Effects of cordycepin on microtubules of cultured mammalian cells

Printed I” Sweden Copyright @ 1978 by Academic PRY\, Inc. All rights of reproduction in any form rexwed 0014.4R27/79/0lOilO-13$O?.M)/O

Experimental Cell Research 118 (1979) 1-13

EFFECTS

OF CORDYCEPIN

OF CULTURED ARLINE Department

ON MICROTUBULES

MAMMALIAN

D. DEITCH and STANLEY

of Pathology,

Columbia

University,

CELLS G. SAWICKI’

New York, NY 10032, USA

SUMMARY Metaphase cells accumulate in HeLa and Vero cultures exposed in G2 to cordycepin (3’deoxyadenosine, 3’dA) at doses of 25-100 pg/ml. The arrested cells have small spindle zones and reduced numbers of interpolar or non-chromosomal spindle microtubules. While these concentrations of 3’dA have been used to suppress mRNA synthesis, the mitotic arrest does not appear to result from inhibition of RNA synthesis. Synchronized Vero cells treated in G2 with sufficient concentrations of actinomycin D to suppress transcription of virtually all RNA do not subsequently become arrested in mitosis. It is proposed that cordycepin interferes with the formation or normal functioning of the mitotic spindle. The microtubules of interphase cells may also be affected by cordycepin. The amount of microtubular crystals formed after exposure of several different cell types to vinblastine sulfate is markedly reduced after pretreatment with cordycepin.

Cordycepin (3’deoxyadenosine, 3’dA) inhibits the growth of Ehrlich ascites tumors in mice [l] and is cytostatic to cultured tumor cell lines [24]; these effects have been ascribed to interference with nucleic acid synthesis [5]. This nucleoside analog is known to suppress the formation of ribosomal precursor RNA, transfer RNA (tRNA) and mitochondrial RNA (mtRNA), while sparing the transcription of heterogenous nuclear RNA (HnRNA) [6, 71. The post-transcriptional addition of poly(A) to HnRNA is markedly inhibited by 3’dA, resulting in a rapid decrease in polyadenylated messenger RNA (mRNA) [8, 93. Because this last effect occurs promptly upon its application, cordycepin has been frequently used in studies on the formation, processing and decay of mRNA. Studies on Album cepa indicate that cordycepin arrests root tip meristem cells in prophase, and it has been suggested that 3’dA may block the production of a specific nucleic acid necessary for the breakdown

of the nuclear membrane during mitosis [lo, 111. We have found that cordycepin arrests cultured mammalian cells in prometaphase and metaphase [ 121; however, in this report we show that the antimitotic effect does not result from inhibiting the production of a specific RNA necessary for completion of mitosis, but rather may result from blocking the formation or functioning of the mitotic spindle. The decrease in the amount of vinblastine-induced crystals of microtubular protein found after cordycepin pretreatment of interphase cells suggests that cordycepin treatment affects the polymerization of microtubules. MATERIALS

AND METHODS

Culture methods Monolayer cultures of HeLa (American Type Culture Collection), HEp2, Vero (African green monkey kidney) and MDBK (Madin Darby bovine kidney) cells ’ Current address: Department of Microbiology, Medical College of Ohio, Toledo, OH 43699, USA. E.w

Cdl

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Deitch and Sawicki

were grown at 37°C in an atmosphere of 5 % CO, in ait in a growth medium consisting of Eagle’s minimal essential medium (MEM, Auto-Pow, Flow Laboratories. Rockville, Md) supplemented with 10% (vol/vol) newborn calf serum (Grand Island Biological Co., Grand Island, N.Y.). Cultures were maintained in exponential growth and subcultured twice weekly by trypsinization with 0.25% trypsin and 0.15% ethylenediamine tetraacetic acid (EDTA) in Earle’s balanced salt solution (EBSS, SchwarzlMann, Orangeburg, N.Y.) buffered at pH 7.4 with 25 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) replacing sodium bicarbonate.

HELP.

60 c

60

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40 20 0

012

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Fig. I. Abscissa:

Cell enumeration Cell numbers were determined with an electronic particle counter (Model B, Coulter Electronics, Hialeah, Fla). Mitotic indices (mitoses/103 cells) were performed by scoring numbers of mitoses in 2000 cells/ monolayer culture. Differential mitotic indices were obtained by multiplying the mitotic index (MI) by the percentage frequency of each mitotic stage, determined by counting a minimum of 200 mitotic cells.

Cell synchrony HeLa cells were synchronized by the method of Pedersen & Robbins [13]. Mitotic cells, detached from monolayer cultures by shaking, were accumulated in S phase by exposing them to 2 mM thymidine (TdR) for I2 h. Ten hours after relief of the thymidine blockade cordycepin was administered in growth medium to replicate cultures and the MIS determined at hourly intervals. Vero cells were synchronized by trypsinizing stationary phase (predominately GI) cultures and replanting them at subconfluent density in growth medium containing cytosipe arabinoside (ara-C, 2.5X 10m6M). After 14 h incubation, the ara-C block was removed, the cells were refed with deoxycytidine ( 10m4M) in growth medium for 1 h and thereafter refed with fresh growth medium. At 8: h after relieving the ara-C blockade, when about 80% of the cells were in G2, replicate cultures were refed with growth medium containing the appropriate drugs and mitotic indices determined at hourly intervals thereafter.

Microscopy For examination of living cultures, cells were subcultured in Cooper dishes (Falcon Plastics Division, B-D Laboratories, Los Angeles, Calif.) modified to permit high magnification microscopy [ 141. Phase microscopy was performed with a Wild M40 inverted microscope with an air curtain device (Sage Instruments Division, Orion Research, Inc., Cambridge, Mass.) for maintaining the temperature at 37°C. Leitz Differential Interference Contrast optics were used to examine and photograph the extent of the spindle in glutaraldehyde-fixed monolayers. (We are indebted to Dr Manfred Nahmmacher, Director of the Laboratory for Applied Microscopy, E. Leitz, Inc., Rockleigh, N.J., for the use of this equipment.) Cytological observations were made of cells fixed in alcohol : formalin : acetic acid (AFA; 85 : IO : 5, volhol)

time (hours); ordinarr: differential mitotic index. O---O, Prophase; O---O. prometaphase and metaphase; A- - -A, anaphase and telophase. Frequency distribution of mitotic phases in unsynchronized HeLa and Vero cultures treated with 50 pg/ ml 3’dA for the times indicated.

and stained with azure B. Mitotic index determinations were made on AFA-fixed cultures stained with the Feulgen reaction and counterstained with fast green, or stained with azure B after extraction of RNA by brief hydrolysis in 4 N hydrochloric acid in order to permit visualization of mitotic chromosomes. (For a description of the staining procedures used see Deitch

L151.1

For transmission electron microscopy (TEM), cultures grown in plastic vessels were fixed in 2% phosphate-buffered glutaraldehyde (pH 7.2), scraped off the surface, rinsed, osmicated and stained in bulk with 0.25 % aqueous uranyl acetate before being pelleted in agar, and dehydrated, embedded and stained by standard procedures. (We are indebted to Dr Gabriel C. Godman, Department of Pathology, Columbia University, for providing the electron micrographs.)

Cordycepin Cordycepin, 3’deoxyadenosine (3’dA), obtained from Sigma (Sigma Chemical Co., St Louis, MO), was chromatographed together with a sample of authentic cordycepin (kindly furnished by Merck & Co., Rahway, N.J.) and was found to have the same R, value as the reference compound using several solvent systems. (We are indebted to Dr Elinore Brunngraber, Department of Biochemistry, Columbia University, for performing the chromatographic analysis.) A stock solution of I mg/ml was made in glass-distilled water, sterile filtered and stored at 4°C. The usual concentration used in these studies was 50 ~g/ml(200 PM).

Colcemid Colcemid, 10 Kg/ml in Dulbecco’s phosphate-buffered saline (PBS) (Grand Island Biological Co., Grand Island, N.Y.), diluted in growth medium to the appropriate concentrations, was used in experiments designed to compare the mitotic accumulation of cultures treated with colcemid as compared with those treated with cordycepin.

Cordycepin Morphometry microtubular

of cultured cells

3

of vinblastine-induced crystals

Relative amounts of vinblastine-induced microtubular crystals were determined by point sampling [16] of photomicrographs of Zenker fixed, phosphotungstic acid hematoxylin-stained [I71 monolayer cultures. The cultures were pretreated with medium containing SO pg/ml cordycepin or with drug-free medium and were then exposed to 10 @g/ml vinblastine sulfate (1.23~ 10e5M Velban, E. Lilly) for If-3 h to induce microtubular crystal formation [ 18-201. Photomicrographs taken of randomly selected fields, printed at 1040x, were overlaid with a transparent rectangular grid with a 2.7 mm spacing. The number of grid intersections falling on crystals was determined for 100 cells/sample.

RESULTS Mitotic

affects microtubules

Ot-----. 0

2.5 59

IO

25

50

015

03

!x

cont. (&ml); ordinate: MI. Cm, HeLa; A---A, Vero. Mitotic indices of unsynchronized HeLa and Vero cultures treated with increasing concentrations of cordycepin or colcemid for 4 h.

Fig. 2. Abscissa:

arrest induced by cordycepin

The proportion of prometaphase and metaphase cells in the population begins to increase within the first hour after addition of 50 pg/ml cordycepin to unsynchronized, exponentially growing HeLa and Vero cultures (fig. 1). By 311 h, maximum numbers of mitotic cells have accumulated. During this time, cells in anaphase and telophase decline in number while the proportion of cells in prophase remains constant for the first 2-3 h and decreases thereafter. The rise in mitotic index (MI) observed with 50 pglml3’dA could be prevented by simultaneously adding 200 PM adenosine; HeLa and Vero cultures treated with cordycepin plus adenosine for 4 h have MIS similar to those of drug-free control cultures. This observation suggested that the nucleoside analog might arrest mitosis by depleting reserves of acid-soluble high energy phosphate compounds considered to be necessary for spindle maintenance [21] and for progression through metaphase [22]. Treatment with deoxyglucose (DOG) is known to deplete the ATP and GTP content of cells by formation of the slowly metabolized derivative, 2-deoxyglucose-6-phosphate (DOG) [23,24]. However, in contrast to the elevated MI found after cordycepin, we

found that application of 6.7 mM DOG in glucose-free medium for 4 h decreased MIS to 30-35% of those found in control cultures. The extent of the mitotic arrest observed upon cordycepin treatment is dependent on dose and is similar in both cell types (fig. 2). There is a concentration-dependent rise in MI between 5 and 25 pglml 3d’A applied for 4 h; no further increase ensues with higher concentrations. Treatment times longer than 4 h, or higher drug concentrations (greater than 50 pg/ml), result in some decrease in MI from the maximum, and pyknotic cells become evident in the population. The mitotic arrest after cordycepin is comparable in extent to that found after colcemid (fig. 2). The accumulation of mitotic cells for 311 h after cordycepin treatment in unsynchronized cultures suggests that at least a fraction of the population is competent to complete G2 in the presence of the analog. To examine this more closely, we synchronized HeLa populations in S phase by addition of excess thymidine (fig. 3). Ten hours after relief of the thymidine blockade, when the majority of cells were in G2, cordycepin was added to half the cultures. Exp Cd Rcs I18 (19791

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Deitch and Sa\ticki 80 t

20 0

60

t 40

t

Fig. 3. Abscissa: time after removing TdR (hours); ordinafe: % mitoses.

Percent mitotic cells in HeLa cultures synchronized by mitotic shake-off followed by 2 mM thymidine for 12 h [13]. Replicate cultures were exposed to 50 &ml 3’dA (B---U), or drug-free medium (O-O), 10 h after relief of the thymidine blockade.

After 1 h, the MIS of the drug-free control cultures peaks at 20% and then declines to a minimal level of 2 % at 15 h after removing the thymidine blockade. In contrast, the MI of HeLa cultures treated in G2 with cordycepin rises to 27-33 % and remains high for the next 4 h before beginning to decline slowly. Because Vero cells are relatively resistant to the cytotoxic effects of high doses of actinomycin D (AMD) and remain viable for at least 12 h after virtually complete (>99%) suppression of RNA synthesis [25], it is possible to compare the MI of G2 populations of Vero treated with AMD and 3’dA (fig. 4). Stationary phase, predominately Gl, Vero cells were trypsinized and replanted at subconfluent density in medium containing ara-C. Eight and a half hours after removing an ara-C block to completion of S phase, when approx. 85% of Vero cells were in G2,3’dA or AMD was added to the synchronized cultures. A wave of mitoses occurs in both the control (drugfree) and the AMD-treated cultures. Although the AMD present was sufficient to prevent more than 95% of RNA transcription [26], this treatment did not significantly alter the numbers of mitotic cells from those found in drug-free cultures. The suggestion that cordycepin inhibits a species of RNA

Fig. 4. Abscissa: time after removing ara-C (hours); ordinare: % mitoses.

Percent mitotic cells in Vero cultures synchronized with ara-C bv the procedure described in Materials and Methods. Eight and one half hours after relief of the ara-C blockade, replicate cultures were refed with drug-free medium (G-O), 2.5 @g/ml AMD (+-.-+) or 50 uglml3’dA (W--W). Some of the cultures treated with 3’dA for 2 h were refed thereafter with drug-free medium (O---Cl).

necessary for completion of mitosis [ 10, 1l] therefore is not tenable, since virtually complete suppression of RNA synthesis with AMD does not lead to an accumulation of mitotic cells. In contrast, in the cordycepintreated cultures, mitotic cells appear in greater numbers than in drug-free or AMDtreated cultures and they remain present for the next 4-6 h. Removing the cordycepin after 2 h permits only an insignificant decline in the numbers of mitotic cells observed. If 50 pg/ml of cordycepin is applied directly to a mitotic Vero population (obtained by shake-off of mitotic cells after a thymidine block followed by release into drug-free medium), it does not slow their entry into interphase. It would seem therefore that in order to arrest mitosis, this dose of cordycepin has to be present when the cells are in the G2 phase of the cell cycle. Cultures treated with cordycepin for 2-3 h and then refed with drug-free medium gradually resume growth. Arrested metaphases occur during the first 4-6 h of the

Cordycepin

0

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affects microtubules

of cultured cells

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Fig. 5. Abscissa: time after removing 3’dA (hours); ordinate: differential MI. O--O, Prophase; O--O, prometaphase and metaphase; A---A, -anaphase and telophase. Recovery after cordycepin. Unsynchronized Vero cultures treated with 50 @g/ml 3’dA for 3 h were refed with drug-free medium for the time indicated on the abscissa. Frequency distributions of mitotic phases was determined.

recovery period; at the same time the other mitotic stages are reduced in number (fig. 5). By about 9 h after removing cordycepin, the cells resume growth and grow thereafter at normal rates (fig. 6). Morphological aspects of cordycepininduced mitotic arrest

The appearance and fate of arrested mitotic cells differ in the two cell lines. After 3-6 h of arrest in cordycepin, most HeLa cells revert to interphase without completing mitosis or cytokinesis. This is similar to the “restitution” to interphase commonly observed after prolonged colchicine treatment [27]. A typical sequence of events in HeLa treated with cordycepin is shown in fig. 7. Here three cells, seen first in prophase or prometaphase at 24 h -after adding cordycepin, are observed to undergo changes including chromosomal decondensation and reformation of nuclear membranes. By the end of 6 h in cordycepin, two of the cells are flattened and extend broad cytoplasmic processes which contact neighboring cells, while the third cell, although

Fig. 6. Abscissa: time (hours); ordinate: cell no. X IO-‘. Growth curve of Vero cultures recovering from cordycepin treatment: Cultures were exposed to 50 unlml 3’dA for 0 h (O-O), 21 h (A---A), or 5 h (Ok), before refeeding with drug-free medium. Duplicate cell counts were made at the times indicated.

still rounded, has apparently reformed a nuclear membrane and reverted to interphase. Vero cells eventually complete mitosis successfully after a l-6 h arrest in metaphase. Arrested metaphases are seen in the continuing presence of cordycepin and for at least 6 h after removal of the nucleoside analog. Cells entering mitosis from 2-5 h after refeeding with drug-free medium were observed to remain in metaphase for an average of 2 h before completing mitosis. The spindle of cordycepin-treated cells

Spindle microtubules persisting in cordycepin-treated cells are normal in diameter, length and in the presence of an electrondense wall and electron-lucent interior. Some of the spindle microtubules can be observed to be attached to kinetochores. However, the presence of profiles of mitochondria and endoplasmic reticulum within the spindle zone as seen in some cells suggests that interpolar microtubules may be absent or reduced in number (fig. 8), since the presence of such non-chromosomal Exp Cell

Res 118(1979)

Cordycepin affects microtubules of cultured cells

Fig. 8. HeLa cell treated with 50 pg/mI 3’dA for 3 h

before fixation. (a) Spindle microtubules can be seen oriented to both poles (arrows). However, profiles of mitochondria (M) and endoplasmic reticulum (ER) are present within the metaphase spindle area suggesting

Fig. 7. Mitotic arrest induced by 50 pg/ml 3’dA in

HeLa cells. The same field of living HeLa cells observed from 24-6 h after addition of cordycepin, X775. (a) At 2 h 30 min, the upper and lower cells are in prometaphase while the cell at the left is in prophase. Note that paired sister chromatids can be seen in the lower cell (arrow). (b) At 2 h 50 mitt, a113 cells are arrested in prometaphase. The individual chromosomes are no longer distinct. (c) At 3 h 10 mitt, both cells on the right extend narrow processes down toward the substratum. (d) At 3 h 30 min, the chromosomes of both cells on the right have become decondensed. (e) At 4 h 40 min, the cell at top right is flattened and has reformed a nuclear membrane. The chromosomes of the other two cells have become less condensed. v) At 6 h, both cells on the right are flattened and have reverted to an interphase appearance. While the cell on the left is still spherical, a nuclear membrane is clearly evident (arrow) surrounding the now decondensed chromatin mass.

7

that few non-chromosomal spindle fibers are present 1281; (b) enlargement of the area shown enclosed in the rectangle, showing normal-appearing microtubules. (a) x 15000; (b) x28 500.

spindle microtubules ordinarily excludes these organelles from the spindle region [28]. In phase contrast micrographs of HeLa and Vero cells in metaphase in drugfree cultures, a pronounced phase-lucent spindle zone is usually seen surrounded by a peripheral cortical region in which the granular elements of the cytoplasm are congregated. In cordycepin-treated HeLa cells, this central clear spindle zone is virtually absent (fig. 7), while in Vero it is relatively normal in appearance. The extent of the spindle zone can be Exp Cell Res I I8 ( 1979)

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Deitch and Sawicki

Fig. 9. Differential interference micrographs of glutaraldehyde-tixed HeLa cells after growth in SOpg/ml 3’dA or drug-free medium. x 1240. (a) Control metaphase showing “depressed” spindle zone with “ele-

vated” spindle fibers converging upon the poles in the clear spindle zone; (b) metaphase after 3 h of 3’dA. with a reduced spindle area.

seen more clearly using differential interference contrast optics (fig. 9). Metaphase HeLa cells grown in drug-free medium have a clearly demarcated spindle area which appears “depressed” (i.e., having a lower index of refraction) when compared with the rest of the cytoplasm and to the chromosomes. In some cells, where the spindle microtubules are presumably closely laterally aligned, elevated spindle “fibers” are seen to converge upon the poles (fig. 9a). After 2-3 h of cordycepin treatment, the spindle is reduced in area and often does not stand out as an area of lower refractive index (fig. 9b). If cultures are refed with drug-free medium after 2 h of 3’dA, the spindle zone becomes enlarged and spindle fibers appear disoriented. After 3 h of treatment, however, return to drug-free medium rarely leads to the formation of a larger spindle zone. Occasionally, after 3 h of treatment, one can observe cells which have reformed a nuclear membrane around condensed chromosomes.

Effect of cordycepin on the induction of microtubular crystals by vinblastine Treatment of living cells with high condissociates centrations of vinblastine formed microtubules and complexes tubulin into large intracytoplasmic crystals which consist almost entirely of microtubular protein [18-20,291. The rate and extent of crystal formation in vivo is enhanced by colchicine [30]. We examined the effect of cordycepin on the extent of vinblastine-induced crystal formation in an attempt to determine if the nucleoside analog affected crystallization of the microtubular proteins of the interphase cell.

Exp Cell Res 118 (1979)

Fig. 10. Formation of vinblastine-induced microtubular crystals in interphase cells. Monolayer cultures were exposed to drug-free medium (n, c, e) or to 50 pg/ml3’dA (b, d,fl prior to treatment with IO fig/ml VEB. The cultures were then fixed and stained by the phosphotungstic acid hematoxylin procedure outlined in Materials and Methods. Growing Vero cultures (u, b) and confluent Vero cultures (c, d) were treated for 3 h with drug-free medium (a, c) or 3’dA (b, d) prior to a 3 h treatment with VBS. HeLa (e, f) treated with VBS for l& h; (e) or with 3’dA for 13 h and then with VEIS for 14h 0.

IO

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and Snicicki

Table 1. Effect of length of treatment per cell as determined

by point

on amounts counting”

of vinblastine-induced

crystals

jiwmed

Grid intersects overlying crystals Cell type Heki

HEp2

Time in cordycepin (hours)

Time in vinblastine (hours)

no./cell”

0 14 0 3 0 3 0 3

14 I’ 31 3 1; 1: 3 3

8.9kO.4 2.OkO.4 10.3+0.5 7.4kO.4 2.2-1-0.2 0.7kO.2 4.4kO.2 1.9kO.2

% of control 22 72 31 42

” A transparent rectangular grid with 2.7 mm spacing was used to overlay photomicrographs of randomly selected fields enlarged to 1040~. The number of intersections of the grid lying over the images of vinblastineinduced crystals was counted for 100cells/sample. b Mean + S.E.

Vinblastine-induced crystals are visible in living cultured cells with the phase contrast microscope and can be stained by a phosphotungstic acid-hematoxylin procedure which reveals them with great clarity against an almost unstained cytoplasmic background (fig. 10). Long thin crystals are formed during 13-18 h exposure to vinblastine in most cells of growing cultures in all the cell types examined (HeLa, HEp2, Vero and MDBK cell lines). It is noteworthy that they are reduced in number or virtually absent in cells of confluent cultures of Vero and MDBK, cell types which show growth regulation ([31], and our unpublished observations). In these two cell lines, cordycepin pre-treatment almost completely prevents the formation of vinblastine-induced crystals. In HeLa and HEp2 cultures the inhibitory effect of cordycepin on crystals formation is less marked and is reversed with increasing time after removal of the nucleoside. Quantitative morphometric measurements (point-sampling [ 161) were therefore performed to determine whether cordycepin

causes a decrease in crystal formation in these two cell types as well. The data in table 1 indicate that more vinblastine-induced crystals form in HeLa than in HEp2 cells. However, in both cell types, cordycepin pre-treatment brings about a marked reduction of vinblastine-induced crystal formation. This is particularly evident when post-treatment with vinblastine is short (14 h). Upon prolonging the time after removal of cordycepin to 3 h, the inhibition of crystal formation becomes less marked. These data suggest that the amount of microtubular protein available for complexing by vinblastine is greater in HeLa than in HEp2, and that cordycepin reversibly inhibits the ability of vinblastine to aggregate tubulin into crystals.

DISCUSSION Cordycepin arrests cultured mammalian cells in mitosis. G2 cells are susceptible, and the resulting prometaphase-metaphase block lasts for 14-6 h or longer. While cor-

Cordycepin affects microtubules of cultured cells dycepin is known to cause a sharp decrease in the amount of newly synthesized mRNA found on cytoplasmic ribosomes [8, 91, the observed mitostatic effect can not be attributed solely to suppression of formation of mRNAs necessary for completion of mitosis for several reasons. (1) We found that almost total suppression of RNA transcription by a high dose of actinomycin does not result in mitotic arrest (fig. 4). (2) It is generally agreed that the bulk of cytoplasmic polyadenylated mRNA in cultured cells is long-lived with a half-life of 6 or more hours [32]. (3) All available evidence indicates that microtubules exist in a state of dynamic equilibrium with pools of soluble tubulin [33-361. A very considerable excess of soluble tubulin exists in cultured cells; less than 5 % of the total tubulinlcell is believed to be assembled into microtubules in subconfluent cultures [37]. Alternatively, cordycepin may be envisaged as inhibiting the formation or functioning of the mitotic spindle. The kinetic and morphological aspects of mitotic arrest after cordycepin resemble those reported after inhibiting spindle formation with colcemid [38, 391. Kleinfeld & Sisken noted that after a low dose of colcemid (0.1 pg/ml; 2.5~ lo-” M), a lag of 30 min occurred before 97-100% of HeLa cells entering mitosis are blocked from continuing on to anaphase. After 2 h of treatment, cells entering mitosis are blocked in pro-metaphase (“star metaphase”) or in metaphase, and the latter cells had smaller than normal mitotic spindles. After longer treatment times, cells either completed cytokinesis in the presence of the drug, or are observed to form a “membrane” around the chromosomes. Upon refeeding with drug-free medium, completion of cytokinesis is accomplished after a delay of 3/4-3 h [38]. A higher dose of colcemid (0.06 pg/ml; 1.5~

11

lo-’ M) was found to permit the formation of normal chromosomal microtubules (kinetochore microtubules), but prevents the formation of interpolar (non-chromosomal) spindle microtubules [39]. Differential sensitivity of these two classes of spindle microtubules has been recorded for a number of treatments including exposure to cold, vinca alkaloids and increased hydrostatic pressure [40], and has been reported here for cordycepin. Tobey et al. [41] proposed that progress through mitosis requires the continuous production of energy from a respirationlinked process. Cordycepin enters the cell competitively with adenosine [2] and is rapidly phosphorylated to the metabolically stable derivative, 3’dATP [42]. Klenow has demonstrated that application of 3’dA, like DOG, depletes the cellular pool of acidsoluble high energy compounds [5]. If this were the primary basis for the cordycepininduced mitotic arrest, then we might expect that exposure to other inhibitors of oxidative phosphorylation should also induce a similar rise in mitotic index. However, we observed a decrease rather than an increase in mitotic activity using DOG. Furthermore, dinitrophenol and other uncouplers are known to prevent the formation of the mitotic apparatus of marine eggs [21], and addition of respiratory inhibitors or uncouplers to Chinese hamster cells (with the exception of rotenone) does not lead to increased numbers of mitoses [43, 441. (Rotenone, a potent inhibitor of mitochondrial respiration, was found by Tobey et al. to arrest cells in metaphase [22]. However, this antimitotic effect has more recently been attributed to the ability of this drug to bind to tubulin at the colchicinebinding site [45].) It is therefore improbable that the prolongation of mitosis induced by cordycepin is primarily attributable to deE.vp Cdl Rr., II8 (IY7Y)

12

Deitch and Sawicki

pletion of respiration-linked high-energy phosphorous compounds. The antimitotic effects caused by cordycepin can be prevented by the simultaneous addition of equimolar amounts of adenosine. Klenow observed that cordycepin is rapidly phosphorylated to the 5’-triphosphate; however, this phosphorylation could be prevented by addition of adenosine [5]. Cordycepin triphosphate has been shown to be a powerful competitive inhibitor of ATP in enzymatic reactions involving RNA polymerase, adenosine kinase and cyclic nucleotide-independent nuclear kinases [4649]. While the roles of phosphorylating enzymes in microtubule polymerization, depolymerization and chromosome movement are virtually unknown at present [51-531, it is generally believed that microtubule polymerization and spindle function involve many ATP-dependent enzymes [50]. If true, the mitostatic effect of cordycepin may result from competitive inhibition of such enzymes. The decrease in amount of vinblastineinduced microtubular crystals observed after pretreatment with cordycepin suggests that cordycepin also affects the microtubules of interphase cells. Vinblastine is known to disrupt formed cytoplasmic microtubules and to sequester tubulin into compact crystalline arrays [18, 19, 54-561. The structure of such crystals has been elucidated recently by Fujiwara & Tilney [57], who proposed that the crystals are formed from microtubular protofilaments arranged as closely-packed bihelices. Their model is supported by Erickson [58] and is suggested by earlier studies which show electron micrographs of vinblastine-induced bihelices assembled in vitro [59, 601, and it is consistent with biochemical evidence that vinblastine induces a progressive stepwise aggregation of tubulin [6 1,621. The explanaE.rp CellRes

118 (1979)

tion of the decrease in crystal formation found after treatment with cordycepin may be inhibition of protofilament formation. In contrast to the potentiating effect exerted by colchicine [30], the reduction in vinblastine-induced crystals after cordycepin clearly indicates that the two drugs interact differently with microtubules. This research was supported by Grant Number CA 13835,awarded by the NCI, DHEW and by Training Grant 5TOl GM 02050 awarded by NIGMS, DHEW.

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