[CANCER RESEARCH 43, 3074-3079, July 1983]

Uptake, Initial Effects, and Chemotherapeutic Efficacy of Harringtonine

in Murine Leukemic Cells Sensitive and Resistant to Vincristine andOther Chemotherapeutic Agents1

Ting-Chao Chou,2 Franz A. Schmid, Aaron Feinberg, Frederick S. Philips, and Jui Han3

Memorial Stoan-Kettering Cancer Center. New York 10021 [T-C. C., F. A. S., A. F., F. S. P.], and Mt. Sinai School of Medicine, New York 10029, New York [J. H.]

ABSTRACT

[3H]Harringtonine was shown to be taken up rapidly by L1210/

0 cells using a fast-mixing, fast-separating technique and was

retained with a slow rate of limited release to the medium. Cellsresistant to vincristine (L1210/VCR) showed impaired capabilityto take up the drug at 20°.Its initial uptake in L1210 sublines in

vitro was: L1210/0 > L1210/cyclophosphamide, L1210/1-/8-D-arabinofuranosylcytosine, L1210/6-mercaptopurine > L1210/5-fluorouracil, L1210/Adriamycin > L1210/VCR. In [3H]hamngton-

ine-preloaded cells, L1210/0 retained significantly more radio

activity than did L1210/VCR cells after repeated washing withfresh medium at 37°.The radioactivity appeared to be predominantly bound to the microsomal fractions. [3H]Leucine incorpo

ration into protein in L1210/0 cells was inhibited 90% within 15min by harringtonine (0.5 ^g/ml); incorporation of [3H]thymidineinto DNA and [3H]cytidine into RNA was much less inhibited and

showed an apparent lag of onset for 5 and 10 min, respectively.The relative potency of harringtonine to inhibit [3H]leucine incor

poration in the above sublines in vitro follows an order similar totheir rates of uptake of harringtonine by these sublines of cells.The efficacy of harringtonine, 2.4 or 3.6 mg/kg i.p., in increasingthe life span of C57BL/6 x DBA/2 F, mice bearing the sublinesof teukemic cells, on the average, was: L1210/0 > L1210/cyclophosphamide, L1210/6-mercaptopurine > L1210/1-/8-D-ar-abinofuranosylcytosine, L1210/5-fluorouracil > L1210/Adriamy-

cin, L1210/VCR. These results suggest that: (a) protein synthesisis the major initial target for the effect of harringtonine; (£>)harringtonine bound more tightly to the cellular components ofVCR-sensitive leukemic cells than to VCR-resistant cells; and (c)

cellular uptake of harringtonine and the relative potency of inhibiting protein synthesis in sublines have a rank order similar tothe Chemotherapeutic efficacy of harringtonine in these cells.

INTRODUCTION

Powell et al. (18, 19) reported that they had isolated severalalkaloids from Cephalotaxus harringtonia and that some of thesealkaloids showed significant activity in experimental tumors (17).These findings are consistent with the beneficial effects of Ce-

phalotaxus fortune! Hook F in the treatment of human malignanttumors that had been described by folk medicine practitioners inFujian Province, China. A systematic study was carried out inChina which has led to a chemotaxonomical-botanical survey,

1This study was supported by Grants CA 18856 and CA 27569 from the

National Cancer Institute, NIH, USPHS, and from the Elsa U. Pardee Foundation.The preliminary results of this study have been reported (8).

2To whom requests for reprints should be addressed.3On leave of absence from the Institute of Materia Medica, Chinese Academy

of Medical Sciences, Beijing. China.Received June 28, 1982; accepted March 18, 1983.

isolation and purification of active principles, chemical partialsynthesis, and toxicological, pharmacological, and clinical studies(4-6,10-13,16,23). Harringtonine (Chart 1), an active ingredient

of the alkaloid isolated from the bark of Cephalotaxus hainanensisLi plants that are indigenous to a Hainan island of southernChina, has been used in clinical trials. It was shown that harringtonine, as a single agent, is effective in treating acute granulocyticleukemia which resulted in 28% complete and 55% partial remissions. It was also effective in treating chronic granulocytic leukemia, erythroleukemia, and myelomonocytic leukemia (4-6,12).Further clinical trials of harringtonine or its analogue, homohar-

ringtonine, in China and in the United States (Baltimore and NewYork) are in progress.

Early pharmacological studies on harringtonine were carriedout in vivo mostly and reported mainly in journals written inChinese (10,12,16,23). Radioautographic studies showed thatharringtonine lowered the dThd4 labeling and mitotic indices of

L1210 cells. Fluorescence cytophotometric determination (22)indicated that harringtonine decreased S-phase cells and in

creased the proportion of cells in Gì.The DNA contents of thesecells were lowered, and karyorrhexis was observed. Harringtonine appeared to be an antileukemic agent which strongly inhibitedthe traverses from d to S phase and from G2 to M phase. Italso strongly inhibited the cellular incorporation of [3H]asparagineand [3H]deoxythymidine as shown by autoradiographic studies

(11).The biochemical effects of harringtonine on HeLa cells and

rabbit reticulocytes have been described by Huang (14). It wasshown that harringtonine induced breakdown of polyribosomesto monosomes with concomitant release of completed globinchains. Evidence was presented that the principal effect ofharringtonine was on the initiation of protein synthesis (14). Sinceharringtonine is an effective antitumor agent, the present studyexamines its uptake retention and initial biochemical effects inmurine leukemic cells. Different sublines of L1210 leukemia cellsresistant to various Chemotherapeutic agents are used to testthe relative susceptibility of these cells to the inhibitory effectsof harringtonine on macromolecular synthesis. An attempt hasbeen made to correlate the uptake, binding, and biochemicaleffects of harringtonine in vitro to the Chemotherapeutic effectsof harringtonine in vivo.

MATERIALS AND METHODS

Chemicals and Radiochemicals. Harringtonine was isolated andpurified from C. hainanensis Li and was supplied by the Institute of

4The abbreviations used are: ara-C, 1-/3-o-arabinofuranosylcytosine; 5-FUra, 5-

fluorouracil; 6-MP, 6-mercaptopurine; CPA, cyclophosphamide: ADR, Adriamycin;VCR, vincristine. The cell lines postscribed by the above compounds represent thecells that are chemotherapeutically resistant to these compounds.

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Harringtonine Uptake and Effects of LI210 Sublines

OCH3

OH OH 0R=CH3-C-CH2-CH2-C-CH2-C-OCH3

CH3 C02~

Chart 1. Chemical structure of harringtonine.

Materia Medica, Chinese Academy of Medicinal Sciences, Beijini China.[3H]Harringtonine (0.92 Ci/mmol) was synthesized by the same istitute6

and was purified in the present laboratory by high-pressure liq*:id chro-matography using a Model 8000 Spectra-Physics instrument, empiete

with data system, and a Model SP 8400 variable wavelength detector(Spectra-Physics, Piscataway, N. J.). An ODS-3, 5-pm particle column

was used together with a guard column packed with Copell (WhatmanLaboratory Products, Inc., Clifton, N. J.). Samples were injected with asyringe into a 100-¿illoop attached to a Valco valve (Glenco, Houston,Texas). Peaks were monitored at 245 nm. [3H]Harringtonine was elutedat 45° with a gradient using 0.1 M formate (pH 6.7):methanol. The

gradient was 25 min from 20% methanol to 50% methanol (v/v) followedby isocratic elution at 50%. The retention time for harringtonine with thissystem was 21.0 min. The fractions containing the radioactivity peakthat had the same retention time as harringtonine were combined andrepurified at 45° using the same solvents but with a gradient of 25%

methanol to 40% methanol over 25 min. The compound then had aretention time of 18.5 min. The radioactive fractions were combined andevaporated to dryness. A minute amount (30,000 cpm) was reinjectedonto the high-pressure liquid chromatography with unlabeled harringtonine. A 25-min gradient of 30% methanol to 40% methanol in the 0.1 M

formate was used. The labeled compound was 99% pure. The totalpurity of the tritiated harringtonine from the original samples was approximately 20%. The UV spectra of [3H]harringtonine and unlabeled

harringtonine both gave Xâ„¢,at 242 and 288 nm and Xâ„¢,at 263 nm atpH 7.0 with < = 2153 at 288 nm. [mef/7y/-3H]dThd (2.0 Ci/mmol), [5-3H]-cytidine (26.8 Ci/mmol), i_-[4,5-3H]leucine (51.6 Ci/mmol), [G-3H]inulin

(100 mCi/g), and 3H2O (1.32 /iCi/ml) were purchased from New England

Nuclear, Boston, Mass. Radioactivity was determined with a PackardTri-Carb Model 3775 liquid scintillation spectrometer using Liquiscintscintillation fluid (National Diagnostics, Somerville, N. J.). Internal standard ratio and the fixed amount of 3H2O were used to correct the

quenching of tritium counts by samples. ara-C, 5-FUra, 6-MP, CPA, and

ADR were obtained from the Research and Development Program,Division of Cancer Treatment, National Cancer Institute. Vincristine sulfate was obtained from Eli Lilly & Co., Indianapolis, Ind.

Leukemic Cell Lines. L1210/0, its sublines resistant to various chem-

otherapeutic agents (see below), and P815/0 leukemia were maintainedby passage in C57BL/6 x DBA/2 (hereafter called BD2F!) mice. Approximately 1 x 106 cells were inoculated i.p. every 6 days for each passage.

For in vitro studies, Day 5 ascites cells were collected and used forexperiments. The resistant cell lines were derived by treating BD2F,mice bearing L1210/0 leukemia with a sublethal dose of ara-C, 5-FUra,

5 Institute of Materia Medica, Chinese Academy of Medicinal Sciences, Beijing,

China, unpublished results.

CPA, 6-MP, ADR, or VCR i.p. for 2 to 20 treatment cycles as specified

below in Table 1. The cell lines resistant to each chemotherapeutic agentwere maintained by giving a sublethal dose of each agent on Day 1 oron Days 1 and 2. P815/VCR cells were developed from P815/0 cellsand maintained in the same manner as were L1210/VCR cells. The invivo treatment was discontinued at least 1 week prior to the use of theseresistant cell lines.

Assays of Precursor Incorporation. The incubation mixture wasmade in Eagle's basal medium (1 ml) containing 10% fetal calf serum, 2

mM L-glutamine, and leukemic cells. The mixtures were incubated at 37°

for a specified time interval. The reaction was terminated by adding 3 mlof cold 10% perchloric acid. Incorporations of [3H]cytidine into RNA,[3H]dThd into DNA, and [3H]leucine into protein in acid-insoluble fractions

were measured with a method described previously (7), except that theacid-insoluble fraction that was resistant to hot acid (10% trichloroacetic

acid) digestion is referred to here as the protein fraction.Uptake and Release of [3H]Harringtonine. A rapid mixing and a rapid

separation technique described by Wohlhueter ef a/. (26) was used foranalyzing uptake of [3H]harringtonine by L1210/0, P815/0, and theirsublines of leukemic cells. The cell suspension was mixed with [3H]-harringtonine at 20° and 2° using the double-syringe apparatus as

specified by Wohlhueter ef al. (26). At specified time intervals, themixtures were dispensed directly into 12 tubes mounted in an EppendorfModel 5412 microcentrifuge and containing a mixture of mineral oil(Sigma Chemical Co., St. Louis, Mo.) and silicone oil (J. T. Baker ChemicalCo., Phillipsburg, N. J.) which had a final density of 1.032 g/ml. Micro-

centrifugation attained 12,000 x g within 5 sec and was tunned off after30 sec. The cell-free medium in supernatant was aspirated, and 0.1 ml

was counted for radioactivity and set aside for later use for calculatingextracellular water space in the cell pellet. To wash off residual radioactivity above the oil layer, 1 ml of water was layered over the oil and wasthen removed and discarded. The radioactivity in the cell pack, usually 6to 10 x 107 cells, was measured for the uptake of [3H]harringtonine (the

residual oil in the cell pack contained no radioactivity). The values werecorrected for substrate trapped in the extracellular water space in thecell pellet which was estimated by the use of [G-3H]inulin. The extracel

lular water space represented 15 to 20% of the total water space in thepellet which was estimated by the use of 3H2O. The net uptake of [3H]-harringtonine was obtained by subtracting 2°values from 20°values.

The number of cells was counted by hemocytometer. In some experiments, mixing of cells and [3H]harringtonine was carried out with a

Vortex mixer and immediately dispensed onto the top of the oil layerwith a Finnpipette prior to microcentrifugation at specific times aftermixing. Comparable results were obtained with either the Vortex-Finn-pipette or double-syringe procedure; however, the uptake within 12 sec

could not be measured by the Vortex procedure because of the timerequired for mixing and pipetting. The release of [3H]harringtonine fromcells was carried out by preloading the cell with [3H]harringtonine at 20°

for 30 min and centrifuging to collect the cell pellet. The pellet was thenrapidly suspended into 100 volumes of Eagle's basal medium at 20°and

pipeted with a Finnpipette at the specified intervals onto the top of theoil layer for centrifugation as described above. The radioactivity remainingin the cell pack was then measured. The binding of [3H]harringtonine tocellular components was carried out by preincubating the cell with [3H],harringtonine, 1 fig and 0.4 jiCi/ml, at 37°for 30 min. After centrifugation

at 650 x g for 5 min, the cell pack was resuspended in fresh mediumand incubated at 37°for 10 min. The incubation-centrifugation-resuspen-

sion washing procedure was repeated several times as specified. Insome experiments, the labeled washed cell pack was homogenized in10 volumes of 0.25 M sucrose with a Potter-Elvehjem homogenizer, and

the subcellular fractions were obtained by centrifugation as described byShibkoefa/. (21).

RESULTS

Uptake and Release of [3H]Harringtonme. The net uptake of

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T-C. Chou et al.

Table 1

Development and maintenance of U 210 sublinesAll leukemia lines were maintained in passage in BD2F, mice. Approximately 10e cells were injected i.p.

every 6 days for each passage.

Development of resistant sublines Maintenance of resistantsublines

TreatmentL1

21O/ara-CL1210/5-FUraU 210/VCRL1210/CPAL1210/ADRL1210/6-MPCells

i.p.on Day099,9,9,9,9,mg/kgi.p.60

280.25

2007.5 or 15

56Day1-6

1-61-61

1 or 21-6Treatment

cycles until resist

ance5

68

1020

2TreatmentCells

i.p.on Day09,9,999,9,mg/kg¡•P.60

281

2001556Day1,2

1.21111,2

is r L12IO/O release

LI2IO/O uptake

LI2IO/VCR uptake

MinutesChart 2. Initial uptake and release of [3H]hamngtonine by L1210/0 and L1210/

VCR cells. Methods for measuring uptake and release are described in "Materialsand Methods." Each aliquot of incubation mixture contained 62 x 10" cells and0.02 jiCi, 0.1 »ig,of [3H]harringtonine in Eagle's basal medium. Experiments werecarried out at 20°and 2°.Data obtained at 2°were used as blanks and weresubtracted from the 20°values. The uptake of [3H]harringtonine by L1210/0 (•)and L1210/VCR (A) are shown. Release of preloaded [3H]harringtonine from L1210/0 cells was measured at 20°(D). A slow rate of release, similar to those of L1210/

0 cells, was observed in L1210/VCR cells.

[3H]harringtonine ¡nL1210/0 and its resistant subline, L1210/

VCR, is given in Chart 2. L1210/VCR cells had severely impairedcapability to take up harringtonine at 20°when compared with

the parent cells. The uptake in both cell lines was temperaturedependent; at 2°,uptake was about 200 cpm/108 cells in 5 min.Uptake of [3H]harringtonine in P815/0 cells at 20°was also foundto be greater than that in P815/VCR cells. The preloaded [3H]-

harringtonine in L1210/0 cells showed a slow rate of release tothe medium at 20°when compared with the rate of uptake ofthis alkaloid. The preloaded [3H]harringtonine in L1210/VCR cellsattained a net accumulation of 100 to 200 cpm/108 cells in 5 min(after subtracting 2°blank). The rate of release of radioactivity

to the medium (data not shown) occurred at a rate similar to thatin L1210/0 cells.

Effect on Precursor Incorporation into Macromolecules. Asshown in Chart 3, [3H]cytidine incorporation into RNA of L1210/

0 cells was inhibited 20% by harringtonine (5 Mg/ml) in 90 min,and the incorporation of [3H]dThd was inhibited 60% during the

100

80

60

40

20

Cyd— RNA

oTrid —ONA

20 40 60Minutes

80 100

Chart 3. Early time course of inhibition of precursor incorporation into protein,DNA, and RNA by harringtonine. Reaction mixture in Eagle's basal medium (1 ml)

contained 10% fetal calf serum; 1 DIM glutamine; 5 fig of harringtonine or 0.9%NaCI solution; radioactive tracer [/7)ef/jy/-3H]dThd (1 pCi. 0.5 nmol), or [5-3H]cytidine(4 ,iCi, 0.14 nmol), or [4,5-3H]teucine (0.2 ^Ci. 50 nmol); and 17 x 10»L1210/0cells. Incubation was initiated by adding cells at 37°in the presence of 95% 02

and 5% CO,, in capped tubes. Reaction was terminated at different time intervalsby adding 2 ml 10% cold perchloric acid; and the incorporations of labeled cytidineinto RNA, dThd into DNA, and lenone into protein in the presence and absence ofharringtonine were determined by methods described previously (7). At the end of90 min, the incorporations of radioactivity in controls for labeled cytidine, dThd,and leucine were 13,200, 2,820, and 4,090 cpm, respectively.

same period. By contrast, incorporation of [3H]leucine into pro

tein was inhibited over 90% within 15 min by the same concentration of harringtonine. In addition, there was an apparent lagof onset of 5 and 10 min, respectively, for the inhibition of [3H]-cytidine and [3H]dThd incorporation, but no lag period wasdetected for the inhibition of [3H]leucine incorporation by harring

tonine. From the onset of action and the degree of inhibition, itis apparent that inhibition of protein synthesis is the primaryeffect exerted by harringtonine in L1210/0 cells. Similar resultswere also observed in this laboratory with P815/0 leukemic cells.

Chemotherapeutic Effects in L1210/0 and Its Sublines.L1210/0 and its sublines resistant to ara-C, 5-FUra, 6-MP, CPA,

ADR, and VCR (Table 1) have been used to compare their relativesensitivity to harringtonine in vivo in BD2Fi mice. Proper dosesand regimens were selected so that all of these agents inducedincreases in life span in L1210/0 cells. At doses specified inTable 1, the median life span of mice bearing L1210/Ó leukemiawas increased 88, 88, 112, 143, 180 and 75%, respectively, byara-C, 5-FUra, VCR, CPA, ADR, and 6-MP. All mice bearing

resistant leukemic cell sublines failed to respond to corresponding agents except L1210/ADR, where residual sensitivity to ADR

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Harringtonine Uptake and Effects of U 210 Sublines

could be detected, which caused a 14% increase in median lifespan at a single dose of 15 mg/kg i.p. Harringtonine at 2.4 andat 3.6 mg/kg i.p. daily for 6 days increased the life span 63%,on the average, in L1210/0 cells, whereas, in its sublines, thechemotherapeutic effects of harringtonine, on the average indecreasing order, were: L1210/CPA, L1210/6-MP > L1210/ara-C, L1210/5-FUra > L1210/ADR, L1210/VCR (Table 2). It should

be noted that harringtonine at the dose of 3.6 mg/kg/day for i.p.for 6 days showed apparent toxicity as indicated by averagebody weight decreases in treated mice.

Comparison of Uptake and Biochemical Effects in L1210Sublines. The rate of uptake of [3H]harringtonine at 20°by

L1210 sublines resistant to various chemotherapeutic agentswas generally lower than that of L1210/0 parent cells (Table 3).They were, in decreasing order: L1210/CPA, L1210/ara-C,L1210/6-MP > L1210/5-FUra, L1210-ADR > L1210/VCR. Therates of uptake of [3H]harringtonine in L1210/0, L1210/ara-C,and L1210/VCR were also tested at 37°which gave a much

more rapid initial uptake with the relative differences of the ratesof uptake among different sublines being reduced. Inhibition of[3H]leucine incorporation into protein by harringtonine (0.5 jug/

Table 2

Chemotherapeuticeffect of harringtoninein BD2F, mice bearingsublines ofL1210 leukemias

The procedure for development and maintenanceof L1210 sublines is given inTable 1. Drug was administered i.p. on Day 1 after transplantation of leukemiccells. Mice that had received 0.9% NaCI solution served as controls. Each groupof test and control consisted of 5 to 8 mice. Averageweight changewas measuredon Day 6. The median life span was used for calculating percentageof increase inlife span after drug treatment.

Harringtoninedose

2.4 mg/kg i.p. for 6 days 3.6 mg/kg i.p. for 6days

L1210sublinesL1210/0

L1210/VCRL1210/ara-CL1210/6-MPL1210/5-FUraL1210/CPAL1210/ADRMedian

life span(days)7

787867Av.

wtchange (g;

Test/control)+1

.2/+3.8+2.2/+3.S+3.6/+3.6+2.0/+3.1+2.3/+S.9+4.8/+3.9+3.8/+3.S%

of increasein lifespan67

02557385014Av.

wtchange(g;

Test/control)-0.2/+3.6

-0.2/+2.2-2.0/+2.6-1.0/+2.8-1 .0/+3.8+0.2/+1.8+0.0/+3.6%

of increasein lifespan57

14382911500

Tabte3

Relativeinitial uptake of fHJharringtonine and relativepotency of inhibitingpHJIeucine incorporation into protein by harringtoninein sublines ofL12W

leukemiccells

CelllinesL1210/0

L1210/CPAL1210/ara-CL1210/6-MPL1210/5-FUraL1210/ADRL1210/VCR[3H]Hamngtonine

uptake8(cpm/10«cells/5min)1780

±243e

875 ± 51656 ± 45633 ± 25450 ± 30450 ± 15276 ± 63%

of inhibitionof[3H]leucineincorporation byharringtonine645.6

±1.336.2 ±5.743.4 ±3.644.7 ±2.242.4 ±2.533.3 ±0.214.5 ±7.0

a Vortex-Finnpipetteprocedurewas used prior to microcentrifugationof sampleson the oil layer (see "Materials and Methods"). The incubation mixture contained 6to 11 x 107cells, 0.04 iiC\ (0.2 ^g) of ¡3H]harringtonineper ml of Eagle's basalmedium.The uptake during the 5-min period was measured.Uptakedata obtainedfrom 2°were used as blank and were subtracted from the valuesobtained at 20°.The net uptake is given in cpm/108cells/5 min.

'' Incubationmixture in Eagle's basal mediumcontained |:'H|leucme.0.2 iiCi (0.2nmol)/ml; harringtonine,0.5 /ig/ml and 34 to 71 x 10" cells. Incubationwas carriedout at 37°for 5 min.

0 Mean ±S.D., from 3 to 4 measurements.

Table4Binding of pHJharringtonine radioactivity to cellular components

Cells, 85 x 106/ml,were incubated with [3H]harringtonine(0.4 »iCi/Mg/ml)at 37"

for 30 min.A smallaliquotof cell suspensionwas removedfor radioactivitycounting.The remainder was incubated at 37°for 10 min prior to centrifugation to obtain

cell pack. The cell pack was resuspendedin a fresh medium to the original volume.The procedure of radioactivity counting, 10-min incubation, centrifugation, cellpack, and resuspensionwas repeated twice more before the cell pack for the thirdwashing was obtained.

cpm/106 cells

Cell suspension withoutwashingCell

suspensionafterfirstwashingCell

suspensionaftersecondwashingCell

suspensionafterthirdwashingCell

pack afterthirdwashingL1210/01005

±54a216

±70216

±23179

±4474

+ 26L1210/VCR1051

±14217

±41121

±20*82

±13°22

± 8CP815/01167

±19304

±51253

±40217

±36114±

19P815/VCR1135

±19229

±33116±11659

±3C24

± 9°

" Mean ±S.D. Except for the cell pack after the third washing, cpm include

medium of the suspension.6 Differencebetween the meansof VCR-sensitiveand -resistant cells,p < 0.05.c Differencebetween the meansof VCR-sensitiveand -resistant cells,p < 0.02.

ml) was studied at 37°during the 5-min incubation period (Table

3). The percentage of inhibition in decreasing order was: L1a10/0; L1210/6-MP; L1210/ara-C; L1210/5-FUra; L1210/CPA;

L1210/ADR; and L1210/VCR. The relative potency of harringtonine in inhibiting leucine incorporation was similar in percentageof inhibition in all sublines except L1210/ADR and L1210/VCR.

Relative Binding of [3H]Harringtonine to Cellular Compo

nents. Although L1210/0 or P815/0 cells take up much more[3H]harringtonine than do L1210/VCR or P815/VCR cells at 20°,

this difference was reduced to 1.5-fold when experiments werecarried out at 37°in which the rates of uptake were increased.These results at 37°may be related in part to lack of markeddifference in inhibiting [3H]leucine incorporation by harringtonine

in some sublines (Table 3). In order to further investigate thenature of harringtonine uptake, these cells were preloaded with[3H]harringtonine at 37°for 30 min and were then repeatedlywashed with fresh medium at 37°.Thus, in the procedure for

binding experiments, the time period and temperature weredifferent from those used in Chart 2 and Table 3 for initial uptakeand release studies. Experiments for Chart 2 were carried outat 20°in order to slow down the initial rate of uptake, to minimize

the complication of concurrent efflux, and to minimize the possibility of metabolism of harringtonine. L1210/0 and P815/0 cellsretained significantly more [3H]harringtonine radioactivity than

did L1210/VCR or P815/VCR cells (Table 4), suggesting that[3H]harringtonine or its metabolite(s) was bound more tightly to

the cells that were sensitive to harringtonine. Analysis of thedistribution of [3H]harringtonine in subcellular fractions of L1210/

0 cells revealed that the radioactivity was concentrated in themicrosomal fractions, especially in the light microsomal fraction(Table 5), when the data were calculated in cpm/mg.

DISCUSSION

This paper indicates that the effect of harringtonine in murineleukemic cells, like those in HeLa cells and rabbit reticulocytes(14), is primarily inhibition of protein synthesis. The relativepotency of harringtonine in inhibiting [3H]leucine incorporation

among different L1210 sublines of leukemic cells resistant tovarious chemotherapeutic agents is in a rank order similar to the

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TablesSubcellular distribution of pHJharringtonine radioactivity in U 210/0 cells

L1210/0 cells were incubated at 37°for 30 min with [3H]hamngtonine, 1 ;iCi

(1.1 f/mol). in a final volume of 7 ml, and then washed repeatedly with fresh mediumas described in Table 4. Experiment 1 used 1015 x 106 cells, washed 4 times afterloading with [3H]hamngtonine, and homogenized at 2°for 15 sec with 4.5 ml of

0.25 M sucrose prior to centrifugaron for subcellular components. Experiment 2used 510 x 10* cells, washed 3 times after loading with [3H]harringtonine, andhomogenized at 2°for 30 sec with 2 ml of 0.25 ml of 0.25 M sucrose prior to

centrifugaron for subcellular components.

Experiment1FractionsUnbroken

cells and nuclear fraction (650g)Mitochondria!

fraction (4,000 xg)Lightmitochondrialfraction(1

4,000xg)Heavymicrosomalfraction(20,000

xg)Lightmicrosomalfraction(100,000

xg)SupernatantTotalcpm6.9604042113011,07211,184cpm/mg1.311.614.123.2238.23.2"ExperimentsTotalcpm9,3151,3021,9248927,10240,345cpm/mg2.629.738.5136.2298.624.1"

rate of uptake of harringtonine in these sublines (Table 2). Therelative rates of uptake of harringtonine by the cells and therelative potency of inhibition of protein synthesis, in turn, aresimilar to the rank order of the chemotherapeutic effects ofharringtonine in mice bearing L1210 sublines of leukemias. Although no direct cause-consequence relationship among uptake,

inhibition of protein synthesis, or therapeutic effect can beproved, the factors may be indirectly related.

The wild-type L1210/0 and P815/0 cells took up more [3H]-

harringtonine radioactivity than did their tested sublines. L1210/VCR and P815/VCR cells took up the least radioactivity. AmongL1210 sublines studied, L1210/VCR and L1210/ADR appearedinsensitive to harringtonine in chemotherapeutic studies in vivo.L1210 cells resistant to cyclophosphamide, ara-C, 6-MP, and 5-

FUra were moderately sensitive to harringtonine.Although both harringtonine and VCR are alkaloids and

showed cross-resistance to L1210/VCR cells, their chemical

structures are quite different. The major common feature is thatthey have relatively large molecules with heterocyclic structures.The cross-resistance between harringtonine and ADR is some

what unexpected. However, earlier reports indicated that Vincaalkaloids and anthracyclines are cross-resistant in Ehrlich ascites

tumor (9), Chinese hamster cell lines (2), leukemic P388 (24) andP815 leukemia (15). Those cells resistant to VCR are also cross-

resistant to actinomycin D in P388 leukemic cells (15, 24) and inChinese hamster cells (2) but are not cross-resistant to ara-Cand 6-MP in Ehrlich ascites tumor (9).

VCR blocks the organization and polymerization of microtu-

bules (20), and ADR and actinomycin D form complexes withDMA (21). Presumably, these interactions are responsible formost of their biological effects. The cross-resistance among

these structurally unrelated drugs has led to the study of common factors of alteration of membrane structure in resistantcells. Comparison of membrane glycopeptides and gangliosidesof cells sensitive and resistant to VCR and actinomycin D hasrevealed alterations in those glycoconjugates. In all Chinesehamster cell sublines examined, there is a selective increase inthe M, 150,000 and a decrease in the M, 100,000 glycopeptidespecies which is quantitatively related to the degree of drugresistance (2). These types of membrane alterations may be

related to the impaired uptake and decreased biochemical effectsof harringtonine in L1210/VCR cells presented in this paper.Beck ef al. (1) showed the presence of altered surface membraneglycoprotein in Vinca alkaloid-resistant human leukemic lymph-

oblasts. In P388 cells resistant to VCR, there was impaireduptake and binding of VCR within these cells (3). These resultsare also consistent with the present suggestion that uptake andbinding of harringtonine are important determinants of its efficacyin L1210 sublines.

The finding that [3H]harringtonine or its metabolite(s) predom

inantly binds to the light microsomal fraction (Table 5) furthersupports the observation that harringtonine inhibits protein synthesis (Chart 3; Table 3) by inducing breakdown of polysome tomonosome as suggested previously (14).

It is of interest to note that the homologue of harringtonine,homoharringtonine, has also been found to be cross-resistant toVCR and ADR but not to ara-C in P388 cells (25). Thus, it seems

reasonable to expect that harringtonine and homoharringtoninemay share the same biochemical and pharmacological mode ofaction.

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2. Siedler, J. L., and Peterson, R. H. F. Altered plasma membrane glycoconjugates of Chinese hamster cells with acquired resistance to actinomycin D,daunorubicin, and vincristine. In: A Sartorelli (ed.), Molecular Action and Targetsfor Cancer Chemotherapeutic Agents, pp. 453-482. New York: Academic

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9. Daño. K. Cross resistance between Vinca alkaloids and anthracyclines inEhrlich ascites tumor in vivo. Cancer Chemother. Rep., 56: 701-708,1972.

10. Department of Pharmacology, Institute of Materia Medica, Chinese Academyof Medical Sciences. Antitumor effects and pharmacological studies of harringtonine. Chinese Traditional and Herbal Drugs, 4:172-176,1976.

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12. Han, J., and Gi, S. J. Antitumor ingredients of Cephalotaxus hainanensis U:pharmacological and clinical studies. Chinese J. Oncol., 1:176-181,1979.

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1983;43:3074-3079. Cancer Res   Ting-Chao Chou, Franz A. Schmid, Aaron Feinberg, et al.   to Vincristine and Other Chemotherapeutic AgentsHarringtonine in Murine Leukemic Cells Sensitive and Resistant Uptake, Initial Effects, and Chemotherapeutic Efficacy of

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