effects of 9-oh-ellipticine on cell survival ... · macromolecular syntheses, and cell cycle...

[CANCER RESEARCH 45, 4229-4236, September 1985]

Effects of 9-OH-Ellipticine on Cell Survival, Macromolecular Syntheses, and

Cell Cycle Progression in Sensitive and Resistant Chinese Hamster LungCells1

Jean-Yves Charcosset, Jean-Pierre Bendirdjian, Marie-FranÃ§oise Lantieri, and Alain Jacquemin-Sablon2

UnitÃ©de Biochimie et Enzymologie, Institut Gustave Poussy, 94800 Villejuif, France

ABSTRACT

In an effort to understand the mechanism of action of theDNA-intercalating antitumor agent 9-hydroxyellipticine (9-OH-E),

we have examined the effects of this drug on the cell survival,macromolecular syntheses, and cell cycle progression in sensitive and resistant cells. Our results show that 9-OH-E toxicity on

sensitive and resistant cells involves different mechanisms ofaction: the drug toxicity in the sensitive cells appears to resultfrom lethal lesions mediated through the interaction of the drugwith an intracellular protein, independently of any effect of thedrug on the macromolecular syntheses; in the resistant cells, thecell death occurs concomitantly with the inhibition of thesesyntheses. Cell cycle progression analysis after 9-OH-E treat

ment showed that, in the sensitive cells, the drug is inducing aGÃ¬and a G2 block, which are both released in the presence of 1HIM caffeine, without any effect on the 9-OH-E toxicity. In the

resistant cells, a G2 block was also observed but only when thecells were resuming their growth after about a 30- to 40-h growth

arrest. Caffeine release of this block, which again had no effecton 9-OH-E toxicity, was only observed when it was added from40 to 60 h after 9-OH-E treatment, when the cells resumed their

growth. Finally in the sensitive cells, cycloheximide exerted aninhibitory effect on 9-OH-E toxicity when it was added before

and during the cell exposure to the drug. This effect was interpreted as indicating that 9-OH-E toxicity in the sensitive cells

relies on a protein which is not induced by the drug but has tobe present in the cells when the drug is added. The possibleimplication of DMA topoisomerases in 9-OH-E toxicity mecha

nism is discussed.

INTRODUCTION

Several DNA-intercalating agents in the ellipticine series areendowed with antitumor properties: one of them, 9-OH-E,3 elicits

high antitumor activity in L1210 mouse leukemia (1, 2); andanother one, 2-A/-methyl-9-hydroxyellipticinium (NSC 264137,

celiptium), was recently introduced in human cancer chemotherapy (3-5). Numerous studies on the mechanism of action ofthese molecules have been reported. Ellipticine and its derivatives kill cells in all phases of the cell cycle, with eventually a

1Supported by the Centre National de la Recherche Scientifique (L. A. 147), the

Institut National de la SantÃ©et de la Recherche MÃ©dicale(U.140), and thÃ©Commissariat Ã l'Energie Atomique.

2To whom requests for reprints should be addressed, at the UnitÃ©de Biochimie

et Enzymologie, Institut Gustave Roussy, 94800 Villejuif, France.3The abbreviations used are: 9-OH-E, 9-hydroxyellipticine; MEM, Eagle's mini

mal essential medium modified; FCS. fetal calf serum; PBS, phosphate-bufferedsaline [glucose (1.1 g)/NaCI (8 g)/KCI (0.4 g)/Na2HPOÂ«/12 H20 (0.39 g)/KH2P04(0.15 g/liter)]; EDÂ»,the dose which reduces either the cell number or the cloningefficiency by 50%.

Received 12/12/84; revised 5/2/85; accepted 5/13/85.

preferential activity on cells in M and GÃ¬(6). DNA, RNA, andprotein syntheses (6, 7), processing of ribosomal precursor RNA(8, 9), and RNA methylation (10) are inhibited by ellipticine.Ellipticine and celiptium, as well as other DNA-intercalatingagents, produced protein-associated DNA single-strand breaksand double-strand breaks with, in both cases, a double/single-strand break ratio higher than that produced by X-ray or other

intercalators in similar conditions (11). Finally these drugs blockasynchronously growing cells in the premitotic (G2) phase of thecell cycle (12). Nevertheless, despite this bulk of information, theprecise mechanism responsible for the cytotoxicity of thesemolecules and the nature of the corresponding intracellular targetare presently unknown.

Isolation of drug-resistant cells has often proven to be a useful

approach toward the analysis of the mechanism of action ofantitumor drugs. We previously described (13) the isolation ofChinese hamster lung cells resistant to 9-OH-E, which were

selected in vitro by adding stepwise increasing drug concentrations to the growth medium. The development of 9-OH-E resist

ance was accompanied by several changes: among them weremodifications of the cell morphology and growth parameters; adecreased oncogenic potential; and a cross-resistance to a

variety of antitumoral agents. Detailed drug uptake and retentionstudies did not reveal any significant difference between thesensitive parental cells and the resistant cells (13,14). Thereforethe resistance to ellipticine derivatives does not result from adecreased cellular drug accumulation and should rather be related to its mechanism of action.

In this work we have compared the effects of 9-OH-E on cell

survival, macromolecule syntheses, and cell cycle progression inboth sensitive and resistant cells. In addition, the influence ofeffectors, such as caffeine, cycloheximide, and hydroxyurea, onthese parameters was also examined. The results indicate thatkilling of sensitive and resistant cells by 9-OH-E involves different

mechanisms.

MATERIALS AND METHODS

Cells and Culture Medium. The Chinese hamster lung cells DC-3Fand the 9-OH-E-resistant subline have been previously described (13).

Monolayer culture were maintained in MEM, supplemented with 7%PCS, streptomycin (50 ng/ml), and penicillin (100 ID/ml). The resistantsubline DC-3F/9-OH-E 0.6 was permanently grown in the presence of 9-

OH-E (0.6 fig/ml).Drugs and Chemicals. 9-OH-E is a 6H-pyridocarbazole derivative,

the structure of which has been previously shown (13). This drug waskindly provided by Sanofi, Paris, France, and further purified by Dr. G.Muzard. Caffeine, cycloheximide, and hydroxyurea were from SigmaChemical Co., St Louis, MO. The radioactive precursors to DNA andRNA and protein syntheses were from the Commissariat Ã l'Energie

CANCER RESEARCH VOL. 45 SEPTEMBER 1985

4229

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EFFECTS OF 9-OH-E ON SENSITIVE AND RESISTANT CELLS

Atomique, Saclay, France. [1-"C]9-OH-E (58 mCi/mmol), prepared by

Dr. Chenu, was kindly provided by Sanofi. All other chemicals were ofreagent grade and obtained from commercial sources.

Drug Exposure and Cell Survival. All the experiments were carriedout on exponentially growing cells.

The day before drug treatment, 60-mm-diameter Retri dishes (Falcon,Becton Dickinson) were seeded with either 4 x 105 DC-3F cells or 6 x105 DC-3F/9-OH-E 0.6 cells in 5 ml of MEM/FCS and incubated at 37Â°C

for 24 h in a 5% CO2-humidified atmosphere. The medium was then

replaced with 3 ml of MEM/FCS containing the drug at increasingconcentrations. After incubation for 3 h at 37Â°C, the drug was washed

off by rinsing the dishes once with 3 ml of MEM/FCS, and the cells weretrypsinized. For the determination of the colony-forming ability, about 2x 102 to 4 x 10* cells were plated in triplicate in 35-mm-diameter Retri

dishes containing 2 ml of MEM/FCS. DC-3F and DC-3F/9-OH-E 0.6

colonies were counted after 6 and 8 to 9 days, respectively. In theseconditions, the cloning efficiencies of the controls were about 50%.

Caffeine, Cycloheximide, and Hydroxyurea Treatments. To studythe effect of caffeine on 9-OH-E toxicity, the cells were incubated with 1rriM caffeine for 20 h after the 9-OH-E treatment. In these conditions,

caffeine did not display any significant toxicity on either one of these cell

lines.The effect of cycloheximide was examined in the following conditions.

Prior to 9-OH-E treatment, the cells in 60-mm-diameter Retri dishes wereincubated for 4 h at 37Â°Cin 3 ml of MEM/FCS containing cycloheximide

(0.4 /Â¿g/ml)-The medium was then replaced with 3 ml of MEM/FCScontaining cycloheximide (0.4 ^g/ml) and 9-OH-E at different concentrations. After incubation for 3 h at 37Â°C in this medium, the cell cloning

efficiency was determined. In other experiments, the 4-h incubation withcycloheximide alone was earned out after 3-h treatment with the 9-OH-

E/cydoheximide mixture.Similarly hydroxyurea was added to the cell growth medium for 4 h

prior to 9-OH-E and then for 3 h with 9-OH-E. The concentrations used

were 0.15 and 0.10 mw for the sensitive and resistant cells, respectively.These concentrations inhibit the DNA synthesis by 70% in each cell line

without any significant effect on protein synthesis.Incorporation of Radioactive Precursors into DNA, RNA, and Pro

teins. The labeled precursors were: [6-3H]thymidine (26 Ci/mmol; 1 fid/ml of cell suspension); [5,6-3H]uridine (37 Ci/mmol; 1 Â¿iCi/mlof cellsuspension); and o-[14C]leucine (54 mCi/mmol; 0.1 Â¿iCi/mlof cell suspen

sion). Sixteen-mm wells of 24-well Costar dishes (Costar, Cambridge,MA; No. 3524) were seeded with 1 x 10s cells in 2 ml of MEM/FCS andincubated in a humidified 5% CO2 atmosphere at 37Â°Cfor about 20 h.

The medium was then replaced with either 1 ml of fresh medium (controls)or 1 ml of medium containing the drug at the indicated concentrations.After 3 h of incubation at 37Â°C, the drug was washed off by rinsing the

cells twice with 1 ml of MEM. The cells were then incubated for 1 h with1 ml of medium containing the a-[14C]leucine and either the [3H]thymidine

or the [3H]uridine. After washing twice with 1 ml of MEM and trypsini-

zation, the cell suspension (1 ml) was mixed with 1 ml of cold 10% (w/v) trichloroacetic acid. The precipitate was collected on a 2.5-cm-diameter

glass microfiber filter (Whatman; GF/C) and washed 3 times with 2 ml ofcold 5% trichloroacetic acid and twice with 2 ml of ethanol. After drying,the filters were transferred to scintillation vials with 4.5 ml of PermafluorIII (Packard Instruments Co.), and the radioactivity was determined.

Flow Cytometry. For analysis of DNA content by flow cytometry, 60-mm-diameter Petri dishes, containing 5x105to2x106 cells, werewashed with 5 ml of PBS and incubated for 15 min at 37Â°C with cell

dispersal solution [PBS containing EDTA (0.5 mw) and trypsin (0.1 mg/ml)]. The cell suspension was then mixed in an equal volume of MEM/PCS and centrifuged. The pellet was washed with 5 ml of PBS. After

centrifugation, the pellet was resuspended in 10 to 15 ml of watercontaining RNase (1 mg/ml) (Worthington) and incubated at 37Â°Cfor 45

min. After centrifugation, the pellet of cell nuclei was washed with 5 ml

of PBS and finally resuspended in about 5 ml of 1.12% sodium citratesolution containing ethidium bromide (50 Â¿ig/ml).The shape and purityof the isolated stained nuclei were checked by phase contrast andfluorescence microscopy. DNA content analysis was carried out using a

Model 4800 Cytofluorograf flow cytometer (Bio/Physics Systems) coupled to a Model 2100 distribution analyzer (Bio/Physics Systems). Thedata were plotted using a Digital Mine 11/23 coupled to a digital plotter(Tektronix Model 4662).

In several experiments, DNA content analysis was also carried out asdescribed by Crissman et al. (15). Both techniques gave identical results.

On the different charts, only results of a typical experiment are shown.However, each experiment was carried out at least in duplicate. Therelative proportions in the different phases were determined accordingto the model of Dean and Jett (16). In order to analyze the profilecorresponding to the 3-h time on Chart 3, this model was adapted to

trimodal distribution analysis by Dr. J. B. Le Pecq and Dr. M. Le Bret.As an indication of the reproducibility of these experiments, the relativeproportions corresponding to this profile in two independent experimentswere: GÃ¬= 11.1 and 9.6%, S = 72.1 and 73.9%, and G2 plus M = 17.2

and 17.3%.Uptake Studies. Uptake of [1-14C]9-OH-E was determined after the

3-h treatment with the drug as previously described (14).

RESULTS

Variation of the Cellular Resistance Level with the DrugExposure Time. In previous works (10,17), the response of theparental DC-3F cells and of the 9-OH-E-resistant DC-3F/9-OH-E 0.6 subline to 9-OH-E and other ellipticine derivatives wasdetermined by either one of two methods: a 72-h assay procedure based on determination of cell number in control and drug-

treated cultures, which has been described in detail elsewhere(13,18); or the determination of the cell cloning efficiency in thepresence of increasing drug concentrations (17). The resistancelevel was then defined as the ratio of the ED50of the resistantcells over the dose which has the same effect on the sensitivecells. Both methods, which imply a prolonged exposure of thecells to the drug, yielded similar resistance levels of about 12- to15-fold. For technical reasons, based on uptake studies (14), the

time of drug exposure was reduced in the present work to 3 h.The cells were then plated in drug-free medium for determination

of the residual cloning efficiency. In these conditions, the apparent level of resistance increased about 100-fold. As shown on

Table 1, this unusual difference may be accounted for by thefact that the 9-OH-E ED50value on the sensitive line was constant

whether the cells were exposed to the drug for 3 h or cloned inthe presence of the drug. On the contrary, 9-OH-E toxicity on

Table 19-OH-Esensitivity of DC-3Fand DC-3F/9-OH-E0.6 cells at different drug

exposuretimesThe cell-cloningefficiency was determined either after a 3-h drug exposure or

in the presence of various drug concentrations (continuous). Each EDÂ»value isthe averageof n independentexperiments.

EDÂ»valueskg/ml)DC-3F

DC-3F/9-OH-E0.6Resistance level3h0.025

Â±0.008" (11)6

2.48 Â±0.33 (6)100Continuous0.024

Â±0.008 (4)0.34 Â±0.08 (4)

14" Mean Â±SO.* Numbers in parentheses,number of independentexperiments.


4230




the resistant cells was time dependent, with an ED.Â»value at 3h much higher than that obtained when the cells were cloned inthe presence of the drug.

Effect of 9-OH-E on Cell Survival and MacromolecularSyntheses. The effects of 9-OH-E on DMA and protein synthesesin sensitive and resistant cells were determined after a 3-h

exposure to the drug at different concentrations. Chart 1 showsthat, in the sensitive DC-3F cells, DNA, RNA, and protein

syntheses, as judged by the inhibition of incorporation of theprecursors into acid-insoluble material, were arrested at about

the same doses. All these syntheses were 50% inhibited in thepresence of 9-OH-E at about 4 x 10~6 M. In contrast, 50%

inhibition of the cloning efficiency was observed at a drug concentration 80- to 100-fold lower (4 to 5 x 10"8 M), indicating that

induction of lethal lesions by 9-OH-E in this cell line occurred

independently of its effect on macromolecular metabolism.In resistant cells, DNA, RNA, and protein syntheses were 50%

inhibited at doses very close to that observed in the parentalsensitive line (2.5, 3, and 6 x 10"6 M, respectively). This is

consistent with the fact that the cellular accumulation of ellipticinederivatives is identical in both sensitive and resistant cells (14).However, in this subline, the cloning efficiency was inhibited onlyat doses similar to those required to inhibit macromolecularsyntheses. This suggested that 9-OH-E cytotoxicity in the re

sistant cells might be related to the inhibition of these syntheses.Effect of Caffeine on the Toxicity and Cell Cycle Progres-

100

BO

ZOu

uce

SO

H h H J-

10- 10-"

9-OH-E [M]

10-

Chart 1. Effect of 9-OH-E on Å“il survival and macromolecular syntheses as afunction of drug concentration in sensitive (A) and resistant (B) cells. After 9-OH-Etreatment, the cell survival was measured by cloning efficiency determination andmacromolecular syntheses by incorporation for 1 h of Â«-[14C]leucine(54 mCi/mmol,1 >iCi/ml) and either [6-3H]thymidine (26 Ci/mmol. 1 fiCi/ml) or [5,6-3H]uridine (37

Ci/mmol, 1 (iCi/ml). Each point represents an average of 6 independent experiments: O, cell survival; â€¢,uridine; Ã‰,leucine, A, thymidine.

sion Effects of 9-OH-E. In mammalian cells, caffeine was shown

to potentiate the toxicity of a number of toxic agents. Amongthese agents are a variety of alkylating agents (19). To explainthis caffeine-potentiated lethality, it was recently proposed (20)

that caffeine prevents the G2arrest induced by these drugs, thusallowing the cells to divide without finishing the repair of the DNAlesions. Intercalating agents, such as actinomycin D, anthracy-

clines, and ellipticine, also induce a G2 arrest of the cell cycleprogression (12, 21, 22), and caffeine was shown to potentiatethe ellipticine toxicity in L1210 cells (23). We then examined theeffect of caffeine on the toxicity and cell cycle progression effectsof 9-OH-E on both sensitive and resistant cells.

After treatment for 3 h with various concentrations of 9-OH-E, DC-3F and DC-3F/9-OH-E 0.6 cells were incubated for 20 h

with 1 mu caffeine. Caffeine, which has no effect in theseconditions on the viability of our cell lines, did not change thetoxicity of 9-OH-E on either of them. Longer treatment or con

centrations higher than 1 ITIMrevealed caffeine toxicity, especiallyon the DC-3F/9-OH-E 0.6 subline (result not shown).

Chart 2 shows the effects of 9-OH-E and caffeine, either

separately or in combination, on the cell cycle progression of thesensitive cells. Caffeine by itself, at 1 ITIMfor 20 h, has no effecton the rate of cell progression through the different phases ofthe cycle. In contrast, at a 0.025-Â¿tg/mlconcentration of 9-OH-Efor 3 h, corresponding to about 50% cloning inhibition, DC-3F

cells accumulated in the G2 plus M phases by about 5.5 h. By20 h, cells appeared to have passed through mitosis into GÃ¬,asindicated by the increased amount of G, cells relative to the 5.5-

h sample. After 48 h, the distribution was that expected for cellsin normal asynchronous growth. When caffeine was added at 1ITIMfor 20 h at the end of the 9-OH-E treatment, a large fraction

of the cell population was already in G1 at 5.5 h, and the profilewas back to normal at 20 h. This demonstrates that caffeine,although it has no effect on 9-OH-E toxicity in DC-3F cells, is

able to release the G, arrest induced by this drug.The profile at 5.5 h after 9-OH-E treatment also showed a

shoulder on the G2 peak, suggesting that other cell cycle alterations might have been provoked by the drug. We then furtherinvestigated the effects of 9-OH-E and caffeine on the DC-3Fcell cycle progression during the next 5 h after 9-OH-E treatment.

Chart 3 shows that, during this period of time, most of the cellswhich were in G, at the time of drug exposure migrate synchronously, as a discrete peak, through S phase. This indicates that,in addition to the G2 block, 9-OH-E is also arresting the cells

somewhere in G,. However, whereas the G2 block remainseffective for several hours after removal of the drug, the GÃ¬blockis rapidly released, thus accounting for the synchronous migration of the cells from G! through S phase. Comparison on Chart3 of the relative amount of cells in S phase, at 2 or 3 h after 9-OH-E treatment, also shows that caffeine is able to release this

G, block as well as the G2 block.Chart 4 shows that, when the resistant cells are treated for 3

h with 9-OH-E (3 M9/ml) (50% cloning inhibition), a G2 arrest wasalso observed, but only after a 48-h delay. This G2 block was

maintained after 72 h and slowly reversed by 96 h, being stilldetectable at 120 and 144 h after treatment (profiles not shown).In these cells, caffeine had no significant effect on the G2 blockwhen it was added during the next 20 h after 9-OH-E incubation.

However, when caffeine was added at the beginning of the G2block (from 40 to 60 h after 9-OH-E treatment), this block was


4231




Ohr 5.5hr 20hr 48hr 72hr

C

Caff

9-OH-E

9 OHE

Caff

eUJm

LUO

DNA CONTENTChart 2. Effect of 9-OH-E and caffeine (Caff) on cell cycle progression of the sensitive cells. Flow cytometric analysis of the ethidium bromide-stained cell nuclei was

carried out at different times after drug treatment as described in "Materials and Methods." Time 0 h corresponds to the end of treatment with 9-OH-E. C, control cells;

C + Caff, cells incubated with 1 RIM caffeine from 0 to 20 h; 9-OH-E, cells treated for 3 h with 9-OH-E (0.025 (ig/ml); 9-OH-E + Caff, cells treated for 3 h with 9-OH-E(0.025 Â¿ig/ml)and then incubated with 1 ITIMcaffeine for 20 h.

Ohr 1hr 2hr 3hr 4hr 5hr

9 OH E

9 OH E

Caff

KtuCO

DZ

U

DNA CONTENT

Chart 3. Early cell cycle progression of sensitive cells after 9-OH-E treatment. DNA histograms were obtained by measuring ethidium bromide (50 ^g/ml) fluorescenceof stained nuclei with flow cytometry at the indicated times. Time 0 h corresponds to the end of treatment with 9-OH-E. C, control cells; 9-OH-E, cells treated for 3 hwith 9-OH-E (0.025 ng/m\); 9-OH-E + Caff, cells treated for 3 h with 9-OH-E (0.025 /ig/ml) and then incubated with 1 row caffeine until the end of experiment (5 h).

released (Chart 4, inset).Chart 5 shows the growth kinetics of the sensitive and resist

ant cells after 9-OH-E treatment. Although the conditions were

defined to have the same cytotoxicity on both lines, the curveswere markedly different. The sensitive cells resumed their growthafter a mitotic delay of about 6 h, whereas the resistant cells


4232




Ohr 5.5 hr 20hr 48 hr 96 hr 168 hr

C

Caff

9 OHE

9 OHE

Caff I A

ocUJco

60hr 90hr

DNA CONTENT

Chart 4. Effect of 9-OH-E and caffeine (Caff) on cell cycle progression of the resistant cells. DNA histograms were obtained by measuring ethidium bromide (50 >ig/ml) fluorescence of stained nuclei with flow cytometry at the indicated times. Time 0 h corresponds to the end of treatment with 9-OH-E. C, control cells; C + Caff, cellsincubated with 1 mm caffeine from 0 to 20 h; 9-OH-E. cells treated for 3 h with 9-OH-E (3 ^g/ml); 9-OH-E + Caff, cells treated for 3 h with 9-OH-E (3 M9/ml) and thenincubated with 1 mw caffeine for 20 h. Lower inset, cells treated for 3 h with 9-OH-E (3 ng/ml), incubated with fresh medium for the next 40 h, and then incubated with1 mw caffeine from 40 to 60 h.

50 100

TIME (hr)

50 100

Charts. Growth curves of sensitive (A) and resistant (B) cells treated with 9-OH-E. At time 0, cells were treated for 3 h with 9-OH-E, and the drug containing

medium was replaced by fresh medium. The cells were then counted at theindicated times. A, â€¢,control cells; O, 9-OH-E (0.025 ,,g/ml); A, 9-OH-E (0.050 Mg/ml). B, â€¢,control cells; O, 9-OH-E (3 Â»ig/ml).

remained arrested for about 48 h. In the presence of caffeine,this 9-OH-E-induced mitotic delay was almost abolished in the

sensitive cells and reduced to about 40 h in the resistant cells(results not shown). This growth arrest of the resistant cells wasconfirmed by determination of the macromolecular syntheses atthe different times after 9-OH-E treatment. During the first 20 h,

DNA and protein syntheses were 80 to 90% inhibited, and RNAsynthesis was about 50% inhibited. All these syntheses onlybegan to resume about 30 hr after 9-OH-E treatment and re

turned to an almost normal level by 50 h, which is consistentwith the growth curve shown on Chart 5.

The growth inhibition then explains the arrest of cell cycleprogression and the lack of G2 accumulation which is observedin the resistant cells during the first 30 h ater 9-OH-E treatment.

The G2 block is only developing when the cells resume theirgrowth and becomes evident 48 h after drug treatment. Caffeineis only efficient in releasing this block when it is added on thegrowing cells but has no effect when present prior to growthresumption.

Effect of Cycloheximide on the Toxicity and Cell CycleProgression Effects of 9-OH-E. In order to determine whetherthe 9-OH-E toxicity would rely on the presence or the induction

of a peculiar protein, we examined the effect of cycloheximide,which according to its mechanism of action is a specific proteinsynthesis inhibitor, on the 9-OH-E toxicity in both sensitive and


4233




resistant cells. Cycloheximide was used at a concentration of0.4 fiQ/m\ for 7 h. In these conditions, cycloheximide inhibitedprotein synthesis by about 80% and DNA synthesis by about70% on both cell lines, without any cytotoxic effects. Cycloheximide was first present for 4 h in the growth medium before theaddition of 9-OH-E and was maintained for 3 h in the presenceof 9-OH-E. Chart 6 shows that, in the presence of cycloheximide,the 9-OH-E toxicity on the sensitive cells was about 2-fold

decreased. In contrast, an increased toxicity, with an ED50valueabout 40% lower, was observed on the resistant cells.

We examined whether changes in the cellular accumulation ofthe drug could account for these cycloheximide-induced modifications of 9-OH-E toxicity. Table 2 shows that the cycloheximide

treatment did not provoke any significant change of the cellularaccumulation of 9-OH-E in the sensitive cells. A less then 10%decrease of the uptake by cycloheximide-treated cells is ob

served in the resistant subline, which obviously cannot accountfor the increase of 9-OH-E toxicity on these cells.

In an alternative schedule, cycloheximide was also added for3 h with 9-OH-E followed, after 9-OH-E removal, by an additional

incubation for 4 h. In these conditions, cycloheximide had noeffect on 9-OH-E toxicity on either cell line.

Therefore cycloheximide is effective in modifying 9-OH-E tox

icity on both sensitive and resistant cells only when it is addedbefore and with the drug. The increased toxicity observed onthe resistant cells might result from a potentiation of the inhibitoryeffect of 9-OH-E on protein synthesis. However, in the reverseschedule (cycloheximide with and after 9-OH-E), the multipleeffects of 9-OH-E alone on macromolecule metabolism do not

allow a precise interpretation of the absence of potentiationobserved in the resistant cells. In the sensitive cells, the results

100

-i 10

I + CIM

+ CIM

0.1 0.2 0.3 0.4 1 2 49-OH-E [|ig /ml]

Charte. Effect of cycloheximide (CHM) on 9-OH-E toxicity in sensitive (A) andresistant (B) cells. Prior to 9-OH-E treatment, the cells were incubated for 4 h withcycloheximide (0.4 //g/ml). This medium was then replaced with medium containingcycloheximide (0.4 jig/ml) and 9-OH-E at different concentrations. After 3-h incubation in this medium, cell survival was measured by cloning efficiency determination. â€¢and â€¢9-OH-E-treated cells without cycloheximide; O and D, 9-OH-E/cycloheximide-treated cells.

Table 29-OH-E uptake in cells treated with cycloheximide

Uptake (nM/KPceHsf

DC-3F DC-3F/9-OH-E 0.6

9-OH-E9-OH-E + cycloheximide

1.12 Â±0.03"1.11 Â±0.04

37.4 Â±0.7431.7 Â±1.24

" Cells were treated as described in the legend to Chart 6 using [1-14C]9-OH-E

at concentrations of 0.4 and 8 Â«Â¿g/mlfor sensitive and resistant cells, respectively.b Mean Â±SD (3 independent experiments).

indicate that 9-OH-E toxicity does not involve the induction of

protein(s) but rather relies on protein(s) which have to be presentin the cell at the time of drug exposure.

We also analyzed the effect of cycloheximide on the cell cyclealterations provoked by 9-OH-E. Cycloheximide was used, asdescribed above, 4 h before and 3 h with 9-OH-E. Chart 7 shows

that cycloheximide by itself, which is known to delay the entryinto S phase (24), provoked in the sensitive cells a slight GÃ¬synchronization, demonstrated by the appearance of a smalldiscrete peak in S phase at 4.5 h after removal of the drugs. Incells treated with both cycloheximide and 9-OH-E, the macro-

molecular syntheses were 80 to 90% inhibited at the end of thedrug exposure time, and 5 h later, DNA synthesis remainedabout 60% inhibited. This inhibition accounts for the apparentrelease of 9-OH-E-induced d and G2 blocks observed in Chart7. By 48 h after incubation with either 9-OH-E alone or thecombination of cycloheximide and 9-OH-E, the cell cycle distri

bution was back to normal. The same experiment was alsocarried out on the resistant cells. As expected, since the cellgrowth and macromolecular metabolism were already arrestedby 9-OH-E alone (Chart 5), cycloheximide had no effect on the

cell cycle distribution of the resistant cells after treatment with9-OH-E (data not shown).

Effect of Hydroxyurea on the Cytotoxicity of 9-OH-E. Asmentioned above, in our experimental conditions, the protein

Ohr 4.5hr 21hr

C

CHM

9-OH-E

9 OH E+

CHM

ocLU00

3Z

DNA CONTENT

Chart 7. Effect of 9-OH-E and cycloheximide (CHM) on cell cycle progressionof sensitive cells. DNA histograms were obtained by measuring ethidium bromide(50 Â¿ig/ml)fluorescence of stained nuclei with flow cytometry at the indicated times.Time 0 h corresponds to the end of the treatment with the drugs. C, control cells;C + CHM, cells treated for 7 h with cycloheximide (0.4 jig/ml); 9-OH-E, cells treatedfor 3 h with 9-OH-E (0.025 jig/ml); 9-OH-E + CHM, cells treated for 4 h withcycloheximide (0.4 ng/ml) and then incubated for 3 h with medium containing 9-OH-E (0.025 Mg/ml) and cycloheximide (0.4 M9/ml).


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synthesis inhibition by cycloheximide was associated with a 70%DMA synthesis inhibition. In order to ascertain the effects ofcycloheximide on 9-OH-E toxicity to the protein synthesis inhibition, we examined the effect of hydroxyurea, a specific DNAsynthesis inhibitor, on 9-OH-E toxicity. Hydroxyurea was usedfor 7 h (4 h before and 3 h with 9-OH-E) at concentrations

resulting in a 70% inhibition of DNA synthesis in both sensitiveand resistant cells, without any detectable toxicity. Chart 8shows that, in these conditions, hydroxyurea had no effect on9-OH-E cytotoxicity on either sensitive or resistant cells. Thisresult demonstrates that the effects of cycloheximide on 9-OH-

E toxicity are specifically related to the inhibition of proteinsynthesis. It should also be noticed that, contrasting with ourdata, Minford ef al. (25) recently showed that hydroxyurea canpotentiate the cytotoxicity of the DNA intercalator 4'-(9-acri-

dinylamino)methanesulfon-m-anisidide. The reasons for this dis

crepancy are presently unclear; it reflects the differences in eitherthe experimental conditions or the properties of the intercalator.

DISCUSSION

To gain an understanding of its mechanism of action, we havecompared the effects of the DNA-intercalating antitumor agent9-OH-E on two cell lines, either sensitive or resistant to this drug.

Beyond about 2 h, the 9-OH-E toxicity on the sensitive cells

was found to be independent of the drug exposure time (Table1). This observation suggested that, in this cell line, a lowintracellular concentration was able to induce enough lethallesions to kill the cells, so that subsequent exposure to the drugwould not have any additional toxic effects. Furthermore theexperiment on Chart 1 shows that the induction of these lesionsoccurs independently of any detectable effect of the drug onmacromolecular syntheses. On the contrary, in the resistant cells,the 9-OH-E toxicity, which only appears concomitantly with the

inhibition of these syntheses, was time dependent. From theseresults, we conclude that 9-OH-E toxicity on sensitive and re

sistant cells involves different mechanisms of action. The lethal

SO 100(tig/ml)

0.1

S-OH-C

-1- i

Chart 8. Effect of hydroxyurea on 9-OH-E toxicity in sensitive (/eft) and resistant(fight) cells. Prior to 9-OH-E treatment, the cells were incubated for 4 h with 0.15mM (sensitive cells) or 0.1 mw (resistant cells) hydroxyurea. This medium was thenreplaced with medium containing hydroxyurea at the same concentrations and 9-OH-E at different concentrations. After 3-h incubation in this medium, cell survivalwas measured by cloning efficiency determination. â€¢and â€¢9-OH-E-treated cellswithout hydroxyurea; O and G, 9-OH-E/hydroxyurea-treated cells.

lesions in the sensitive line would be mediated through theinteraction of the drug with a specific target. In the resistantcells, this target would be modified so that it is no longerrecognized by the drug, and the cell death would result from theinhibition of macromolecular syntheses.

Detailed analysis of the effects of 9-OH-E on cell cycle pro

gression is also in agreement with the existence of differentmechanisms of action on the sensitive and resistant cells. In thesensitive cells, 9-OH-E is inducing both a d and a G2 arrest of

the cell cycle traverse. However, whereas the GÃ¬block is veryrapidly released after removal of the drug, the G2 block ismaintained for several hours after drug treatment, and, althoughduring this period DNA synthesis was not inhibited (Charts 1 and3), a 5- to 7-h mitotic delay was observed (Chart 5). The physiological significance of these G-, and G2 blocks, especially with

regard to an eventual protective role against drug toxicity, remains presently unclear. Their release in the presence of caffeinedoes not increase the 9-OH-E toxicity. This is in contrast with

previously reported results on L1210 cells, where ellipticinetoxicity was increased in the presence of caffeine (23). We alsoobserved this effect of caffeine on ellipticine toxicity in L1210cells, but caffeine neither changed the toxicity of 9-OH-E inL1210 cells nor that of ellipticine in DC-3F cells (data not shown).

Therefore, whereas the G2 block is constantly observed, theeffect of caffeine on the toxicity of ellipticine derivatives appearsto depend on which derivatives and which cell lines are used.

In the resistant cells, the 9-OH-E-induced G2 block was only

observed more than 40 h after the end of drug treatment andlasted for more than 50 h (Chart 4). During the next 20 h after9-OH-E exposure, the macromolecular syntheses were arrested,

thus preventing the cell cycle progression and hence the G2block. Therefore the G2 arrest signal, acquired by the resistantcells during the drug exposure time, was maintained by the cellsduring the posttreatment growth inhibition period and only became effective after growth resumption. In this case too, thecaffeine-induced release of the G2 block had no effect on 9-OH-

E toxicity. Most cytotoxic agents, which are producing theirbiological effects through interaction with DNA, provoke a G2arrest of the cell cycle progression, the release of which bycaffeine is associated with an increased drug toxicity. Accordingto current hypotheses (19, 20), the absence of such an effect in9-OH-E-treated cells suggests that the lesions produced by thisdrug in DC-3F cells might be insensitive to the cellular DNA repair

processes.Cycloheximide exerts an inhibitory effect on 9-OH-E toxicity in

the sensitive cells, which is specifically observed when cycloheximide is added to the growth medium before and during 9-OH-E

treatment. This effect can be attributed to the inhibition of proteinsynthesis, since specific DNA synthesis inhibition by hydroxyureahas no effect on 9-OH-E toxicity. These results show that theintracellular target recognized by 9-OH-E and responsible for its

toxicity in the sensitive cells is a protein. This protein is notinduced as a consequence of the drug treatment but has to bepresent in the cells when the drug is added.

Treatment of mammalian cells with a variety of DNA-interca

lating agents results in the formation of DNA strand breaks thathave been postulated to result from the action of a DNA topoi-

somerase (26, 27). Recently, using purified mammalian DNAtopoisomerase II, Tewey ef a/. (28) demonstrated that ellipticineand 2-CH3-9-OH-ellipticinium can produce reversible protein-


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linked DMA strand breaks. These authors then proposed thatthe mechanism of DNA breakage induced by these molecules islikely to be due to the drug stabilization of a cleavable complexbetween DNA and topoisomerase II. A similar mechanism ofaction was proposed by Pommier ef al. (29) for 4'-{9-acridinyla-

mino)methanesulfon-m-anisidide, another DMA-intercalatingagent to which 9-OH-E-resistant cells are cross-resistant. Type

II DNA topoisomerase then appears as a possible target for invivo toxicity of 9-OH-E. Cellular resistance to this drug wouldthen involve a qualitative and/or quantitative alteration of this

enzyme.

ACKNOWLEDGMENTS

The expert technical assistance of C. Delaporte and S. GuÃ©rineauis gratefullyacknowledged.

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effects of 9-oh-ellipticine on cell survival ... · macromolecular syntheses, and cell cycle...

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