Comparative Study of Early Effects of Epipodophyllotoxin … · [CANCER RESEARCH 37, 2998-3005,...

[CANCER RESEARCH 37, 2998-3005, September 1977]

Comparative Study of Early Effects of EpipodophyllotoxinDerivatives and Other Cytostatic Agents on MastocytomaCultures

Alfons Grieder,1 Richard Maurer, and Hartmann StÃ¤helin

Biological and Medical Research Division, Sandoz Ltd.. CH-4002 Basel, Switzerland

SUMMARY

The effects of various cytostatic agents (mechloretham-ine, 1-/3-D-arabinofuranosylcytosine, 6-mercaptopurine,methotrexate, colchicine, vincristine, a podophyllotoxinderivative, bleomycin, 1,2-bis(3,5-dioxopiperazin-1-yl)pro-pane, the epipodophyllotoxin derivatives 4'-O-demethyl-1-O-[4,6-O-2-thenylidene-ÃŸ-D-glucopyranosyl]epipodophyllo-toxin and 4'-O-demethyl-1-O-[4,6-O-ethylidene-/3-D-gluco-pyranosyljepipodophyllotoxin, and X-rays) on cell proliferation, mitotic index, cellular DNA, RNA, and protein contentand on the incorporation of their respective isotope-labeledprecursors were studied in cultures of mastocytoma cellline P-815X2 during the first 6 hr of exposure to the variousagents.

Most of these agents exhibited effects compatible withtheir accepted mechanisms of action. An exception to thiswas the finding that 1-/3-D-arabinofuranosylcytosine depressed the mitotic index to less than one-half of the control value within 90 min of drug addition; this effect is notcompatible with an exclusive action in G, or S phase of thecell cycle, but it suggests an additional block in G-2.

The two epipodophyllotoxin derivatives 4'-O-demethyl-1-O-[4,6-O-2-thenylidene-/S-D-glucopyranosyl]epipodophyl-lotoxin and 4'-O-demethyl-1-O-]4, 6-O-ethylidene-0-D-gluco-

pyranosyljepipodophyllotoxin exhibited qualitatively identical actions. On the other hand, of the other agents only X-rays exerted some effects comparable to those of the epipo-dophyllotoxins. We thus conclude that, of the drugs tested,4'-O-demethyl-1-O-[4,6-O-2-thenylidene-ÃŸ-D-glucopyrano-syljepipodophyllotoxin and 4'-O-demethyl-1-O-[4,6-O-eth-ylidene-/3-D-glucopyranosyl]epipodophyllotoxin possess aunique mechanism of action.

INTRODUCTION

The podophyllotoxin derivatives VM 262 and VP 16-213have been shown to prevent cells from entering mitosis (5,

1To whom requests for reprints should be addressed2 The abbreviations used are: VM 26, 4'-O-demethyl-1-O-(4,6-O-2-theny-

lidene-/3-D-glucopyranosyl]epipodophyllotoxin (NSC 122819); VP 16-213, 4'-

O-demethyl-1-O-[4,6-O-ethylidene-/:J-D-glucopyranosyl]epipodophyllotoxin(NSC 141540); PBG, podophyllotoxin-/3-o-benzylidene glucoside; ara-C, 1-/3-o-arabinofuranosylcytosine (NSC 63878); HN2, nitrogen mustard (me-chlorethamine, NSC 762); VCR, vincristine (NSC 67574); BUM, bleomycin(NSC 125066); 6-MP, 6-mercaptopurine (NSC 755); MTX. methotrexate(NSC 740); ICRF 159, 1,2-bis(3.5-dioxopiperazine-1-yl)propane (NSC 129943).

Received July 6. 1976; accepted May 27. 1977.

17). This is in contrast to the arrest in metaphase producedby podophyllotoxin itself and its earlier derivatives (18). VP16-213, which is less active in vitro than is VM 26 but whichshows a more pronounced therapeutic effect in leukemicmice (18, 19), was investigated more extensively as to itseffects on mitosis and macromolecular synthesis in murinemastocytoma cells in vitro (5). It was found to arrest cells inlate S or G2phase of the cell cycle immediately. At the sametime, incorporation of exogenous thymidine into DMA wasmarkedly inhibited, while the synthesis of DMA was slowedto a lesser degree. The discrepancy between [3H]thymidine

incorporation and DNA synthesis became more pronouncedwith increasing drug concentration. Similar results havebeen obtained with both VM 26 and VP 16-213/n vitro (7-9,24), using other cell types. In in vivo experiments, Krishan efal. (8) have shown by pulse cytophotometry that cells accumulate in Gz after treatment with the epipodophyllotoxinderivatives. In several of these studies, the earlier observation (17) of a low degree of reversibility of the effect of thistype of compound was confirmed.

Although the biochemical basis underlying the main effect of VM 26 and VP 16-213, i.e., inhibition of entry intomitosis, is still unknown, it seemed of interest to comparethe effects of these drugs on mitosis, precursor incorporation, and macromolecular synthesis with those of othercytostatic agents and X-rays. It was considered that such acomparison may be of value for selecting combinations ofagents for therapy in patients. PBG was also included in thecomparison (Chart 1); this compound is chemically verysimilar to VM 26 in that it is also an aldehyde condensationproduct, but it differs from VM 26 in 2 important characteristics in that it is not demethylated in position 4' and its

glucose is in a different steric position in relation to thepodophyllotoxin ring structure (18).

MATERIALS AND METHODS

Cell Line and Culture Technique. Cells of the transplant-able murine mouse mastocytoma line P-815X2 were grownas stationary suspension cultures in Erlenmeyer flasks. Thecomposition of the Medium 72 P used for cultivating thesecells as well as the culture technique were described indetail in a preceding publication (5).

Preparation of Drug Solutions. ara-C (Nutritional Bio-chemicals Corp., Cleveland, Ohio), HN2, colchicine (bothfrom Ciba-Geigy Ltd., Basel, Switzerland), VCR (Eli Lilly andCo., Indianapolis, Ind.), and BLM (Bristol Laboratories, Syr-

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Early Effects of Cytostatic Agents in Vitro

â€”OH

12 ,R2= H3c_

OCH3

OCH3

Chart 1. /, podophyllotoxin; //, PBG; ///, VM 26; IV, VP 16-213.

acuse, N. Y.) were dissolved directly in Medium 72 P. Theother drugs are insoluble in aqueous solutions and weredissolved as follows. 6-MP (Fluka Ltd., Buchs, Switzerland)and VP 16-213 were dissolved in dimethyl sulfoxide (5 mg/

0.05 ml). VM 26, 1 mg, or PBG, 3 mg (Sandoz Ltd., Basel,Switzerland), were dissolved in 0.1 ml dimethyl sulfox-

ide:Tween 80 (10:1). MIX (Lederle Laboratories, PearlRiver, N. Y.) was solubilized in 1% sodium bicarbonate (0.1mg/ml), and ICRF 159 (kindly provided by Dr. K. Hellmann,Imperial Cancer Research Fund, London, England) wasdissolved in 0.1 N HCI (10 mg/ml). The solutions were diluted to appropriate concentrations with prewarmed Medium 72 P.

Incubation of the Experimental Cell Cultures. The experiments were started at time zero by adding 4.5 ml of theprewarmed drug solutions to 445.5-ml portions of the cell

suspension, giving the final concentrations indicated in thetables. The initial cell density was between 2 and 3 x 10s

cells/ml. Subsequently, the cell cultures were divided intoaliquots of 80 ml each in prewarmed Roux bottles, gassedfor 15 sec with 5% CCX:95% air, and further incubated. At1.5, 3, 4.5, and 6 hr following drug addition, 1 bottle of eachculture was removed and its content was further processedand analyzed as described previously (5).

Determination of Cell Multiplication and Mitotic Index.Cell reproduction was determined on the basis of cellcounts obtained with a Microcellcounter CC-1002 (Toa

Medical Electronics Co., Ltd., Kobe, Japan). The proportionof dead cells was determined with the dye exclusion testusing 0.25% Evans blue. For determination of mitotic indices, aliquots of each culture were fixed and stained according to the method of Schindler (14).

Isotope Incorporation Studies. [6-3H]Thymidine (finalconcentration, 0.5, /Â¿Ci/ml;5 Ci/mmole), [5-3H]uridine (finalconcentration, 0.5 /^iCi/ml; 5 Ci/mmole) and [4,5-3H]leucine

(final concentration, 1 /Â¿Ci/mi; 1 Ci/mmole) were used instudies of incorporation into DNA, RNA, and protein, re

spectively, as described previously (5). All isotopes wereobtained from the Radiochemical Centre, Amersham, England. Scintillation counting was carried out in a Packard Tri-

Carb Model 3320 spectrometer using a liquid scintillationcocktail containing 30% ethanol and 70% tolu-

ene:POPOP:PPO.Determination of DNA, RNA, and Protein. The spectre-

photometric measurements of total DNA, RNA, and proteincontent were performed on duplicate culture samples asdescribed (5), except that the calf thymus DNA used as astandard (Serva, Heidelberg, Germany) was dried over P._,O5for 48 hr before use.

Irradiation. Roux bottles containing 450 ml of cell suspension were irradiated at room temperature with 600 radsusing a Philips RT 225 therapeutic X-ray unit working at 250kV and 12 ma and furnished with a 3-mm copper filter. Cell

samples were irradiated at a distance of 45 cm with a doserate of 47 rads/min as determined in air with a Simplex-Universal-Dosimeter (Physikalisch-Technische WerkstÃ¤tten,

Dr. Pychlau, Freiburg i Breisgau, Germany). Control measurements in an empty Roux bottle at the level of the cellsuspension showed that the whole field was irradiatedhomogeneously with a deviation of Â±5%.

Calculations. Data reported are the mean values of atleast 2 independent samples per point. All experimentswere repeated at least once. In some cases the mean valuesÂ±S.E. of data from 3 to 12 experiments are shown. S.E.'s

for data consisting of less than 3 repeat experiments perpoint were not calculated.

RESULTS

Effect on Cell Multiplication and Mitotic Index. All drugsused in this study had an inhibitory effect on the proliferation of P-815 cells (Table 1). Considerable inhibition or

delay of cell multiplication was observed after 6 hr, with theexception of the lower doses of PBG and BLM. Cessation of

SEPTEMBER 1977 2999



A. Grieder et al.

cell proliferation had already occurred 1.5 hr after additionof most of the drugs or exposure to X-rays (not shown). Incontrast, 6-MP inhibited cell proliferation by only about 33%within the 1st 6 hr at a relatively high concentration.

Table 1Influence on multiplication of P-815 cells in vitro

AgentControlHN2ara-C6-MPMTXColchicmeVCRPBGBLMICRF

159VM

26VP

16-213X-raysConcentration

(/ig/ml)0.05

0.10.05

0.51

100.110.05

0.50.01

0.1330101001100.1111600

radsFactor

of cell multiplication after a 6-hr

exposure"1

.63 Â±0.04"1.11

1.171

.25 Â±0.031.10 Â±0.031.44

1.421.22

Â±0.091.23 Â±0.111.15

1.151.26

Â±0.121.23 Â±0.171.58

1.021.52

1.211.37

0.971.15

1.041.10

Â±0.011.03 Â±0.020.93

" The cell number at the beginning of incubation (time zero) wasset as 1.0.

" Mean Â±S.E.

Table 2 shows the time course of mitotic activity duringthe incubation period. For simplicity, results are given inthis and all subsequent tables for a single active concentration. The effects on cell division, although not in all casesconcentration dependent, were mostly in agreement withthe generally proposed mechanism of action of the agents.The antimetabolites ara-C, 6-MP, and MTX produced amarked reduction of the mitotic index with time as did HN2,an alkylating agent. On the other hand, in the presence ofthe mitotic inhibitors colchicine, VCR, and PBG, cells accumulated in metaphase (metaphase stop was clearly visiblemicroscopically but is not indicated in Table 2). X-irradia-tion greatly decreased proliferation up to 4 hr, but after 6 hrthe mitotic index was only slightly reduced. A similar effectwas obtained with the high dose of BLM. ICRF 159 caused amoderate increase of the mitotic index, apparently blockingsome cells in prophase or early metaphase [this latter assumption is based not on counting of the mitotic phases buton findings in lymphocytes with this compound (16)]. The 2closely related podophyllotoxin derivatives VM 26 and VP16-213 gave rise to a marked and immediate decrease in themitotic index.

DNA Synthesis. Results concerning the effect of theagents on thymidine incorporation and DNA content of 106

cells during the 1st 6 hr of treatment are given in Tables 3and 4. The alkylating agent and the 3 antimetabolites allreduced thymidine incorporation by about 80% or morewithin 1.5 hr of drug addition. DNA content remained quiteclose to the control level with these 4 compounds. Themitotic blocking agents colchicine and VCR slightly inhibited thymidine incorporation within 2 hr and evokedsubsequently a progressive decrease (see "Discussion").

The initial increase in DNA content induced by colchicinewas somewhat slower than anticipated considering the increase in cell number in the controls and the increase inmitotic index in treated cultures (Table 2). PBG, on theother hand, showed a marked increase in cellular DNAcontent in the 1st 3 hr but strongly depressed thymidineincorporation throughout the experiment. BLM produced aprogressive reduction in thymidine incorporation, whereasDNA content gradually increased. A very regular increase inDNA content was produced by ICRF 159, corresponding in

Table 2Mitotic index of P-815 cells after addition of various cytostatic agents

Mitotic index"

AgentHN2ara-C6-MPMTXColchicineVCRPBGBLMICRF

159VM26VP

16-213X-rayswviwi

in a LI Â»uiii(Ã-Â¿g/ml)0.10.5100.10.50.130100100.11600

rads1.5

hr0.860.81

Â±0.22"1.51.5

Â±0.861510

Â±1.59.71.25.20.140.25

Â±0.10.183

hr0.510.36

Â±0.011.20.23

Â±0.032522

Â±0.9151.35.30.0570.13

Â±0.060.234.5

hr0.720.21

Â±0.010.710.19

Â±0.024138

Â±4.4221.27.30.0860.17

Â±0.030.966

hr0.210.160.950.0904544282.0110.0450.0581.8Â±

0.01Â±

0.01Â±

1.9Â±

0.01

" Mitotic index = % cells in mitosis. The mean mitotic index of untreated control cells was 2.4 Â±0.06.6 Mean Â±S.E.





Table 3Effect of cytostatic agents upon incorporation of [3H]thymidine into DNA of 706cells

% incorporation relative to control at different times after drugaddition"


159VM26VP16-213X-raysWl

WVI in HMVIi(/ig/ml)0.10.5100.10.50.130100100.11600

rads1.5

hr168

Â±1.5"1217

Â±2.39087

Â±1.722851326056

Â±7.7833

hr197

Â±1.21315

Â±2.27272

Â±3.524731315451

Â±6.01114.5

hr227

Â±1.71423

Â±1.24739

Â±6.021521215849

Â±7.577157173423191639904541206

hrÂ±

2Â±

2.0Â±

0.7Â±

6.8

" [3H]Thymidine was added to aliquote of the cultures at times indicated. Pulse length was 30 min." Mean Â±S.E.

Table 4Effect of cytostatic agents upon cellular DNA content

DNA content" per 106cells in % relative to control


159VM26VP16-213X-raysVWI

PW* in OLIISIi(/ig/ml)0.01001510.50.30100100.1600.11rads1989710410010610911310011710410488.5

hrÂ±

0.9"Â±

1.2Â±

2.7Â±

0.5Â±2.03

hr10396

Â±10397

Â±117124

Â±133106127111

Â±115Â±12233.92.24.83.44919610295128138124110142122128128.5hrÂ±

1Â±

2Â±

1Â±

6Â±3.5.1.6.4.76

hr8599

Â±105100

Â±132140

Â±126120159128

Â±134Â±135213.86.55.2

" DNA content was determined at times indicated after exposure to the drugs. DNA content of 106untreated controlcells was 9.0 Â±0.13 t*.g.

" Mean Â±S.E.

magnitude to the increase in cell number in the controlcultures (both increased by a factor of about 1.6); thymidineincorporation was increased for up to 4.5 hr. The 2 epipodo-phyllotoxin derivatives, VP 16-213 and VM 26, both slowedthymidine incorporation to 60 to 40% of controls. At thesame time DNA synthesis was not or only slightly reducedfor the 1st 3 hr, thus leading, in conjunction with inhibitionof cell division, to an increased DNA content per 106 cellscompared with controls. X-irradiation produced an increasein DNA content similar to that produced by theepipodophyl-lotoxins, but thymidine incorporation was not reduced untilat least 4.5 hr after exposure.

RNA Synthesis. Table 5 shows the effects of drug treatment on incorporation of uridine, and Table 6 shows thoseon the RNA content, both calculated for 106cells. It may be

seen that HN2 slightly decreased uridine incorporationthroughout the period of incubation whereas RNA contentwas diminished only after 6 hr. Of the antimetabolites, ara-C produced a continuous increment in uridine incorporation and an augmentation of the RNA content during the 1st3 hr. 6-MP and MTX, on the other hand, both stronglyinhibited precursor incorporation but induced little changeIn cellular RNA content. Colchicine and VCR, agents that

inhibit spindle formation, produced a continuous decreasein the accumulation of uridine-derived radioactivity,whereas RNA content increased. The latter was also true forPBG, but this drug induced marked inhibition of uridineincorporation (about 80%) within the 1st 30 min followingdrug addition, as it did with thymidine incorporation (resultsnot shown). Incubation with BLM resulted in an increase inboth parameters, but neither effect was time dependent.ICRF 159 also augmented both uridine incorporation andRNA content in a more regular fashion than did BLM and asdid both the epipodophyllotoxin derivatives and X-irradiation; with these latter 4 treatments, however, uridine incorporation tended to plateau or decrease after 6 hr.

Protein Synthesis. Drug-induced deviation from controlvalues with respect to leucine incorporation and proteincontent of 106 mastocytoma cells is shown in Tables 7 and8. HN2 and ara-C influenced both parameters of proteinsynthesis similarly by the way they affected RNA synthesis.6-MP slightly inhibited leucine incorporation but did notchange protein content. MTX, while also diminishing precursor incorporation, led to a late augmentation of proteincontent. Colchicine, VCR, and PBG did not alter leucineincorporation but increased the protein content per cell.

SEPTEMBER 1977 3001



A. Grieder et al.

Table 5Effect of cytostatic agents upon incorporation of [3H]uridine into RNA of JO6ce//s



159VM26VP16-213X-raysConcentration

(/Â¿g/ml)0.10.5100.10.50.130100100.11600

rads1781062022909221121127102102103.5

hrÂ±

1.0*Â±

3.2Â±

5.1Â±

2.63

hr79116

Â±0.42127

Â±9.48081

Â±7.021161132100113

Â±3.61374.5

hr72132

Â±0.72325

Â±7.96669

Â±2.818110142130123

Â±2.21396

hr541612928545614142142119117135Â±

5.5Â±

7.3Â±

3.1Â±

9.8

" [5-3H]Uridine was added to aliquots of the cultures at times indicated. Pulse length was 30 min." Mean Â±S.E.

Table 6

Effect of cytostatic agents upon cellular RNA content



(/Â¿g/ml)0.10.5100.10.50.130100100.11600

radsRNA

content" per 106cells in % relative tocontrol1.5

hr100104

Â±1.3*98110

Â±3.6109112

Â±2.8108125120104104

Â±3.71003

hr97125

Â±092108

Â±3.4117124

Â±1.4131124135111122

Â±2.81354.5

hr98129

Â±0.59395

Â±4.3122130

Â±0.9122108157129132

Â±8.41466

hr76129

Â±9390

Â±117134

Â±122138159134140

Â±1530.71.34.35.5

" RNA content was determined at times indicated after exposure to the drugs. RNA content of 106untreated controlcells was 24.5 Â±0.7 /ig.

" Mean Â±S.E.

Table 7Effect of cytostatic agents upon incorporation of [3HÂ¡leucine into protein of JO6cells



159VM26VP16-213X-rays\sui

iwd ILI a 11u ii(MO/rnl)0.10.5100.10.50.130100100.11600

rads1.5

hr77112

Â±3.0"8989

Â±0.79097

Â±2.49311412795106

Â±4.3993

hr73120

Â±1.07780

Â±6.38199

Â±3.3108104125108124

Â±4.3138461142707597102102106144123141139.5

hrÂ±

1.9Â±

5.3Â±

5.5Â±2.56

hr41170

Â±7987

Â±10099

Â±95127138147138

Â±1440.81.29.93.6

[4,5-3H]Leucine was added to aliquots of the cultures at times indicated. Pulse length was 40 min.Mean Â±S.E.

BLM induced variable increases in both values. ICRF 159enhanced incorporation leading to a steady increase in theprotein content per cell during 6 hr. The maximum increasein protein content induced by this drug was the same as thatcalculated for increases in RNA and DNA content and also

for the increase of cell number in control cultures. More orless the same holds true for X-irradiation and for the 2epipodophyllotoxin derivatives VM 26 and VP 16-213 which,however, produced a slower increase in RNA content thandid ICRF 159 or X-rays.





Table 8

Effect of cytostatic agents upon cellular protein content



(ng/ml)0.10.5100.10.50.130100100.11600

radsProtein

content" per 106 cells in %relative1.5

hr9597

Â±1.6"98105

Â±10105108

Â±2.2112121121100101

Â±4.81093

hr97115

Â±2.495105

Â±5.0117112

Â±5.113112413297110

Â±3.91384.5

hr115118

Â±3.091112

Â±1.5122117

Â±3.2120116151119128

Â±5.7148to

control6

hr87124104120129131124141164138132164Â±

2.2Â±

8.8Â±

6.4Â±

6.5

Â°Protein content was determined at times indicated after exposure to the drugs. Protein content of 106 untreated

control cells was 160 Â±5.5 /Â¿g.6 Mean Â±S.E.

DISCUSSION

It was the purpose of this investigation to study the in vitroeffects of various cytostatic agents, including VM 26 and VP16-213, on nucleic acid and protein metabolism. Althoughwe will discuss some aspects concerning the mechanism ofaction, attention will be restricted primarily to a comparisonof the newer, chemically related podophyllotoxin derivatives with the other agents and to discrepancies betweenour results and those appearing in other publications. Aprevious report (5) dealt with the effects of VP 16-213 ongrowth and mitotic index of P-815 mastocytoma cells invitro, its influence on incorporation of radioactive precursors, and its influence on the amount of cellular DNA, RNA,and protein.

Early effects are more relevant in elucidating the primarybiochemical mechanism of drug action, although it is recognized that in the clinical use of the drugs late effects maybe equally as or even more important than those that occurin the 1st 6 hr. In interpretation of these results, it is helpfulto consider the duration of the cell cycle phases in P-815cells (15), i.e.. G,, 2.0 hr; S, 4.8 hr; G2, 1.7 hr; and mitosis,0.4 hr, which add up to a generation time of 8.9 hr. Theinvestigation was restricted to the 6-hr period followingdrug addition, i.e., roughly two-thirds of a full cell cycle.Extrapolation of the increase in cell number in controlcultures after 6 hr (Table 1) to a factor of 2 results in adoubling time of about 8.5 hr which is in agreement withearlier observations (15).

It has been known for some time that podophyllotoxin,like colchicine and the Vinca alkaloids, vinblastine andVCR, induces mitotic arrest of cells (for review see Ref. 13).Zweig and Chignell (30) and Wilson ef al. (28) have shownmore recently that podophyllotoxin binds to the same siteon microtubular proteins as does colchicine. Consequently,the assembly of microtubules and development of mitoticspindles are prevented and cells, unable to divide, accumulate in metaphase. Like most podophyllotoxin derivatives,PBG (which is chemically closely related to VM 26) is shownto act somewhat like the mitotic inhibitors colchicine andVCR. In spite of the differences in concentrations used, all 3

drugs inhibited cell multiplication and arrested cells duringmitosis (Tables 1 and 2). The fact that the increase in themitotic index with colchicine and VCR was linear for the 1st4.5 hr indicates that manipulation of the cell cultures duringdrug addition did not result in cell synchronization. Although the intracellular content of nucleic acids and proteincontinued to increase, but perhaps at a reduced rate, incorporation of thymidine and uridine was obviously inhibited.Leucine incorporation was not affected. The apparent discrepancy between increased DMA and RNA content per celland decreased incorporation of labeled precursors (4) isprobably due to pool alterations of the nucleotide triphos-phates directly participating in the synthesis of nucleicacids or to inhibition of the precursor transport into thecells (28). This demonstrates that precursor incorporationstudies alone may be an insufficient and unreliable index ofnucleic acid synthesis and may lead to incorrect interpretations.

In contrast to the typical mitotic blocking agents, treatment with the 2 other podophyllotoxin derivatives, VM 26and VP 16-213, quickly reduced the mitotic index of thenonsynchronized mastocytoma cells to almost zero. In thisrespect these drugs differ from colchicine, VCR, and PBGand also from BLM and ICRF 159. At the same time, proliferation of cells ceased, while the amount of DMA, RNA, andprotein per cell continued to accumulate throughout theexperiment, as one would expect from cells that are arrested before entering mitosis. Incorporation of leucine increased concomitantly with the accumulation of protein,whereas the continuously decreasing rate of thymidine incorporation again did not reflect the gradual accumulationof intracellular DNA. In this respect the 2 compounds resemble PBG to which they are related chemically. This lattereffect seems to be due to that part of the podophyllotoxinmolecule which is common to all 3 derivatives. Incorporation of labeled uridine into RNA, on the other hand, wasdependent on the concentration of VM 26 and VP 16-213.Lower drug concentrations produced a rise of incorporatedradioactivity, while higher concentrations inhibited uridineincorporation into RNA, indicating an additional mode ofaction that may come into play at the higher concentration.

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A. Grader et al.

VM 26 produced quantitatively effects almost identical withthose of VP 16-213 at a 10-fold higher dose. These resultsconfirm earlier suggestions (5, 9, 17, 24) that these compounds arrest cells in the late S or G>phase of the cell cycle.

A remarkable resemblance was detected between the effects of VM 26 and VP 16-213 and those of ionizing radiation. We found that P-815 cells exposed to X-rays immediately stopped proliferation and mitotic activity, which is inaccordance with other reports (11, 12). After a certain period of time, cells recovered and apparently regained theirnormal growth pattern. During this period of delay in division, there was continual increase in the amount of DNA,RNA, and protein per 106cells, indicating an accumulationin G-, phase of the cell cycle. Correspondingly, incorporation of uridine and leucine also gradually increased. However, except for a transient recovery around the 3rd hr afterirradiation, incorporation of labeled thymidine was stronglyinhibited (6). These results lead to the conclusion that X-ray-induced delay in cell division results in a G2accumulation,similar to the G-,arrest of cells induced by VM 26 or VP 16-213. However, the drug-induced G2arrest is poorly reversible (17, 20), whereas irradiated cells soon resume proliferation for 1 ora few generations (11).

BLM, a glycopeptide-containing antibiotic, has beenshown to inhibit DNA synthesis without affecting RNA andprotein synthesis in Escherichia coli, Ehrlich ascites, andHeLa cells (21, 23). Other investigators have reported aBLM-induced increase in DNA content of VX-2 carcinomaand Ehrlich ascites carcinoma cells following their arrest inthe 1st half of G2 (10). It seems that BLM-induced effectsdiffer with concentration, duration of treatment, and type ofcells. In our in vitro system with P-815 mastocytoma cells,BLM at the relatively high concentration of 100 ^g/ml considerably decreased cell proliferation without significantlyincreasing the mitotic index over control values, indicatingthat only a few cells reach mitosis. Thymidine incorporationwas strongly reduced, while uridine and leucine incorporation were in general unaffected with the exception of alower rate between the 3rd and 5th hr of incubation. Intra-cellular levels of DNA, RNA, and protein increased throughout the experiment, which seems compatible with the proposed late S or early G-, arrest of cells. However, othermechanisms must also be involved to account for the persistence of some mitotic cells (or entry of new cells intomitosis).

ICRF 159, a cytostatic agent with biochemically unknownmode of action, revealed some interesting features. In ourpresent investigation with nonsynchronized mastocytomacells, we have found that DNA, RNA, and protein contentper cell increased more or less steadily with ICRF 159 and tothe same extent as the cell number in control cultures.These results are in contrast to those of Creighton andBirnie (1), who found ICRF 159 to be a potent inhibitor ofDNA synthesis in growing mouse embryo fibroblasts. Theirinterpretation, however, was based on incorporation of labeled precursors following 22 hr of treatment with the substance, which may explain the discrepancy. Incorporationof uridine and leucine were not impaired under our conditions, while thymidine incorporation initially continued unaltered until the 5th hr of treatment, after which it de

creased. It would seem that this compound neither interferes with macromolecular synthesis nor changes the pyri-dine nucleotide pool sizes. At 10 Â¿Â¿g/ml,it completely prevents cell division. The permanent increase of the mitoticindex reflects at least a partial block of cells in mitosis,although most of the cells seemed to be arrested in G2(dataobtained from an analysis of DNA distribution by means ofpulse cytophotometry). Such an interpretation seems compatible with the findings of Sharpeef al. (16), who observeda block in late G2and early mitosis and noticed the appearance of large multinuclear cells after treatment with ICRF159.

The antimetabolite ara-C is a powerful inhibitor of theDNA polymerase and, as a consequence, of DNA synthesis(2). It reduces grossly the rate of progression from G, into Sphase (22). We confirmed these findings. Cell proliferationwas stopped, and thymidine incorporation as well as DNAaccumulation were completely inhibited. However, ara-Cdid not interfere with synthesis of RNA and protein asjudged by the absence of an effect on incorporation of theirlabeled precursors and levels of RNA and protein. Interference with DNA synthesis alone, however, would not explainwhy the mitotic index decreased within 1.5 hr after additionof ara-C. This suggests that ara-C also inhibits passage ofcells through G2 into mitosis. This is in agreement withYataganas ef a/. (29), who studied the effect of ara-C on theprogression of human lymphoid cells through the cell cycledetermining the mitotic index, the [3H]thymidine labeling

index, and the distribution of the relative DNA content usingflow microfluorometry. Their results revealed that most ofthe cells were blocked upon entering S phase. Cells thatcompleted S phase accumulated in G2, while a minor portion of the population was blocked in G,.

The antimetabolites MTX and 6-MP produced similar inhibitory effects upon P-815 mastocytoma cells. Both agentshave been shown to inhibit the de novo synthesis of purineribonucleotides and, accordingly, DNA and RNA synthesis(27). Fundamentally, we obtained similar results, althoughthe slower decline of mitotic cells in our cultures treatedwith 6-MP suggests that this compound delays all phases ofthe cell cycle. Under our conditions in which the drug wascontinuously present during 6 hr of incubation, 6-MP produced an inhibition of incorporation of labeled nucleic acidand protein precursors, while the intracellular content ofDNA, RNA, and protein remained constant. This indicatesthat the cells discontinued macromolecular synthesis. Inaddition to interfering with the production of purine ribonucleotides, MTX acts as an inhibitor of dihydrofolate reduc-tase (25). Treatment with this drug stopped cell proliferationand provoked a marked drop of the mitotic index. Precursorincorporation as well as the intracellular level of DNA wasdecreased, pointing to a block in G, and S phases. RNA andprotein synthesis were also affected, but to a lesser extentafter 6 hr of incubation in MTX.

HN2 produces a variety of effects because of its alkylatingactivity (26). In our study, too, HN2 affected a broad spectrum of parameters in that it considerably inhibited cellproliferation, mitotic activity, and all metabolic events, asmight be expected from its mode of action.

In conclusion, our comparative investigation revealed





that cytostatic drugs of various categories exert their owncharacteristic effects on cellular and biochemical parameters. Although there are some similarities, no agent used inthis study exhibited the same effects as did VM 26 and VP16-213. The effects differed in 1 or more parameters, or atleast the degree of interference was different. With respectto the measured responses, the effects of the epipodophyl-lotoxins were closest to those of X-rays. On the basis ofthese findings, we believe that the mechanism of action ofboth epipodophyllotoxin derivatives is unique and differsfrom that of the other drugs tested in this investigation.Their effects are, among others, compatible with a block inG2and agree with those observed by others (3, 8, 9).

ACKNOWLEDGMENTS

The authors wish to thank Professor R. Weil (Geneva, Switzerland), for hisadvice and for many helpful discussions, and A. Bordmann and H. Lehmann.for their skilled technical assistance.

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SEPTEMBER 1977 3005



1977;37:2998-3005. Cancer Res Alfons Grieder, Richard Maurer and Hartmann Stähelin CulturesDerivatives and Other Cytostatic Agents on Mastocytoma Comparative Study of Early Effects of Epipodophyllotoxin

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