[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.

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(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 *,\- -1vÎI 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. Although the results obtained with this technique do mitosis with increasing drug concentration until a plateau not directly enable us to distinguish between drug concen region was attained where the transition point no longer tration-dependent blockade occurring at various ages in G2 decreased with increasing concentration. The linear portion and drug concentration-dependent induction of blockade of the curve was calculated over a concentration range as a function of 62 age but with the actual block at a single from 0.8 (transition point corresponding to the S-G2bound point late in 62, small differences in G2 cell sensitivity are ary) to 40 /tig/ml (transition point corresponding to pro- quantifiable (25). metaphase) and exhibited a slope constant (m) of 46 (see Table 1). For concentrations of actinomycin D greater than RESULTS 40 pig/ml, the transition point remained at 42 ±1 min prior to the end of karyokinesis. For comparison, Dewey and Actinomycin D. The effect of continuous exposure to Highfield (8, 12), using the same CHO cell line and tech various concentrations of actinomycin D (1.0 to 125 /*g/ml) nique, reported G2transition points of 120, 79, and 52 min on the selection of mitotic CHO cells is shown in Chart 1. prior to the end of karyokinesis for 2, 5, and 15 /*g Mitotic cells continued to enter the selection window at the actinomycin D per ml, respectively. These data (8, 12) control rate for a period of 20 to 60 min after exposure to correlated well with Chart 2. actinomycin D and then declined to a concentration-de- Adriamycin. A response similar to that obtained with

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CONCENTRATION, »M CONCENTRATKDN,

10 100 1000 K> 100 WOO

uo Actmomycin-D 140 Adriamycin

120

. 100 100

80 80

60

40 40

0.1 1 10 100 1000 0.1 1 10 no 1000

CONCENTRATION, pg/ml CONCENTRATION, vg/mi Chart 2. Effect of actinomycin D concentration on the transition point Chart 3. Effect of Adriamycin concentration on the transition point. The (i.e., the time prior to karyokinesis, after which cells are refractory to drug different symbols refer to different experiments; A, •.•continuous treatment and proceed through mitosis). •,•different experiments. exposure to Adriamycin; A, O pulsed exposure of 10 to 30 min.

Table 1 CONCENTRATION, uM Parameters of concentration-dependent G2transition point 10 ICO 1000 regression line T = m log C/Co + T0° 140 tionrange1-500.3-501-200.1-3001-505-1000.4-40m46254225416836C^g/rnl504820*500MM429550T» DrugActinomycin (min)41454547 £ 120

DAdriamycinLucanthoneMitomycin 5 _. roo

CBleomycin0Concentra z m

i" 60

" T, G2transition point (min) before completion of karyokinesis produced by a drug concentration (C); m, slope constant; C0, 40 minimum drug concentration yielding the plateau transition point, T"A Solubility limitation of lucanthone, 20 pg/ml. OJ 1 10 100 1000 c Three separate lots of bleomycin; see Chart 6. CONCENTRATION, pg/ml Chart 4. Effect of lucanthone concentration on the transition point. The actinomycin D was observed with Adriamycin. As shown in different symbols refer to different experiments; •,•continuous exposure Chart 3, the location of the Adriamycin G2 transition point to lucanthone; D, O, pulsed exposure of 60 to 120 min. was also dependent upon drug concentration. There is a linear relationship between the transition point and the tration and the G2transition point (Chart 4) is similar to the logarithm of the concentration of adriamycin between 0.3 other intercalating antibiotics. Although it was not possible and 50 /u.g/ml. The transition point decreased from =105 to test concentrations of lucanthone greater than 20 ¿ig/ml min (S-G2boundary) at 0.3 /ug/ml to 45 min (prometaphase) because of solubility limitations, the results of an experi at 50 /^g/ml with a slope constant (m) of 25 (see Table 1). ment (not shown) with a slurry of 50 ¿tg/mlindicated that a Concentrations of Adriamycin greater than 50 /ng/ml did constant mimimum value of the transition point existed at not further alter the transition point, which was located 45 45 min prior to the end of karyokinesis. The slope constant ±3 min prior to the end of karyokinesis. Results of treat of the transition point plotted as a function of logarithm of ments pulsed for 10 to 30 min were similar to continuous lucanthone concentration was 42 (Table 1). treatment. Mitomycin C. The dependence of the G2transition point Lucanthone. Lucathone is a thiaxanthenone analog of on mitomycin C concentration is shown in Chart 5. Again, actinomycin D and Adriamycin that intercalates into DNA because of solubility limitations, it was impossible to and inhibits RNA and DNA synthesis and also inhibits the achieve high enough concentrations of the drug to demon accumulation and repair of sublethal radiation damage (6, strate a minimum plateau for the transition point. However, 17). Unlike actinomycin D and Adriamycin, which bind the transition point varied with concentration of mitomycin irreversibly to DNA, lucanthone only loosely binds to DNA, C over the range of 1 to 300 ¿¿g/mlandexhibited a slope and consequently its effects are completely reversible (6, constant of 25 (Table 1). 17). However, the relationship between lucanthone concen- Bleomycin. The relationship between the G2 transition

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CONCENTRATION, uM

10 100 woo 140

™ 120 Z K»

* 80

g 60

40

Ql 1 K> 100 woo woo CONCENTRATION, ug/ml CONCENTRATION . ug/ml Chart 6. Effect of bleomycin concentration on the transition point. •.• Chart 5. Effect of mltomycin C concentration on the transition point. •, A, 3 experimentsiriments with 3 different lots of bleomycinbleomvcin •different experiments. point and the concentration of bleomycin for 3 separate lots •Cycloheximide of bleomycin is shown in Chart 6. All 3 curves shown in 140 Puromycin Chart 6 exhibit a concentration dependence of the G-, . Hydroxyurea transition point, but the slope constants of the curves vary 120 by a factor of 2 (Table 1). Although 1 lot of bleomycin was suspect because it was out of date and had been stored under unknown conditions (curves composed of solid tri angles), the other 2 lots, gifts directly from Bristol Labora 80 tories, were also outdated but were guaranteed to have full clinical potency. Considering the number of constituents in 60 bleomycin, it is not surprising that a difference in the concentration dependence of the G2transition point occurs 40 between lots. The minimum value of the transition point induced by the highest concentration of bleomycin was 47 min prior to the end of karyokinesis, and the slope con 0.1 l wo 1000 stants for the 3 lots were 36, 41, and 68 (Table 1). CONCENTRATION, ug/ml Hydroxyurea. When cells were exposed to either 1.0 or Chart 7. Effect of cycloheximide. puromycin. and hydroxyurea concen 10 IHM hydroxyurea (Chart 7), the rate at which mitotic cells tration on the transition point. The G2 transition point is independent of entered the selection window remained equal to the control concentration with these antimetabolites. Cycloheximide data from 6 exper iments, puromycin data from 3 experiments, and hydroxyurea data from 6 for =20 min and then declined to 0 at about 220 min after experiments. initiation of treatment. The transition point was at 116 ±3 min prior to the end of karyokinesis (average of 19 experi tein-synthesis inhibitor produced complete blockade of G-, ments). After a 10-min pulse of tritiated thymidine to label S progression with a constant transition point of 58 ±1 min phase cells, the number of selected unlabeled mitotic cells (Chart 7). At lower concentrations there was incomplete (i.e., cells in G, or M during the 10-min pulse) was equal to suppression, but cells that were blocked exhibited the the number of cells refractory to hydroxyurea treatment. In same transition point. The puromycin and cycloheximide addition, no radioactivity was detected in cells selected transition points were indistinguishable. when hydroxyurea was added coincident with tritiated thy midine. These results indicate that the location of hydrox DISCUSSION yurea transition point is at the S-G. boundary. At a concen tration of 0.1 rriM hydroxyurea there was incomplete block By monitoring the rate at which mitotic cells were selec ade of cells, and a transition point determination could not tively detached from an asynchronously growing mono- be accurately made (26). layer, we were able to determine the point in the cell cycle Cycloheximide. Treatment with cycloheximide at con (the transition point) at which various concentrations of centrations between 0.1 and 100 /ug/ml completely sup DNA-active agents and antimetabolites act to interfere with pressed the progression of mitotic cells into the selection the progression of cells into mitosis. window with a transition point of 56 ±1 min prior to the The antimetabolic drugs, hydroxyurea, an inhibitor of end of karyokinesis (Chart 7). For each concentration, the DNA synthesis, and cycloheximide and puromycin, inhibi mitotic yield was equal to the control for approximately 30 tors of protein synthesis, have transition points represent min and then declined to 0 over the next 50 min. ing the last point in the cell cycle when synthesis of an Puromycin. At concentrations above 50 ¿¿g/ml,thispro- essential precursor for progression occurs (19). Since cy-

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1978 American Association for Cancer Research. Concentration-dependent G2 Transition Points cloheximide and puromycin inhibit protein synthesis, any Dewey and Highfield (8,12), also using the mitotic selection cell in which the last protein(s) essential for mitosis had procedure, were able to discern different transition points been synthesized would continue through division without for 3 concentrations of the drug. delay in the presence of these drugs, while a cell that had Epifanova ef a/. (10) observed a time- and concentration- not synthesized that protein (i.e., any cell earlier in the cell dependent decrease in the mitotic index of CHO cells after cycle than =57 min prior to the end of karyokinesis) would the addition of lucanthone; however, they could not differ be unable to progress further. Agents that act by such a entiate G2transition points. Nowell (21) was able to show a mechanism are characterized by single, concentration-in similar decrease in the mitotic index after treatment with dependent transition points (Chart 7). mitomycin C, but again, differentiation of the transition CHO cells treated with intercalating antibiotics (actino- points could not be observed. Several investigators have mycin D, Adriamycin, and lucanthone) and the DNA-active reported that bleomycin causes a G2block and an accumu agents (bleomycin and mitomycin C) displayed concentra lation of cells in G2 (5, 13, 20, 27). Although different tion-dependent G2transition points. At low concentrations, positions for the G2block were reported, no concentration the transition point was at or near the S-G2boundary. Over effect was observed. Our data showing the lot-to-lot varia the intermediate-to-high concentration ranges, there was tion in the efficacy of bleomycin should be taken as a complete suppression of cell progression in G2.The number warning both to the research and clinical communities of of cells refractory to the drug decreased with increasing the possible heterogeneity of different preparations for concentrations, and the transition points for the high drug bleomycin. concentrations occurred later in G2.The slope constant (m Finally, we have shown that for the intercalating antibiot in Table 1) of the regression line produced when the ics and for bleomycin there exists a common minimum transition point is plotted against the logarithm of drug transition point at approximately 45 min prior to the end of concentration is a measure of the relative effectiveness of a karyokinesis that cannot be altered by high concentrations particular drug in limiting the number of refractory cells. of drug. The commonality of the minimum transition point Thus, actinomycin D (m = 46) is more effective on a per at high concentrations suggests that at this point in the cell molecular basis than lucanthone (m = 42), which in turn is cycle (probably prometaphase)5 the cell is refractory to more effective than Adriamycin (m = 25) or mitomycin C (m drug-induced blockade. Since the transition point for inhi = 25). The slope constants for 3 lots of bleomycin ranged bition of DMA synthesis (hydroxyurea transition point) or from 36 to 68, indicating a nearly 2-fold difference in inhibition of protein synthesis (cycloheximide or puromycin efficacy between lots of bleomycin that were stated to transition point) is concentration independent, it is unlikely possess full clinical potency. that the anticancer drugs studied block cell progression For the intercalating antibiotics and 1 lot of bleomycin, only by inhibition of macromolecular synthesis. Therefore, there was a constant minimum transition point (7,,) once a it seems reasonable that the intercalating antibiotics, by certain drug concentration (Cn) had been exceeded (see binding to DNA, or mitomycin C, by cross-linking DNA, or Table 1). Because of insufficient information concerning bleomycin, by causing DNA strand breaks, induce concen uptake rates and the intracellular concentrations that might tration-dependent structural defects in DNA that interfere be reached for these drugs, it is not possible to use d, as a with either chromatin condensation, spindle fiber forma definitive parameter. However, the constant value of Tufor tion, or attachment of spindle fibers to the centromere or each drug, with a narrow range between 41 and 47 min centriole and thus prevent normal progression of G2 cells prior to the completion of karyokinesis, indicates a common towards or into mitosis. temporal event such that, once a cell has progressed beyond it, further progression through mitosis cannot be REFERENCES prevented, regardless of drug concentrations. 1. Barlogie. B., Drewinko, B., Benjamin, R. S., and Freireich, E. J Kinetic Our results indicate at least 2 different mechanisms by Response of Human Lymphoid Cells to Adriamycin-DNA Complex in which drugs block the progression of mammalian cells into Vitro. Cancer Res.,36: 2450-2454, 1976. mitosis. The first mechanism is inhibition of the synthesis 2. Barlogie, B., Drewinko, B., Johnston, D. A., Freireich. E. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1978 American Association for Cancer Research. Induction of Concentration-dependent Blockade in the G2 Phase of the Cell Cycle by Cancer Chemotherapeutic Agents

Bruce F. Kimler, Martin H. Schneiderman and Dennis B. Leeper

Cancer Res 1978;38:809-814.

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