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[CANCER RESEARCH 38, 809-814, March 1978] Induction of Concentration-dependent Blockade in the G2 Phase of the Cell Cycle by Cancer Chemotherapeutic Agents1 Bruce F. Kimler,2 Martin H. Schneiderman,3 and Dennis B. Leeper Laboratory of Experimental Radiation Oncology, Department of Radiation Therapy and Nuclear Medicine, Thomas Jefferson University Hospital, Philadelphia, Pennsylvania ABSTRACT for detailed analysis. Thus, results have been limited to describing the location of a block in terms of general The mitotic cell selection procedure for cell cycle anal phases of the cell cycle, i.e., G,, S, G,,, M, or at the ysis was utilized with Chinese hamster ovary fibroblasts boundary between 2 phases. Even when greater precision to determine the transition points in G,, i.e., the age in G, was attained, seldom was a concentration dependence of at which cells become refractory to drug-induced progres the location of the block observed (29). sion blockade, for several cancer Chemotherapeutic Using the mitotic cell selection procedure for cell cycle agents and antimetabolites over a 1000-fold concentration analysis (26), we have determined the number of cells range. refractory to drug-induced G-,blockade after treatment with The G transition points for five anticancer drugs (acti- various concentrations of several cancer Chemotherapeutic nomycin D, Adriamycin, lucanthone, mitomycin C, and agents and antimetabolites. We were able to calculate the bleomycin) varied linearly as a function of the logarithm time in G2 at which a particular concentration of drug of the drug concentration between the S-G_,boundary at inhibited progression or induced a delay. low concentrations and prometaphase (45 min prior to the end of karyokinesis) at high concentrations. Very low concentrations produced an incomplete blockade with MATERIALS AND METHODS some cells continuing to progress through the cycle. Cell and Culture Conditions. Chinese hamster ovary Above a certain concentration the transition point did not CHO4 fibroblasts were maintained in exponential growth as decrease further but attained a minimum value at 45 min monolayer cultures in 75-sq cm Falcon plastic flasks at 37° prior to the end of karyokinesis, implying that once a cell in a humidified atmosphere of 5% CO2in modified McCoy's has entered prometaphase it is completely refractory to Medium 5A (Grand Island Biological Co., Grand Island, N. drug action and proceeds through mitosis without pertur Y.). The medium was supplemented with 10% fetal calf bation. serum (Flow Laboratories, Rockville, Md.) and antibiotics. In contrast, the age at which cells are refractory to Stock cultures were maintained in antibiotic-free medium treatment with three antimetabolites did not show a drug and were found to be Mycoplasma free by the method of concentration dependence. Over wide ranges of concen Levine (18). Under these conditions the cells exhibited a tration, hydroxyurea, cycloheximide and puromycin re generation time of approximately 12 hr, with Gì= 3 hr, S sulted in transition points of 116 (the S-G boundary), 56, = 7 hr, G2 = 1.2 hr, and M = 0.7 hr. and 58, min respectively, prior to the end of karyokinesis. Mitotic Selection Technique. The progression of cells We interpret these results to indicate a physical mech into mitosis was monitored by means of the mitotic cell anism, distinct from pleotrophic action, by which the selection procedure for cell cycle analysis as described by anticancer antibiotics block the progression of G cells. Schneiderman ef al. (26). The specific techniques utilized have been published previously (16). Briefly, the rate at INTRODUCTION which cells progress into a narrow selection window [22-4 min, with 16 min as the average, prior to the end of Many of the cancer-chemotherapeutic agents currently in karyokinesis (reformation of the nuclear membrane in the clinical use share the property of inhibiting the progression daughter cells)] was determined by counting the number of of cells through the growth cycle in addition to causing mitotic cells dislodged by a 20-sec selection shake repeated cytotoxicity. If the cell cycle blockade induced by these every 10 min. Cell volume was routinely monitored by a agents is reversible and blockade lasts only as long as the Coulter Channelizer to verify that the selected cells were intracellular titer of the drug is sufficiently high, then one mitotic in size. In addition, the mitotic index was periodi should be able to take advantage of the induced synchrony cally determined microscopically to verify that the selected in scheduling subsequent treatment. Although many re cells were 2:95% mitotic. If the mitotic index of the control, ports have documented the effects of Chemotherapeutic scored microscopically, was not 95% or greater, an experi agents on cell cycle kinetics (1-5, 7-16, 19-31); most of the ment was discarded. Prescott (23) has recently given the techniques used lacked the necessary sensitivity required name retroactive synchrony to this technique. Drug Treatment. Hydroxyurea (NSC 32065), cyclohexi ' Supported by USPHS Grants CA 11602 and CA 16110. mide (NSC 185), puromycin (NSC 3055), actinomycin D 2 Present address: Department of Radiation Oncology, University of Kan (NSC 3053), lucanthone (NSC 14574), and mitomycin C sas Medical Center, Kansas City, Kans. 66103. 3 To whom requests for reprints should be addressed. Received June 13, 1977; accepted December 8, 1977. 4 The abbreviation used is: CHO. Chinese hamster ovary. MARCH 1978 809 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1978 American Association for Cancer Research. S. F. Kim 1er et al. (NSC 26980) were obtained commercially from Calbiochem, 292Concentration San Diego, Calif. Adriamycin (NSC 123127) was provided by the Drug Development Branch, Division of Cancer Treat ment, National Cancer Institute. Bleomycin (NSC 125066) 20.0 as Bleoxane was a gift from Bristol Laboratories, Syracuse, Actinomycin-Dio Midpoint—..-v N. Y. The drugs were dissolved in complete medium prior to the start of an experiment. Treatment was accomplished 10.0 by replacing control medium with drug-containing medium °. °o °o °°° at the desired concentration. o°o.S.;?• 5.0 °\«\a Analysis of Data. When a drug that blocks G; progression S is added to an asynchronous population of cells, the yield X IYV-««ll\\1min«1.0 ftg/mi 90 of mitotic cells (the number of cells selected at mitosis) a remains constant for a short period of time and then 2.0 1\•-•1 declines to a level dependent upon drug concentration. min-a20 jjg/ml 83 When the number of mitotic cells selected per shake is *,\- -1vÃŽI plotted as a function of time after treatment, the resultant 0 1.0 »i ' curve can be resolved into 3 parts (e.g., utilizing actinomy- m,nv50 pg/ml 80 cin D; see Chart 1): (a) an upper plateau equal to the control 0.5 ll^: rate of selection; (b) a log-normal decline in the number of 10 pg/ml 60 min selected cells; and (c) a lower plateau, less than 5% of the control value, representing a background level of inter- \{•'.E«p.min•• 25 )J9/ml 32 min•SO >ig/ml 25 phase cells and debris. 0.2 The mean time that a particular concentration of a drug m,n1 125 «/mi 25 takes to prevent cell progression is estimated graphically by determining the time from the initiation of treatment to o.i i i 100 200 400 500 the point representing 50% of the control number of mitotic 300 600 cells. This time is subsequently referred to as the midpoint. MINUTESAFTERINITIATIONOf TREATHENT The transition point, operationally defined as the last point Chart 1. Effect of various concentrations of actinomycin D on the rate of selection of mitotic cells. At time 0, the control medium was replaced with in the cell cycle at which cells can be delayed by a particular medium containing the indicated concentrations of actinomycin D for the agent, is then estimated by adding the average time of duration of the experiment. Exp , experiment. selection, i.e., 16 min prior to the end of karyokinesis, to the midpoint. pendent lower plateau. The midpoint the mitotic yield In the charts the calculated value of the transition point curves decreased with increasing actinomycin D concentra was plotted as a function of logarithm of the drug concen tion (up to 50 pig/ml). The increasing concentration also tration (/¿g/mland ¿tM).Different symbols in the charts resulted in a progressive shortening of the upper plateau represent different experiments. The straight line portions region. Concentrations of 1 to 5 ¿¿g/mldidnot completely of the curves in Charts 2 to 6 were fitted by linear regression reduce the mitotic yield to 0. In this experiment the transi analysis, and the curved portion was fitted by eye. tion point (midpoint plus 16 min) for G2 blockade by acti To determine whether the effect of the drugs were due to nomycin D shifted from 106 min (1 ^g/ml) to 42 min (50 and an alteration of the "selectability" of mitotic cells, we 125 /¿g/ml,respectively) before the end of karyokinesis. analyzed the mitotic index after a 5-hr Colcemid accumula The G2transition point as a function of the logarithm of tion in the presence of each drug. The results showed no actinomycin D concentration is plotted in Chart 2. The accumulation of mitotic cells. This indicates that the effect composite curve for actinomycin D (and the curves in on cell progression determined by mitotic selection was not Charts 3 to 6 for the other drugs tested) was described by a due to drug-induced changes of surface properties of the straight line as the transition point occurred closer to cells.

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