[CANCER RESEARCH 39, 31 52-31 55, August 1979]
`0008-5472/79/0039-0000$02.0O
`
`Cell Cycle Phase-specific Cytotoxicity of the Antitumor Agent
`Maytansine1
`
`PoW N. Rao,2 Emil J. Freireich, Marion L. Smith, and Ti Li Loo
`
`Department of Developmental Therapeutics, The University of Texas System Cancer Center, M. 0. Anderson Hospital and Tumor Institute, Houston, Texas 77030
`
`ABSTRACT
`
`MATERIALS AND METHODS
`
`The objective of this investigation was to study the effects of
`maytansine on the cell cycle kinetics of HeLa cells. The results
`of this study indicate that maytansine is a very potent mitotic
`inhibitor and that it has no effect on macromolecular synthesis.
`Maytansine-induced cytotoxicity was dependent upon the p0-
`sition of the cell in the cell cycle. Mitotic and G2cells are most
`sensitive to this agent, while G1 phase cells are the most
`resistant, with S-phase cells being intermediate. Small (0.82
`x 10-8 M)fractionated
`doses given at an interval of 8 hr have
`been found to be more cytotoxic than was a large (1.64 x 10_a
`M) single
`dose.
`In evaluating
`the drug
`combinations,
`we ob
`served that the schedule in which I -fJ-D-arabinofunanosylcy
`tosine treatment was followed by maytansine treatment ex
`hibited greater cell kill than the reverse sequence. No schedule
`dependent effects were observed when maytansine was tried
`in combination with Adniamycin.
`
`INTRODUCTION
`
`isolated
`Maytansine, a naturally occurring ansa macrolide,
`from the East African shrub Maytenus serrata (4, 5), has been
`reported to have significant antitumor activity against several
`experimental animal tumors, including P388 lymphocytic leu
`kemia, Bi 6 melanoma, and Walker 256 carcinoma (1 1). The
`antitumor activity of maytansine appears primarily to be due to
`its stathmokinetic effects, as in the case of Vinca alkaloids
`(12). Phase 1 clinical trials with maytansine in our department
`at M. D. Anderson Hospital and Tumor Institute appear to be
`promising because the antitumor activity of maytansine in
`patients with melanoma, breast carcinoma, and head and neck
`clear cell carcinoma is associated with little or no myelo
`suppression (2). Responses were also observed by other in
`vestigators in patients with acute lymphocytic leukemia, non
`Hodgkin's lymphoma, ovarian cancer (3), and carcinoma of the
`breast (1). Maytansine is now in Phase 2 clinical trials.
`Since maytansine is associated with some dose-dependent
`gastrointestinal
`toxicity (2, 3), we decided to study the effects
`of scheduling, dose, and dose fractionation of maytansine
`alone and in combination with ara-C3 or Adniamycin on HeLa
`cells in vitro.
`
`,ThisresearchissupportedinpartbyGrantsCA-i1520,CA-i4528,andCA
`19856 and Contract CM-53773
`from the National Cancer Institute, and Grant
`GM-23252
`from the National
`Institute for General Medical Sciences, NIH, De
`partment of Health, Education, and Welfare.
`2 To whom
`requests
`for
`reprints
`should
`be sent.
`3 The
`abbreviations
`used
`are:
`ara-C,
`1 -fl-o-arabinofuranosylcytosine;
`thymidine.
`Received July 24, 1978; accepted May 15, 1979.
`
`dThd,
`
`Cells. HeLa cellsusedinthisstudywere grownin Luxplastic
`dishes as monolayer cultures in McCoy's Medium 5A supple
`mented with 16% heat-inactivated fetal calf serum and 1%
`penicillin (10,000 units/ml):stneptomycin
`(10,000
`sg/ml).
`These cells have a cell cycle time of approximately 22 hr and
`a plating efficiency of about 90%.
`Cell Synchrony. HeLa cells were synchronizedin S phase
`by the excess dThd (2.5 mM)double-block method (8). Cells in
`S and G2 phases were obtained by trypsinizing monolayer
`cultures at 1 and 7 hr, respectively, after reversal of the second
`dThd block. A pulse label of 30 mm with [3HJdThd gave a
`labeling index of 95% for S-phase population and 15% for the
`G2 population.The mitoticindex in these populationswas less
`than 1%. Mitotic HeLa cells of 98% purity or better were
`obtained by selective detachment after the exposure to nitrous
`oxide (at 80 psi and 37°)of a monolayer culture that was
`partially synchronized into S phase by a single dThd block (7).
`G1 phase cells were obtained by incubatingthe N2O-arrested
`mitotic cells under regular culture conditions for 3 hr. During
`this 3-hr incubation, the mitotic index decreased from 98% to
`below 5%,
`indicating the successful completion of mitosis
`following reversal of the N2O block.
`Drugs. Maytansine(NSC 153858), ara-C (NSC 63878), and
`Adriamycin (NSC 1231 27) were supplied by the Drug Devel
`opment Branch, National Cancer Institute, NIH. Stock solutions
`of these drugs were freshly prepared just before use and then
`serially diluted in complete culture medium to obtain the desired
`concentrations.
`Cell Cycle Kinetics. A culture in exponential growth was
`trypsinized and plated in a number of Lux 35-mm plastic dishes
`at 2 x 1O@cells/dish about 20 hr before the experiment. The
`experiment was begun by replacing the medium in the dishes
`with fresh medium containing maytansine. The drug concentra
`tions studied were 0, 0.5, 1.0, 2.0, 4.1, 8.2, 16.4, and 32.8
`nM. For each
`concentration,
`there were
`2 dishes,
`one for an
`18-hr continuous treatment and the other for a pulse treatment
`of 60 mm (followed by a wash to remove the drug) and a
`posttreatment
`incubation of 17 hr in regular medium. At the
`end of this period, the cells were collected by trypsinization,
`deposited directly on clean slides by the use of a cytocentri
`fuge, fixed in absolute methanol:glacial acetic acid (3:1 ,
`stained with acetoorcein, and scored for the percentage of
`cells in mitosis. Five hundred cells were scored for each point.
`The mitotic accumulation was plotted as a function of dose.
`The data presented represent an average of 3 experiments.
`Dose-Survival Studies. The procedures for drug treatment
`and the determination of plating efficiency have been described
`previously (9). HeLa cells in exponential growth, which were
`trypsinized and plated in a number of dishes the day before
`
`31@2
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`the experiment, were exposed to various concentrations of the
`drug for 1 hr or more, depending upon the purpose of the
`experiment. At the end of the treatment, medium containing the
`drug was removed, and cells were washed with drug-free
`medium, trypsinized, plated for colonies, and incubated for 10
`days. The number of colonies observed in the treatments was
`expressed as a percentage of
`the value for the untreated
`control. The plating efficiency of the controls was 85 ±7%
`(S.D.).
`Evaluation of Drug Combinations. For the in vitroevaluation
`of combined drug effects, a random population of HeLa cells
`was exposed first
`to one drug that was then removed by
`washing before the second drug was added to the medium.
`Soon after the drug treatments, cells were washed with regular
`medium, trypsinized, and plated for colonies. In this study, 2-
`drug combinations, maytansine:ara-C and maytansine:Adnia
`mycin, were examined. The various schedules included may
`tansine followed by either ara-C or Adniamycin and the reverse
`sequence. The duration of exposure of cells to maytansine and
`Adriamycin was 60 mm. However, with ara-C, the treatment
`was 16 hr because it has been shown that a 16-hr incubation
`of a random population of HeLa cells with a sublethal dose (0.8
`@sg/ml)of ara-C reversibly blocked about 90% of the cells in S
`phase (9).
`
`RESULTS
`
`Effect on Cell Cycle Traverse and Macromolecular Synthe
`sis. The purpose of this set of experiments was to determine
`the optimum dose and duration of treatment of HeLa cells with
`maytansine to produce the maximum cytotoxic effects. The
`primary effect of maytansine on HeLa cells was the arrest of
`cells in metaphase. The degree of mitotic accumulation in a
`random population of HeLa cells after 18 hr of continuous
`exposure or after 17 hr of incubation following a 1-hr treatment
`with maytansine is presented in Chart 1. In general, the effects
`of maytansine on mitotic accumulation were similar to those of
`Colcemid. The effects of a pulse (60 mm) exposure of cells to
`the drug were reversible at lower (0.05 to 0.2 x 10_a M) but
`
`Phase-specific Cytotoxicity
`
`of Maytansine
`
`not at higher concentrations.
`Maytansmnehas no inhibitory effect on the rate of incorpo
`ration of tritium-labeled precursors into DNA, ANA, and protein
`during a 3-hr period (Table 1). However,
`there was some
`increase in the incorporation of [3Hjleucine in the presence of
`Colcemid or maytansine in the medium.
`Effect on Cell Survival. The effect of a 1-hr treatment with
`various concentrations of maytansine on the plating (cloning)
`efficiency of HeLa cells was studied. Initially, the plating effi
`ciency decreased with an increase in dose, but it soon reached
`a plateau (Chart 2). Further increase in dose had little or no
`effect on survival until the concentration reached 6.56 x 10@
`M. However,
`an increase
`in the duration
`of
`treatment
`resulted
`in a decrease in the plating efficiency (Chart 3).
`Effect of Dose Fractionation on Survival. Based on the
`dose-survival curve in Chart 2, a maytansine concentration of
`1.64 x 10_a M was selected. This dose was fractionated into
`2 doses(0.82 x 10_a M each), and each was applied for 1 hr,
`with or without an interval between them. These results indicate
`that the longer the interval between the split doses (up to a
`maximum of 8 hr studied) the greater was the decrease in cell
`survival (Chart 4).
`Cell Cycle Phase-Specific Effects of Maytanslne on Plating
`Efficiency. When HeLa cells synchronizedin various phases
`of the cell cycle were exposed to maytansine (1.64 x 10_a
`M)
`for 1 hr.
`the percentage
`of survival
`varied
`depending
`upon
`the phase of the synchronized population (Chart 5). The great
`est drug sensitivity was observed in mitotic populations fol
`lowed by G2, 5, and G1, in order of decreasing sensitivity.
`In Vitro Evaluation of Drug Combinations.
`In view of the cell
`
`Table1
`Effect of maytansine on the incorporation of tritium-labeled
`precursors
`in DNA, RNA, and protein
`
`Thedatarepresentthe averageof 4experiments.TreatmentRelative
`cells[3H)dThd[3H@Jridine[3H]LeucineControl
`
`cpm/1 O@
`
`@
`
`Colcemid(0.05100
`11.10pg/mI)
`
`106.4 ±439a100 945
`
`7.20iOO121.2 ±
`
`±8.10111.3
`
`±10.60152.0
`
`±
`
`13.8a
`Maytansine(O.656
`x 10'M;O.S
`ng/mI)102.7
`
`Mean
`
`± S.D.
`
`I00
`
`z0I
`
`-
`(.3
`
`q L
`
`@.
`
`3<
`Lu
`
`0 ‘
`
`3
`I-.
`
`0I
`
`-.
`
`@
`
`20
`
`DOSE (XIcI8M)
`Chart 1. Effect of various concentrations of maytansine on the mitotic accu
`mulation of HeLa cells in exponential growth. 0, a pulse treatment; cells were
`exposed to maytansine for 60 mm; the drug was removed by washing and then
`incubated in regular medium for 17 hr. •,cells were incubated with maytansine
`for 18 hr. Cells exposed to Colcemid (1.37 x i07 M or 0.05 pg/mI) served as
`a control to monitor the antimitetic effects of maytansine. U, 18 hr continuous
`Colcemid treatment; 0, 1 hr Celcemid treatment followed by 17 hr incubation in
`regular medium. Bars, S.D.
`
`-i
`4
`
`Cl)
`
`328
`DOSE (Xl1@M)
`Chart 2. Effect of a 1-hr treatment with various concentrations of maytansine
`(0) on the plating efficiency of HeLa cells in exponential growth. Bars, S.D.
`
`AUGUST 1979
`
`31S3
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`cancerres.aacrjournals.org
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`Research.
`
`IMMUNOGEN 2054, pg. 2
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`IPR2014-00676
`
`

`

`effects are not observed if the duration of treatment is limited
`to 1 hr. A 60-mm treatment with concentrations of maytansine
`above 0.82 x 1
`M produces a mitotic block in HeLa cells
`that remained irreversible up to 18 hr. At concentrations of 0.2
`x 10-@Mor lower, the antimitotic effects are quickly reversible
`by washing and resuspending the cells in drug-free medium.
`Similar results were obtained with munine leukemia cells by
`Wolpert-Defilippes et a!. (12).
`A 3-hr incubation of a random culture of HeLa cells with
`maytansine at concentrations of 6.6 x 10@ M had no effect
`on the incorporation of [3H]dThd and [3H]uridmneinto DNA and
`RNA, respectively. A measurable increase in the uptake of
`[3H]Ieucine into both Colcemid- and maytansine-treated cells is
`unexpected. Probably, these agents, due to their disorganizing
`effects on the cytoskeleton, may increase the permeability of
`
`I00
`
`;@80
`
`0
`
`Li.
`
`60
`
`-J 40
`
`:3
`(I,
`
`0
`
`A
`
`B
`
`C
`
`0
`
`E
`
`F
`
`Chart 5. Effect of maytansine (1.64 x 108 M for 60 mm) on the plating
`efficiency of HeLa cells synchronized in various phases of the cell cycle. The
`number of colonies observed in the treatment is expressed as a percentage of
`the untreated control
`for each phase of the cell cycle. A, control, a random
`culture. Maytansine treatments: B, random culture; C, G phase; 0, 5 phase; E,
`G2 phase; F. mitotic populations. Bars, S.D.
`
`0I
`
`..
`(3
`
`4 I
`
`i.
`
`-J
`
`4 :
`
`3C
`
`l)
`
`ABCDE
`
`FGH
`TREATMENTS
`Chart 6. Effect of maytansine in combination with ara-C or Adriamycin on the
`survival of HeLa cells in exponential growth. The treatments included ara-C (0.8
`tiM)
`for 16 hr. Adriamycin
`(0.2 gig/mI)
`for 1 hr. and maytansine
`(1 .64 x 10'
`M)
`for 1 hr. A, untreated control; B, ara-C treatment; C, ara-C followed by maytan
`sine; 0, maytansine followed by ara-C; E, maytansine alone; F, maytansine
`followed by Adriamycin; G, Adriamycin followed by maytansine; H, Adriamycin
`alone. Bars, S.D.
`
`P. N. Rao et al.
`
`0 (
`
`3
`
`4 I
`
`A.
`
`-J
`
`Cl)
`
`DURATION OF TREATMENT (HOURS)
`
`Chart 3. Effect of the duration of maytansine (1.64 x 10@' M) (0) treatment
`on the plating efficiency of HeLa cells in exponential growth. Bars, S.D.
`
`.-
`
`r@1@
`
`@L
`
`1
`
`1
`
`I
`
`.:
`
`L@HL@]
`
`A
`
`B
`
`C
`
`0
`
`E
`
`F
`
`G
`
`H
`
`Chart 4. Effect of dose fractionation and the duration of interval between the
`treatments en the survival of HeLa cells in exponential growth. A, untreated
`control; B, maytansine treatment (0.82 x i 0_8 M) for 1 hr; C, maytansine
`treatment (1.64 x 108 hi) for 1 hr. For dose fractionation studies, 2 treatments
`of maytansine at 0.82 x 108 N for 60 mm each were applied at zero (0), 2- (E),
`4- (F), 6- (G), and 8- (H) hr intervals. Bars, S.D.
`
`cycle phase-specific cytotoxicity of maytansine, we decided to
`study whether
`there would be any increase in cell kill by
`synchronizing with a low dose of ara-C and then exposing the
`cells to maytansine. Schedule-dependent cytotoxic effects
`were observed in the combination of maytansine with ana-Cbut
`not with Adniamycin (Chart 6). Exposure of cells to ara-C for
`16 hr followed by a 1-hr treatment with maytansine reduced
`the plating efficiency to about 25%, as compared with 41 %
`survival when the sequence was reversed. The data presented
`are the averages of 3 experiments.
`
`DISCUSSION
`
`The results of this study indicate that maytansine is primarily
`a mitotic inhibitor. As a mitotic inhibitor,
`it is effective over a
`wide range of concentrations (Chart 1). The lowest effective
`concentration (0.5 nM) for maytansine in HeLa cells is about
`200 times smaller than that for Colcemid (1 .37 x 1O@ M).
`Similarly, maytansine has been shown to be at least 100 times
`more potent as an antimitotic agent
`than vincnistine in sea
`urchin eggs (10).
`Continuous treatment of HeLa cells with higher doses of
`maytansine may slow down the progression of cells through
`the cell cycle to some extent, as indicated by a slight reduction
`in the degree of mitotic accumulation (Chart 1). Such retarding
`
`31S4
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`thus facilitating greater
`the cell membranes to [3H]leucine,
`uptake and consequently greater incorporation of label into the
`protein than occurs in the control. These results are at variance
`with those of Wolpert-Defilippes et a!. (12), who reported that,
`in murine leukemic cells, DNA synthesis was inhibited to the
`greatest extent. However, they measured the uptake of labeled
`precursors into DNA, RNA, and protein by pulse-labeling of
`cells that were incubated with maytansine over a period of 12
`hr. A 12-hr exposure to maytansine at 10@ M would certainly
`block more than 50% of these cells in mitosis. The arrest of a
`significant fraction of the cell population in mitosis would result
`in a considerable reduction in the incorporation of label
`into
`DNA, RNA, and protein when compared with untreated control
`cultures.
`The dose-survival curve for maytansine (Chart 2) quickly
`reaches a plateau with an increase in the concentration, mdi
`cating that there are 2 cell types in the population, one sensitive
`and the other relatively resistant. However, with a fixed dose,
`there is a direct correlation between the duration of treatment
`and the percentage increase in cell kill (Chart 3). Dose frac
`tionation studies reveal that small fractionated doses are more
`cytotoxic than is a large single dose (Chart 4). The longer the
`interval between the fractionated doses, the lower is the plating
`efficiency up to 8 hr. These results suggest that most of the
`sensitive fraction in the cell population is killed by the first
`treatment, whereas the second treatment kills those that move
`from a resistant to a more sensitive phase of the cell cycle.
`Studies with synchronized populations reveal that cells in
`mitosis are the most sensitive and those in G, are the most
`resistant to this agent(Chart 5). The closer the cell is to mitosis,
`the more sensitive it is to the cytotoxic effects of maytansine.
`Depolymenization and inhibition of polymerization oftubulin has
`been shown to account for the antimitotic effects of maytansine
`(10). Since oxidation of the sulfhydryl groups in tubulin inhibits
`its polymerization,
`the effect of maytansine may be due to its
`binding to these groups (10). It is also conceivable that tubulin,
`which is the most important constituent of the mitotic appara
`tus, accumulates gradually during the cell cycle reaching a
`peak at the beginning of mitosis (6). Thus, cells in G2 and
`mitosis would have a full complement of
`these proteins,
`whereas those in G1 would have the least proteins, with S
`phase cells being intermediate. As soon as the cells were
`exposed to cytotoxic doses of maytansine, the spindle protein
`(tubulin) would be inactivated (depolymenized) by the inrevens
`ible binding of the drug. If the drug were removed after a brief
`exposure of 60 mm, cells in G1 and to some extent those in S
`could synthesize new tubulin and thus overcome the antimitotic
`effects of maytansine. Hence, G1cells would be more resistant
`to maytansine than those in other phases of the cell cycle, as
`we have observed. In the light of these observations, we can
`
`Phase-specific Cytotoxicity
`
`of Maytansine
`
`the dose-survival curve (Chart 1) as
`explain the pattern of
`follows. The initial sharp decrease in the plating efficiency
`represents the killing of cells that were in the sensitive phases
`of the cell cycle by low concentrations of maytansine. On the
`other hand, the plateau is represented by the more resistant
`G1fraction,whichconstitutesabout 50% of the populationand
`remains unaffected over a relatively wide range of drug con
`centrations.
`the cytotoxic effects of maytansine in
`In vitro evaluation of
`combination with ara-C revealed schedule-dependent effects
`(Chart 6). Administration of maytansine following a 16-hr ara-C
`treatment
`is more cytotoxic than the reverse sequence. This
`could be due to the synchronization of cells in S phase by the
`ara-C treatment. Our results (Chart 5) indicate that S-phase
`cells are more sensitive than those in G,. Since Adriamycin
`does not induce cell synchrony, the sequence of administration
`of Adriamycin and maytansine made no difference in cell sun
`vival.
`
`ACKNOWLEDGMENTS
`
`We thank Dr. Howard Thames for his assistance in statistical analysis.
`
`REFERENCES
`
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`with antltumer activIty. Cancer Treat. Rep., 62: 435—438,1978.
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`10. Remlllard, S., and Rebhun, L.
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`Inhibitor maytansine. Science, 189: 1002—1005, 1975.
`11. Sieber, S. M., Wolpert, M. K., Adamson, R. H., Cysyk, R. L., Bone, V. H.,
`and Johns, D. G. Experimental studies with maytansine—anew antitumer
`agent. Bibl. Haematol., 43: 495—500,1976.
`12. Wolpert-Defilippes, M. K., Adamson, A. H., Cysyk, R. L., and Johns, D. G.
`Initial studies on the cytetoxic action of maytansine, a novel ansa macrelide.
`Blechem. Pharmacel., 24: 751-754, 1975.
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`tumor
`
`AUGUST 1979
`
`3155
`
`
`
`cancerres.aacrjournals.org Downloaded from
`
`on December 8, 2014. © 1979 American Association for Cancer
`Research.
`
`IMMUNOGEN 2054, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Cell Cycle Phase-specific Cytotoxicity of the Antitumor Agent
`Maytansine
` 
`Potu N. Rao, Emil J. Freireich, Marion L. Smith, et al.
`Cancer Res  
`
`1979;39:3152-3155.
`
`Updated version
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`Access the most recent version of this article at:
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`
`IMMUNOGEN 2054, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`