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Heterogeneity in drug sensitivity must, in part, account for the relative lack of success with single agent chemotherapy for glioblastoma multiforme (GBM). In order to develop in vitro model systems to investigate this, clones derived from the VM spontaneous murine astrocytoma have been characterised with regard to drug sensitivity. Six clonal cell lines have been tested for sensitivity to a panel of cytotoxic drugs using an intermediate duration 35S-methionine uptake assay. These lines have previously been extensively characterised with regard to morphological, antigenic, kinetic, tumourigenic potential in syngeneic animals and chromosomal properties and display considerable heterogeneity. The present study indicates that heterogeneity extends to sensitivity to all classes of cytotoxic drugs. The greatest difference in sensitivity between the clones was seen in response to cell cycle-specific drugs like the Vinca alkaloids (14-fold and 20-fold for vincristine (VCR) and vindesine (VIND) respectively), while the nitrosoureas, CCNU and BCNU displayed a smaller fold difference in sensitivity (4.3 and 3.6-fold difference respectively). All the clones were considerably more resistant to the adriamycin (ADM), cis-platinum (C-PLAT) and the Vinca alkaloids than the parental cell line although the difference in sensitivity between the clones and parental cell line were less marked for the nitrosoureas and procarbazine (PCB). It has also been possible to examine the relationship between drug sensitivity and the phenotypic and genotypic properties of these clonal cell lines. There is a relationship between chromosome number and sensitivity of a wide variety of cytotoxic drugs including the nitrosoureas, Vinca alkaloids, PCB, C-PLAT, BLEO but not ADR or 5-FU. Clones with small numbers of chromosomes were more resistant than clones with gross polyploidy. Similarly, sensitivity to Vinca alkaloids and ADM, but not other classes of drugs, was greatest in cells with numerous cytoplasmic processes and which did not express large amounts of cell surface fibronectin. Preliminary experiments have been conducted on reconstituting clonal mixtures of cells with different sensitivity to Vinca alkaloids and results from these studies indicate that the drug resistance phenotype is dominant, with clonal mixtures of sensitive and resistant cell adopting the sensitivity of the more resistant partner. These cell lines should prove to be useful models for examining the cell biological basis of drug resistance in glioma and may lead to the identification and exploitation of novel cellular targets in new therapies for GBM.

In this study we assessed the influence of patient- and drug-specific parameters in the short-term MTT-chemosensitivity assay in 150 primary cell cultures derived from human brain tumors. In 45 patients the MTT assay was directly compared with the CFA (Colony Forming Assay). Resistance was 10-20% higher in the MTT assay than in the CFA, but there was a good agreement in both assays, that more malignant gliomas had a higher in vitro chemosensitivity against ACNU and BCNU. Overall the results demonstrate, that there is no uniform correlation between the in vitro chemosensitivity and the histopathological classification of the tumors, which corresponds well to the clinical situation. On the basis of this study we suggest prospective clinical trials with the MTT assay in human brain tumors.

The aim of this study was to optimize the experimental conditions of the MTT assay for primary cultures of human brain tumors. This assay is based on the mitochondrial reduction of MTT-(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) salt to formazan crystals by living cells. Formazan can be quantified spectrophotometrically. This assay measures the antimetabolic and, by using an adequate recovery period for the cells, also the antiproliferative effects of cytotoxic drugs. Our results suggest the following experimental design for its application as an chemosensitivity assay for human brain tumors: 1-h drug exposure followed by a seven days recovery period without drugs. Then tumor cells are incubated 4 hours with 1 mg MTT/ml and final absorbance readings are performed at 550 nm and 630 nm as test and reference wavelengths respectively. In this way, the assay seems to be a reliable and simple method for rapid chemosensitivity testing in human brain tumors.

A cell line, 497-P(1), derived from the VM spontaneous murine astrocytoma has been used to develop an in vitro therapeutic model of human glioma. In this study we describe the preparation of MTS from this cell line. The in vitro chemosensitivity of 497-P(1) MTS has been examined and compared to the sensitivity of the monolayer culture. BCNU and CCNU both produced growth delay in MTS at doses below the ID50 of the monolayer culture. MTS, however, were considerably more resistant to vincristine and procarbazine when compared to the monolayer culture.

The effects of cyclic nucleotides, dibutyryl cyclic adenosine monophosphate and dibutyryl cyclic guanosine monophosphate (db-cAMP and db-cGMP), on the growth rate of multicellular tumour spheroids were evaluated by comparing the growth delay and colony forming efficiency in vitro. Multicellular tumour spheroids were derived directly from human brain tumours. To compare the chemotherapeutic effect of cyclic nucleotides, CCNU was used as a known effective cytotoxic drug on malignant gliomas. Significant growth delay was obtained by db-cAMP (p less than 0.001) while CCNU was tumouricidal rather then producing a delay in growth of the tumour spheroids. Db-cGMP found not to be effective in decreasing the growth rate of the tumour spheroids in vitro (p greater than 0.2). The role of cyclic nucleotides in brain tumours is discussed on a review basis.