symptoms, such as body weight loss or paraplegia as a result of the meningeal gliomatosis within 8 days after tumour inoculation, 80% of the mice treated with IL-12 at 0.5 microg i.p. were cured. Many lymphocytes, mostly CD4+ and CD8+ cells, infiltrated to the tumours of IL-12-treated mice. The numbers of these cells increased in the cervical lymph nodes, into which the cerebrospinal fluid drains, and there they secreted a considerable amount of interferon-gamma. Mice cured by IL-12 rejected subcutaneous or i.t. rechallenge with their original glioma cells, but the same mice were not able to reject other syngeneic tumour cells. These results indicate that the immune system recognizes malignant glioma cells in the subarachnoid space of the CNS and that systemic IL-12 may produce effective anti-tumour activity and long-lasting tumour-specific immunity.

Meningeal neoplasms associated with cerebral vascular malformations

Two patients with meningeal neoplasms and nearby vascular anomalies are reported. The lesions were excised and histologically confirmed. One patient had a meningotheliomatous meningioma and an arteriovenous malformation involving the right frontal lobe; the other, a hemangiopericytoma and an arteriovenous malformation in the right parietooccipital region.

Transduction of glioma cells using a high-titer retroviral vector system and their subsequent migration in brain tumors.

The intracranial migration of transduced glioma cells was investigated in order to improve the treatment of malignant glioma by gene therapy using retroviral vectors. In this study, about half the volume of the tumor mass could be transduced in 14 days after only a single implantation of 3 × 105 retrovirus-producing cells into a tumor mass with a diameter of 5 mm. Moreover, we were able to follow the migration of glioma cells transduced by the lacZ-harboring retroviruses originating from the high-titer retrovirus-producing cells. Besides the importance of using a high-titer retroviral vector system, our results also indicate that the implantation site of the virus-producing cells and the interval between the implantation of the virus-producing cells and the subsequent administration of ganciclovir are important factors for the efficient killing of glioma cells.

Anticonvulsant-induced Suppression of IFN-γ Production by Lymphocytes Obtained from Cervical Lymph Nodes in Glioma-bearing Mice

It is well known that phenytoin can cause impairment of cellular immunity. The authors investigated the potential role of other anticonvulsant drugs in the development of antitumor immunity in murine malignant glioma models.The survival rate was determined in murine glioma models using syngeneic 203 glioma cells following treatment with four anticonvuisants, which are most commonly administered to glioma patients, i.e., phenytoin, phenobarbital, valproate and zonisamide. In a second set of experiments, we further examined the effect of these drugs on interferon-γ (IFN-γ) secretion by lymphocytes prepared from cervical lymph nodes (CLN) in the same models. The IFN-γ production of CLN lymphocytes as measured by ELISA method was markedly impaired in the early stage of tumor-bearing mice treated with phenytoin or zonisamide, and the median survival time (MST) of controls and of mice treated with either phenytoin or zonisamide was 13, 10 and 11 days, respectively, which was not a statistically significant difference. Phenobarbital and valproate did not affect either IFN-γ production or their survival rate. In addition, immunohistochemistry showed a reduction in tumor-infiltrating lymphocytes containing CD4 and CD8 antigens in the mice treated with phenytoin and zonisamide.Two anticonvulsants, phenytoin and zonisamide, showed a significant inhibitory effect on IFN-γ production by CLN lymphocytes in murine glioma models, although there was no statistically significant difference in MST between controls and the anticonvulsant-treated mice. These drugs might have some detrimental influence on the prognosis of brain tumor patients when combined with the latent immune dysfunction accompanying the tumor-bearing state.

Biological Characterization of Human Medulloblastoma (ONS-76 and ONS-81) Cell Lines

Medulloblastomas are the most common malignant brain tumors in children. The origins of medulloblastoma cells are controversial. Most neuropathologists believe them to have originated from immature precursor cells which have the potentiality to differentiate into either neuronal or glial cells [1]. Although it is very difficult to establish a cell line of human medulloblastoma [2,3], we have established 2 cell lines with neuronal characteristics.

Murine Models with Leptomeningeal Dissemination of Human Medulloblastoma Cells

The 5-year survival rate of patients with medulloblastoma has improved as much as 70% in the past 15 years. This dramatic improvement is mainly due to advances in radiotherapy [1–3]. Although medulloblastomas are certainly radiosensitive, they are not always radiocurable because of limited irradiation dosages and adverse effects on the whole neural axis. Sooner or later, tumor regrowth takes place, and the meningeal dissemination of the tumor cells then results in the demise of most patients with medulloblastoma [4].