Effect of ~~~tetrahydrocannaliinol on cyclic nucleotides in synchronously dividing Tetrah ymena

Divisiorl of Natural Science, Clesldon College, York University , Torotzto, Qnt., and Departme,tt of Zoology, Ut~ivursiby of Toronto, Torouto, Brat., Carlhld~

Zimerman, S., Zimerman, A. M. & Laurence, H. (198%) Effect of A"tetrahydrocannabino1 on cyclic nencleotides in synchronously dividing Tetrukrymena. Can. J'. Biochem. 59,489-493

Cyclic nucleotide levels were determined in division-synchronized Tetralrymeias and the effect of A@-tetrahydrocannabinol (THC) on the cyclic nucleotide levels was studied. In non- drug-treated division-synchronized cells, there was no statistically significant variation in the level of cAMP and cGMP during the G2 period, preceding the first division. During the free running cell cycle (the interval of time between the first and second synchronous division) the twofold increase in the level of CAMP was statistically significant; however the variation in the level of cGMP was not statistically significant.

THC caused a lowering of cAMP and cGMP levels throughout the 4-h experimental treat- ment. The suppression of cAMP and cGMP levels altered the cyclic nucleotide pattern of the cell cycle. The cAMP pattern was changed particularly in the G2 period preceding the first synchronous division, and immediately after division during the free running cell cycle. THC treatment caused division delays of approximately 8-15 min in the onset of the first and second synchronous division. However, the duration of the free running cell cycle (1 10-820 min) was unchanged. The suppression of cyclic nucleotide levels resulting from THC treatment is dis- cussed in relation to delays in the division schedule.

Zimrnerrnan, S . , Zimerman, A. M. & Laurence, H. (1981) EKect of A$-tetrahydrocannabinol on cyclic nucleotides in synchronously dividing Tetraliyrnetaa. Carl. J. Biockem. 59,489-493

Nous avons determind le taux des nucldotides cycliques chez Tetral~yrnntci se divisant de fagon synchrone et nous avons CtudiC l'effet du A$-tetrahydrocannabinol (THC) sur le taux de ces nuclCotides cycliques. Bans les cellules non traiaCes, se divisant de fagon synchrone, il n'existe aucune variation statistiquement significative dans les teneurs du cAMP et du cGMP durant la pCriode Gz prkCdant la premikre division. Durant le cycle cellulaire sans division (H'intemalle de temps entre la premikre et la seconde division synchrone), la teneur du cAMP augmente de deux fois, une augmentation statistiquement significative; cependant, la variation du taux du cGMP n'est pas statistiquement significative.

Le THC diminue les taux du cAMP et du cGMP durant les 4 h que durent le traitement expkrirnental. La suppression des taux du CAMP et du cGMP altkre le profil des nuclCotIdes cycliques durant le cycle cellulaire. Le profil du cAMP est particulikrement changC durant Ba pCriode G z prkCdant la premikre division synchrone et immddiatement aprks cette division, durant la pCriode sans division du cycle cellenlaire. Le THC retarde d'environ 8 h 15 min le debut de la premikre division et de la seconde division synchrones. Cependant, la d u r k de l'intervalle entre la premikre et la seconde division synchrone(ll0-128 minldemeure inchaaagd. Nous discutons de la suppression des teneurs'des nucldotides cycliques, causCe par le traitement avec le THC, en relation avec les retards dans le cycle des divisions.

[Traduit par le journal]

Introduction Studies on THC, the active psychomimetic can-

nabinoid in marihuana, have shown that it sup- presses macromolecular synthesis in cell cultures (I9 2 ) and in protozoa ( 3 ) . Other experiments have revealed that THC suppresses the humoral im- mune response in rodents (4, 5) and the blasto- genic response of lymphocytes derived from mari- huana users (6). It has been postulated that THC

ABBREVIATI~~NS: THC, A'-tetrahydrocannabinol; RIA, radioinnmunoassay; TCA, trichloroacetic acid.

'Author to whom all correspondence should be addressed at Division of Natural Science, Glendon College, York University, 2275 Bayview Ave., Toronto, Ont., Canada.

acts on the plasma membrane of cells as a conse- quence of its affinity for lipid molecules. In this respect, it is interesting to note that MeIlors a d co-workers demonstrated that THC blocks acceler- ation of phospholipid turnover during lymphocyte transformation ('7) and suppresses lysolecithin acyltransferase, a membrane-bound enzyme that is involved in lymphocyte transformation (8 ) . How- ever, the action of THC on membrane biochemistry still remains elusive.

Cyclic nuclestides are considered to act as mes- sengers interacting between receptor sites on the cell membrane and intracellular activity. According t s Wang et al. (9) CAMP is involved in lympho-

oSer8-4018/81/078489-05$01 .W/O fG 1981 National Research Council of Canada/Conseil national de recherches du Canada

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490 CAN. J. BIBCHEM. VOL. 59, 1381

cyte cell cycle progression; they reported that both a rise and fall in CAMP levels were required for the onset of DNA synthesis. Cyclic nucleotide levels have also been reported to be related t s specific events during the cell cycle in Tetrwhymenw (10- 12).

In the current study we report on the cyclic nucleotide 'levels during the cell cycle sf sgmchro- nously dividing Tetrcirhymena pyuiformfs and the effects sf THC on cyclic nucleotide levels in these synchronously dividing ce'l'ls.

Mate~als and methods

Cukturing o{ organi.~m Axenic cultures of T , pyriformis strain GE-1 were main-

tained in 2% proteose peptone (Difco Chemicals. British Drug Houses, Toronto, Ona.) supplemented with 0.1% liver fraction E (Nutritional Biochemical Corp.. Cleveland, OH). This growth medium is referred to as PPL. Stock cultures of cells were maintained at 28°C.

Cell qwehrsrtieati~nn Division synchrony was achieved by the one thermal

shock per generation method of Zeuthen ( 1 3 ) . This tech- nique consisted of administering a 38-min thermal shock six times at 34°C separated by periods of 159 min at 28°C. The p e r i d of time following the last heat shock is called EH. Cell density after eight treatments ranged from 30 000 to 50 000 ~eIBs~rnL-~. Gel1 counts were determined with a model 22 Coultes counter (Goulter Electronics, Hialeah, FL) fitted with a 286b-pm aperture tube. The test agents were added to cells, at the optimum growth temperature sf 28"C, immediately after the last heat treatment.

Chemicals The THC was generously supplied by Mr. R. A. Gr,&m,

Health Protection Branch, Health and Welfare Canada, Ttanney's Pasture, Ottawa, Ont. Stock THC is supplied as 200 rng -HHL-~ in ethanol. Stock THC (150 pL) was i n t r e duced into 100 mL of cell suspension (final concentration 9.6 p M ) . An equal amount of ethanol was added to the controls. Previous studies (3) have shown that this volume of ethanol does not affect form, movement, growth, division schedule, or macrsmoleculas synthesis of log phase o r division-synchroni%ed cultures of Tebr6shymcna.

The New England Nuclear RIA kit for the determination s f CAMP and cGMP was purchased from New England Nuclear, Boston, MA. The CAMP $%''I tracer consists sf CAMP ~~IJ-2'-Qd-succinyl(idotyrosi~e methyl ester) > 150 Ci .mrnoI-I ( 1 CCi = 37 GBq) . The cGMP '"1 tracer con- sists of cGMP p'T]-2p-O-su~~irnyl(iodotyr~~ine methyl ester) > f 50 @i a mmol-*.

Assay prdse~dkcre At various designated times post EH, duplicate 5-mL

aliquots s f cells ( 3 x BOkeells - rnL-I) were taken from both control and drug flasks. Smples were washed three times by centrifugation with phosphate buffer (pH 6.75). Cells were resuspend& in 0.5 mE of cold 6% TCA, vortexed, and frozen quickly in acetone - dry ice mixture and stored in a freezer. Thawed samples were homogenized with a Teflon glass Potter-EBvehjem homogenizer at 4°C. Hsrnoge- nates were centrifuged at 1800 x g for 15 min. Super- natants were collected and extracted three times with 5 m%

of H20 saturated ether. Final extracts were placed in a HzO bath at 65°C and allowed to evaporate to dryness. The dry residues were dissolved with 1 m L of sodium acetate bulffer (0.05 M , pH 6.2) . A 100-pE aliquot of each sample was assayed for CAMP and cGMP. The immunsassay pro- cedure was adapted from that of Steiner eb a f . (14) as given with the H A kits from New England Nuclear, Montreal, P.Q.

Efiect of THC ore the cell cycle The cell cycle induced in Tetrahynsena after the

one heat shock per generation synchrony method (13) resulted in division peaks which occurred at 88-90 min and 190-21 0 min EH. The interval of time between the first and second synchronous di- vision, designated as the free running cell cycle (dur- ing which there is no thermal treatment), was 110- I20 nain. As previously shown 15) there is no dis- tinct GI phase in the free running cell cycle since ["]thymidine incorporation occurs immediately after the first synchronous division; the length of S phase is approximate'ly 70 min. A representative experiment illustrated in Fig. I - . shows a peak in division index of 86% at 80-min EH and a peak in division index of 48% at 200-min EH. This figure also reveals a sharp twofold increase in cell density immediately after the first division which reflects the synchrony of the division.

A concentration of 9.6 pM THC was used in this study. This dosage was considered a moderate dose on the basis of previous experiments which showed that 9.6 pM THC caused a modest reduction in log growth and a short delay in the first synchronous division of Tetrshyrnenw ( 3 ) . In the present ex- periments, 9.6 pM THC caused delays sf approxi- mately 8-15 min in both tke first and second syn- chronous divisions. In all cases? however, the dura- tion of the free running cell cycle was not altered but remained the same, as in the control cells, i.e., 110-120 min.

Efleek of THC on CAMP and cGMP activity The overall patterns of CAMP and cGMP levels

during the cell cycle of division-sgmchronized, con- trol cells, were similar (Figs. 1B and 1 C; Table 2 ). Cyclic nucleotide levels fluctuated during the G2 period preceding the first division; however, there was no significant variation in the levels of CAMP and cGMP ( p > 0.4). An increase in CAMP level was observed during the period preceding the sec- ond division. During the free running cell cycle, the concentrations of both CAMP and cGMP increased twofold. The increase in the level of CAMP from division 1 to division 2, i.e., 1.82 pmol to 4.4 pmol was statistically significant ( p < 0.81 ) . However, the increase in the 'level of cGMP which rose from

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ZIMMERMAN ET AL. 49 1

0.63 pmol at division 1 to 1.36 pmol at division 2

,, g was not statistically significant ( p > 0.05). In gen- eral, the level sf cGMP was three t s four times lower than that sf CAMP.

The addition of 9.6 pM THC, immediately after " the last thermal treatment, caused a general sup-

pression of CAMP and cGMP levels and reduced oscillation of the cyclic nucleotides throughout the 4-h experimental period. The overall suppression of CAMP and cGMP levels by the drug, across all test times, was statistically significant at the 0.1 % level when the raw data and log transformed data were assessed by two-way analysis of variance. The CAMP pattern in the THC-treated cells was particu- larly altered in the G2 period preceding the first synchronous division, immediately after division 1 and at the time of the second synchronous division. Two-way analysis of variance of the log-transformed data showed that the differences between the CAMP levels of control and THC-treated cells were statis- tically significant at 50-, 1 10-, 140-, and 200-min

C cGM$ EH. Similarly, the cCMP control and THC-treated

A/A-/

/ cells showed statistically significant differences at ~-A/A-A && A---- ,as@@ 5Q-, 1 18-, 148-, 178-, 206)-, and 230-min EH A----A--- -a%----& ---- &----A@-- (Table 1 ). I L I l 1 I I l L I I I I

40 80 120 160 200 240 Time after EH (rnin)

FIG. a. Effects of 9.6 ,uM THC on cell division, CAMP, and cBMP levels. Terrakymena were synchronized by the one heat shock per generation method of Zeuthen (IS) . The time EH represents the time after the last thermal shock. (A) The development of division maxima, the percent of cells dividing ( @ ----- @ ), and the mitotic index as determined by cell density ( x . a - X ) are shown at various times after EH. Stages of the cell cycle are also shown. The arrows indicate the division maxima of Tetra- hymeran continuously exposed to THC commencing im- mediately after the last heat treatment. (B ) The Bevels of CAMP in control cells (W----B) and THC treated cul- tures 48- - -8). ( C ) Levds sf eBMP in controI (A-A) and THC treated cells ( A - - - A ) .

Ht is interesting to note that in a separate pre- liminary study designed to determine drug-dose effects, the level of CAMP at 50-min EH was found to be a dose dependent function of THC. The level sf CAMP was elevated 27% above control values when cells were treated with 3.2 pM THC. As the concentration of THC increased to 9.4, 16, and 32 pM the level of CAMP compared with control values progressively decreased 2 1, 67, and 86 9% , respectively; at the two higher doses some cell lysis was evident. This finding is similar to that found in brain tissue isolated from mice, in which THC caused both elevation and depression of CAMP levels, depending upon the dosage employed ( 16).

TABLE I. Effects of THC on levels of CAMP and cGMP during the ceil cycle

CAMP levels, cCMP levels, pmol - m l cells-' f SE gmol r n l cells-3 f SE

Time after EH, min Experiments Controls THC (9.6 M M ) Cgssatrols THC (9.6 prM)

%ignifisantly different from control value at the I %level using two-way analysis of variance on leg-transformed data. bSignificantly different from control value at the 5 % level using two-way analysis of varianee on Beg-transformed data.

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482 CAN. J. BIBCHEM. VOL. 59, 1981

In the present study, THC caused a lowering of cAMP and cGMP levels throughout the cell cycle of division-synchronized Tetrahymena. This sup- pression of CAMP and cGMP Ievels altered the pattern of cyclic nucleotides with respect to phases of the cell cycle. THC treatment also caused division delays of approximately 8-15 min which may be related to the lowered cyclic nucleotide levels.

Prior studes of cyclic nucleotides in synchro- nized Tetrahymena have revealed fluctuations dur- ing the cell cycle. Dickinson et al. (10) found that cAMP reached maximum values at division, in selection-synchronized Tetrahymena. However, Kassis and Zeuthen ( 12) reported maximum cAMP values at the end of S phase and minimum cAMP values at division in Tetrahymena, synchronized with the one thermal shock per generation method of Zeuthen (13). The apparent contradiction in these results may be a reflection of the different synchrony methods used in the two studies. For ex- ample, different synchrony methods have been noted to result in different patterns of nuclmside phospho- transferase activity ( 15) .

In the current study, cAMP values were ex- pressed as picomoles per millilitre of cell culture. These values were normalized for 106 cells from cell counts taken at the beginning of the experiment (or immediately after the last heat shock). There was no apparent difference in the general pattern of cyclic nucleotides when values were expressed as picomoles per rnillilitre of cultures or picomoles per 10%ells (cf. Ref. H 2) . During the free running cell cycle, cAMP levels reached a high level in G2 and peaked at division, similar to the pattern noted in selection synchronized Tetvakymena ( 10) . The different cAMP patterns at diyisions 1 and 2, noted in our control studies, may be related to biochemical changes which result from the thermal treatment used in the synchrony. In the period preceding the first synchronous division, cells were pulsed with heat during the Batter part of the S phase and divi- sion occurred approximately 80 rnin after the heat pulse. During the free running cell cycle, there was no heat pulse and the duration of the cycle was ap- proximately 1 1 5-1 20 rnin.

Fluctuations in cyclic nucleotides have also been studied in division-synchronized Tetrahymena sub- jected to X-irradiation ( 1 1 ). Although cAMP levels were suppressed in irradiated cultures before and after cell division the level of cGMP markedly in- creased 108-fold immediately after division in both irradiated and nonirradiated cultures. In our study, a similar magnitude of cGMP increase was not ob-

TABLE 2. Cyclic nucleotidelevels determined from T'etmBqvnaensr synchronized (18) by alternating 30-min heat shocks

Nontreated THC-treated controlsa ceHsb

Time cAMP"cGMPVAMPC cGMPe

Cells collected during the last thermal treatment 6 .2 1 .2 - -

30-WBin EH 9.3 1 . 5 7.7 0.9 90-1~1h EH g 0 3 0.8 9 .2 2 .1

15&min EH 4.8 1 . 1 5 .8 1-0 21Grnira EH 5.0 1.2 5.3 1.2

aFirst and second synchronous divisions occur at 70- and 170-min EH. bCells were continuously exposed to 9.6 THC commencing immedi-

ately after the last thermal shock. Division in THC ceBBs was delayed 20-30 min.

Walues expressed in picornotes per Boa cells.

s e n d in nontreated control cells. To establish whether the difference in cGMP values was due to the method of synchrony, cells were synchronized by the method of alternating 30-min heat pulses (1 71, a method used by Charp and Whitson (1 1 ), and cyclic nucleotide levels were determined. The resultant values sf cAMP and cGMP (Table 2) , ex- pressed as picomoles per 106 cells, were of a similar magnitude as those found with the synchrony tech- nique of one heat shock per generation. Thus the difference in the cGMP pattern cannot be attributed to the synchrony method but may be related to the method of assaying for cyclic nucleotides.

Cyclic AMP and cGMP have been purported to play a role in the control of cell division in various cellular systems. It has been proposed that elevated levels of intracellular cAMP inhibit cell division (18) or alternately, that lowered levels of intra- cellular cAMP inhibit cell division ( 19 ) . In addition, it has been suggested that cGMP acts antagonis- tically to cAMP in a variety of cell activities and that increased levels of cGMP promote cell growth (1 1, 20). In our experiments, the pattern of oscilla- tion for both cyclic nucleotides was essentially the same in control cells during cell cycle events, al- though the level of cGMP was always lower than that of CAMP. These observations do not suppof$ the idea that opposing levels of cAMP and cGMP, at specific times during the cell cycle, could initiate cell division ( cf. Ref. 2 1 ) . Nevertheless, THC sup- pression of cyclic nucleotides (e.g., the statistically significant depression of the CAMP peak in G2 (at 50-min EH)) may be related to the division delays found in THC-treated Tetrahymena. It has been suggested that the heat-shock method of synchrony causes changes in surface membrane fluidity and lipid composition which result in conformational

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changes in adenylate cyclase (12). It may be that THC which has a high affinity for surface mem- branes may also affect the activity of adenylate cyclase so that CAMP Ieveis are Iowered. The cur- rent study has not resolved the mechanism of action of THC. However, the investigation has shown that cyclic nucleotide 'levels fluctuate during the cell cycle and the pattern may be influenced by the method of cell synchrony. Moreover, the suppres- sion of cyclic nucleotides following THC treatment is compatible with the studies showing suppression of macromolecular synthesis in THC-treated cells 6 3 ) .

This work was supported in part by a research grant %ram Glendon College, York University (to S.Z.) amad by a grant from the Natural Sciences and Engineering Research Council of Canada (to A.M.Z.) .

1. BBevins, %a. D. & Regan, J. D. (1976) in Marihuana, Chemistry, Biochemistry and Cellular Eflects (Nahas, G. G., ed.), pp. 213-222, Springer-Verlag, New York

2. Carchman, R. A., Harris, E. S. & Mupawn, A. E. ( 1976) Carzcer Rw. 36,95-100

3. MsCIean, D. K. & Zimrnerman, A. M. (1976) Phar- macology 14,304-321

4. Rosenkrantz, H., Miller: A. J . & Esber, H. J. (1975) J . Toxicol. Ef2va'aon. $%ealth 1, 1 1 9-1 25

5. Zhmerman, S., Zimrnerman, A. M.. Cameron, 5. %. & Laurence, H. E. (197'7) Pharmacology 15, 18-23 Nahas, G. G., Sucia-Foca, N., Armand, J. P. & Mori- shinma, A. (1 974) Science (Washington, D.C.) 183, 41 9-420

4. Mellors, A. (1974) in Marih~aana, Chemistry, Bio- chemistry and Cellufar Effects (Nahas, G. G., ed.), pp. 283-298, Springer-Verlag, New York

8. Greenberg, J. H., Saunders, M. E. & MelIors, A. (1977) Science (Wnshirrgtsn, D.C.) 197. 475-476

9. Wang, T., Sheppad, J. W. & Foker, J. E. (1978) Science (Wmhington, DOC.] 201, 155-1 57

10. Diekinson, J. R., Graves, M. G. $s Swoboda, B. E. P. (1976) FEBS Lett. 65, 152-154

18. C h q , P. A. $s Whitson, G. L. ( 1978) Radiat. Res. 74, 323-334

12. Kassis, S. & Zeuthen, E. (1949) Exp. Cell Res. 124, 73-78

13. Zeuthen, E. ( 1971 ) Exp. Cell Wes. 68,49-60 14. Steiner, A, L., Parker, C. W. & Kipnis, D. M. (1972)

J . Biol. Chem. 247, 1 106-1 113 15. Bols, N. C. & Zhmerman, A. M. (1 974) Exp. Cell

R ~ s . 108,259-268 16. Dolby, T. W. & Kleinsmitk, L. J. (1974) Biachem.

Phnrmacsl. 23, 18 17-1 825 1'7. Scherbaurn, 0. & Zeuthera, E. (1954) Exp . CeId Res.

6 , 2 2 1-227 18. Pastan, I. H., Johnson, G. S. & Anderson, W. B.

(1975) An~eu. Rev. Biochem. 44,491-522 19. Schor, S. & Rozengurt, E. (1973) 1. Cell. PhysioI. 81,

339-345 20. Seifert, W. & Rudland, P. S. (1974) Prsc. Natb. AcaH.

Sci. U.S.A. 71, 49264924 21. Miller, Z., Lovelace, E., GaIBo, M. & Pastan, I. (1975)

Science (Washiragton, B E . ) 190, 1213-1215

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