Mouse glioma 261 (Gl261) cells are used frequently in experimental glioblastoma therapy; however, no detailed description of the Gl261 tumor model is available. Here we present that Gl261 cells carry point mutations in the K-ras and p53 genes. Basal major histocompatibility complex (MHC)I, but not MHCII, expression was detected in Gl261 cells. The introduction of interferon-γ γ γ γ-encoding genes increased expression of both MHCI and MHCII. A low amount of B7-1 and B7-2 RNA was detected in wild-type cells, but cytokine production did not change expression levels. Gl261 cells were transduced efficiently by adenoviral vectors; the infectivity of retroviral vectors was limited. Low numbers of transplanted Gl261 cells formed both subcutaneous and intracranial tumors in C57BL /6 mice. The cells were moderately immunogenic : prevaccination of mice with irradiated tumor cells 7 days before intracranial tumor challenge prevented tumor formation in approximately 90% of mice. When vaccination was carried out on the day or 3 days after tumor challenge, no surviving animals could be found. In vitro-growing cells were radiosensitive: less than 2 Gy was required to achieve 50% cell mortality. Local tumor irradiation with 4 Gy X-rays in brain tumor-bearing mice slowed down tumor progression, but none of the mice were cured off the tumor. In conclusion, the Gl261 brain tumor model might be efficiently used to study the antitumor effects of various therapeutic modalities, but the moderate immunogenicity of the cells should be considered. (Cancer Sci 2006; 97: 546-553) M alignant brain tumors have a poor outcome, with a median survival of 9 months and only 5-10% of patients surviving 2 years. The conventional therapies for glioblastoma multiforme are surgical removal of the bulk tumor mass, followed by radiotherapy. As complete surgical removal is almost impossible, radiotherapy continues to play a major role in the treatment of malignant gliomas, but these tumors are often radioresistant. The combination of chemotherapy with other modalities provides only modest improvements in survival rates. (1-3) The application of suitable animal models in glioma research is necessary for the development of new therapeutic approaches. An ideal animal model of human glioma should reproduce the major characteristics of this neoplasm: predictable and reproducible in vitro and in vivo growth patterns; infiltrative, but non-metastatic, tumor growth; and poor immunogenicity. A number of animal brain tumor models are being used; however, no model currently available simulates human high-grade gliomas exactly. Tumor models vary in their immunogenicity, growth patterns and invasiveness. For this reason it is important to choose an appropriate animal model depending on the endpoint examined. Most models are derived from rat and several xenograft models, based on the intracer-ebral transplantation of human brain tumors into immune-deficient nude mice, are also available. Murine models of malignant brain tumors are used much less frequently, mainly beca...

Poor efficiency of gene transfer into cancer cells constitutes the major bottleneck of current cancer gene therapy. We reasoned that because tumors are masses of rapidly dividing cells, they would be most efficiently transduced with vector systems allowing transgene propagation. We thus designed two replicative retrovirus -derived vector systems: one inherently replicative vector, and one defective vector propagated by a helper retrovirus. In vitro, both systems achieved very efficient transgene propagation. In immunocompetent mice, replicative vectors transduced >85% tumor cells, whereas defective vectors transduced < 1% under similar conditions. It is noteworthy that viral propagation could be efficiently blocked by azido -thymidine, in vitro and in vivo. In a model of established brain tumors treated with suicide genes, replicative retroviral vectors ( RRVs ) were approximately 1000 times more efficient than defective adenoviral vectors. These results demonstrate the advantage and potential of RRVs and strongly support their development for cancer gene therapy.

We showed that a U5-U3 junction was reproducibly detected by a PCR assay as early as 1 to 2 h postinfection with a DNase-treated murine leukemia virus (MLV)-containing supernatant in aphidicolin-arrested NIH 3T3 cells, as well as in nonarrested cells. Such detection is azidothymidine sensitive and corresponded to neosynthesized products of the reverse transcriptase. This observation was confirmed in two additional human cell lines, TE671 and ARPE-19. Using cell fractionation combined with careful controls, we found that a two-longterminal-repeat (two-LTR) junction molecule was detectable in the cytoplasm as early as 2 h post virus entry. Altogether, our data indicated that the neosynthesized retroviral DNA led to the early formation of structures including true two-LTR junctions in the cytoplasm of MLV-infected cells. Thus, the classical assumption that two-LTR circles are a mitosis-dependent dead-end product accumulating in the nucleus must be reconsidered. MLV-derived products containing a two-LTR junction can no longer be used as an exclusive surrogate for the preintegration complex nuclear translocation event.

This study is an initial proof of principle for the use of complementary retroviral vectors to deliver and propagate transgenes in vitro and in solid tumors in vivo, but with reduced pathogenicity compared to its parental virus. In-between replication-defective and replication-competent retroviral vectors, this semi-replicative system offers good grounds for its application in in vitro studies and allows envisioning its further development for cancer gene therapy.