Effects of anti-microtubule agents on Paramecium cell culture growth

Eu ropea n Journal of

Europ .J. Proti stol. 27,283-289 (199 1) September 9, 1991

PROTISTOLOGY

Effects of Anti-Microtubule Agents on Paramecium Cell Culture Growth Regina Pape, Roland Kissmehl, Rene Glas-Albrecht and Helmut Plattner Biological Faculty, University of Konstanz, Konstanz, FRG

SUMMARY Since there are no systematic studies available on the effects of anti-microtubule agents on ciliated protozoa, we screened a wide variety of such compounds for their effects on the growth of Paramec ium tetraurelia cell cultures. Compounds tested include agents of widely different chemical compo sition and with reported effects on widely different cell types. We can differentiate between different dru g effects: (a) Rotenone is the only agent without any recognisable effect. (b) Another grou p of compounds (including colchicine) requires very high concentr ations, as compa red to higher animal cells, i.e., rather close to a cytotoxic level; this group also includes tubul ozole (unexpectedly without any difference between the cis- and the trans-stereoisomer). (c) A third group of drugs inhibits cell culture grow th witho ut any lethal effects (benzimidazo les, nocodazole, parbendazole; the (anti- ]fungal antibio tic, griseofulvin; the herbicide, tr ifluralin). (d) Finally a gro up of agents are active in a concentration range also repor ted for plants (the herbicide , APM) or for higher animal cells (including the microtubule sta biliser, taxol ) or for both (vinblastine, vincristine, tr iethyl lead), alth ough they are cytotox ic at higher concentrations (like compo unds of group [bJ). Therefore, in part icular compounds of gro up (c) and possibly of group (d) might be considered further on for a more detailed analysis of a possibly genuine anti -microtubular effect in Param ecium cells. Of pa rticular interest may be nocod azole, parbendazole and trifluralin, since they can inhibit cell cultu re growth (over 24 h tested ) in relatively low concentrations (comparable to other cell types) without any impairment of cell viability.

Introduction In the ciliated protozoon Paramecium tetraurelia microtubule s are importa nt not only for positioning of nuclei [10, 18,60], for nuclear [67, 68] and cell division [65], but also for ciliary locomotion, transcellular transport of some organell es and maintenance of the shape of the cell and of some of its subcellular constituents . The cortex of a Paramecium cell is held rigid by microtubules oriented parallel to the cell sur face, emanating from ciliary basal bodies [65). These also serve as organ isers for a transient populat ion of vertically oriented microtubules which might guide tricho cysts to the cell cortex [18,54]. This probably represents the ultra stru ctur al basis of saltatory transport of trichocysts and mitoch ondria [6]. Microtubular rails also guide discoidal vesicles from the cytoproct to the cytostome dur ing membrane recycling [3]. For the © 1991 by Gustav Fischer Verlag, Stuttgart

display of different microtubule populations in the gullet, see [18]. Finally the cytoproct [5] and the osmoregulatory system [4,32] are enforced by microtubules. For a systematic survey, see [17, 18,29]. These also estimated microtubule s in one Paramecium cell to be of an unexp ected length of up to 200 mm! In conclusion, microtubules appe ar to be of par amount importance for variou s cell function s in paramecia. Nevertheless, there are only a few systematic studies available on the effects of ant i-microtubule agents on protozoa, in particular on Paramecium, whereas such drugs are frequently applied to higher eukaryotic cells. The dat a available for Paramecium concern the inhibitory effect of vinblastine on position ing of macronucl ei and of trichocysts [10, 60]. It has also been known for a long time that these functions, along with lysosomal functions [26] and nuclear division [50], are not affected by colchicine 0932-4739/91/002 7-0283$3.50/0

284 . R. Pape, R. Kissmehl, R. Glas-Albrecht and H. Plattner

[26,50]. Furthermore, nocodazole has been shown to affect cytoplasmic microtubules [66] and nuclear division [17, 18]. Other agents have not been tested with Paramecium before. Therefore, we analysed the effects of a wide variety of agents, among them some introduced fairly recently in cell biology. There are (A) disrupting or (B) stabilising compounds acting on microtubules, among the latter taxol [47]. In short, according to reviews on microtubule functions and anti-microtubule drugs [12,27,53], compounds of group (A) comprise: (a) plant alkaloids (colchicine, vinblastine, vincristine) used mainly to inhibit mitotic spindle formation in mammalian cells; (b) the anti-fungal antibiotic (of fungal origin) griseofulvin, a mitosis inhibitor in higher animal and plant cells; (c) some anti-fungal and antihelminthic benzimidazole compounds, like nocodazole (also anti-mitotic in mammalian cells) and parbendazole; (d) the respiratory chain inhibitor rotenone; (e) some chemically widely different herbicides, trifluralin and APM ("amiprophos-methyl"); see [53]. More recently (f) triethyl lead, also with known effects on plant and mammalian cells [28, 72], has been introduced, as well as (g) tubulozole, whose cis-, but not the trans-stereoisomer acts on microtubules in mammalian cells [69]. Besides these drugs we have also analysed the effects of cold treatment (disruption) and of DzO (stabilisation of microtubules). We investigated the effects on P. tetraurelia cell culture growth since this aspect can be analysed most reliably. It should reflect any disturbance of different microtubule subpopulations (known to have a different turnover [17,68] and, hence, different drug sensitivity [17,61]) and of different vital cell functions depending on them. The data we obtained may serve as a guideline for a more detailed analysis of microtubule dependent functions in Paramecium cells. As we shall show, however, only a few anti-microtubular agents may be considered for further use with Paramecium.

Material and Methods We used wild type cells (a derivative of stock 51 from strain 75) of Paramecium tetraurelia, cultivated at 25°C in a medium prepared from dry salad powder, monoxenically inoculated with Enterobacter aerogenes as a food source. Cells in logarithmic growth phase were processed as follows. The day before the experiments, cells were transferred to a 10-fold volume of fresh medium, again with E. aerogenes added. To test the effect of different compounds, 10 cells per experiment were transferred onto glass plates with 20 depressions (serving for several parallel assays) and adjusted to a final volume of 100 ul each, including the drug solutions. All experiments were carried out at 25°C. Glass plates were kept for up to 24 h (in some cases 2 days) in a moist chamber. For some experiments samples were transferred to O°c. To characterise the compounds and their use, we indicate the source, their M, value (Dalton), the possible addition of dimethylsulfoxide (DMSO, mostly in a final concentration of 0.5 % v/v [only exceptionally slightly higher]) and the highest nominal concentration tested. (Solubility was controlled by visual inspection only.) We used: Colchicine (Merck, Darmstadt, FRG;

459 Da, -DMSO, tested up to 38 mM); vinblastine sulfate (Sigma, St. Louis, Mo., USA; 909 Da, -DMSO, ::s 275 ~M); vincristine sulfate (Serva, Heidelberg, FRG; 923 Da, + DMSO, ::s 270 ~M); nocodazole (Sigma; 303 Da, + DMSO, ::s 19 ~M)j parbendazole (TAD Pharmazeut. Werke, Cuxhaven, FRG; 247 Da, + DMSO, ::s 20 ~M); griseofulvin (Sigma; 353 Da, + DMSO, ::s 570 ~M); rotenone (Sigma; 394 Da, + DMSO, ::s 18 ~M); trifluralin (Dow-Elanco, Indianapolis, Ind., USA; 335 Da, + DMSO, ::s 300 ~M); APM (Bayer AG, Leverkusen, FRG; 305 Da, + DMSO, ::s 160 ~M); triethyl lead (DKZ, Heidelberg; 330 Da, - DMSO, ::s 2 mM); cis- and trans-tubulezole (janssen, Beerse, B; 545 Da, + DMSO, ::s 92 ~M); taxol (Nat!. Cancer Inst., Silverspring, Md., USA; 854 Da, + DMSO, up to 59 ~M tested). D 2 0 from Aldrich (Milwaukee, Wisc., USA) was tested up to 50% v/v. After different time periods we determined the number of living Paramecium cells per depression and plotted the resulting growth curves. From the dose-dependency curves we determined the inhibitory concentrations (IC sD, IC w D, 24 h). In parallel we always determined culture growth without additives or with DMSO only, as used in the respective experiments. Since DMSO effects were small (0.5% DMSO causing only a -7% reduction of culture growth over 24 h), this appeared negligible. Similarly we counted the number of remaining viable cells in order to determine lethal concentrations (LC sD, LC wD, 24 h) of different compounds.

Results We determined cell culture growth curves over up to two days for controls (without or with 0.5% v/v DMSO; see "Material and Methods") and in presence of various anti-microtubule agents. Three kinds of drug effects were observed. (a) Some drugs resulted in a concentration dependent growth reduction, with lethal effects at higher concentrations, as exemplified in Fig. 1. These drugs include colchicine, vinblastine, vincristine, APM, triethyl lead, t- and c-tubulozole, as well as taxo!. (b) Some other drugs caused a reduction of cell culture growth without lethal effects, i.e., cultures closely maintained their original cell density. This is demonstrated for nocodazole (Fig. 2), but holds also for parbendazole, trifluralin. The concentration-response curve for griseofulvin is in between those of group (a) and (b). (c) The only compound without any reduction of cell density was rotenone (tested up to a maximally soluble concentration), as shown in Fig. 3. From the cell culture growth curves of all compounds tested we derived the 50% and 100% inhibitory concentrations (IC sD, IC lOD, 24 h; Figs. 4-6), as summarised in Table 1. Since the effect of nocodazole levels off slightly above the original cell density, a IC lOD value can only be roughly extrapolated in this case. We also determined lethal concentrations (LC so, LC roo, 24 h) for different drugs by counting surviving cells in the samples and then by extrapolation from the concentration dependency curves. The resulting values are presented in Table 1. LC values missing in Table 1 indicate that, e.g., griseofulvin, nocodazole, parabendazole, rotenone and trifluralin exert no lethal effects.

Anti-microtubule Agents in Paramecium . 285 40

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One can also derive from Table 1 that, although vinblastine, triethyllead and taxol exert cytotoxic effects at higher concentrations, they inhibit cell culture growth in a concentration range which is normally applied also to higher eukaryotic animal cells (Table 2; see also "Discussion "). The same holds for APM with regard to plant cells.

With the Colchicum and the Vinca alkaloids, colchicine and vincristine, excessively high concentrations are required to achieve any effect with paramecia (Table 1), in striking contrast to higher animal cells (Table 2). Another remarkable aspect is the practically identical effect of either one of the tubulozole stereoisomers, but again rather

Figs. 1-3. Cell culture growth in dependence of the concentration of colchicine (Fig. 1), nocodazole (Fig. 2) and rotenone

286 . R. Pape, R. Kissmehl, R. Glas-Albrecht and H. Plattner Table 1. Values of rcso, rc tOO, LCso and LC lOo [I-tMoles/L] determined for different anti-microtubule compounds with P. tetraurelia cells Compound Colchicine Vinblastine Vincristine Griseofulvin Nocadazole Parbendazole Rotenone Trifluralin APM Triethyl lead c-Tubulozole r-Tubulozole' Taxol

ICso

IC wo

LCso

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Discussion Depending on their effect on Paramecium cell culture growth, we can now differentiate between different groups of agents (Table 1). Onl y some of them exert no lethal effect on paramecia in any concentration used (griseofulvin, nocodazole, parbendazole, trifluralin). Others become to xic in higher concentrations (colchicine, vinblastine, vincristine, APM, tr iethyl lead, c- or t-tubulozole and taxol). All these compounds also reduce the culture growth rat e of paramecia. Onl y rotenone has no effect at all. Dat a compiled in Table 2 are representative values, to comp are the response of paramecia with cells of similar or of different systematic levels. However, it has to be borne in mind that, even when different mammalian cells [70] or different microtubule populations in one and the same cell are compared [61], the effects of anti-microtubule drug s might be quite variable. In detail, comparison of the IC 100 values determined for par amecia (Table 1) with data from other protists (algae and protozoa; for Stentor, however, see below), plant cells and from metazoan cells (Ta ble 2, with references) reveals the following. As generally kno wn, paramecia are remarkably insensitive to colchicine (see, e.g., [26, 50]), just as are other lower eukaryotes [34], including protists, and plant cells (see below and Table 2). Sensitivity to vincristine is also not very high (Table 1). With vinblastine we observed considerable sensitivity of cell culture growth; similarly even as little as 27 /lM vinblastine disturbs the positioning of macronuclei and of trichocysts [10, 60]. Sensitivity to griseofulvin is low in comp arison to plant cells and

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high concentrations are required. For more details , see below. D 2 0 in concentrations up to 50 % v/v had no effect on cell division. Exposure to 0 °C stopped cell culture growth completely. 50 % lethality was observed within 3 h, 100% within 6 h.

Table 2. IC lOo values (concent ra tions [p.Moles/L] required for maximal inhibition of P. tetraurelia cell culture growth) for the different compounds tested, in comp arison to data (from the literature) on concentrations used in oth er systems to block various microtubule dependent cell functions' Compound Colchicine

IC lOo in P. tetraurelia 34 x 10 3

Vinbl astine

7

Vincristine

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75

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75

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Trierhyl lead

Taxol

0.1

1.9

1-10

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0

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a A wide variety of cell functio ns has been considered. b Th is indicates either no effect (ro tenone) or no IC lOo achieved (see text ). c [43] ; d [59]; e [8]; 1[24]; s No data available to us; h [71 ]; i [39, 40]; j [55]; k [64]; 1[57 ]; m[58]; n [21]; ° [9, 37]; p [31]; q Physarum: [35]; r [45]; s [30]; r [25]; u [28]; v[13]; w [63]; x [23]; y [11] ; z [36]; aa [51]; bb [20]; cc [34]; dd [14]; «[72]; ff [62, 69]; gg [44]; hh [19, 61].

Anti-microt ubule Agents in Paramecium . 287

particularly to metazoan cells. A surprisingly strong response is observed with parbend azole and nocodazole. Th e respon se to the two herbi cides tested , APM and trifluralin, is similar to th at in plant cells; other author s have reported only a slightly higher sensitivity of other protists (Table 2 ). Param ecium cells respond to triethyl lead in a concentration equal to, or lower than indicated for met azo an cells. Paramecia are also quite sensitive to taxol, just as some other prot ists [9, 37]. Much higher taxol con centrations are requ ired to disturb the arrangement of microtubules in heliozoan axonemes [33]. Two main reason s are discussed in the literature, wh y some established anti -microtubule drugs may not be active in different protozoa, notably low permeability [56] or an unfavourable binding con stant, e.g., for tubulin-colchicine interaction [46]. This aspect may be seen in line with some other molecular data, e.g., the limited immunological crossreactivity of anti-tubulin antibodies, when Paramecium is compared with different systematic groups [1,2]. Our dat a compare favourabl y to the few data published so far on Param ecium (fo r vin blastine, see abo ve). For instance, colch icine has little effect (or only in high concentrations) on the po sitioning of macronuclei or of cortical organelles [10,60] , on the biogene sis of the lysosomal apparatus [26] (tho ugh this involves microtubules, see "Introduction ") , or on nuclear division [50]. On the oth er hand our dat a on noco dazole effects also fit well with the relatively high sensitivity of cytoplasmic rnicro tubuies in Paramecium [66]. The results we obtained with different drugs are also in agreement with the effects on different cell functions in Tetrahymena [41, 42 , 59]. Like paramecia, oth er lower euka ryotes are sensiti ve to parbend azole [34 ]. In contrast to our findings, a mu ch higher sensitivity has been reported only for the regeneration of oral cilia in Stentor in pr esence of trifluralin, but surprisingly also with colchicine [8, 48]. One reason for this discrep ancy between members of rather closely related systematic groups might be that some ciliates possess colchicine-sensitive and -insensitive micr otubules [27]. However, in a more recent investigation of ora l cilia regeneration in Stentor, 0.9 M colchicine was requ ired for full inhibition [15]. Also the sensitivity of thi s system to vin blastine [15, 22 J resembl es tha t of Param ecium (Ta ble 1) and of metazo an cells (Table 2). Similar to Plasmodium [21] , Param ecium is affected by both, c- and r-tubulozole, also in comparabl e con centrations. As to the effect of cold tr eatm ent (see " Results"), we have not established this to be relat ed to microtubular fun ctions, although some micro tubular subpopulations are kno wn to be cold sensitive in a va riety of cells [38], including paramecia [66] and some other protists [49]. Any selective effect on the different microtubule subpopulations (see [27,61], and "Introduction") rema ins to be analysed in paramecia, with the different drugs we found to be efficient. Another open question con cerns possible side-effects on non-microtubule related functions, as frequently reported for other cells with different dru gs, particularly at high concentra tio ns (see, e.g., [7, 52 , 62]).

From our own dat a we conclude tha t some of th e agents tested will not be of any particular interest for further wo rk with para mecia. Th is holds especially for colch icine, rotenone and D 20 ( ~ 50% v/v D20 inhibiting mito sis in plant cells [16]). Other dru gs also appear less important, such as vincristine and griseofulvin. Of some interest may be compounds actin g at relat ively low concentration s: Vinbl astin e, parbend azole, nocoda zole, trifluralin, APM , tr ieth yl lead and taxol. Since nocodazole , pa rb end azole and trifluralin are not cytotox ic even beyond IC lOO values, th ese agents might deserve particular interest for further work with paramecia. On e would expect that th ey wo uld exert only little side-effects. Acknowledgements We thank Dr. H. P. Zimmermann (Deutsches Krebsforschungszentrum) for a sample of rrierhyl lead, Dr. J. Douro s (National Cancer Institute) for a gift of taxol and the firms Dow-Elanco as well as Bayer AG for pro viding samples of trifluralin and APM, respectively. We also thank Mr s. M. HarralSchulz for helping us in prepari ng this manu script. Support ed by SFB 156.

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Key words: Cell division - Ciliates - Microtubules - Paramecium - Protozoa Helmut Plattner, Biological Faculty, University of Konstanz, P.O . Box 55 60, D-7750 Konstanz, FRG