- Email: [email protected]
Growth inhibition by competitive inhibitors of 3-hydroxymethylglutarylcoenzyme a reductase in Helianthus tuberosus tissue explants
P l a n t S c i e n c e L e t t e r s , 34 (1984) 269--276
269
Elsevier Scientific Publishers Ireland Ltd.
GROWTH INHIBITION BY COMPETITIVE INHIBITORS OF 3 - H Y D R O X Y M E T H Y L G L U T A R Y L C O E N Z Y M E A R E D U C T A S E IN HELIANTHUS TUBEROSUS TISSUE E X P L A N T S
NELLO CECCARELLI and ROBERTO LORENZI I s t i t u t o di O r t i c o l t u r a e F l o r i c o l t u r a della Universitd degli S t u d i di Pisa, Viale delle Piagge, 2 3 - 5 6 1 0 0 Pisa (Italy)
(Received August 29th, 1983) (Revision received November 21st, 1983) (Accepted November 21st, 1983)
SUMMARY
Compactin and mevinolin, t w o structurally related inhibitors, completely inhibit the growth of Helianthus tuberosus explants at 2.5 × 10 -s M. Mevalonic acid (MVA) restores the growth while sterol precursors like famesol and squalene overcome the inhibition only partially. Growth inhibition was also reversed when a small quantity of MVA was added along with famesol or squalene. The results indicate that the effect o f MVA in preventing the growth induced by compactin or mevinolin goes through the synthesis of two different sets of products: one derived from famesol and squalene and the other o f a non-sterolic nature. Various MVA-derived c o m p o u n d s have been tested for their ability to substitute this small quantity o f MVA.
Key words: Helianthus tuberosus--Growth inhibition - - H M G - C o A reductase -- MVA - - C o m p a c t i n -- Mevinolin INTRODUCTION
A branched p a t h w a y leading to several physiological relevant c o m p o u n d s originates from the metabolism of MVA in plants. The key enzyme in the synthesis of MVA, 3 - h y d r o x y m e t h y l g l u t a r y l c o e n z y m e A (3-HMG-CoA) reductase is competitively inhibited by compactin and mevinolin, t w o structurally related fungal metabolites (Scheme I) [1,2]. The acid form of these Abbreviations: ABA, absciscic acid; BSA, bovine serum albumin; 2,4-D, 2,4-dichlorophenoxyacetic acid; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; GA~, gibberellic acid; 3-HMG-CoA, 3-hydroxymethylglutarylcoenzyme A;Ms. microsomal fraction; Mt, mitochondrial fraction; MVA, mevalonic acid; 2iP, ~2-isopentenyladenine; 2iPA, 2-isopentenyladenosine ; Z, zeatin ; Zr, zeatin riboside. 0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
270 CH3 HO~'~-COOH
S
doA
R=H COMPACTIN R=CH3 MEVINOLIN
3-HMG-CoA
Scheme I. Structures of compactin (ML 236 B), mevinoIin (MK 803) and 3-HMG-CoA.
compounds includes a portion that resembles the HMG moiety of 3-HMG-CoA reductase [3]. The block of MVA-synthesis converts the cells into MVA-auxotroph and can be used for investigating the role of the different products of the MVA-pathway in the growth. Compactin has been successfully employed in the elucidation of a 'multivalent feedback' regulation of 3-HMG-CoA reductase in vertebrates [4]. Such a regulation seems to coordinate isoprenoid synthesis and cell growth. Studies performed in different laboratories with the use of compactin have illustrated the critical role of some non-sterolic products of the MVA-pathway in the regulation of cell replication and growth [4,5]. Compactin inhibition of DNA-synthesis in synchronized cells has also been reported [6]. The inhibition was counteracted only when, in addition to cholesterol, small quantities of MVA were supplied to the cell culture. More interestingly, MVA was replaced by A2-isopentenyladenine (2iP) or zeatin (Z) in restoration of DNA replication blocked by compactin [7 ]. Investigation into the regulatory properties of 3-HMG-CoA reductase in plants using these specific inhibitors have been undertaken only recently [8--10]. In radish plants it has been shown that mevinolin acts as a competitive inhibitor of microsomal 3-HMG-CoA reductase [ 11 ]. Moreover, mevinolin inhibits root elongation in dark-grown radish and wheat seedlings. The root growth inhibition induced by mevinolin in radish plants is reverted by increasing concentrations of MVA [ 12]. We used compactin and mevinolin with H. tuberosus explants as tools for studying the regulatory properties of MVA derived compounds, particularly the growth regulators gibberellins, cytokinins and abscisic acid. MATERIALS AND METHODS
Chemicals The inhibitors compactin (ML-236B) and mevinolin (MK-803) were generous gifts from Professor Akira Endo (Tokyo Noko University) and Dr. A.W.
271 Alberts (Merck, Sharp & D o h m e Research Laboratories, New Jersey), respectively; they were dissolved as described [ 13 ]. MVA lactone, farnesol, squalene, dolichol m o n o p h o s p h a t e , c o e n z y m e Q10, 3-HMG-CoA reductase and the plant hormones were all purchased fzom Sigma Chemicals Co. []4C] 3-HMG-CoA (spec. act. 55 mCi/mmol) was obtained from Amersham. MVA lactone was converted to its sodium salt before use. Tissue culture D o r m a n t tubers of H. tuberosus L. cv. OB1, grown outside by vegetative multiplication were stored in moist sand at 4°C. Tissue disks of medullary p a r e n c h y m a (8.5 mm in diameter, 1 mm thick) were aseptically excised from d o r m a n t tubers as described [14]. The basal medium o f culture was that of Bertossi et al. [15], supplemented with 4% sucrose, 10/aM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.8% Difco agar and adjusted to pH 6. Compactin or mevinolin and MVA were added to the sterilized medium through a millipore filter; all the other c o m p o u n d s were supplied in ethanolic solution. The final concentration o f ethanol in the culture medium, that never exceeded 0.5%o, had no inhibitory effect on explant growth. All experiments were card e d o u t in 5 cm diameter Petri dishes containing 5 ml of culture medium. Immediately after excision four disks o f tissue were placed in each Petri dish and kept in the dark at 25°C for a week, then fresh and dry weights of the explants from each Petri dish were taken. Single treatments were repeated five times. 3-HMG-CoA reductase isolation and assay Preparation o f enzyme solutions. Parenchymatic explants after 4 days of culture were homogenized with a blender in t w o vols. of 50 mM potassium phosphate buffer (pH 7.5) containing 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM dithiothreitol (DTT), 5 mM MgC12, 500 mM sucrose and 8% Polyclar AT. The homogenate was filtered through cheese cloth and centrifuged at 2000 X g for 10 min, the resulting supernatant was centrifuged at 15 000 X g for 30 min and the pellet, resuspended in the homogenization buffer w i t h o u t sucrose and Polyclar AT, was referred to as the 'mitochondrial fraction' (Mt). The supernatant was centrifuged at 105 000 × g for 1 h and the pellet, resuspended as above, was referred to as the 'microsomal fraction' (Ms). Enzyme assay. 3-HMG-CoA reductase activity was assayed b y the m e t h o d described in Ref. 16 with some modifications. The incubation mixture contained 50 mM potassium phosphate buffer (pH 7.5), 10 mM DTT, 10 mM EDTA, 300 pM NADPH, 1 mg bovine serum albumin (BSA), 3-[14C] HMG-CoA, 150 nmol (spec. act. 1450 d p m / n m o l ) and Mt or Ms preparations (equivalent to 0.8 g fresh wt. of tissue) in a total volume of 150 pl. Incubation was carried o u t at 30°C for 30 min and was stopped by adding 30 pl o f 6 N HC1 and 10 pmol of MVA lactone as a carrier. The mixture was then left
272
at 37°C for I h for lactonization. After sedimentation of proteins b y centrifugation 100 t~l of supernatant were streaked o n t o silica gel thin-layer plates, developed with acetone/ethyl acetate/acetic acid (60:30:1) and scanned for radioactivity with a Packard Radiochromatogram Scanner. The silica gel o f the MVA lactone region was scraped into a scintillation vial and 1 ml of water was added. Radioactivity was measured by liquid scintillation counting after the addition of 10 ml of Lumagel. RESULTS
AND
DISCUSSION
Parenchymatic explants isolated from d o r m a n t tubers o f H. tuberosus and placed in a culture medium containing sucrose and an auxin (2,4-D) are stimulated to proliferate [17]. Cell growth occurs only in the o u t e r layers of the explant and the t w o or three first divisions are, at least partially, syncronous [18]. In our experiments the dry weight increase of the explants after 1 week o f culture was a b o u t 100% o f the initial dry weight. Figure 1 shows the effect of different concentrations o f compactin and mevinolin on the fresh and dry weight increase of H. tuberosus explants after 1 week of culture The inhibition effect is a function of the inhibitor concentration and both substances completely inhibit the fresh and dry weight increase at a concentration o f 2.5 × 10 -s M. Since in this plant system the increase in weight is normally due to cell division [17] it can be reasonably argued that compactin and mevinolin block cell division also in plants. Mevinolin has been reported to be slightly more active than compactin in the inhibition of 3-HMG-CoA reductase [19 ]; the dose-response curve repor-
100_ ~
& compactin~ e mevinolin ~ fresh weight
~ ~
"~ ~"~\
0
I
'
'
. compactin'~ o mevinolin .J~dry we,ght
10-7M 10-6M 10-5M 10-4 M Fig. 1. Growth inhibition 0£ H. tuberosus explants by increasing concentrations of mevinolin and compactin.
273
ted in Fig. 1 shows that compactin and mevinolin have very similar inhibitory activity on the growth of H. tuberosus explants. Since response to the various treatments did not show significant differences b y expressing the results as fresh or dry weights, only the latter will be reported u p o n here To verify if the inhibition effect is exerted only at the level of MVA synthesis, different concentrations of MVA were supplied to the explants along with the inhibitors. As can be seen in Fig. 2, 2 mM MVA completely reverts the growth inhibition induced b y compactin or mevinolin. This demonstrates that the growth arrest caused b y these inhibitors in H. tuberosus explants is exclusively exerted by suppressing the synthesis of MVA inside the cells. The result is consistent with the evidence previously obtained in animals [4,20] and with the restoration by MVA of root growth in mevinolin-treated radish plants [ 12]. As is well known, MVA represents a key precursor for sterols that are required for membrane structure and cell growth. It has been reported that compactin totally inhibit the incorporation of acetate into sterols in A c er tissue cultures [12]. To ascertain if the growth inhibition caused by mevinolin or compactin comes from the inability of cells to synthesize sterols we supplemented the growth-medium with farnesol or squalene, two direct precursors o f sterols. While a partial restoration o f growth was obtained when farnesol or squalene were supplied at a concentration of 0.1--0.5 mM, total reversion o f the inhibition was observed only when a small
MEVINOLIN 2,5.10"5M + MEVALONIC ACID 250-
£
COMPACTIN 2,5-105M + MEVALONIC ACID
~0.05 0.1 0.2 1.0 2.0raM
~0,05 0.1 0.2 1.0 2Dml~
C
o
I"1"
200,o
¢2_ I.g
,o
u~
.E
150ac: P,
o
.E
¢0
o.
÷ t
E
100-
50-
1
Fig. 2. Reversal of the growth inhibition induced by compactin and mevinolin by increasing concentrations of MVA. Each point represents the average o f 5 repetitions, mean ± S.E.
274 TABLE I
REVERSAL BY MVA OR STEROL PRECURSORS OF M E V I N O L I N - I N D U C E D I N H I B I T I O N IN H. TUBEROSUS E X P L A N T S
GROWTH
I n i t i a l d r y w e i g h t o f f o u r e x p l a n t s w a s 8 0 rag. E a c h p o i n t r e p r e s e n t s t h e a v e r a g e o f 5 rep e t i t i o n s , m e a n ± S.E. Treatments
Dry wt. (mg)
Dry wt. i n c r e a s e (%)
Control M e v i n o l i n 5 x 10 -5 M MVA2x 10 -3 M M V A 2 X 10 4 M F a r n e s o l 1 0 -4 M S q u a l e n e 5 x 10 -4 M M e v i n o l i n 5 x 10 -5 M M e v i n o l i n 5 x 1 0 -s M M e v i n o l i n 5 x 10 -s M M e v i n o l i n 5 x 10 -s M M e v i n o l i n 5 x 10 -5 M M V A 2 x 10 -4 M M e v i n o l i n 5 x 10 -5 M MVA2x 10-4 M
M V A 2 x 10 -4 M M V A 2 x 10 -3 M F a r n e s o l 10 -4 M S q u a l e n e 5 x 10 -4 M F a r n e s o l 10 -4 M +
245 + 7 91 _+ 5 255+ 7 222+_ 7 2 2 5 _+ 20 242 + 9 179 + 2 239 + 7 1 7 3 _+ 2 5 1 8 6 +_ 9 2 5 3 + 17
100 7 106 86 88 98 60 94 56 64 105
+ S q u a l e n e 5 x 1 0 -3 M +
2 4 0 _+ 13
97
+ + + + +
a m o u n t of MVA (0.2 mM) was also supplied to the explants (Table I). This limited a m o u n t of MVA would be required for the synthesis of some secondary metabolites necessary for total restoration o f explant growth also in the presence of sterol precursors. The effectiveness o f MVA 2.0 mM b u t n o t 0.2 mM to completely restore growth would be ascribable to the availability of enough MVA to s u p p o r t sterol biosynthesis as well as the synthesis of the non-sterol compound(s) (Table I). Recently [22], it has been reported that in b a b y hamster kidney cells MVA plays a dual role in the regulation of the cell cycle. First MVA serves as a precursor for the cholesterol required for membrane structure, cell growth and for the passage of cells through the G1phase; successively, possibly by means o f 2iP or a related cytokinin, MVA is required for the onset of the S-phase DNA replication. Our results suggest that, as in animals, the action o f MVA in counteracting c o m p a c t i n or mevinolin inhibition goes through the synthesis of two different sets o f products: one derived from farnesol and the other o f a non-sterolic nature. In a search for this compound(s) several end-products of the various branches of the terpene p a t h w a y were tested along with farnesol or squalene, for their ability to substitute for the limited a m o u n t of MVA required for total restoration of growth. Various concentrations o f abscisic acid (ABA), gibberellic acid (GA3), dolichol m o n o p h o s p h a t e , c o e n z y m e Q10 were added to the culture medium b u t none of these c o m p o u n d s overcame the requirement for a small a m o u n t of MVA. Also, the effect of cytokinins was particularly investigated in consideration of the ability of 2iP and Z to substitute for MVA in the re-
275 TABLE II EFFECT OF 5 x 10 -7 M MEVINOLIN ON 3-HMG-CoA REDUCTASE ACTIVITY Assay conditions as described in Materials and Methods. Enzyme preparations
M t fraction M s fraction
(dpm) Control
Mevinolin
2370 2135
950 540
storation o f DNA-synthesis inhibited b y compactin in b a b y hamster cells [7]. The addition to the culture m e d i u m of different concentrations of 2iP, A2-isopentenyladenosine (2iPA), Z, zeatin riboside (Zr), 2iPA-5-monophosphate and 2iPA-5-diphosphate did n o t show any significant effect on explant growth inhibition. However, interpretation of these results must be cautious since no evidence is available as to the proper entry and targeting of these substances. Besides, it must be considered that the growth response in tissue explants is a far more complex and lengthy process than cell division in the syncronized cell cultures used in experiments with animals [6,7,22]. It is likely that in prolonged experiments, like those reported here, in the presence of compactin more than one isoprenoid substance could b e c o m e limiting for cell growth. Direct evidence that the growth inhibition induced b y mevinolin in H. tuberosus explants is exerted through the inhibition of 3-HMG-CoA reductase was obtained from in vitro studies. Incubation of both mitochondrial and microsomal preparations with 5 × 10-v M mevinolin resulted in a strong reduction of [~4C]HMG-CoA conversion to MVA {Table II). These results are in accordance with the observations o f Bach and Lichtenthaler in radish plants [11], however, further w o r k is required to better qualify the mechanism of inhibition o f 3-HMG-CoA reductase b y mevinolin and compactin in H. tuberosus explants. As regards this we are n o w trying to improve reliability o f the assay in order to avoid a competitive influence by other 3-HMGCoA utilizing enzymes [23]. REFERENCES 1 A. Endo, M. Kuroda and Y. Tsujita, J. Antibiotics, 29 (1976) 1346. 2 A.W. Alberts, J. Chen, G. Kuron, V. Hunt, J. Huff, C. Hoffman, J. Rothrock, M. Lopez, H. Joshua, E. Harris, A. Patchett, R. Monagan, S. Currie, E. Stapley, G. Alberts-Schonberg, O. Hensens, J. Hirshfield, K. Hoogsteen, J. Liesch and J. Springer, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 3957. 3 A. Endo, Methods Enzymol., 72 (1981) 1684. 4 M.S. Brown and J.L. Goldstein, J. Lipid Res., 21 (1980) 505. 5 I. Kaneko, Y. Hazame-Shimada and A. Endo, Eur. J. Biochem., 87 (1978) 313.
276 6 V. Quesney-Huneeus, M.H. Wiley and M.D. Siperstein, Proc. Natl. Acad. Sei. U.S.A., 76 (1979) 5056. 7 V. Quesney-Huneeus, M.H. Wiley and M.D. Siperstein, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 5842. 8 R.J. Wong, D.K. Mc Cormack and D.W. Russell, Arch. Biochem. and Biophys., 216 (1982) 631. 9 H. Suzuki and I. Uritani, Plant Cell Physiol., 18 (1977) 485. 10 R. Yu°Ito, K. Oba and I. Uritani, Plant Cell Physiol., 20 (1979) 867. 11 T.J. Bach and H.K. Lichtenthaler, Z. Naturforsch., 37c (1982) 416. 12 T.J. Bach and H.K. Lichtenthaler, Naturwissenschaften, 69 (1982) 242. 13 T. Kita, M.S. Brown and J.L. Goldstein, J. Clin. Invest., 66 (1980) 1094. 14 D. Serafini-Fracassini, N. Bagni, P.G. Cionini and A. Bennici, Planta, 148 (1980) 332. 15 F. Bertossi, N. Bagni, G. Moruzzi and C.M. Caldarera, Experientia, 22 (1965) 81. 16 D.J. Shapiro, J.L. Nordstrom, J.J. Mitschelen, V.W. Rodwell and R.T. Schimke, Biochim. Biophys. Acta, 370 (1974) 369. 17 M.M. Yeoman, A.F. Dyer and A.I. Robertson, Ann. Bot., 29 (1965) 265. 18 M.M. Yeoman and P.K. Evans, Ann. Bot. N.S., 31 (1967) 323. 19 A. Endo, J. Antibiot., 33 (1980) 334. 20 A. Endo, Trends Biochem. Sci., 6 (1981) 10. 21 N.S. Ryder and J. Goad, Biochim. Biophys. Acta, 619 (1980) 424. 22 V. Quesney-Huneeus, H.A. Galick, M. Siperstein, S.K. Erickson, T.A. Spencer and A. Nelson, J. Biol. Chem., 258 (1983) 378. 23 R. Yu-Ito, K. Oba and I. Uritani, Agric. Biol. Chem., 46 (1982) 2091.
269
Elsevier Scientific Publishers Ireland Ltd.
GROWTH INHIBITION BY COMPETITIVE INHIBITORS OF 3 - H Y D R O X Y M E T H Y L G L U T A R Y L C O E N Z Y M E A R E D U C T A S E IN HELIANTHUS TUBEROSUS TISSUE E X P L A N T S
NELLO CECCARELLI and ROBERTO LORENZI I s t i t u t o di O r t i c o l t u r a e F l o r i c o l t u r a della Universitd degli S t u d i di Pisa, Viale delle Piagge, 2 3 - 5 6 1 0 0 Pisa (Italy)
(Received August 29th, 1983) (Revision received November 21st, 1983) (Accepted November 21st, 1983)
SUMMARY
Compactin and mevinolin, t w o structurally related inhibitors, completely inhibit the growth of Helianthus tuberosus explants at 2.5 × 10 -s M. Mevalonic acid (MVA) restores the growth while sterol precursors like famesol and squalene overcome the inhibition only partially. Growth inhibition was also reversed when a small quantity of MVA was added along with famesol or squalene. The results indicate that the effect o f MVA in preventing the growth induced by compactin or mevinolin goes through the synthesis of two different sets of products: one derived from famesol and squalene and the other o f a non-sterolic nature. Various MVA-derived c o m p o u n d s have been tested for their ability to substitute this small quantity o f MVA.
Key words: Helianthus tuberosus--Growth inhibition - - H M G - C o A reductase -- MVA - - C o m p a c t i n -- Mevinolin INTRODUCTION
A branched p a t h w a y leading to several physiological relevant c o m p o u n d s originates from the metabolism of MVA in plants. The key enzyme in the synthesis of MVA, 3 - h y d r o x y m e t h y l g l u t a r y l c o e n z y m e A (3-HMG-CoA) reductase is competitively inhibited by compactin and mevinolin, t w o structurally related fungal metabolites (Scheme I) [1,2]. The acid form of these Abbreviations: ABA, absciscic acid; BSA, bovine serum albumin; 2,4-D, 2,4-dichlorophenoxyacetic acid; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; GA~, gibberellic acid; 3-HMG-CoA, 3-hydroxymethylglutarylcoenzyme A;Ms. microsomal fraction; Mt, mitochondrial fraction; MVA, mevalonic acid; 2iP, ~2-isopentenyladenine; 2iPA, 2-isopentenyladenosine ; Z, zeatin ; Zr, zeatin riboside. 0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
270 CH3 HO~'~-COOH
S
doA
R=H COMPACTIN R=CH3 MEVINOLIN
3-HMG-CoA
Scheme I. Structures of compactin (ML 236 B), mevinoIin (MK 803) and 3-HMG-CoA.
compounds includes a portion that resembles the HMG moiety of 3-HMG-CoA reductase [3]. The block of MVA-synthesis converts the cells into MVA-auxotroph and can be used for investigating the role of the different products of the MVA-pathway in the growth. Compactin has been successfully employed in the elucidation of a 'multivalent feedback' regulation of 3-HMG-CoA reductase in vertebrates [4]. Such a regulation seems to coordinate isoprenoid synthesis and cell growth. Studies performed in different laboratories with the use of compactin have illustrated the critical role of some non-sterolic products of the MVA-pathway in the regulation of cell replication and growth [4,5]. Compactin inhibition of DNA-synthesis in synchronized cells has also been reported [6]. The inhibition was counteracted only when, in addition to cholesterol, small quantities of MVA were supplied to the cell culture. More interestingly, MVA was replaced by A2-isopentenyladenine (2iP) or zeatin (Z) in restoration of DNA replication blocked by compactin [7 ]. Investigation into the regulatory properties of 3-HMG-CoA reductase in plants using these specific inhibitors have been undertaken only recently [8--10]. In radish plants it has been shown that mevinolin acts as a competitive inhibitor of microsomal 3-HMG-CoA reductase [ 11 ]. Moreover, mevinolin inhibits root elongation in dark-grown radish and wheat seedlings. The root growth inhibition induced by mevinolin in radish plants is reverted by increasing concentrations of MVA [ 12]. We used compactin and mevinolin with H. tuberosus explants as tools for studying the regulatory properties of MVA derived compounds, particularly the growth regulators gibberellins, cytokinins and abscisic acid. MATERIALS AND METHODS
Chemicals The inhibitors compactin (ML-236B) and mevinolin (MK-803) were generous gifts from Professor Akira Endo (Tokyo Noko University) and Dr. A.W.
271 Alberts (Merck, Sharp & D o h m e Research Laboratories, New Jersey), respectively; they were dissolved as described [ 13 ]. MVA lactone, farnesol, squalene, dolichol m o n o p h o s p h a t e , c o e n z y m e Q10, 3-HMG-CoA reductase and the plant hormones were all purchased fzom Sigma Chemicals Co. []4C] 3-HMG-CoA (spec. act. 55 mCi/mmol) was obtained from Amersham. MVA lactone was converted to its sodium salt before use. Tissue culture D o r m a n t tubers of H. tuberosus L. cv. OB1, grown outside by vegetative multiplication were stored in moist sand at 4°C. Tissue disks of medullary p a r e n c h y m a (8.5 mm in diameter, 1 mm thick) were aseptically excised from d o r m a n t tubers as described [14]. The basal medium o f culture was that of Bertossi et al. [15], supplemented with 4% sucrose, 10/aM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.8% Difco agar and adjusted to pH 6. Compactin or mevinolin and MVA were added to the sterilized medium through a millipore filter; all the other c o m p o u n d s were supplied in ethanolic solution. The final concentration o f ethanol in the culture medium, that never exceeded 0.5%o, had no inhibitory effect on explant growth. All experiments were card e d o u t in 5 cm diameter Petri dishes containing 5 ml of culture medium. Immediately after excision four disks o f tissue were placed in each Petri dish and kept in the dark at 25°C for a week, then fresh and dry weights of the explants from each Petri dish were taken. Single treatments were repeated five times. 3-HMG-CoA reductase isolation and assay Preparation o f enzyme solutions. Parenchymatic explants after 4 days of culture were homogenized with a blender in t w o vols. of 50 mM potassium phosphate buffer (pH 7.5) containing 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM dithiothreitol (DTT), 5 mM MgC12, 500 mM sucrose and 8% Polyclar AT. The homogenate was filtered through cheese cloth and centrifuged at 2000 X g for 10 min, the resulting supernatant was centrifuged at 15 000 X g for 30 min and the pellet, resuspended in the homogenization buffer w i t h o u t sucrose and Polyclar AT, was referred to as the 'mitochondrial fraction' (Mt). The supernatant was centrifuged at 105 000 × g for 1 h and the pellet, resuspended as above, was referred to as the 'microsomal fraction' (Ms). Enzyme assay. 3-HMG-CoA reductase activity was assayed b y the m e t h o d described in Ref. 16 with some modifications. The incubation mixture contained 50 mM potassium phosphate buffer (pH 7.5), 10 mM DTT, 10 mM EDTA, 300 pM NADPH, 1 mg bovine serum albumin (BSA), 3-[14C] HMG-CoA, 150 nmol (spec. act. 1450 d p m / n m o l ) and Mt or Ms preparations (equivalent to 0.8 g fresh wt. of tissue) in a total volume of 150 pl. Incubation was carried o u t at 30°C for 30 min and was stopped by adding 30 pl o f 6 N HC1 and 10 pmol of MVA lactone as a carrier. The mixture was then left
272
at 37°C for I h for lactonization. After sedimentation of proteins b y centrifugation 100 t~l of supernatant were streaked o n t o silica gel thin-layer plates, developed with acetone/ethyl acetate/acetic acid (60:30:1) and scanned for radioactivity with a Packard Radiochromatogram Scanner. The silica gel o f the MVA lactone region was scraped into a scintillation vial and 1 ml of water was added. Radioactivity was measured by liquid scintillation counting after the addition of 10 ml of Lumagel. RESULTS
AND
DISCUSSION
Parenchymatic explants isolated from d o r m a n t tubers o f H. tuberosus and placed in a culture medium containing sucrose and an auxin (2,4-D) are stimulated to proliferate [17]. Cell growth occurs only in the o u t e r layers of the explant and the t w o or three first divisions are, at least partially, syncronous [18]. In our experiments the dry weight increase of the explants after 1 week o f culture was a b o u t 100% o f the initial dry weight. Figure 1 shows the effect of different concentrations o f compactin and mevinolin on the fresh and dry weight increase of H. tuberosus explants after 1 week of culture The inhibition effect is a function of the inhibitor concentration and both substances completely inhibit the fresh and dry weight increase at a concentration o f 2.5 × 10 -s M. Since in this plant system the increase in weight is normally due to cell division [17] it can be reasonably argued that compactin and mevinolin block cell division also in plants. Mevinolin has been reported to be slightly more active than compactin in the inhibition of 3-HMG-CoA reductase [19 ]; the dose-response curve repor-
100_ ~
& compactin~ e mevinolin ~ fresh weight
~ ~
"~ ~"~\
0
I
'
'
. compactin'~ o mevinolin .J~dry we,ght
10-7M 10-6M 10-5M 10-4 M Fig. 1. Growth inhibition 0£ H. tuberosus explants by increasing concentrations of mevinolin and compactin.
273
ted in Fig. 1 shows that compactin and mevinolin have very similar inhibitory activity on the growth of H. tuberosus explants. Since response to the various treatments did not show significant differences b y expressing the results as fresh or dry weights, only the latter will be reported u p o n here To verify if the inhibition effect is exerted only at the level of MVA synthesis, different concentrations of MVA were supplied to the explants along with the inhibitors. As can be seen in Fig. 2, 2 mM MVA completely reverts the growth inhibition induced b y compactin or mevinolin. This demonstrates that the growth arrest caused b y these inhibitors in H. tuberosus explants is exclusively exerted by suppressing the synthesis of MVA inside the cells. The result is consistent with the evidence previously obtained in animals [4,20] and with the restoration by MVA of root growth in mevinolin-treated radish plants [ 12]. As is well known, MVA represents a key precursor for sterols that are required for membrane structure and cell growth. It has been reported that compactin totally inhibit the incorporation of acetate into sterols in A c er tissue cultures [12]. To ascertain if the growth inhibition caused by mevinolin or compactin comes from the inability of cells to synthesize sterols we supplemented the growth-medium with farnesol or squalene, two direct precursors o f sterols. While a partial restoration o f growth was obtained when farnesol or squalene were supplied at a concentration of 0.1--0.5 mM, total reversion o f the inhibition was observed only when a small
MEVINOLIN 2,5.10"5M + MEVALONIC ACID 250-
£
COMPACTIN 2,5-105M + MEVALONIC ACID
~0.05 0.1 0.2 1.0 2.0raM
~0,05 0.1 0.2 1.0 2Dml~
C
o
I"1"
200,o
¢2_ I.g
,o
u~
.E
150ac: P,
o
.E
¢0
o.
÷ t
E
100-
50-
1
Fig. 2. Reversal of the growth inhibition induced by compactin and mevinolin by increasing concentrations of MVA. Each point represents the average o f 5 repetitions, mean ± S.E.
274 TABLE I
REVERSAL BY MVA OR STEROL PRECURSORS OF M E V I N O L I N - I N D U C E D I N H I B I T I O N IN H. TUBEROSUS E X P L A N T S
GROWTH
I n i t i a l d r y w e i g h t o f f o u r e x p l a n t s w a s 8 0 rag. E a c h p o i n t r e p r e s e n t s t h e a v e r a g e o f 5 rep e t i t i o n s , m e a n ± S.E. Treatments
Dry wt. (mg)
Dry wt. i n c r e a s e (%)
Control M e v i n o l i n 5 x 10 -5 M MVA2x 10 -3 M M V A 2 X 10 4 M F a r n e s o l 1 0 -4 M S q u a l e n e 5 x 10 -4 M M e v i n o l i n 5 x 10 -5 M M e v i n o l i n 5 x 1 0 -s M M e v i n o l i n 5 x 10 -s M M e v i n o l i n 5 x 10 -s M M e v i n o l i n 5 x 10 -5 M M V A 2 x 10 -4 M M e v i n o l i n 5 x 10 -5 M MVA2x 10-4 M
M V A 2 x 10 -4 M M V A 2 x 10 -3 M F a r n e s o l 10 -4 M S q u a l e n e 5 x 10 -4 M F a r n e s o l 10 -4 M +
245 + 7 91 _+ 5 255+ 7 222+_ 7 2 2 5 _+ 20 242 + 9 179 + 2 239 + 7 1 7 3 _+ 2 5 1 8 6 +_ 9 2 5 3 + 17
100 7 106 86 88 98 60 94 56 64 105
+ S q u a l e n e 5 x 1 0 -3 M +
2 4 0 _+ 13
97
+ + + + +
a m o u n t of MVA (0.2 mM) was also supplied to the explants (Table I). This limited a m o u n t of MVA would be required for the synthesis of some secondary metabolites necessary for total restoration o f explant growth also in the presence of sterol precursors. The effectiveness o f MVA 2.0 mM b u t n o t 0.2 mM to completely restore growth would be ascribable to the availability of enough MVA to s u p p o r t sterol biosynthesis as well as the synthesis of the non-sterol compound(s) (Table I). Recently [22], it has been reported that in b a b y hamster kidney cells MVA plays a dual role in the regulation of the cell cycle. First MVA serves as a precursor for the cholesterol required for membrane structure, cell growth and for the passage of cells through the G1phase; successively, possibly by means o f 2iP or a related cytokinin, MVA is required for the onset of the S-phase DNA replication. Our results suggest that, as in animals, the action o f MVA in counteracting c o m p a c t i n or mevinolin inhibition goes through the synthesis of two different sets o f products: one derived from farnesol and the other o f a non-sterolic nature. In a search for this compound(s) several end-products of the various branches of the terpene p a t h w a y were tested along with farnesol or squalene, for their ability to substitute for the limited a m o u n t of MVA required for total restoration of growth. Various concentrations o f abscisic acid (ABA), gibberellic acid (GA3), dolichol m o n o p h o s p h a t e , c o e n z y m e Q10 were added to the culture medium b u t none of these c o m p o u n d s overcame the requirement for a small a m o u n t of MVA. Also, the effect of cytokinins was particularly investigated in consideration of the ability of 2iP and Z to substitute for MVA in the re-
275 TABLE II EFFECT OF 5 x 10 -7 M MEVINOLIN ON 3-HMG-CoA REDUCTASE ACTIVITY Assay conditions as described in Materials and Methods. Enzyme preparations
M t fraction M s fraction
(dpm) Control
Mevinolin
2370 2135
950 540
storation o f DNA-synthesis inhibited b y compactin in b a b y hamster cells [7]. The addition to the culture m e d i u m of different concentrations of 2iP, A2-isopentenyladenosine (2iPA), Z, zeatin riboside (Zr), 2iPA-5-monophosphate and 2iPA-5-diphosphate did n o t show any significant effect on explant growth inhibition. However, interpretation of these results must be cautious since no evidence is available as to the proper entry and targeting of these substances. Besides, it must be considered that the growth response in tissue explants is a far more complex and lengthy process than cell division in the syncronized cell cultures used in experiments with animals [6,7,22]. It is likely that in prolonged experiments, like those reported here, in the presence of compactin more than one isoprenoid substance could b e c o m e limiting for cell growth. Direct evidence that the growth inhibition induced b y mevinolin in H. tuberosus explants is exerted through the inhibition of 3-HMG-CoA reductase was obtained from in vitro studies. Incubation of both mitochondrial and microsomal preparations with 5 × 10-v M mevinolin resulted in a strong reduction of [~4C]HMG-CoA conversion to MVA {Table II). These results are in accordance with the observations o f Bach and Lichtenthaler in radish plants [11], however, further w o r k is required to better qualify the mechanism of inhibition o f 3-HMG-CoA reductase b y mevinolin and compactin in H. tuberosus explants. As regards this we are n o w trying to improve reliability o f the assay in order to avoid a competitive influence by other 3-HMGCoA utilizing enzymes [23]. REFERENCES 1 A. Endo, M. Kuroda and Y. Tsujita, J. Antibiotics, 29 (1976) 1346. 2 A.W. Alberts, J. Chen, G. Kuron, V. Hunt, J. Huff, C. Hoffman, J. Rothrock, M. Lopez, H. Joshua, E. Harris, A. Patchett, R. Monagan, S. Currie, E. Stapley, G. Alberts-Schonberg, O. Hensens, J. Hirshfield, K. Hoogsteen, J. Liesch and J. Springer, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 3957. 3 A. Endo, Methods Enzymol., 72 (1981) 1684. 4 M.S. Brown and J.L. Goldstein, J. Lipid Res., 21 (1980) 505. 5 I. Kaneko, Y. Hazame-Shimada and A. Endo, Eur. J. Biochem., 87 (1978) 313.
276 6 V. Quesney-Huneeus, M.H. Wiley and M.D. Siperstein, Proc. Natl. Acad. Sei. U.S.A., 76 (1979) 5056. 7 V. Quesney-Huneeus, M.H. Wiley and M.D. Siperstein, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 5842. 8 R.J. Wong, D.K. Mc Cormack and D.W. Russell, Arch. Biochem. and Biophys., 216 (1982) 631. 9 H. Suzuki and I. Uritani, Plant Cell Physiol., 18 (1977) 485. 10 R. Yu°Ito, K. Oba and I. Uritani, Plant Cell Physiol., 20 (1979) 867. 11 T.J. Bach and H.K. Lichtenthaler, Z. Naturforsch., 37c (1982) 416. 12 T.J. Bach and H.K. Lichtenthaler, Naturwissenschaften, 69 (1982) 242. 13 T. Kita, M.S. Brown and J.L. Goldstein, J. Clin. Invest., 66 (1980) 1094. 14 D. Serafini-Fracassini, N. Bagni, P.G. Cionini and A. Bennici, Planta, 148 (1980) 332. 15 F. Bertossi, N. Bagni, G. Moruzzi and C.M. Caldarera, Experientia, 22 (1965) 81. 16 D.J. Shapiro, J.L. Nordstrom, J.J. Mitschelen, V.W. Rodwell and R.T. Schimke, Biochim. Biophys. Acta, 370 (1974) 369. 17 M.M. Yeoman, A.F. Dyer and A.I. Robertson, Ann. Bot., 29 (1965) 265. 18 M.M. Yeoman and P.K. Evans, Ann. Bot. N.S., 31 (1967) 323. 19 A. Endo, J. Antibiot., 33 (1980) 334. 20 A. Endo, Trends Biochem. Sci., 6 (1981) 10. 21 N.S. Ryder and J. Goad, Biochim. Biophys. Acta, 619 (1980) 424. 22 V. Quesney-Huneeus, H.A. Galick, M. Siperstein, S.K. Erickson, T.A. Spencer and A. Nelson, J. Biol. Chem., 258 (1983) 378. 23 R. Yu-Ito, K. Oba and I. Uritani, Agric. Biol. Chem., 46 (1982) 2091.