Cell Cycle Phase Specificity of Antitumor Agentsl
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[CANCER RESEARCH 32, 398-407, February 1972] Cell Cycle Phase Specificity of Antitumor Agentsl B. K. Bhuyan, L. G. Scheldt, and T. J. Fraser Research Laboratories, The Upjohn Company, Kalamazoo, Michigan 49001 SUMMARY correlating the biochemical effect of the drug to its lethal effect. For example, many drugs are maximally toxic to cells The sensitivity to drugs of synchronous and asynchronous at the G]-S boundary, and a correlation between the lethal populations of DON cells was studied. Agents that were and biochemical effects of the drug may help to delineate the cytotoxic at a specific phase of the cell cycle gave dose-survival biochemical reaction that triggers the cell across the Gi-S curves that decreased to a constant saturation value. DNA boundary, (b) They may help in designing combinations of synthesis inhibitors such as 1-0-D-arabinofuranosylcytosine, drugs on a rational basis. Thus, 2 drugs affecting the same 5-azacytidine, 5-hydroxy-2-formylpyridinethiosemicarbazone phase of the cell cycle would not give additive effects, if (NSC 107392), sodium camptothecin (NSC 100880), combined. 5-fluorodeoxyuridine, and pseudourea (NSC 56054) were Our studies were intended to determine the phase most cytotoxic to cells in the S phase. However, the DNA specificity of several clinically active agents and of agents with synthesis inhibitor, neocarzinostatin, was most cytotoxic to known biochemical activity. Parts of this study were reported cells in the G! phase. The protein synthesis inhibitors, previously (7). pactamycin and sparsomycin, were also most cytotoxic to cells in the S phase. Cells in the Gi-S border region were most MATERIALS AND METHODS sensitive to the RNA synthesis inhibitors, actinomycin D and nogalamycin, and to the alkylating agents, Cell Culture. DON cells, from a Chinese hamster fibroblast 1 ,3-bis(2-chloroethyl)-l-nitrosourea, and line (ATCC CCL16), were grown at 37° in McCoy's 1-(2-chloroethyl)-3-cyclohexyl-1 -nitrosourea. Streptozotocin 5A medium modified by the addition of lactalbumin and tubercidin, which markedly inhibit the synthesis of DNA, hydrolysate (0.8 g/liter) and fetal calf serum (200 RNA, and protein, were cytotoxic to cells in all phases of the ml/liter). The medium was obtained from the Grand cell cycle. 5-Fluorouracil was also not phase specific. Cells in Island Biological Company, Grand Island, N. Y. The mitosis and in the Gt phase were most sensitive to cells were grown in 8-oz prescription bottles planted with chlorambucil, L-phenylalanine mustard, and ellipticine. about 2 X IO6 cells in 25 ml of medium and were maintained in logarithmic growth by subculture every 2 days. With renewal of the medium every 2 days, an 8-oz bottle could INTRODUCTION support logarithmic cell growth, with a generation time of 10 to 12 hr, until there were about IO8 cells/bottle. For Bruce et al. (12) have classified a number of subculturing, the cell monolayer was detached from glass by chemotherapeutic agents into 3 groups. The 1st group, consisting of -y-radiation and nitrogen mustard, killed both treatment with a 0.1% trypsin solution; the cells were then dispersed in medium, and an aliquot was planted in bottles. proliferative and resting cells in all portions of the generation Synchronous DON Cells. A modification of the method of cycle. In contrast, the agents in the 3rd group killed only Stubblefield et al. (42) was used to obtain a population of proliferative cells in all phases of the cell cycle. Agents mitotic cells. Logarithmically growing cells were planted in belonging to both of these groups gave exponential curves, roller bottles (11 x 28.5 cm; 840-sq cm growth surface; Bélico when survival was measured at different drug concentrations. Glass, Inc., Vineland, N. J.) at about IO7 cells in 200 ml of The 2nd group of agents gave dose-survival curves that medium. The bottles were gassed with a 5% C02 :95% air decreased to a constant saturation value at high doses, mixture and were rotated for 2 days on a roller apparatus indicating that they killed cells in 1 portion of the cell cycle; (Bélico)at 0.6 rpm. After 2 days of incubation, Colcemid i.e., these agents were phase specific. (demecolcine; Ciba Pharmaceutical Company, Summit, N. J.) The variation in the sensitivity of cultured mammalian cells was added to the medium to give 0.06 Mg/ml, and the bottles through the division cycle to different agents has been were incubated for 3 more hr. The medium was then poured reported. The recent paper by Mauro and Madoc-Jones (33) off, and we selectively removed the mitotic cells by rolling the summarizes the results obtained with a large number of agents. bottles with 40 ml of trypsin (at 4°;0.125 mg/ml) and 1 ml of Such studies are useful for 2 reasons: (a) They may enable us NaHC03 (7.5%). The mitotic cells were centrifuged and better to understand the parameters of the cell cycle by resuspended in medium at 4°, and about IO6 cells were planted in 3-oz bottles containing medium at 37°.The mitotic 'This study was supported in part by Contract PH43-68-1023 with cells constituted between 85 and 95% of the harvested cells. Chemotherapy, National Cancer Institute, NIH, Bethesda, Md. 20014. Determination of Percentage of Cell Survival after Exposure Received December 21, 1970; accepted November 3, 1971. of DON Cells to Drug. Cells growing in synchrony were 398 CANCER RESEARCH VOL. 32 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1972 American Association for Cancer Research. Cell Cycle Phase Specificity exposed to drug for 1 hr at different times after the planting cycloheximide) were developed by The Upjohn Company, of mitotic cells to expose cells in different parts of the cell Kalamazoo, Mich. The solubilities and structures of the drugs cycle. Asynchronous cells were also exposed to the drug for 1 are as follows. FUdR, neocarzinostatin (NSC 69856; Ref. 32), hr. thio-TEPA (NSC 6396), cycloheximide, ara-C, 5-azaCR, After exposure to drug, the supernatant medium was sodium camptothecin (alkaloid; NSC 100880; Ref. 20), and poured off, and the cells were detached with trypsin and streptozotocin (22) were soluble in H2O, Tu, and sparsomycin resuspended in medium at 37°.When it was suspected that the (45) were soluble in hot H2O. FU was soluble at 20 mg/ml in drug might also detach cells, the supernatant medium was also 7.5% NaHC03. Nogalamycin (46) and actinomycin D were centrifuged, and these cells were added to the total cell pool. dissolved in acetone at 1 mg/ml. The cells were diluted in medium at 37°,and 2 ml of cells 5-Hydroxy-2-formylpy ridine thiosemicarbazone (NSC were planted in plastic Petri plates (Linbro Chemical Co., New 107392), pactamycin (44), BCNU (NSC 409962), CCNU (NSC Haven, Conn.) to give 10 to 100 colonies per plate. Twelve 79037), and chlorambucil [4 {p- [bis(2-chlore- plates were planted for each sample. After incubation for 7 to thyl)amino] phenyl} butyric acid; NSC 3088] were dissolved 8 days in an atmosphere of 8% C02:92% air at 37°, the in ethanol at 5 to 10 mg/ml. Pseudourea (NSC 56054; Ref. 13) medium was poured off, and the colonies were stained with was dissolved at 10 mg/ml dimethyl sulfoxide. Ellipticine 0.2% méthylèneblue in 70% ethanol. The colonies were (NSC 71795; Ref. 20) and L-phenylalanine mustard counted with a Quebec colony counter (Spencer Lens Co., [3-(p-[bis(2-chloroethyl)amino]phenyl-L-alanine; NSC 8806] Buffalo, N. Y.). The plating efficiencies were about 50% for were dissolved at 10 mg/ml in 0.1 N HC1. synchronous cells and 70% for asynchronous cells. For When we used solvents for solutions of drugs, we attempted calculation of the percentage of survivals, the control (no drug to prepare a 10 mg/ml solution. Thus, it was possible to dilute treatment) samples were normalized to 100% survival. In these out the solvent in the process of diluting the drug to the experiments, the coefficient of variation in determining cell desired concentration. The drug concentrations used to survival was about 15%, the coefficient of variation being the determine sensitivity of different phases were chosen to show standard deviation expressed as a percentage of the mean. a well-defined age response. In many cases, 2 or 3 different Thus, if the percentages of survival of 2 different samples were levels of the drug were used (see Charts 2, 4, etc.), but the 50 and 30%, respectively, then they would be statistically sensitivity of different phases to the drug pertains only to the significantly different at the 95% confidence level. Almost all concentrations used and is not necessarily true at much higher experiments were repeated, and the results were found to be drug concentrations. reproducible; in duplicate determinations, the individual values were within 10% of the mean value. Determination of Macromolecule Synthesis. Asynchronous DON cells were planted at IO6 cells/3-oz bottle in 10 ml of medium. After overnight incubation, the cells were refed with fresh medium, and drug and labeled precursors were added for g 2 1 hr. To stop uptake of radioactivity, cells were detached with l-H- 0.1% trypsin containing unlabeled precursors (100 jug/ml), and the cells were suspended in 0.9% NaCl solution. One aliquot "•1 was counted in the Coulter counter to give the cell number, while another (1-ml) aliquot of cells was filtered through WO 0.45-M Millipore filters. The filter was washed 4 times with cold 10% trichloroacetic acid and once with ethanol. The filter was then incubated with 0.5 ml of 0.5 N perchloric acid at 70° 80 for 20 min.