The anti-glioma effect of suicide gene therapy using BMSC expressing HSV/TK combined with overexpression of Cx43 in glioma cells.

The disseminated neoplastic foci of malignant gliomas are essentially responsible for the limited efficacy of current available therapeutic modalities. Bone marrow-derived stem cells (BMSCs) have the ability to migrate into these tumors and even track infiltrating tumor cells, making them to be promising cellular vehicles for delivering therapeutic agents to glioma cells. The herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) suicide gene therapy with a potent bystander effect has been considered as one of the most promising therapeutic strategies for malignant gliomas. In this study, we evaluate the anti-glioma effect of suicide gene therapy using BMSCs expressing HSV-TK combined with overexpression of connexin 43 (Cx43), which can restore the gap junction of intercellular communication and may enhance the bystander effect of suicide gene therapy. To assess the potential of BMSCs to track glioma cells, a spheroid co-culture system in matrigel was used to show that some BMSCs migrated to C6 glioma cell microspheres. Transwell assay showed the tumor tropic property of BMSCs. In addition, BrdU-labeled BMSCs injected directly into the cerebral hemisphere opposite to the established C6 rat gliomas were capable of migrating into the xenograft gliomas. C6 cell growth was more intensively inhibited by HSV-TK/GCV treatment mediated by BMSCs, and could be further enhanced by combination with Cx43 transfection into glioma cells. The same result was observed in vivo by the growth of C6 gliomas and the survival analysis of rats bearing C6 glioma. In conclusion, Cx43 combined with HSV-TK/GCV gene therapy using BMSCs as vehicles was highly effective in a rat glioma model and therefore hold great potential as a novel approach for the gene therapy of human malignant gliomas.

Suppression of EGFR expression by antisense or small interference RNA inhibits U251 glioma cell growth in vitro and in vivo.

Epidermal growth factor receptor (EGFR) had been reported as one of the major responsible genes for malignant progression and phenotype reversion of gliomas, and has been used as one of the most important therapeutic targets. In the present study, small interference RNA (siRNA) and antisense EGFR expression constructs, which target sequences of human EGFR catalytic domain (2400-2420) and the 3'-coding region, respectively, were used to examine the growth inhibition effects on U251 glioma cells. Cell growth was significantly inhibited and G2/M arrest was observed in antisense- and siRNA-treated groups. Matrigel matrix demonstrated spotted cell clustering pattern in antisense- and siRNA-transfected U251 cells, indicating poor cell growth activities. In addition, the tumor volumes in U251 subcutaneous mice model treated with antisense and siRNA were significantly smaller than those treated with control siRNA and phosphate-buffered saline. Also, glial fibrillary acidic protein expression was upregulated in antisense- and siRNA-treated groups than the control groups. Our results demonstrated that antisense- or siRNA-targeting intracellular region of EGFR can inhibit EGFR expression, exerted growth inhibition effect on U251 glioma cells in vitro and in vivo. Consequently, siRNA expression plasmid-mediated gene therapy would be a new strategy in treatment of gliomas.

Study of the antioxidant enzymes in human brain tumors.

The activities of antioxidant enzymes i.e. Cu, Zn-SOD, Mn-SOD, CAT, and GSH-Px in the normal brain and brain tumors, as well as the two varieties of SOD in the mitochondria were examined and correlated to the histopathological diagnosis and the degree of malignancy of tumors. It was found that these scavenging enzymes of oxygen free radicals were expressed with great regularity in brain tumors. Both Cu, Zn-SOD and Mn-SOD were decreased in descending order in meningiomas, low grade astrocytomas, high grade astrocytomas and medulloblastomas. Furthermore, the reduction of Mn-SOD in mitochondria was proportionate to that of the whole tissues. While in contrast to the SODs, the CAT levels were significantly increased in ascending order in high grade astrocytomas, low grade astrocytomas and meningiomas. GSH-Px increased in meningiomas but not in gliomas.

Chromosome number and carmustine sensitivity in human gliomas.

BACKGROUND: Although some patients with malignant gliomas respond to treatment with chemotherapeutic agents like BCNU, tumor recurrence inevitably occurs, heralding the development of chemoresistance. Treating and/or preventing chemoresistance requires distinguishing newly developed resistance from the presence of intrinsically resistant cells in the primary tumor population. This study relates the chromosomal complements of freshly resected astrocytomas to the cells' chemosensitivity and ultimately to the patients' response to treatment. METHODS: The authors dissociated 31 freshly resected human gliomas (5 astrocytomas, 10 anaplastic astrocytomas, 16 glioblastomas multiforme) into single cells, and performed cytogenetic analysis and BCNU sensitivity testing using the colony-forming assay (CFA) on first division cells from these tumors. RESULTS: The major cytogenetic abnormalities involved the loss of a sex chromosome in all three classes of gliomas and the gain of chromosome 7 in anaplastic astrocytoma and glioblastoma multiforme; clonal marker chromosomes were observed in only anaplastic astrocytoma and glioblastoma multiforme with no common rearrangement observed among the tumors. The five astrocytomas were near-diploid (2n+/-, 35-57 chromosomes/cell), and all were resistant to BCNU. Seven of ten anaplastic astrocytomas were composed primarily of 2n+/- cells and were BCNU resistant. Three other anaplastic astrocytomas had a 39% or greater representation of 4n+/- cells (88-101 chromosomes/cell), and these tumors were sensitive to BCNU. Ten of 16 glioblastomas multiforme were composed predominantly of 2n+/- cells and were resistant to carmustine. Six other glioblastomas multiforme had at least 41% 3n+/- (58-87 chromosomes/metaphase) and 4n+/- cell populations and were sensitive to carmustine. Thus, gliomas demonstrating BCNU sensitivity were more than 60% hyperdiploid (60 or more chromosomes/metaphase) with 1 to 8 clonal marker chromosomes and multiple clonal populations involving complex karyotypic deviations. In contrast, all 22 resistant tumors were composed primarily of near-diploid cells. Only 4 of 22 tumors had a clonal marker, and the chromosome ploidy changes were less extensive. CONCLUSIONS: In freshly resected untreated human gliomas, BCNU is most effective against hyperdiploid cells that have extensive ploidy changes and chromosome rearrangement, whereas resistance to carmustine is characteristic of near-diploid populations with few ploidy changes and rearranged chromosomes. This observation was consistent for all three classes of gliomas.

BCNU-sensitivity in parental cells and clones from four freshly resected near-diploid human gliomas: an astrocytoma, an anaplastic astrocytoma and two glioblastomas multiforme.

We compared the BCNU sensitivity of 4 freshly resected tumors (astrocytoma WM, and malignant gliomas MK, MB, and AM) and their clones to their karyology. The majority of primary cells in all 4 tumors had near-diploid chromosome numbers (2n +/-) and all were resistant to concentrations of BCNU exceeding 10 micrograms/ml. Following in vitro cultivation, the cells from tumors WM and MB retained their 2n +/- modal chromosome number with little change in the complexity of the karyotype. In contrast, tumors MK and AM demonstrated a more unstable genome. The modal chromosome number of MK shifted from 45 to 86 and that of tumor AM from 45 to 90. Karyotyping demonstrated additional ploidy changes and new marker chromosomes in both tumors. The colony forming assay (CFA) performed on the in vitro cultivated cells demonstrated little change in the sensitivity to BCNU in tumors WM and MB, while tumors MK and AM exhibited greater than a one log cell kill at 10.0 micrograms/ml and 15.0 micrograms/ml BCNU, respectively. The modal chromosome number and BCNU sensitivity followed a similar pattern in the 30 clones that were isolated; 21 clones with near-diploid and pseudodiploid chromosome numbers were all resistant to BCNU doses at or greater than 10 micrograms/ml. In contrast, 9 clones isolated from the 3 malignant gliomas with 3n +/- and 4n +/- modal chromosome numbers were sensitive to this concentration of BCNU. The karyotypes of the hyperdiploid clones were more complex; they contained 5 or more ploidy changes and/or had marker chromosomes. These studies confirm the association of diploidy and BCNU-resistance in freshly resected malignant gliomas.

A survey of 790 cases of astrocytoma.

790 cases of cerebral astrocytoma, which consisted of 181 cases of astrocytoma grade I, 282 cases of grade II, and 327 cases of grade III-IV, histopathologically verified, are presented. The sex and age distribution, topographical distribution of the tumours, and clinical manifestations are analyzed. A total of 862 operations were performed, and 69 cases (8.6%) were operated on more than twice for recurrence. The operative mortality was 7.7%. 179 of the survivors following operation received a combined treatment with radiotherapy and/or chemotherapy. Based on the follow-up study of 170 cases, the factors which may correlate with the length of survival, are discussed.