Development of Resistance to Antitumor Chloroethylnitrosoureas in Vitro in Brain Tumor Cells

Abstract

Rat brain tumor cell lines (9L, C6-1, C6-2), human brain tumor cells (T98G), and HeLa S3 cells were studied to assess their acquired resistance to the chloroethylnitrosoureas (CENUs), 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-ni~osourea hydrochloride (ACNU) and methyl-6-[ 3 4 2-chloroethyl) -3-nitrosoureido] -6deoxy-a-D-glucopyranoside (MCNU), after 10 repeated exposures of a panel of different drug concentrations. Assay end-point was colony-forming ability after 24-h drug exposure. Intrinsic resistance was tested at the 10% survival dose (SD10) and CGl, T98G, and HeLa S3 cell lines were 3 to 16 times more resistant to ACNU than 9L and C6-2 cell lines. After repeated exposures to ACNU, 9L and C6-2 cells acquired 2-and 5-fold resistance to ACNU respectively, whereas C6-1 and T98G cells retained a resistance almost equivalent to the respective parent cells. HeLa S3 cells also acquired resistance to ACNU, as evidenced by a 3.5-fold increase. The SDlO of the cells to MCNU ranged from 4.3 p M (C6-2 cells) to 151.7 pM (T98G cells). After long-term exposure to MCNU, all five cell lines became significantly resistant compared to their respective parent cells. The easily obtained acquired resistance to CENUs suggests a clinical disadvantage of continual and repeated adjuvant monochemotherapy with these agents.Chloroethylnitrosoureas (CENUs) permeate the bloodbrain barrier and are not cell cycle specific and therefore often used for chemotherapy of human brain tumors. Their therapeutic effectiveness, however, remains quite uncertain, and clinical surveys have shown unsatisfactory results (1). Besides intrinsic drug resistance also acquired resistance can explain the poor effectiveness. The latter phenomenon suggests a clinical disadvantage of longstanding adjuvant CENU therapy.With this background, we have studied acquired resistance to 1 -( 4-amino-2-methyl-5-pyrimidinyl)methyl-3-( 2- glucopyranoside ( MCNU), after long-term exposure using an in vitro colony formation assay. Material and MethodsCell strains and culture. The cell strains included rat cell lines (9L, C6-1, C6-2), a human glioma cell line (T98G).and HeLa S3 cells. The 9L cell line derived from a Fisher 344 rat brain tumor (2). The C6 glioma was originally induced by N-methylnitrosourea in a Wistar rat (3): the culture of the C6-1 cell line has been maintained in our laboratory for some 10 years, and the C6-2 cell line was supplied by the Japanese Cancer Research Resources Bank (JCRB, Tokyo. Japan). The T98G cells were also given by the JCRB, and the HeLa S3 cells were kindly provided by Dr. K. Ishizaki, Radiation Biology Center, Kyoto University, Japan.All strains were maintained in Eagle's minimal essential medium (MEM, Flow Lab.. McLean. VA., U.S.A.) supple-mented with 10% (vol./vol.) fetal bovine serum, penicillin G (50 units/ml), streptomycin (50 pglml), and fungizone (2.5 pg/ml). The cells were grown in a humidified atmosphere of 95% air and 5% carbon dioxide at 37°C.Long-term culture in CENU-containimg medium. After digestion of cel...

Enhancing effect ofO6-alkylguanine derivatives on chloroethylnitrosourea cytotoxicity toward tumor cells

“…C6-1 and 9L rat brain tumor cells have been maintained in our laboratory for some 10 years (Mineura et al, 1992a,b). C6-2/ ACNU cells were established as a cell strain resistant to ACNU after repeated exposures of ACNU to C6-2 parent cells (Mineura et al, 19926). The malignant astrocytoma cell line AM was established in our laboratory from specimens of a 40-year-old male patient.…”