Doctoral Dissertations

Date of Award

5-1996

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Donita L. Frazier

Committee Members

William H. Adams, Karla J. Matteson, Tuan Vo-Dinh

Abstract

High-grade astrocytomas are the most common primary malignant brain tumors in adults. They constitute a significant cancer problem in the U.S.A, accounting for nearly 11,000 deaths annually. Despite improvements in surgical techniques and radiotherapy, the prognosis of these patients remains dismal with median survival of less than 1 year. Recent data suggest that new techniques of intra-arterial and intratumoral administration of cytotoxic drugs may result in improved efficacy against these tumors. This research evaluated the interaction of γ irradiation, vincristine and methotrexate as single agents and in combination in treatment of two human glioma cell lines: U-87, glioblastoma multiforme and U-373, astrocytoma. a a high grade Cytotoxicity resulting from treatment was evaluated by determination of mitochondrial function (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide enzyme reduction) cell survival 3 or 5 days after treatment using a protein assay. Additive cytotoxicity was seen when radiation (20 Gy) immediately followed a 48-hour incubation with methotrexate at a concentration concentrations of of 40 mM for U-373 cells and 0.4-40 mM for U-87 at cells. Additionally, cytotoxicity in both cell lines was additive when cells were treated with radiation immediately preceded a 48-hour methotrexate exposure (4) and 40 mM), but additivity was lost methotrexate. at 0.4 mM Results of the study indicate that the interaction of methotrexate with radiation is both dose- and schedule-dependent. Cytotoxicity resulting from methotrexate as a single agent was less than 26% for both cell lines. Examination of the mechanisms responsible for the low methotrexate sensitivity showed no differences comparing [3H] methotrexate uptake of a known methotrexate-sensitive cell line, CCRF-CEM, and the glioma cells. However, the intracellular concentration of dihydrofolate reductase, the main target of methotrexate, was greater (p<0.0001) in both glioma cell lines than in the CCRF-CEM leukemia cell line. Combination treatment of glioma cells with radiation and vincristine demonstrated an additive effect only when radiation immediately followed a 48-hour incubation with 4.3 µM vincristine. When radiation preceded a 48-hour incubation with vincristine (0.4) 40 µM), significantly less cytotoxicity was seen than with either treatment modality alone for the U-87 cell line. In an attempt to explain this radiation-induced modulation Of vincristine cytotoxicity, drug incorporation studies at various intervals up to 120 minutes following single or combination modality treatment were determined. The results were inconclusive but suggested that steady-state concentrations were reached in the combination group but not in the group treated with drug alone. It is hypothesized that the plateau phase in the combination group may be due to increased drug efflux as a result of radiation-altered cell membrane integrity or secondary to a radiation-induced decrease in the number of intracellular binding sites. Cell cycle studies indicated that radiation decreased the percentage of cells in the synthetic (S) phase of the cell cycle, the most vincristine-sensitive phase, and increased the percentage of cells in the G2 phase. These cell cycle alterations may have contributed cytotoxicity when vincristine followed irradiation. to decreased treatment immediately This study supports the investigation of adjuvant chemotherapy with vincristine but not methotrexate in the treatment of human gliomas.

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