[CANCER RESEARCH 41, 3840-3843, October 1981] 0008-5472/81 /0041-OOOOS02.00 Reversal of Resistance to Methotrexate by Hyperthermia in Chinese Hamster Ovary Cells1 Terence S. Herman, Anne E. Cress, Cynthia Sweets, and Eugene W. Gerner Departments of Internal Medicine [T. S. H., C. S.], and Radiology [A. E. C., E. W. G.], University oÃ-Arizona College of Medicine, Tucson, Arizona 85724 ABSTRACT Our Chinese hamster ovary cells are extremely resistant to methotrexate (MIX) (100% survival after 500 /ig/ml for 13 hr). However, exposure to 43° (but not 41° or 42°) for 1 hr sensitizes the cells to MIX so that a 50% cell kill in excess of that due to hyperthermia occurs. Treatment of cells at 43° increases net MIX uptake by about 30% at 30 min but causes a substantial reduction after 1 hr. This negative effect is greater in cells continually heated at 43° than in those exposed for only 1 hr. Treatment at 43°for 1 hr also markedly increases efflux of MTX out of cells over the first 2 hr. Dihydrofolate reducÃ-ase activity was found to decrease to about 50% of control values by 4 to 5 hr after exposure to 43°.The biological half-life of dihydrofolate reductase in Chinese hamster ovary cells was determined to be about 4.5 hr, indicating that hyper- thermia-induced cessation of protein synthesis may explain both the decrease in dihydrofolate reductase activity and the sensitization to MTX observed with heat exposure. In sched uling experiments, lethality due to exposure to 43°for 1 hr in conjunction with MTX was maximum when 1-hr drug exposure began just at the end of heat treatment. INTRODUCTION There is now considerable clinical interest in the treatment of cancer with a combination of chemotherapeutic drugs and hyperthermia (9,13). In general, however, early clinical results have been less than outstanding when patient morbidity and the difficulties in delivering the hyperthermia (at least whole body) are considered (4, 16, 19). In vitro and in vivo studies conducted in this laboratory and others (9,17) have suggested that the scheduling of chemotherapeutic drugs with heating may be critical to maximization of cellular lethality. Better clinical results might be anticipated if these factors were un derstood. Because other studies had shown that exposure to temperatures greater than 43°could transiently shut off protein synthesis, causing significant decreases in the levels of impor tant intracellular enzymes (8, 10), we chose to investigate the interaction between exposure of cells to hyperthermia and MTX,2 since MTX acts by inhibiting one such enzyme, DHFR (1 ). Our strain of CHO cells proved an excellent model because it is resistant to MTX due to high levels of this enzyme (11 ). We further attempted to define the best scheduling of this drug with heat to better plan for eventual clinical application. MATERIALS AND METHODS Method of Cell Culture. CHO cells, originally obtained in 1974 from ' Supported in part by Grants CA 26220 and CA 17343 and in part by Research Advisory Group, Veterans Administration, Tucson, Ariz. 85723. 2 The abbreviations used are: MTX, methotrexate; DHFR, dihydrofolate reduc tase: CHO, Chinese hamster ovary. Received October 23. 1980; accepted June 29. 1981. Dr. Ronald M. Humphrey, University of Texas Cancer Center, M D. Anderson Hospital, Houston, Texas, and RT9 cells, originally obtained in 1975 from Dr. Kenneth Wheeler, University of Rochester, Rochester, N. Y., were both grown in McCoys' Medium 5A supplemented with 20% (v/v) fetal calf serum, penicillin (11 units/ml), streptomycin (100 fig/ml), and Fungizone (0.25 /ig/ml) (all from Grand Island Biological Co., Grand Island, N. Y.). Cells were maintained in logarithmic growth phase at 37°in a 5% CO?-95% air atmosphere. Doubling time was 13 to 14 hr for the CHO cells and 15 to 16 hr for the RT9 cells. Cell numbers were determined with an electronic particle counter (Coulter Electronics, Hialeah, Fla.). Cells were replated approximately 12 hr before experimental manipulation. Cell Viability Measurements. Cell viability was measured by the ability of single cells to form colonies in vitro. For this assay, known numbers of cells were plated in plastic Falcon No. 3002 Petri dishes (Falcon Plastics, Oxnard, Calif.) containing 5 ml of fresh medium. Three dilutions of the known cell number were made for each drug concen tration, and each was plated in triplicate. After incubation for 7 to 8 days, colonies were fixed and stained with 0.5% crystal violet (Fisher Scientific Co., Fair Lawn, N. J.) in 95% ethanol. Viable cells were those that produced colonies of more than 50 cells upon examination under a dissecting microscope. The plating efficiencies of control cultures of CHO cells were between 80 and 90%. The results from colony forma tion experiments, described in Charts 1 and 6, represent 3 to 5 replicate experiments per data point. Heat Treatments. Exponentially growing cells were grown in T2S flasks (Falcon Plastics). Flasks were then immersed in a specially designed plexiglass water tank with continuous flow of heated water for a Haake Model FK water circulator (temperature variability, ±0.1") (Haake, Inc., Saddle Brook, N. J.). Five ml of fresh media or media plus drug were added directly prior to heating. Thermometry was accomplished with mercury and glass thermometers calibrated by the method of Cetas and Conner (6). Drug Treatments. Exponentialiy growing cells were exposed to varying concentrations of MTX (Lederle Laboratories, Pearl River, N. Y.) in 60-mm Petri dishes for 1 hr at 37°, or in T25 flasks as above at 41 °,42°,or 43°.Drug solutions were always prepared immediately prior to use by dissolving the drug in sterile water. Appropriate dilutions of drugs were added to fresh medium, with the volume of drug solution never exceeding 20% (1 ml) of the total volume of 5 ml. After treatment, the medium was removed, and the cultures were washed twice with warmed Puck's Saline A (Grand Island Biological Co.) and trypsinized (0.05% for 2 to 3 min). Following this, known numbers of cells were plated into plastic Petri dishes for colony growth as described above. Measurement of DHFR. The activity of DHFR was measured by the method of Mathews and Huennekens (18). Twenty million cells were used for each determination. CHO and RT9 cells were removed from monolayer cultures by scraping with a rubber policeman. These cells were suspended (1 .Ox 10' cells/ml) in 0.1 M Tris HCI buffer, pH 7.4, and were disrupted by sonication (E/MC Corp. ultrasonic cell disruptor; 4.5-in probe, 20-sec pulse). The resulting cell homogenate was cen- trifuged at 12,000 x g for 10 min. The pellet was discarded, and the supernatant extract was placed on ice. The reaction mixture included 40 mM potassium phosphate (pH 7.4), 8 rriM 2-mercaptoethanol, 0.08 HIMdihydrofolate, 0.08 rnw NADPH, and 0.20 ml of sample supernatant in a total volume of 1.00 ml. DHFR activity was then determined at 37° by measuring the decrease in absorbance at 340 nm of the solution as 3840 CANCER RESEARCH VOL. 41 Research. on January 28, 2016. © 1981 American Association for Cancer cancerres.aacrjournals.org Downloaded from