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Miracil D: Effects on nucleic acid synthesis, protein synthesis, and enzyme induction in Escherichia coli
44 °
BIOCHIMICAET BIOPHYSICAACTA
BBA 95647
MIRACIL D: E F F E C T S ON NUCLEIC ACID SYNTHESIS, P R O T E I N SYNT H E S I S , AND ENZYME I N D U C T I O N IN E S C H E R I C H I A COLI
I. BERNARD WEINSTEIN, RICHARD CARCHMAN, EMILY MARNER AND ERICH HIRSCHBERG Departments o/Medicine and Biochemistry, College o/Physicians and Surgeons, Columbia University, and Francis Delafield Hospital, New York, N.Y. (U.S.A .)
(Received January i3th, 1967)
SUMMARY
I. When added to a culture of intact Escherichia coli, Miracil D inhibits growth, causes virtually complete inhibition of total cellular RNA synthesis and partial inhibition of DNA and protein synthesis, and blocks the induction of/%galactosidase. 2. All of these toxic effects are prevented by simultaneous addition to the culture of spermine. Evidence is presented that spermine acts b y blocking cellular uptake of Miracil D. An additional effect of spermine on the formation of the MiraciI D - D N A complex is suggested. 3- Miracil D also inhibits DNA-directed RNA synthesis with purified E. coli RNA polymerase but in the usual assay system it does not inhibit subcellular amino. acid incorporation. 4- These results indicate that Miracil D inhibits bacterial growth mainly b y complexing with DNA, thereby blocking cellular RNA synthesis. The drug provides an interesting comparison to actinomycin D in terms of specificity and mode of action.
INTRODUCTION Miracil D, a Io-thiaxanthenone, is an effective agent in the treatment of schistosomiasis in man 1 and prolongs the survival of mice bearing a variety of transplantable tumors 2. We have recently demonstrated that this drug also inhibits the growth of Bacillus subtilis and in this organism produces immediate and complete inhibition of RNA synthesis, a lesser inhibition of protein synthesis and no inhibition of DNA synthesis 3. Evidence was presented that these effects in vivo reflect an ability of the drug to complex with nucleic acids and thereby inhibit the template activity of DNA with respect to RNA synthesis ~. In this respect the drug appears to be similar to actinomycin D (refs. 4, 5)- In contrast to actinomycin D, however, Miracil D Abbreviations: TMG, methyl-fl-D-thiogalactoside; pyranoside; poly U, polyuridylic acid. Biochim. Biophys. Acta, 142 (i967) 44o-449
ONPG,
O-nitrophenyl-D-galacto-
MIRACIL D AND E. coli DNA, RNA AND PROTEIN
441
also inhibits the growth of Escherichia coli (ref. 3). Because of the paucity of specific inhibitors of RNA synthesis in E. coli it was of interest to determine the effects of this drug on nucleic acid and protein synthesis in intact E. coli as well as in subcellular extracts. We have also studied the effects of Miracil D on induction of fl-galaetosidase in E. coli and have demonstrated that several of the toxic effects of the drug are prevented b y spermine.
MATERIALS AND METHODS
Miracil D (I-diethylaminoethylamino-4-methyl-Io-thiaxanthenone, Lucanthone, Nilodin) was a gift from Burroughs Wellcome Co. through the courtesy of Dr. G. HITCHINGS. The following materials were obtained from commercial sources: spermine tetrahydrochloride (Nutritional Biochemicals); salmon-sperm DNA (Worthington Biochemical Corp.); ~2-1*C3uridine, ~aHlthymidine, randomly labeled I14Clleucine or E14Clphenylalanine, and [8-14CIATP (Schwarz BioResearch, Inc.); methyl-fl-D-thiogalactoside (TMG) (Sigma Chemical Co.); O-nitrophenyl-D-galactopyranoside (ONPG) (Mann Research Labs.); GTP, CTP, UTP, ATP (P-L Biochemicals); and polyuridylic acid (poly U) (Miles Laboratories). Membrane filters, type A medium, were purchased from Carl Schleieher and Schuell Co. and used in a Tracerlab filtration tower. E. coli B (Hill strain) was obtained from Dr. E. BOREK and, unless specified otherwise, was grown in Penassay broth (Difco) supplemented with 0.5 g % glucose. Cells were grown in side-arm flasks as previously described 3. In isotope-incorporation experiments, E2-14Cluridine (28 mC/mmole), ESHJthymidine (15 C/mmole), or ~14C~1eucine (200 mC/mmole) were added to the medium at final concentrations of o.I #C/ml, 2/~C/ml and 0.5/~C/ml, respectively. Isotopes were added at the mid-exponential phase of growth to control cultures or to cultures which had received drug 30 sec previously. I-ml aliquots were removed at subsequent time intervals and added to I ml of cold IO % trichloroacetic acid. For measurements of RNA synthesis, the precipitates were dispersed b y vigorous mixing, deposited on membrane filters, washed under suction with three 3-ml volumes of cold 5 % trichloroacetic acid. The membrane filters were mounted on planchets, dried and counted in a gas-flow counter as described previously 6. For measurement of protein synthesis, the samples in 5 % trichloroacetic acid were heated at 9 °0 for 30 rain, then deposited on membrane filters, washed with three 3-ml volumes of a i % trichloroacetic a c i d - 1 % casamino acid solution, then washed with 2 ml of 7 ° % ethanol, and counted on planchets as described above. For DNA, the samples in 5 ~o trichloroacetic acid were centrifuged at 200o ×g for IO min, the supernatant fluid was discarded, and the precipitate washed once in 5 °?o trichloroacetic acid. The precipitate was then solubilized in 2 ml of I M K O H and incubated at 37 ° for 12 h. The sample was neutralized with 0.4 ml of 6 M HC1 and the DNA precipitated b y adding 2.4 ml of IO % trichloroacetic acid. The precipitate was deposited on membrane filters and washed with three 3-ml volumes of 5 °Jo trichloroacetic acid. The filters were transferred to scintillation vials, IO ml of Brays solution and 2.5 ml of 5 % NH4OH were added, and the samples then counted b y liquid scintillation in a Packard Tri-Carb liquid scintillator Model 4322. For studies on fl-galactosidase, I mM TMG was added at the mid-exponential Biochim. Biophys. Acta, 142 (1967) 440-449
B. WEI~STEIN et al.
442
phase of growth to control cultures or to cultures which simultaneously received Miracil D. I-ml aliquots were removed at subsequent time intervals and ~-galaetosidase activity was assayed on toluenized cells by the procedure of R E V E L , LURIA AND ROTMAN7. Since Miracil was extracted b y the toluene it did not interfere with the colorimetric assay. RNA polymerase was purified from commercially grown E. coli B (Grain Processing Corp.) cells which had been harvested in the early phase of logarithmic growth. The enzyme was assayed according to the procedure of CHAMBERLIN AND BERGs. The S-3o and iS-3o fractions of E. coli B were prepared and assayed for subcellular amino acid incorporation b y methods described previously 9.
RESULTS
E][ect o[ Miracil D on the growth o[ E. coli Preliminary experiments 3 indicated that when E. coli was grown in LB broth and Miracil D was added at the mid-logarithmic phase of growth, the drug at 60 #M (2o/~g/ml) was without effect, whereas at o.15 mM it caused complete growth inhibition for at least 45 rain. Subsequent studies indicated that the medium has a considerable influence on the sensitivity of E. coli. In a Tris-tryptone-glucose medium 1°, Miracil at o.15 mM was without detectable effect and at 0.3 mM produced only transient growth inhibition. Cells grown in Penassay broth plus 0.5 g % glucose were considerably more sensitive to growth inhibition by Miracil D. The drug at 60/,M caused 50-75 °/o growth inhibition, and at o.12 mM caused complete growth inhibition as well as partial ]ysis of the cells. The reason for this marked effect of the medium is not known. Penassay broth and cells in the mid-logarithmic phase of growth were employed in all the present studies, because these conditions permitted the use of relatively low concentrations of drug.
E[[ects o[ Miracil D on R N A , DNA and protein synthesis in E. eoli (Fig. z). When added at a final concentration of 60 ~M, Miracil D caused immediate and almost complete inhibition of the incorporation of E2-1~C]uridine into total cellular rb 4
2O
t g
L
} ,° .g
E o
.g
%
I 4
o
8
~J
4
Time (rain)
~J
4
Fig. I. E f f e c t of Miracil D on R N A (a), D N A (b), a n d p r o t e i n (c) s y n t h e s i s . 0 - 0 , O - O , d r u g a t 60/~M a d d e d at t i m e zero. Biochim. Biophys. Acta, 142 (1967) 44o-449
8
control;.
MIRACIL D AND E. coli DNA, RNA AND PROTEIN
443
RNA. RNA synthesis was inhibited 7 6 % 2 min after the addition of drug, and 9 6 % at 8 min, when compared to a culture without drug. Measurements at later timepoints suggested some escape from drug inhibition. In contrast to its marked effect on RNA synthesis, Miracil D caused only a partial inhibition of the incorporation of [aHlthymidine into DNA. DNA synthesis was not significantly inhibited at 2 rain, though at 8 rain it was inhibited 47 070-Under the same conditions, Miracil D caused a partial inhibition of the incorporation of [l*C]leucine into protein. This inhibition was also less than that occurring in RNA synthesis but increased with time from 2 1 % at 2 min to 4 6 % at 8 rain. These results in E. coli provide both similarities with and contrasts to the earlier findings in B. subtilis (ref. 3). In both bacterial systems, RNA synthesis was the primary site of inhibitory action of Miracil D. However, DNA synthesis in B. subtilis was completely unaffected b y the drug whereas some inhibition was observed consistently in E. coli. In related studies n it has been found that DNA synthesis is even more readily inhibited than RNA synthesis when cells of mouse leukemia LI2IO are exposed to Miracil D under similar conditions in vitro.
E//ect o[ Miracil D on fi-galactosidase induction (Fig. 2) In view of the ability of Miracil D to cause essentially complete inhibition of RNA synthesis in intact E. coli while inhibiting protein synthesis only partially, it
b
8
i'
30
5
) ao
0
,oi
~2
5
~D I0
20
~0
I0
~0
Time(rain)
3O
I0
20
30
Fig. 2. Effect of Miracil D on /5-galactosidase induction (a), R N A (b) a n d protein (c) synthesis. Q - Q , control; 71-[N, d r u g at 3 ° / z M added at time zero (To) ; O - O, drug at 60 #M added at T 0. All flasks received TMG at T 0. The curves for fl-galactosidase activity are n o t e x t r a p o l a t e d to T o because of the k n o w n lag in induction 1~.
was of interest to determine the effect of this drug on the induction of a specific enzyme. In the absence of Miracil D, the addition of TMG to a mid-exponential phase culture of E. coli (grown in Penassay broth and in the absence of glucose to minimize catabolite repression) resulted in the induction of fl-galactosidase activity. Although under our conditions the level of activity was low, it was readily demonstrable b y IO min after addition of inducer and increased in amount thereafter. When Miracil D at 30 t*,M was added together with the inducer, enzyme induction was unimpaired. Biochim. Biophys. Acta, 142 (1967) 440-449
B. WEINSTEIN et al.
444
At 6o FM, however, Miracil D completely blocked the appearance of fl-galactosidase activity. Simultaneous measurements of RNA and protein synthesis during the 3o-min period following the addition of TMG indicated that with drug at 30 FM RNA synthesis was only slightly inhibited and there was no detectable inhibition of protein synthesis. On the other hand, drug at 60/zM inhibited RNA synthesis by over 85 % and protein synthesis was inhibited approx. 65 %. Control studies indicated that the presence of TMG itself did not significantly influence total RNA or protein synthesis in the absence or presence of Miracil D.
Reversal by spermine o/the e[/ects o~ Miracil D on intact E. coli (Figs. 3 and 4) The fact that the dialkylaminoalkylamino side chain of Miracil D resembles the aliphatic polyamines 3, as well as evidence that polyamines can antagonize the action of proflavine TMand quinacrine13,1t led us to test the possibility that spermine might prevent the toxic effects of Miracil D in E. coli. Fig. 3 indicates the ability of spermine 600
400
200
~ I00
50
L
£o
'
40
TIME IN MINUTES
Fig. 3. Effect of s p e r m i n e on g r o w t h inhibition b y Miracil D. Spermine a n d / o r Miracil added at Klett 7 o. O - O , control; A - / x , s p e r m i n e (8 mM) plus Miracil D (60/zM); & - A , spermine (8 mM) plus Miracil D (o.12 raM); © - - - © , Miracil D (6o/zM); B - - - B , Miracil D (o.12 mM). Spermine (8 nlM) alone gave the same curve as s p e r m i n e (8 raM) plus Miracil D (60/~M).
b so
o_
40
X
D ~lO
v~
c~ to
~_b r~ ~
/// ~ o @
L9
30
rime(rain)
60
30 Time ( m i n )
60
Fig. 4. Effect of s p e r m i n e (8 raM) on Miracil D (o.12 raM) inhibition of R N A synthesis (a) and fl-galactosidase induction (b). D - O , control; & - A , Miracil D plus s p e r m i n e at time zero (To); × - × , Miracil D at T o plus s p e r m i n e a t I rain; ( D - G , Miracil D a t T o plus spernfine at io min; B - B , Miracil D alone at T 0. All flasks received TMG at T 0. Spermine alone at T o gave the same curve as the control.
Biochim. Biophys. Acta, 142 (1967) 440-449
MIRACIL D AND E. coli DNA, RNA AND PROTEIN
445
to protect cells against the growth-inhibitory effect of Miracil D. In the absence of spermine, Miracil at 60/~M produced greater than 50 % growth inhibition and at o.12 mM produced complete inhibition as well as lysis of cells. Spermine (8 raM), when added simultaneously with the inhibitor, completely protected cells against growth inhibition b y Miracil at 60/~M and protected cells against lysis by Miracil at o.12 raM, permitting growth at approx. 50 % of the control rate. Spermine alone (8 mM) produced some inhibition of growth and additional studies indicated that this inhibition was more pronounced at higher concentrations of spermine. This is consistent with previous evidence that spermine is toxic to a variety of microorganisms (see MILLS AND DUBIN 15 and review b y TABOR AND TABORI~). When instead of adding the two agents simultaneously, spermine was added either I, 2, 5 or IO rain after Miracil D there was a progressive decrease in the ability of spermine to protect cells from growth inhibition b y Miracil. The ability of spermine to prevent growth inhibition b y Miracil was paralleled b y effects on RNA synthesis and fl-galactosidase induction. Fig. 4 indicates that whereas Miracil (o.12 mM) completely inhibited RNA synthesis and the induction of /5-galactosidase, the simultaneous addition of spermine restored RNA synthesis and enzyme induction. When spermine was added either i or IO min after Miracil there was a lesser protection of RNA synthesis and fl-galactosidase induction. It is of interest that with delayed addition of spermine there was a pronounced lag between the addition of the polyamine and the appearance of detectable enzyme. PARDEE AND PRESTIDGE17 described a normal lag of 3-4 min in the induction of fl-galactosidase and studies b y NAKADA AND MAGASANIK18 indicate that this lag represents the time required to synthesize fl-galactosidase messenger RNA. Though more detailed kinetic studies are required, the present results suggest that in addition to a general effect on protein synthesis, Miracil D interferes with an early event in the induction process, presumably the synthesis of this messenger RNA. To determine the mechanism by which spermine protects E. coli from Miracil D, a culture was incubated for 30 rain in the usual medium with Miracil D, in the absence and presence of spermine, and the cells were then harvested by centrifugation. In the absence of spermine the bacterial pellet was bright yellow (the color of Miracil D) whereas in the presence of spermine it was colorless. When the cell-free media were extracted and assayed for Miracil D b y the method of BRINDLE, HIRSCHBERG AND GROSS 19, the medium with spermine contained approx, seven times as much Miracil D as the medium without spermine. These results clearly indicate that spermine interferes with the cellular uptake of Miracil D. However, the possibility of an additional site of spermine antagonism must be considered in view of the results obtained in subcellular systems.
E[/ect o/Miracil D on subcellular R N A and protein synthesis (Fig. 5, Tables I and I I ) The present studies with intact E. coli as well as our previous studies with B. subtilis (ref. 3) indicate that Miracil D inhibits the incorporation of labeled precursors into RNA and protein. To exclude the possibility that the drug affects precursor utilization, it was necessary to obtain direct evidence in subcellular systems. Fig. 5- indicates that 30 #M Miracil D produced greater than 50 % inhibition of [14C]ATP incorporation into RNA in a system containing purified E. coli B RNA Biochim. Biophys. Acta, 142 (1967) 440-449
446
B. WEINSTEIN et al.
polymerase and a limiting amount of DNA as primer. Supplementation of the system with excess DNA reversed Miracil inhibition, whereas supplementation with excess enzyme did not, indicating that the target of drug action is DNA and not enzyme. The effect of spermine on RNA synthesis in vitro, in the absence and presence of Miracil D, was determined in a separate experiment (Table ]). Whereas Miracil
I00
75 z 0
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25
*
t
i
i
i
I
2
3
4
5
MIRACIL D
(XI~SM)
Fig. 5. Miracil D i n h i b i t i o n of t h e R N A p o l y m e r a s e reaction. R e a c t i o n s y s t e m s c o n t a i n e d pe r ml: 7 ° / ~ g of s a l m o n - s p e r m DNA, 75 u n i t s of E. coli R N A p o l y m e r a s e , a n d Miracil D a t t h e final conc e n t r a t i o n s i n d i c a t e d . T h e r e m a i n i n g c o n d i t i o n s are d e s c r i b e d u n d e r METHODS. The c ont rol s y s t e m , i.e., in t h e a b s e n c e of drug, i n c o r p o r a t e d 12.5 m # m o l e s of [14C]ATP. The d a t a i n d i c a t e t h e m e a n of t h r e e e x p e r i m e n t s .
TABLE I EFFECT
OF
SPERMINE
ON
D
MIRACIL
INHIBITION
OF
THE
RNA
POLYMERASE
REACTION
R e a c t i o n s y s t e m s c o n t a i n e d p e r ml: 7 ° / , g of s a l m o n - s p e r m D N A a n d 75 u n i t s of E. coli R N A p o l y m e r a s e . Miracil D a n d / o r s p e r m i n e were a d d e d a t t h e fi na l c o n c e n t r a t i o n s i n d i c a t e d . The r e m a i n i n g c o m p o n e n t s w e r e as d e s c r i b e d u n d e r METHODS. Since t h e a d d i t i o n of s p e r m i n e s t i m u l a t e d t h e r e a c t i o n , i n h i b i t i o n b y Miracil D w a s c a l c u l a t e d in ea c h p a r t of t h e e x p e r i m e n t as pe r c e n t of t h e v a l u e in t h e c o n t r o l t u b e c o n t a i n i n g t h e c o r r e s p o n d i n g c o n c e n t r a t i o n of s p e r m i n e .
Miracil (M)
Spermine (M)
Incorporation o[ EI*C~A T P (ml~moles) 12. 5
_
_
3"
IO-S 5" to-5
---
3-9 0.3
--
io
3"t° 5 5" t o s
io 5 lO -5
__
10-4
3" 5" _ 3" 5"
lO-5 to-~ to-S lO-5
5
lO -4 1°-4 lO-3 io 3 lO -3
Biochim. Biophys. $cta, 142 (1967) 44o-449
Inhibition by 3liracil D
(%)
--
69 98
13.3
_
2.8 1.5 23.1 8.8 3.4 43.1 25.1 16.9
79 89 --
62 85 -42 61
MIRACIL D AND TABLE
E . coli DNA, R N A AND PROTEIN
447
I1
EFFECT OF MIRACIL D ON PROTEIN SYNTHESIS IN A SUBCELLULAR SYSTEM FROM E .
coli B
A s s a y s y s t e m s f o r tb_e e n d o g e n o u s r e a c t i o n c o n t a i n e d o . I m l o f t h e S-3o f r a c t i o n of E. coli B; f o r t h e p o l y U r e a c t i o n , o . i m l o f t h e iS-3o f r a c t i o n a n d p o l y U, i o o / ~ g / o . 4 ml. M i r a c i l D w a s a d d e d a t t h e f i n a l c o n c e n t r a t i o n s i n d i c a t e d . R e a c t i o n s w e r e i n c u b a t e d a t 37 ° f o r 3 ° rain. T h e r e m a i n i n g c o m p o n e n t s of t h e a s s a y s y s t e m a n d t h e p r o c e s s i n g of s a m p l e s a r e a s d e s c r i b e d u n d e r METHODS. I n c o r p o r a t i o n is e x p r e s s e d a s / H ~ m o l e s p e r a s s a y s y s t e m .
Miracil (M)
None 1.3" IO -~ 6 . 6 . lO -5 1.3" lO -4 2.6- lO -4
[l~Cl Phenylalanine incorporation Endogenous reaction (l~ltmoles)
plus poly U (l~#rnoles)
2.5 2.2 2. 4 2.7
163 154 196 189
2.2
18 5
inhibited incorporation of ATP into RNA, spermine alone (lO-5-1o -~ M) actually caused a concentration-dependent stimulation of RNA synthesis. Spermine stimulation has been previously described in the DNA-directed RNA polymerase reaction 2°,21. RNA synthesis in the presence of spermine was more resistant to inhibition b y Miracil D than RNA synthesis in the absence of spermine. Thus, whereas RNA synthesis in the presence of i mM spermine was inhibited 6 1 % by 5o/zM Miraeil D, the same concentration of Miracil produced 98 % inhibition of RNA synthesis in the absence of spermine when each was calculated against the appropriate control. This effect of the polyamine might be explained by assuming that: (I) spermine enhances the utilization of portions of the DNA primer which do not contain Miracil D or (2) spermine prevents the attachment of Miracil D to DNA, perhaps b y competing with the drug for phosphate residues on DNA. The first postulate is consistent with the suggestion of Fox, GUMPERT AND WEISS 2° that the RNA polymerase reaction in vitro is limited b y product inhibition (because newly synthesized RNA remains complexed with the enzyme) and that polyamines stimulate the reaction b y dissociating this complex. Attempts to demonstrate by spectrophotometric methods ~that spermine blocks the attachment in vitro of Miracil D to DNA have thus far been unsuccessful because addition of spermine at concentrations greater than o.I mM caused precipitation of the DNA. In the RNA polymerase reactions (Table I), spelmine did not cause precipitation of the DNA apparently because of the presence of the enzyme and/or the nucleotide triphosphates. In contrast to the pronounced effect of Miraeil D on RNA synthesis in the subcellular E. coli system, the drug has no effect on amino acid incorporation under comparable conditions, as summarized in Table II. When the incorporation of L14C]phenylalanine was directed b y either native messenger RNA's or by poly U, the reaction was not appreciably inhibited by concentrations of Miracil D in the range of 13 /,M to 0.26 mM. When assayed in a previously described rat-liver subcellular system ~2, these concentrations of Miracil D also failed to inhibit phenylalanine incorporation. Biochim. Biophys. Acta, 142 (1967) 4 4 o - 4 4 9
448
B. WEINSTEIN ct al.
DISCUSSION
The present studies establish Miracil D as an inhibitor of RNA synthesis in intact E. coli as well as in the subcellular RNA polymerase reaction. The results provide strong support for our hypothesis that Miracil D inhibits bacterial growth b y complexing with DNA and thereby inhibiting RNA synthesis 3. Miracil D provides an interesting contrast to actinomycin D, another threering heterocyclic compound with pronounced biological activity. The latter drug is a potent inhibitor of RNA synthesis in intact mammalian cells and gram-positive bacteria4,L Intact E. coli, however, are extremely resistant to actinomycin, apparently because they are not permeable to the drug unless treated with E D T A (refs. 4, 23, 24). The molecular weight of Miracil D, approx, one-third that of actinomycin D, or the lesser complexity of its side chain, m a y explain its ability to penetrate intact E. coli. Miracil, nevertheless, is in general a less potent drug since concentrations in the range of 50/~M are required to inhibit RNA synthesis, whereas in sensitive systems actinomycin is effective in the range of o.I/tM (refs. 4, 5). Based on previously published results 4,2~, as well as parallel studies in our own laboratory (unpublished), Miracil is somewhat more potent than proflavine as an inhibitor of purified E. coli RNA polymerase. A major disadvantage of Miracil as a tool for the further elucidation of macromolecular synthesis is the fact that it lacks the specificity of actinomycin D. Concentrations which inhibit RNA synthesis in intact E. coli also partially inhibit DNA and protein synthesis. Preliminary results with purified E. coli DNA polymerase indicate that Miracil can inhibit DNA synthesis directly though this reaction is more resistant than the RNA polymerase reaction. Studies in progress on the nature of the Miracil D - D N A complex 26 should help to explain the differential effects of this drug on the two template functions of DNA. The early inhibition of protein synthesis in intact cells m a y be due to binding of Miracil D to RNA since in vitro the drug can complex with both DNA and RNA (ref. 3). Later inhibition of protein synthesis m a y be explained by depletion of cellular messenger RNA. Our results on subcellular amino acid incorporation indicate that protein synthesis is much more resistant to Miracil D than the RNA polymerase reaction. A precise quantitative comparison of the relative response of the two reactions is not possible, however, because of the differences in ionic and nucleic acid composition of the two assay systems; extrapolation from the subcellular systems to the intact cell is complicated by the same factors. Concentrations of Miracil D which block RNA synthesis also inhibit the induction of fl-galactosidase. This is consistent with the concept that enzyme induction requires RNA synthesis de novo 2v. The effect on enzyme induction is not simply due to general inhibition of amino acid incorporation since induction of fl-galactosidase is completely blocked at concentrations of drug which only partially inhibit total protein synthesis. Our results do not exclude the possibility that catabolite repression, resulting from partial inhibition of protein synthesis TM, plays a role in the effect of Miracil D on fl-galactosidase induction. The effects of Miracil D on growth, RNA synthesis and enzyme induction in E. coli were dramatically prevented by spermine. Measurements of the concentration of Miracil D in ceils and media, in the presence and absence of the polyamine, indicate that spermine interferes with cellular uptake of the drug. At this time it is Biochim. Biophys. Acta, 142 (t967) 440-449
MIRACIL D AND E. coli DNA, RNA AND PROTEIN
449
not clear whether spermine also exerts a direct antagonism at the DNA level, although this is suggested by the experiments carried out in the subcellular system (Table I). During the preparation of this manuscript SIMON,COHEN AND RAINA2s reported that levorphanol inhibition of RNA synthesis in E. coli is reversed by spermidine and it is possible that this effect is also mediated at the level of drug uptake. The mechanism by which spermine affects Miracil D uptake is not known. It is possible that spermine competes with the basic side chain of Miracil D for receptor sites, perhaps phospholipids, in the cell membrane. Previous studies indicate that spermine can stabilize bacterial membranes15,16 and it can also alter membrane function with respect to potassium fluxes 15. Regardless of the mechanism, the findings provide an interesting example of drug competition at the level of cellular uptake and suggest the possibility that genetic resistance to certain growth inhibitors may be mediated by an increased endogenous synthesis of polyamines.
ACKNOWLEDGEMENTS
This work was supported in part by U. S. Public Health Service Research Grant R i o CA 02332 from the National Cancer Institute. One of us (I.B.W.) is Career Scientist of the Health Research Council of the City of New York (I-19o) . REFERENCES i D. M. BLAIR, Bull. World Health Organ., 18 (I958) 989. 2 E. HIRSCHBERG, A. GELLHORN, M. R. MURRAY AND E. F. ELSLAGER, J. Natl. Cancer Inst., 22 (1959) 567 . 3 I. B. WEINSTEIN, R. CHERNOFF, I. FINKELSTEIN AND E. HIRSCHBERG, Mol. Pharmacol., 1 (1965) 297. 4 J. HURWlTZ, J. S. FURTH, M. MOLANEY AND M. ALEXANDER, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1222. 5 I. H. GOLDBERG AND E. REICH, Federation Proc., 23 (1964) 958. 6 M. OCHOA, Jr. AND I. B. WEINSTEIN, J. Biol. Chem., 239 (1964) 3834 . 7 H. R. REVEL, S. E. LURIA AND B. ROTMAN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1956. 8 M. CHAMBERLIN AND P. BERG, Proc. Natl. Acad. Sci. U.S., 48 (1962) 81. 9 S. M. FRIEDMAN AND I. B. WEINSTEIN, Biochim. Biophys. Acta, 114 (1966) 593. IO A. GAREN AND C. LEVlNTHAL, Biochim. Biophys. Acta, 38 (196o) 471. I I E. HIRSCHBERG, I. ]3. WEINSTEIN AND R. CARCHMAN, Abstr. 9th Internatl. Cancer Congr., Tokyo, 1966, p. 347. 12 D. KAY, Biochem. J., 73 (1959) 149. 13 M. SILVERMAN AND E. A. EVANS, Jr., J. Biol. Chem., 154 (1944) 521. 14 A. K. MILLER AND L. PETERS, Arch. Biochem., 6 (1945) 281. 15 J. MILLS AND D. T. DOBIN, Mol. Pharmacol., 2 (1966) 311. 16 H. TABOR AND C. W. TABOR, Pharmacol. Rev., 16 (1964) 245. 17 A. B. PARDEE AND L. S. PRESTIDGE, Biochim. Biophys. Acta, 49 (1961) 77. 18 D. NAKADA AND g . MAGASANIK, J. Mol. Biol., 8 (1964) lO 5. 19 S. D. BRINDLE, E. HIRSCHBERG AND P. R. GROSS, Cancer Res., 24 (1964) 1738. 20 C. F. F o x , R. I. GUMPERT ANn S. ]3. WEISS, J. Biol. Chem., 24o (1965) 21Ol. 21 R. L. O'BRIEN, J. G. OLENICK AND F. E. HAHN, Proc. Natl. Acad. Sci. U.S., 55 (1966) 1511. 22 I. B. WEINSTEIN, M. OCHOA, Jr. AND S. M. FRIEDMAN, Biochemistry, 5 (1966) 3332. 23 :B. MACH AND E. L. TATUM, Science, 139 (1963) lO51. 24 L. LEIVE, Biochem. Biophys. Res. Commun., I8 (1965) 13. 25 ]3. NICHOLSON AND A. PEACOCKE, Biochem. J., IOO (1966) 5 o. 26 I. B. WEINSTEIN, N. GERSTEN, E. MARNER AND E. HIRSCHBERG, Proc. Am. Assoc. Cancer Res., 7 (1966) 74. 27 F. JACOB AND J. MONOD, J. Mol. Biol., 3 (1961) 318. 28 E. J. SIMON, S. S. COHEN AND A. RAINA, Biochem. Biophys. Res. Commun., 24 (1966) 482.
Biochim. Biophys. Acta, 142 (1967) 440-449
BIOCHIMICAET BIOPHYSICAACTA
BBA 95647
MIRACIL D: E F F E C T S ON NUCLEIC ACID SYNTHESIS, P R O T E I N SYNT H E S I S , AND ENZYME I N D U C T I O N IN E S C H E R I C H I A COLI
I. BERNARD WEINSTEIN, RICHARD CARCHMAN, EMILY MARNER AND ERICH HIRSCHBERG Departments o/Medicine and Biochemistry, College o/Physicians and Surgeons, Columbia University, and Francis Delafield Hospital, New York, N.Y. (U.S.A .)
(Received January i3th, 1967)
SUMMARY
I. When added to a culture of intact Escherichia coli, Miracil D inhibits growth, causes virtually complete inhibition of total cellular RNA synthesis and partial inhibition of DNA and protein synthesis, and blocks the induction of/%galactosidase. 2. All of these toxic effects are prevented by simultaneous addition to the culture of spermine. Evidence is presented that spermine acts b y blocking cellular uptake of Miracil D. An additional effect of spermine on the formation of the MiraciI D - D N A complex is suggested. 3- Miracil D also inhibits DNA-directed RNA synthesis with purified E. coli RNA polymerase but in the usual assay system it does not inhibit subcellular amino. acid incorporation. 4- These results indicate that Miracil D inhibits bacterial growth mainly b y complexing with DNA, thereby blocking cellular RNA synthesis. The drug provides an interesting comparison to actinomycin D in terms of specificity and mode of action.
INTRODUCTION Miracil D, a Io-thiaxanthenone, is an effective agent in the treatment of schistosomiasis in man 1 and prolongs the survival of mice bearing a variety of transplantable tumors 2. We have recently demonstrated that this drug also inhibits the growth of Bacillus subtilis and in this organism produces immediate and complete inhibition of RNA synthesis, a lesser inhibition of protein synthesis and no inhibition of DNA synthesis 3. Evidence was presented that these effects in vivo reflect an ability of the drug to complex with nucleic acids and thereby inhibit the template activity of DNA with respect to RNA synthesis ~. In this respect the drug appears to be similar to actinomycin D (refs. 4, 5)- In contrast to actinomycin D, however, Miracil D Abbreviations: TMG, methyl-fl-D-thiogalactoside; pyranoside; poly U, polyuridylic acid. Biochim. Biophys. Acta, 142 (i967) 44o-449
ONPG,
O-nitrophenyl-D-galacto-
MIRACIL D AND E. coli DNA, RNA AND PROTEIN
441
also inhibits the growth of Escherichia coli (ref. 3). Because of the paucity of specific inhibitors of RNA synthesis in E. coli it was of interest to determine the effects of this drug on nucleic acid and protein synthesis in intact E. coli as well as in subcellular extracts. We have also studied the effects of Miracil D on induction of fl-galaetosidase in E. coli and have demonstrated that several of the toxic effects of the drug are prevented b y spermine.
MATERIALS AND METHODS
Miracil D (I-diethylaminoethylamino-4-methyl-Io-thiaxanthenone, Lucanthone, Nilodin) was a gift from Burroughs Wellcome Co. through the courtesy of Dr. G. HITCHINGS. The following materials were obtained from commercial sources: spermine tetrahydrochloride (Nutritional Biochemicals); salmon-sperm DNA (Worthington Biochemical Corp.); ~2-1*C3uridine, ~aHlthymidine, randomly labeled I14Clleucine or E14Clphenylalanine, and [8-14CIATP (Schwarz BioResearch, Inc.); methyl-fl-D-thiogalactoside (TMG) (Sigma Chemical Co.); O-nitrophenyl-D-galactopyranoside (ONPG) (Mann Research Labs.); GTP, CTP, UTP, ATP (P-L Biochemicals); and polyuridylic acid (poly U) (Miles Laboratories). Membrane filters, type A medium, were purchased from Carl Schleieher and Schuell Co. and used in a Tracerlab filtration tower. E. coli B (Hill strain) was obtained from Dr. E. BOREK and, unless specified otherwise, was grown in Penassay broth (Difco) supplemented with 0.5 g % glucose. Cells were grown in side-arm flasks as previously described 3. In isotope-incorporation experiments, E2-14Cluridine (28 mC/mmole), ESHJthymidine (15 C/mmole), or ~14C~1eucine (200 mC/mmole) were added to the medium at final concentrations of o.I #C/ml, 2/~C/ml and 0.5/~C/ml, respectively. Isotopes were added at the mid-exponential phase of growth to control cultures or to cultures which had received drug 30 sec previously. I-ml aliquots were removed at subsequent time intervals and added to I ml of cold IO % trichloroacetic acid. For measurements of RNA synthesis, the precipitates were dispersed b y vigorous mixing, deposited on membrane filters, washed under suction with three 3-ml volumes of cold 5 % trichloroacetic acid. The membrane filters were mounted on planchets, dried and counted in a gas-flow counter as described previously 6. For measurement of protein synthesis, the samples in 5 % trichloroacetic acid were heated at 9 °0 for 30 rain, then deposited on membrane filters, washed with three 3-ml volumes of a i % trichloroacetic a c i d - 1 % casamino acid solution, then washed with 2 ml of 7 ° % ethanol, and counted on planchets as described above. For DNA, the samples in 5 ~o trichloroacetic acid were centrifuged at 200o ×g for IO min, the supernatant fluid was discarded, and the precipitate washed once in 5 °?o trichloroacetic acid. The precipitate was then solubilized in 2 ml of I M K O H and incubated at 37 ° for 12 h. The sample was neutralized with 0.4 ml of 6 M HC1 and the DNA precipitated b y adding 2.4 ml of IO % trichloroacetic acid. The precipitate was deposited on membrane filters and washed with three 3-ml volumes of 5 °Jo trichloroacetic acid. The filters were transferred to scintillation vials, IO ml of Brays solution and 2.5 ml of 5 % NH4OH were added, and the samples then counted b y liquid scintillation in a Packard Tri-Carb liquid scintillator Model 4322. For studies on fl-galactosidase, I mM TMG was added at the mid-exponential Biochim. Biophys. Acta, 142 (1967) 440-449
B. WEI~STEIN et al.
442
phase of growth to control cultures or to cultures which simultaneously received Miracil D. I-ml aliquots were removed at subsequent time intervals and ~-galaetosidase activity was assayed on toluenized cells by the procedure of R E V E L , LURIA AND ROTMAN7. Since Miracil was extracted b y the toluene it did not interfere with the colorimetric assay. RNA polymerase was purified from commercially grown E. coli B (Grain Processing Corp.) cells which had been harvested in the early phase of logarithmic growth. The enzyme was assayed according to the procedure of CHAMBERLIN AND BERGs. The S-3o and iS-3o fractions of E. coli B were prepared and assayed for subcellular amino acid incorporation b y methods described previously 9.
RESULTS
E][ect o[ Miracil D on the growth o[ E. coli Preliminary experiments 3 indicated that when E. coli was grown in LB broth and Miracil D was added at the mid-logarithmic phase of growth, the drug at 60 #M (2o/~g/ml) was without effect, whereas at o.15 mM it caused complete growth inhibition for at least 45 rain. Subsequent studies indicated that the medium has a considerable influence on the sensitivity of E. coli. In a Tris-tryptone-glucose medium 1°, Miracil at o.15 mM was without detectable effect and at 0.3 mM produced only transient growth inhibition. Cells grown in Penassay broth plus 0.5 g % glucose were considerably more sensitive to growth inhibition by Miracil D. The drug at 60/,M caused 50-75 °/o growth inhibition, and at o.12 mM caused complete growth inhibition as well as partial ]ysis of the cells. The reason for this marked effect of the medium is not known. Penassay broth and cells in the mid-logarithmic phase of growth were employed in all the present studies, because these conditions permitted the use of relatively low concentrations of drug.
E[[ects o[ Miracil D on R N A , DNA and protein synthesis in E. eoli (Fig. z). When added at a final concentration of 60 ~M, Miracil D caused immediate and almost complete inhibition of the incorporation of E2-1~C]uridine into total cellular rb 4
2O
t g
L
} ,° .g
E o
.g
%
I 4
o
8
~J
4
Time (rain)
~J
4
Fig. I. E f f e c t of Miracil D on R N A (a), D N A (b), a n d p r o t e i n (c) s y n t h e s i s . 0 - 0 , O - O , d r u g a t 60/~M a d d e d at t i m e zero. Biochim. Biophys. Acta, 142 (1967) 44o-449
8
control;.
MIRACIL D AND E. coli DNA, RNA AND PROTEIN
443
RNA. RNA synthesis was inhibited 7 6 % 2 min after the addition of drug, and 9 6 % at 8 min, when compared to a culture without drug. Measurements at later timepoints suggested some escape from drug inhibition. In contrast to its marked effect on RNA synthesis, Miracil D caused only a partial inhibition of the incorporation of [aHlthymidine into DNA. DNA synthesis was not significantly inhibited at 2 rain, though at 8 rain it was inhibited 47 070-Under the same conditions, Miracil D caused a partial inhibition of the incorporation of [l*C]leucine into protein. This inhibition was also less than that occurring in RNA synthesis but increased with time from 2 1 % at 2 min to 4 6 % at 8 rain. These results in E. coli provide both similarities with and contrasts to the earlier findings in B. subtilis (ref. 3). In both bacterial systems, RNA synthesis was the primary site of inhibitory action of Miracil D. However, DNA synthesis in B. subtilis was completely unaffected b y the drug whereas some inhibition was observed consistently in E. coli. In related studies n it has been found that DNA synthesis is even more readily inhibited than RNA synthesis when cells of mouse leukemia LI2IO are exposed to Miracil D under similar conditions in vitro.
E//ect o[ Miracil D on fi-galactosidase induction (Fig. 2) In view of the ability of Miracil D to cause essentially complete inhibition of RNA synthesis in intact E. coli while inhibiting protein synthesis only partially, it
b
8
i'
30
5
) ao
0
,oi
~2
5
~D I0
20
~0
I0
~0
Time(rain)
3O
I0
20
30
Fig. 2. Effect of Miracil D on /5-galactosidase induction (a), R N A (b) a n d protein (c) synthesis. Q - Q , control; 71-[N, d r u g at 3 ° / z M added at time zero (To) ; O - O, drug at 60 #M added at T 0. All flasks received TMG at T 0. The curves for fl-galactosidase activity are n o t e x t r a p o l a t e d to T o because of the k n o w n lag in induction 1~.
was of interest to determine the effect of this drug on the induction of a specific enzyme. In the absence of Miracil D, the addition of TMG to a mid-exponential phase culture of E. coli (grown in Penassay broth and in the absence of glucose to minimize catabolite repression) resulted in the induction of fl-galactosidase activity. Although under our conditions the level of activity was low, it was readily demonstrable b y IO min after addition of inducer and increased in amount thereafter. When Miracil D at 30 t*,M was added together with the inducer, enzyme induction was unimpaired. Biochim. Biophys. Acta, 142 (1967) 440-449
B. WEINSTEIN et al.
444
At 6o FM, however, Miracil D completely blocked the appearance of fl-galactosidase activity. Simultaneous measurements of RNA and protein synthesis during the 3o-min period following the addition of TMG indicated that with drug at 30 FM RNA synthesis was only slightly inhibited and there was no detectable inhibition of protein synthesis. On the other hand, drug at 60/zM inhibited RNA synthesis by over 85 % and protein synthesis was inhibited approx. 65 %. Control studies indicated that the presence of TMG itself did not significantly influence total RNA or protein synthesis in the absence or presence of Miracil D.
Reversal by spermine o/the e[/ects o~ Miracil D on intact E. coli (Figs. 3 and 4) The fact that the dialkylaminoalkylamino side chain of Miracil D resembles the aliphatic polyamines 3, as well as evidence that polyamines can antagonize the action of proflavine TMand quinacrine13,1t led us to test the possibility that spermine might prevent the toxic effects of Miracil D in E. coli. Fig. 3 indicates the ability of spermine 600
400
200
~ I00
50
L
£o
'
40
TIME IN MINUTES
Fig. 3. Effect of s p e r m i n e on g r o w t h inhibition b y Miracil D. Spermine a n d / o r Miracil added at Klett 7 o. O - O , control; A - / x , s p e r m i n e (8 mM) plus Miracil D (60/zM); & - A , spermine (8 mM) plus Miracil D (o.12 raM); © - - - © , Miracil D (6o/zM); B - - - B , Miracil D (o.12 mM). Spermine (8 nlM) alone gave the same curve as s p e r m i n e (8 raM) plus Miracil D (60/~M).
b so
o_
40
X
D ~lO
v~
c~ to
~_b r~ ~
/// ~ o @
L9
30
rime(rain)
60
30 Time ( m i n )
60
Fig. 4. Effect of s p e r m i n e (8 raM) on Miracil D (o.12 raM) inhibition of R N A synthesis (a) and fl-galactosidase induction (b). D - O , control; & - A , Miracil D plus s p e r m i n e at time zero (To); × - × , Miracil D at T o plus s p e r m i n e a t I rain; ( D - G , Miracil D a t T o plus spernfine at io min; B - B , Miracil D alone at T 0. All flasks received TMG at T 0. Spermine alone at T o gave the same curve as the control.
Biochim. Biophys. Acta, 142 (1967) 440-449
MIRACIL D AND E. coli DNA, RNA AND PROTEIN
445
to protect cells against the growth-inhibitory effect of Miracil D. In the absence of spermine, Miracil at 60/~M produced greater than 50 % growth inhibition and at o.12 mM produced complete inhibition as well as lysis of cells. Spermine (8 raM), when added simultaneously with the inhibitor, completely protected cells against growth inhibition b y Miracil at 60/~M and protected cells against lysis by Miracil at o.12 raM, permitting growth at approx. 50 % of the control rate. Spermine alone (8 mM) produced some inhibition of growth and additional studies indicated that this inhibition was more pronounced at higher concentrations of spermine. This is consistent with previous evidence that spermine is toxic to a variety of microorganisms (see MILLS AND DUBIN 15 and review b y TABOR AND TABORI~). When instead of adding the two agents simultaneously, spermine was added either I, 2, 5 or IO rain after Miracil D there was a progressive decrease in the ability of spermine to protect cells from growth inhibition b y Miracil. The ability of spermine to prevent growth inhibition b y Miracil was paralleled b y effects on RNA synthesis and fl-galactosidase induction. Fig. 4 indicates that whereas Miracil (o.12 mM) completely inhibited RNA synthesis and the induction of /5-galactosidase, the simultaneous addition of spermine restored RNA synthesis and enzyme induction. When spermine was added either i or IO min after Miracil there was a lesser protection of RNA synthesis and fl-galactosidase induction. It is of interest that with delayed addition of spermine there was a pronounced lag between the addition of the polyamine and the appearance of detectable enzyme. PARDEE AND PRESTIDGE17 described a normal lag of 3-4 min in the induction of fl-galactosidase and studies b y NAKADA AND MAGASANIK18 indicate that this lag represents the time required to synthesize fl-galactosidase messenger RNA. Though more detailed kinetic studies are required, the present results suggest that in addition to a general effect on protein synthesis, Miracil D interferes with an early event in the induction process, presumably the synthesis of this messenger RNA. To determine the mechanism by which spermine protects E. coli from Miracil D, a culture was incubated for 30 rain in the usual medium with Miracil D, in the absence and presence of spermine, and the cells were then harvested by centrifugation. In the absence of spermine the bacterial pellet was bright yellow (the color of Miracil D) whereas in the presence of spermine it was colorless. When the cell-free media were extracted and assayed for Miracil D b y the method of BRINDLE, HIRSCHBERG AND GROSS 19, the medium with spermine contained approx, seven times as much Miracil D as the medium without spermine. These results clearly indicate that spermine interferes with the cellular uptake of Miracil D. However, the possibility of an additional site of spermine antagonism must be considered in view of the results obtained in subcellular systems.
E[/ect o/Miracil D on subcellular R N A and protein synthesis (Fig. 5, Tables I and I I ) The present studies with intact E. coli as well as our previous studies with B. subtilis (ref. 3) indicate that Miracil D inhibits the incorporation of labeled precursors into RNA and protein. To exclude the possibility that the drug affects precursor utilization, it was necessary to obtain direct evidence in subcellular systems. Fig. 5- indicates that 30 #M Miracil D produced greater than 50 % inhibition of [14C]ATP incorporation into RNA in a system containing purified E. coli B RNA Biochim. Biophys. Acta, 142 (1967) 440-449
446
B. WEINSTEIN et al.
polymerase and a limiting amount of DNA as primer. Supplementation of the system with excess DNA reversed Miracil inhibition, whereas supplementation with excess enzyme did not, indicating that the target of drug action is DNA and not enzyme. The effect of spermine on RNA synthesis in vitro, in the absence and presence of Miracil D, was determined in a separate experiment (Table ]). Whereas Miracil
I00
75 z 0
-~ 5 0 Z
25
*
t
i
i
i
I
2
3
4
5
MIRACIL D
(XI~SM)
Fig. 5. Miracil D i n h i b i t i o n of t h e R N A p o l y m e r a s e reaction. R e a c t i o n s y s t e m s c o n t a i n e d pe r ml: 7 ° / ~ g of s a l m o n - s p e r m DNA, 75 u n i t s of E. coli R N A p o l y m e r a s e , a n d Miracil D a t t h e final conc e n t r a t i o n s i n d i c a t e d . T h e r e m a i n i n g c o n d i t i o n s are d e s c r i b e d u n d e r METHODS. The c ont rol s y s t e m , i.e., in t h e a b s e n c e of drug, i n c o r p o r a t e d 12.5 m # m o l e s of [14C]ATP. The d a t a i n d i c a t e t h e m e a n of t h r e e e x p e r i m e n t s .
TABLE I EFFECT
OF
SPERMINE
ON
D
MIRACIL
INHIBITION
OF
THE
RNA
POLYMERASE
REACTION
R e a c t i o n s y s t e m s c o n t a i n e d p e r ml: 7 ° / , g of s a l m o n - s p e r m D N A a n d 75 u n i t s of E. coli R N A p o l y m e r a s e . Miracil D a n d / o r s p e r m i n e were a d d e d a t t h e fi na l c o n c e n t r a t i o n s i n d i c a t e d . The r e m a i n i n g c o m p o n e n t s w e r e as d e s c r i b e d u n d e r METHODS. Since t h e a d d i t i o n of s p e r m i n e s t i m u l a t e d t h e r e a c t i o n , i n h i b i t i o n b y Miracil D w a s c a l c u l a t e d in ea c h p a r t of t h e e x p e r i m e n t as pe r c e n t of t h e v a l u e in t h e c o n t r o l t u b e c o n t a i n i n g t h e c o r r e s p o n d i n g c o n c e n t r a t i o n of s p e r m i n e .
Miracil (M)
Spermine (M)
Incorporation o[ EI*C~A T P (ml~moles) 12. 5
_
_
3"
IO-S 5" to-5
---
3-9 0.3
--
io
3"t° 5 5" t o s
io 5 lO -5
__
10-4
3" 5" _ 3" 5"
lO-5 to-~ to-S lO-5
5
lO -4 1°-4 lO-3 io 3 lO -3
Biochim. Biophys. $cta, 142 (1967) 44o-449
Inhibition by 3liracil D
(%)
--
69 98
13.3
_
2.8 1.5 23.1 8.8 3.4 43.1 25.1 16.9
79 89 --
62 85 -42 61
MIRACIL D AND TABLE
E . coli DNA, R N A AND PROTEIN
447
I1
EFFECT OF MIRACIL D ON PROTEIN SYNTHESIS IN A SUBCELLULAR SYSTEM FROM E .
coli B
A s s a y s y s t e m s f o r tb_e e n d o g e n o u s r e a c t i o n c o n t a i n e d o . I m l o f t h e S-3o f r a c t i o n of E. coli B; f o r t h e p o l y U r e a c t i o n , o . i m l o f t h e iS-3o f r a c t i o n a n d p o l y U, i o o / ~ g / o . 4 ml. M i r a c i l D w a s a d d e d a t t h e f i n a l c o n c e n t r a t i o n s i n d i c a t e d . R e a c t i o n s w e r e i n c u b a t e d a t 37 ° f o r 3 ° rain. T h e r e m a i n i n g c o m p o n e n t s of t h e a s s a y s y s t e m a n d t h e p r o c e s s i n g of s a m p l e s a r e a s d e s c r i b e d u n d e r METHODS. I n c o r p o r a t i o n is e x p r e s s e d a s / H ~ m o l e s p e r a s s a y s y s t e m .
Miracil (M)
None 1.3" IO -~ 6 . 6 . lO -5 1.3" lO -4 2.6- lO -4
[l~Cl Phenylalanine incorporation Endogenous reaction (l~ltmoles)
plus poly U (l~#rnoles)
2.5 2.2 2. 4 2.7
163 154 196 189
2.2
18 5
inhibited incorporation of ATP into RNA, spermine alone (lO-5-1o -~ M) actually caused a concentration-dependent stimulation of RNA synthesis. Spermine stimulation has been previously described in the DNA-directed RNA polymerase reaction 2°,21. RNA synthesis in the presence of spermine was more resistant to inhibition b y Miracil D than RNA synthesis in the absence of spermine. Thus, whereas RNA synthesis in the presence of i mM spermine was inhibited 6 1 % by 5o/zM Miraeil D, the same concentration of Miracil produced 98 % inhibition of RNA synthesis in the absence of spermine when each was calculated against the appropriate control. This effect of the polyamine might be explained by assuming that: (I) spermine enhances the utilization of portions of the DNA primer which do not contain Miracil D or (2) spermine prevents the attachment of Miracil D to DNA, perhaps b y competing with the drug for phosphate residues on DNA. The first postulate is consistent with the suggestion of Fox, GUMPERT AND WEISS 2° that the RNA polymerase reaction in vitro is limited b y product inhibition (because newly synthesized RNA remains complexed with the enzyme) and that polyamines stimulate the reaction b y dissociating this complex. Attempts to demonstrate by spectrophotometric methods ~that spermine blocks the attachment in vitro of Miracil D to DNA have thus far been unsuccessful because addition of spermine at concentrations greater than o.I mM caused precipitation of the DNA. In the RNA polymerase reactions (Table I), spelmine did not cause precipitation of the DNA apparently because of the presence of the enzyme and/or the nucleotide triphosphates. In contrast to the pronounced effect of Miraeil D on RNA synthesis in the subcellular E. coli system, the drug has no effect on amino acid incorporation under comparable conditions, as summarized in Table II. When the incorporation of L14C]phenylalanine was directed b y either native messenger RNA's or by poly U, the reaction was not appreciably inhibited by concentrations of Miracil D in the range of 13 /,M to 0.26 mM. When assayed in a previously described rat-liver subcellular system ~2, these concentrations of Miracil D also failed to inhibit phenylalanine incorporation. Biochim. Biophys. Acta, 142 (1967) 4 4 o - 4 4 9
448
B. WEINSTEIN ct al.
DISCUSSION
The present studies establish Miracil D as an inhibitor of RNA synthesis in intact E. coli as well as in the subcellular RNA polymerase reaction. The results provide strong support for our hypothesis that Miracil D inhibits bacterial growth b y complexing with DNA and thereby inhibiting RNA synthesis 3. Miracil D provides an interesting contrast to actinomycin D, another threering heterocyclic compound with pronounced biological activity. The latter drug is a potent inhibitor of RNA synthesis in intact mammalian cells and gram-positive bacteria4,L Intact E. coli, however, are extremely resistant to actinomycin, apparently because they are not permeable to the drug unless treated with E D T A (refs. 4, 23, 24). The molecular weight of Miracil D, approx, one-third that of actinomycin D, or the lesser complexity of its side chain, m a y explain its ability to penetrate intact E. coli. Miracil, nevertheless, is in general a less potent drug since concentrations in the range of 50/~M are required to inhibit RNA synthesis, whereas in sensitive systems actinomycin is effective in the range of o.I/tM (refs. 4, 5). Based on previously published results 4,2~, as well as parallel studies in our own laboratory (unpublished), Miracil is somewhat more potent than proflavine as an inhibitor of purified E. coli RNA polymerase. A major disadvantage of Miracil as a tool for the further elucidation of macromolecular synthesis is the fact that it lacks the specificity of actinomycin D. Concentrations which inhibit RNA synthesis in intact E. coli also partially inhibit DNA and protein synthesis. Preliminary results with purified E. coli DNA polymerase indicate that Miracil can inhibit DNA synthesis directly though this reaction is more resistant than the RNA polymerase reaction. Studies in progress on the nature of the Miracil D - D N A complex 26 should help to explain the differential effects of this drug on the two template functions of DNA. The early inhibition of protein synthesis in intact cells m a y be due to binding of Miracil D to RNA since in vitro the drug can complex with both DNA and RNA (ref. 3). Later inhibition of protein synthesis m a y be explained by depletion of cellular messenger RNA. Our results on subcellular amino acid incorporation indicate that protein synthesis is much more resistant to Miracil D than the RNA polymerase reaction. A precise quantitative comparison of the relative response of the two reactions is not possible, however, because of the differences in ionic and nucleic acid composition of the two assay systems; extrapolation from the subcellular systems to the intact cell is complicated by the same factors. Concentrations of Miracil D which block RNA synthesis also inhibit the induction of fl-galactosidase. This is consistent with the concept that enzyme induction requires RNA synthesis de novo 2v. The effect on enzyme induction is not simply due to general inhibition of amino acid incorporation since induction of fl-galactosidase is completely blocked at concentrations of drug which only partially inhibit total protein synthesis. Our results do not exclude the possibility that catabolite repression, resulting from partial inhibition of protein synthesis TM, plays a role in the effect of Miracil D on fl-galactosidase induction. The effects of Miracil D on growth, RNA synthesis and enzyme induction in E. coli were dramatically prevented by spermine. Measurements of the concentration of Miracil D in ceils and media, in the presence and absence of the polyamine, indicate that spermine interferes with cellular uptake of the drug. At this time it is Biochim. Biophys. Acta, 142 (t967) 440-449
MIRACIL D AND E. coli DNA, RNA AND PROTEIN
449
not clear whether spermine also exerts a direct antagonism at the DNA level, although this is suggested by the experiments carried out in the subcellular system (Table I). During the preparation of this manuscript SIMON,COHEN AND RAINA2s reported that levorphanol inhibition of RNA synthesis in E. coli is reversed by spermidine and it is possible that this effect is also mediated at the level of drug uptake. The mechanism by which spermine affects Miracil D uptake is not known. It is possible that spermine competes with the basic side chain of Miracil D for receptor sites, perhaps phospholipids, in the cell membrane. Previous studies indicate that spermine can stabilize bacterial membranes15,16 and it can also alter membrane function with respect to potassium fluxes 15. Regardless of the mechanism, the findings provide an interesting example of drug competition at the level of cellular uptake and suggest the possibility that genetic resistance to certain growth inhibitors may be mediated by an increased endogenous synthesis of polyamines.
ACKNOWLEDGEMENTS
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