Inhibition of Nuclear Envelope Nucleoside

[CANCER RESEARCH 44, 3812-3819, September 1984]

Inhibition of Nuclear Envelope Nucleoside Triphosphatase-regulated Nucleocytoplasmic Messenger RNA Translocation by 9-ÃŸ-o- Arabinofuranosyladenine S'-Triphosphate in Rodent Cells1

Heinz C. SchrÃ¶der,2Doris E. Nitzgen, August Bernd,3 Branko Kurelec, Rudolf K. Zahn, Monika Gramzow, and Werner E. G. MÃ¼ller4

Institut fÃ¼rPhysiologischeChemie, UniversitÃ¤t,Duesbergweg,6500 Mainz, WestGermany[H. C. S., D. E, N., A. B., fÃ.K.Z., M. G., W. E. G. M.], and Center for Marine Research,Institut Ruder BoskoviÃ³,52210Rovini, Yugoslavia[B. K.]

ABSTRACT direct mutagens nor premutagens as determined by the Salmo- ne//a-mammalian microsome mutagenicity test. Nucleocytoplasmic translocation of polyadenylated messenger RNA is an energy-dependent process which is regulated by a INTRODUCTION nuclear envelope nucleoside triphosphatase; this enzyme was found to be stimulated by the 3'-terminal polyadenylic acid It is assumed that the antibiotic ara-A5 (8), which has been [poly(A)] tail of messenger RNA (Bernd, A., SchrÃ¶der, H. C., shown to be a potent anti-herpes virus (47) and carcinostatic Zahn, R. K., and MÃ¼ller,W. E. G. Eur. J. Biochem., 729: 43-49, (42, 43, 48) agent, acts on a molecular basis primarily on the 1982). level of DNA synthesis (43,47). ara-ATP, which is formed readily RNA efflux from isolated mouse lymphoma (L5178Y) cell nuclei from ara-A after entering the cell, was found to be a competitive is strongly reduced if 9-Â£-D-arabinofuranosyladenine 5'-triphos- inhibitor of DNA-dependent ONA polymerases a and ÃŸwith phate (ara-ATP) is present in the transport medium. Half-maximal respect to dATP in vitro (24, 43) as well as in intact cell systems inhibition of RNA efflux occurs with 120 U.Mara-ATP. Most likely, (25, 42); most sensitively inhibited is the herpes virus-induced the inhibitory effect of ara-ATP is caused by inhibition of nuclear enzyme (38). On a cellular level, ara-A treatment results in a envelope nucleoside triphosphatase; this enzyme was found to marked increase of cell volume (17) and in a reduction of cell be highly sensitive to inhibition by this antibiotic. The inhibition proliferation (42). type of the nucleoside triphosphatase of rat liver nuclear ghosts Besides inhibition of DNA polymerases, ribonucleotide reduc is competitive with respect to ATP; the K,:Â«â„¢ratiowas deter Ãase(16), and S-adenosylhomocysteine hydrolase (27) by ara-A mined to be 0.27. Besides nucleoside triphosphatase, nuclear or ara-ATP, which primarily affect DNA synthesis, ara-ATP has envelopes contain a protein phosphokinase modulating the affin been reported recently also to inhibit poly(A) polymerase(s) [po- ity of pore complex laminae to poly(A). This enzyme was also lynucleotide adenylyltransferase (EC 2.7.7.19)] (52, 53). The found to be strongly inhibited by ara-ATP in a competitive way synthesis of the 3'-terminal poly(A) tails of hnRNA or mRNA with respect to ATP (KjiK^, 0.056) and could therefore also catalyzed by poly(A) polymerase(s) (22) has been shown to be contribute to the overall inhibition of RNA transport. an essential step during posttranscriptional processing of hnRNA The polyadenylation of endogenous RNA by poly(A) polymer- (41), and it was suggested that the poly(A) segment is involved ase(s) in intact rat liver nuclei as well as in nuclear matrices in determining mRNA stability (35), gene splicing (14), and nu- isolated from the same source was found to be markedly sup cleocytoplasmic mRNA transport (41). pressed in the presence of ara-ATP. The inhibitions of both In this paper, we describe for the first time the nucleocyto- poly(A) polymerase activities (contained in whole nuclei or nuclear plasmic translocation step of poly(A)-containing mRNA as a matrix bound) are of the competitive type with respect to ATP. further site of action of ara-ATP. The transport of poly(A)-con- In in vitro assays, nuclear envelope nucleoside triphosphatase taining mRNA through the nuclear pore complexes is an energy- is inhibited by microtubule protein. Of the 2 ATP-dependent requiring process (1, 69) which is thought to be regulated by a enzyme activities associated with microtubule protein (cyclic adenosine 3':5'-monophosphate-dependent protein kinase and nuclear envelope nucleoside triphosphatase (EC 3.6.1.15) (4); the activity of this enzyme has been shown to be stimulated in adenosine triphosphatase), only the kinase was slightly affected vitro by synthetic poly(A) (3) as well as by the poly(A) segment by ara-ATP. of isolated poly(A)-containing mRNA (12). Moreover, poly(A)- Cellular uptake of adenosine 5'-monophosphate and perhaps 9-/3-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) is associated proteins (41) and microtubule proteins (41, 57) were demonstrated to play possibly a critical part in controlling nucleo- facilitated by a cellular membrane-bound 5'-nucleotidase. Our cytoplasmic poly(A)-containing mRNA transport. Nuclear envel studies revealed that neither cleavage of ara-AMP nor inhibition opes contain besides nucleoside triphosphatase a protein phos of the enzyme activity by ara-AMP occurs. phokinase (EC 2.7.1.37) (61) and a phosphoprotein phosphohy- 9-/3-D-Arabinofuranosyladenine and ara-AMP represent neither drolase (EC 3.1.3.2) activity (62); these enzymes, which are

1Supported by grants from trie Deutsche Forschungsgemeinschaft(Mu 348/7- modulated by poly(A) in an opposite manner (10), have been shown recently not to be constituents of the nucleoside triphos- 4) and from the Fonds der Chemischen Industrie. 2Recipientof a Liebig stipend from the Fonds der ChemischenIndustrie. 3Present address: Zentrum der Dermatologie und Venerologie,Theodor-Stern- 5The abbreviations used are: ara-A, 9-,8-D-arabinofuranosyladenine;ara-AMP, Kai 7, J.-W. Goethe-UniversitÃ¤t,6000Frankfurt (M) 70, West Germany. 9-0-D-arabinofuranosyladenine5'-monophosphate; ara-ATP, 9-/3-o-arabinofurano- 4To whom requests for reprints should be addressed. syladenine5'-triphosphate; cAMP, cyclic adenosine 3':5'-phosphate; poly(A),po ReceivedFebruary 27,1984; accepted June 6,1984. lyadenylicacid; hnRNA, heterogeneous RNA.

3812 CANCER RESEARCH VOL. 44

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1984 American Association for Cancer Research. Inhibition of Nucleoside Triphosphatase by ara-ATP phatase cycle (10) as proposed previously (2). et al. (30) as described earlier (12). After treatment with DNase I and In addition to the inhibition of nuclear envelope nucleoside RNase A, followed by extraction with buffer of low magnesium ion triphosphatase and protein phosphokinase by ara-ATP, we stud concentration, the nuclear ghosts were suspended at a concentration of 5 x 108/ml in a 10 mM Tris-HCI buffer (pH 7.4; 0.2 mM MgSO4,1 mM Â«- ied the effect of the antibiotic on poly(A) synthesis in isolated phenyl-methylsulfonyl fluoride, and 2.1 M sucrose). The preparation of nuclei and nuclear matrix preparations from rat liver. Since pore laminae from nuclear ghosts was performed as described by Dwyer microtubule protein in vitro has been shown to inhibit nucleoside and Blcbel (19). The pore laminae were washed 3 times by resuspension triphosphatase activity (12), the influence of ara-ATP on ATP- and centrifugation (20,000 x g; 10 min; 4Â°)with 50 mM Tris-HCI and 1 dependent enzymes associated with microtubule protein (63) is mM MgC>2,pH 8.O. examined. Furthermore, the effect of ara-ATP on cell membrane- Preparation of the Nuclear Matrix. The nuclear matrix was prepared bound 5'-nucleotidase [5'-ribonucleotide phosphohydrolase (EC from freshly isolated rat liver nuclei as described by Kaufmann ef al. (30); 3.1.3.5)] responsible for cellular nucleotide uptake (18), and the the digestion of DNA was performed at a concentration of about 3.5 x mutagenicity of ara-A and ara-AMP are tested. 10s nuclei/ml with DNase I (220 /Â¿g/ml)for 60 min at 4Â°.After this step, 1 mM a-phenylmethylsulfonyl fluoride and 0.1 mM sodium tetrathionate were added to all the matrix isolation buffers used (11 ). DNase I (Boehringer; grade I) was purified over an agarose 5'-[(4- MATERIALS AND METHODS aminophenyl)phosphoryl]uridine 2'(3')-phosphate column (Miles) to re Materials. ara-ATP (No. A-6642; purity, 97%), a-phenylmethylsulfonyl move residual amounts of RNase (66). fluoride, O-phospho-OL-serine, spermidine, podophyllotoxin, and If the sum of the masses of DNA, RNA, protein, and phospholipid benzo(a)pyrene were from Sigma Chemical Co., St. Louis, MO; [2,8-3H]- contained in the nuclear matrix preparations is set at 100%, the per ATP (specific activity, 42 Ci/mmol), [7-Å“P]ATP (specific activity, 3 Ci/ centage of composition of the nuclear matrices was as follows: DNA, mmol), [2-3H]AMP (specific activity, 15 Ci/mmol), and [5-3H]uridine (spe 1.3 Â±0.6 (S.D.); RNA, 10.9 Â±1.8; protein, 53.3 Â±3.2; and phospholipid, cific activity, 5 Ci/mmol) were from The Radiochemical Centre, Amer- 34.4 Â±4.8. sham, England; poly([3H]A) (specific activity, 18 mCi/mmol) was from Measurement of RNA Efflux. RNA efflux from isolated nuclei was Schwarz/Mann, Orangeburg, NY; ATP (No. 127531), poly(A) (single measured according to the method of Agutter ef al. (1, 5) with modifica stranded; with an average molecular weight of 248,000), DNase I (EC tions. Briefly, after 2 washes in transport medium [50 mM Tris-HCI (pH 3.1.21.1; bovine pancreas; grade I; specific activity, 2000 units/mg). 7.6), 25 mM KCI, 250 HIM sucrose, 2.5 mM MgCI2, 0.3 mM MnCI2, 5 mM RNase A (EC 3.1.27.5; bovine pancreas; specific activity, 50 units/mg), 2-mercaptoethanol, 5 mM spermidine â€¢HCI,and yeast RNA (0.3 mg/ml), phosphoendpyruvate, pyruvate kinase, minimum essential medium (Ea freed from nuclease activities by heat treatment], the labeled nuclei were gle's), and horse serum were from Boehringer/Mannheim, Mannheim, resuspended in transport medium to a concentration of about 2.5 x 106/ Germany; yeast RNA (SM = 8; RNA content higher than 97%), actinc- ml and preincubated with or without ara-ATP for 10 min at 0Â° as mycin D, and Triton X-100 were from Serva, Heidelberg, Germany; described in "Results." Subsequently, the suspension was incubated methyl methanesulfonate was from E. Merck, Darmstadt, Germany; under vigorous shaking for 0 to 30 min at 37Â°in the presence of 2.5 mM DEAE-cellulose, DE81 discs (diameter, 2.3 cm; Whatman), and GF/C ATP, 5 mM Na2HPO4, 5 mM phosphoenolpyruvate, and pyruvate kinase filters were from Hormuth and Vetter, Heidelberg, Germany; nitrocellu (35 units/ml). In control experiments, ATP was replaced by 2.5 mM lose filters (type HA; pore size, 0.45 *im; diameter, 2.3 cm) were from EDTA, and Na2HPO4, and the ATP-regenerating system were omitted. Millipore, Neu-lsenburg, Germany; Aguasol was from New England Nu After cooling in ice-water, the nuclei were pelleted by centrifugation clear, Boston, MA. ara-AMP (chromatographically pure) was a gift of H. (1500 x g; 10 min; 4Â°),and the RNA released was obtained from the Mack, lliertissen, Germany. Oligo(pA)io was prepared by degradation of supernatant by trichloroacetic acid precipitation as described (5). The poly(A) by endoribonuclease IV (57). pelleted material was counted after hydrolysis in 100 /Â«11NNaOH (2 hr; Cell Culture. L5178Y mouse lymphoma cells were grown in Eagle's 56Â°) and addition of 100 n\ 1 N HCI in 10 ml Aquasol. The total minimum essential medium (20), supplemented with 10% horse serum radioactivity incorporated into nuclei was determined by disintegration of in spinner culture (43). Cell concentration was measured with a Model B the nuclei in Tissusol-Roth and counting as before. Coulter Counter with a size distribution plotter (Coulter Electronics, Cell Membranes. The cell membranes were isolated from mouse liver Hiateah, FL). Cultures (500 ml) were initiated by inoculation of 2 x 10* according to the method of Warren ef al. (67). The membrane preparation cells/ml and incubated for 48 hr; after this period, the cell concentration was dialyzed (24 hr; 4Â°)against 50 mM Tris-HCI, pH 7.2. was 5.2 x 1o5/mi. Subsequently, the cells were collected by centrifuga- Preparation of Microtubule Protein. Microtubule protein was isolated tion (700 x g; 5 min; 37Â°)and resuspended in 320 ml culture medium at from bovine brain by 3 cycles of temperature-dependent assembly- a concentration of 3.9 x 10s cells/ml. For RNA efflux experiments, 2 /tCi disassembly using the glycerol-free buffer system described by Asnes of [5-3H]uridine/ml were added, and the cultures were incubated in roller and Wilson (9). tubes at 37Â°for 2 hr; then the cells were harvested. Purification of Terminal Riboadenylate Transferase. Terminal ri- Isolation of Nuclei. After 2 washings by resuspension in 20 ml of Tris boadenylate transferase [Mn2+-dependent poly(A) polymerase] was pu buffer [10 mw Tris-HCI, 1 mM MgCI2, 1 mM a-phenylmethylsulfonyl rified nearly to homogeneity from calf thymus gland as described by fluoride, and 1 HIM ethyleneglycol bis(j3-aminoethyl ether)-A/,/V,A/',A/'- Tsiapalis ef al. (64); Fraction VIII with a specific activity of 1070 units/mg tetraacetic acid, pH 7.4] and centrifugation (700 x g; 10 min; 4Â°),the protein (protein concentration, 0.12 mg/ml) was used for the experi cells were resuspended in 4 ml of the same buffer and ruptured in a ments. One unit of enzyme is equal to 1 nmol of AMP incorporated per Dounce potter by 10 strokes with an S pestle. Subsequently, 20 ml of hr under the assay conditions used. 20 mM Tris-HCI [pH 7.4; containing 1 mM MgCI2 and 60% (w/v) sucrose] Enzyme Assays. The reaction mixture to determine nuclear envelope were added, resulting in a final sucrose concentration of 50%. Then, the nucleoside triphosphatase activity was composed as follows (final vol suspension was layered on top of a 8-ml cushion of the sucrose solution ume, 6.4 ml) (5, 12): 25 mM Tris-HCI (pH 8.0); 2.5 mM MgCI2; 2.5 mM described above and centrifuged (130,000 x g; 1 hr; 4Â°). ATP; 130 mM sucrose; and 400 //I nuclear ghost suspension (142 Â»g Rat liver nuclei were isolated according to the method of Blobel and protein). Incubation was performed for 0 to 15 min at 37Â°, and after Potter (15); the homogenization buffer was supplemented with 5 mM 2- different time intervals 1-ml aliquots were taken for determination of the mercaptoethanol. P. released (70). Nuclear Ghosts and Pore Laminae. Nuclease-treated nuclear ghosts The nuclear envelope-associated protein kinase activity was deter were prepared from liver of male Wistar rats (body weight, 250 to 270 mined in 100-jjl assays consisting of 25 mM Tris-HCI (pH 8.0), 2.5 mM g; Ivanovas, Kisslegg, Germany) according to the method of Kaufmann MgCI2,150 mM NaCI, 1 mM 2-mercaptoethanol, 10 mM NaF, 10 mM O-

SEPTEMBER 1984 3813

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1984 American Association for Cancer Research. H. C. Schroder et al. phospho-DL-serine,40 /Â«M[y-^PJATP(about 1500 dpm/pmol), and 5 x as standard, and phospholipidphosphate by treatment of the chloroform- 106nuclear ghosts (10, 37). After incubation for 0 to 20 min at 20Â°,the methanolextract (23) as described by Ames (6). phosphate incorporated was determined by trichloroacetic acid precipi tation as described (10). The terminal riboadenylate transferase [Mn2+-dependentpoly(A) po- RESULTS lymerase] activity was determined essentially as described (64). The Nuclear Envelope Nucleoside Triphosphatase Activity. Nu reaction mixture contained in a final volume of 50 pi 200 mM Tris-HCI (pH 8.3), 1 mM MnCfe,0.2 mw dithiothreitol, 20 pM [3H]ATP(5.5 x 102 clear envelope nucleoside triphosphatase activity was deter dpm/pmol), 5 Â¡Â¿gofoÃ¼goÃ•pA)â„¢,bovineserum albumin(10 /ig/ml), and 10 mined in nuclear ghosts from rat liver, which were freed from ill of enzyme (containing 0.2 unit). After incubation at 37Â°for 30 min, a endogenous polyribonucleotides by RNase A treatment. Using 40-/il aliquot was taken and spotted onto a GF/C filter disc to determine this preparation, an apparent Km of 0.452 Â±0.038 mw and a the acid-precipitableradioactivity as described (44). maximal reaction velocity of 3.3 ^mo\ ATP hydrolyzed per hr per In intact rat liver nuclei, poly(A) polymerase activity was measured by mg protein were found [in the presence of 150 mM KCI in the suspending the nuclei at a concentration of 2.5 x 107/ml(175 ng DNA/ reaction mixture (2)]. ml) in an incubation medium which contained in a final volume of 200 n\ The nucleoside triphosphatase activity was found to be 50 mMTris-HCI(pH 8.0), 5 mM MgCI2,0.5 mM MnCI2,0.5 mMCaCI2,10 strongly reduced in the presence of ara-ATP; using an ara-ATP . mM KCI, 2 mM dithiothreitol, 10 mM phosphoenolpyruvate, pyruvate concentration of 0.8 mw, the enzyme activity was inhibited by kinase (10 units/ml), 5 mM spermidine, actinomycin D (25 Â¿Â»g/ml),and4 ,iM [3H]ATP(9.2 x 10sdpm/nmol). Reactions were performed at 30Â°for about 50%. As shown in Chart 1, ara-ATP inhibits nucleoside 0 to 30 min and terminated by chilling in ice and addition of 1 ml of ice- triphosphatase in a competitive way with respect to ATP; the K, cold 10% (w/v) trichloroacetic acid containing 1% (w/v) sodium pyro- was determined to be 0.122 Â±0.014 mw. The relative affinity phosphate. After standing on ice for 15 min, precipitates were collected (KÂ¡:Km)ofara-ATP for nucleoside triphosphatase was calculated on Millipore filters (type HA; pore size, 0.45 urn). According to Ref. 26, to be 0.27; this figure is 1.7 times lower than that observed with filters were washed with about 30 ml of 5% trichloroacetic acid-1% the nuclear poly(A) polymerase (see below). sodium pyrophosphate and then incubated separately in vials containing The nucleoside triphosphatase is able to hydrolyze ara-ATP 5% trichloroacetic acid, 1% sodium pyrophosphate, and 1 M KCI. The (Table 1). The rate of nucleoside triphosphate cleavage in the dried filters were counted in 5 ml Aguasol. case of ara-ATP was about 5 times lower than that obtained in Nuclear matrix-associated poly(A)polymerase activity was measured the presence of ATP. in a final reaction volume of 200 n\ containing 50 mM Tris-HCI (pH 8.0), Nuclear Envelope-associated Protein Kinase Activity. Nu 5 mM MgCl;>.0.5 mM MnCI2, 10 mM KCI, 2 mM dithiothreitol, 5 mM phosphoenolpyruvate, pyruvate kinase (3 units/ml), actinomycin D (50 clear envelopes contain a protein phosphokinase activity (37,61) pg/ml), 4 MM[3H]ATP(9.2 x 10Â«dpm/nmol),and 20 ^ (230 /ig) of the which phosphorylates 2 protein species (M, 106,000 and M, nuclear matrix preparation. After incubation for 0 to 15 min, the acid- 64,000) in the nuclear envelope independently of the nucleoside precipitable radioactivity was determinedas describedfor nuclearpoly(A) triphosphatase cycle (10). This enzyme was found to be inhibited polymerase activity. by poly(A) (10). As shown in Table 2, ara-ATP strongly reduces Microtubule-associatedprotein kinase activity (autophosphorylationof the protein phosphokinase activity. After addition of 5 MM ara- microtubule protein) was determined, as described previously(55),in the ATP, the enzyme activity in the standard reaction mixture was absence or the presence of 5 //McAMP. decreased by 43%. The inhibition was of the competitive type The assay mixture to determine microtubule-associatedATPase (EC with respect to ATP (data not shown). Using an inhibitor concen 3.6.1.3) activity was composed as outlined earlier (55). The amount of tration of 20 tiM, the KI of the protein kinase (Ki,TP= 308 ^M) phosphate liberated was measured using the DEAE-celiuloseOE81 disc technique (45). was found to be 17.2 ^M, giving a K,:Kn,ratio of 0.056. The 5'-nucleotidase activity was determined by the DE filter paper technique described previously (45). The standard assay mixture (100 3.0- ,il) contained 100 mM Tris-HCI (pH 8.2), 4 mM MgCI2,100 pM [3H]AMP (4x10* dpm/nmol), and 20 //I of the cell membranefraction. The samples were incubated at 27Â°for 20 min. One unit of enzyme is 1 nmol of 2.5- adenosine formed per 10 min. Bindingof Poly(A) to Pore Laminae. Poly(A)-bmdingexperimentsin the presence of ara-ATP were performed using the microdetermination t 2.0 assay developed previously (13). The binding mixture contained per 100 iÂ¡\,in a 50 mM Tris-HCI buffer (pH 8.0; 1 mM MgCI2),23 pg of pore p. 1.5- laminae (obtained from 5.4 x 107 nuclear ghosts) and 1 nmol of poly- ([3H]A)(4 x 103dpm/nmol). Pore laminae-boundpoly(A)was separated from free poly(A) by centrifugation through a sucrose cushion as de 1.0- â€¢, scribed (13). Mutagenicity Testing. The mutagenicresponseof the nucleosides and nucleotides was measured with the SalmonellatyphimuriumTA100- sjar- Ames test (7, 31). The compounds, dissolved in dimethyl sulfoxide, were tested directly or with the S-9 fraction, isolated from the immature carp liver, treated with methylcholanthrenei.p. (50 mg/kg), as described (51). The number of /ws+revenants was counted automatically by Dynatech 0 0.1 0.2 0.3 0.4 0.5 Model-980 counter. A premutagen benzo(a)pyrene served as positive ara-ATP [mM] Chart 1. Competitive inhibition of nuclear envelope nucleoside triphosphatase control and methyl methanesulfonateserved as a direct mutagen. by ara-ATP. Dixon plot. The reaction conditions were described in "Materials and Miscellaneous. DNA was determinedaccordingto the method of Methods." Increasing concentrations of ara-ATP were added to the reaction mix Labarca and Paigen (33), RNA by the procedure of Webb (68), protein tures containing 0.2 mM (D), 0.5 mm (O), and 2.5 mm ATP (â€¢).Abscissa, ara-ATP according to the method of Lowry ef al. (34) using bovine serum albumin concentration; ordinate, reciprocal values of the reaction velocity.

3814 CANCER RESEARCH VOL. 44

Table 1 ATP, the radioactivity bound to pore laminae was reduced to Hydrolysis of ara-ATP in comparison to that of ATP by nuclear envelope almost the same extent (33.9%; Table 3). nucleoside triphosphatase The reactions were performed in the presence of the indicated nucleotide Nuclear Poly(A) Polymerase Activity. In mammalian tissues, concentrations under the conditions described in "Materials and Methods." at least 2 different types of poly(A) polymerases are distin guished: a cytoplasmic Mn2+-dependent enzyme, also called Nucleoside triphosphatase activity (ttmot nucteoside tri- terminal riboadenylate transferase (64); and a nuclear Mg2+- phosphate hydrolyzed/hr/mg Substrate Concentration (^M) protein) dependent enzyme (21). The latter poly(A) polymerase was sug gested to exist in 2 functional states: (a) as chromatin-associated ATP 100 Â±0.07a 200 1.12 + 0.15 enzyme; and (o) in a soluble, nucleoplasmic form (54). Our 250ara-ATP 1.29Â±0.160.11 inhibition studies with ara-ATP were performed with the homo

100 Â±0.02 geneous, cytoplasmic terminal riboadenylate transferase from 200 0.23 Â±0.03 calf thymus and the poly(A) polymerase contained in intact rat 2500.59 0.25 Â±0.03 a Mean Â±S.D. of 4 experimental determinations.

Table 2 Inhibition of protein kinase activities in nuclear ghost preparations from rat liver and in microtubule protein by ara-ATP Microtubule protein was obtained from bovine brain (55). The reactions were performed as described in "Materials and Methods* after a 10-min preincubation at 4Â°with or without the indicated amounts of ara-ATP. Microtubule-associated protein kinase activity was determined with or without cAMP in the absence of exogenous substrate (autophosphorylation of microtubule protein). protein-associated ara-ATP ghost-asso (%)-CAMP100protein kinase activity concentra ciated protein kinase tion (pM)0 activity(%)100 CAMP100 Â±7.1* Â±3.2 Â±5.7 5 56.7 Â±3.4 97.9 Â±1.9 96.5 Â±5.9 10 40.1 Â±5.0 95.1 Â±4.0 95.0 Â±3.3 50 23.9 Â±1.7 91.0 Â±4.1 90.1 Â±1.3 100Nuclear 17.9 Â±2.2Microtubule 85.9 Â±4.0+ 83.1 Â±2.9 8 Mean Â±S.D. of 4 experimental series. Timt (min) Chart 2. Inhibition of RNA efflux from isolated nuclei by ara-ATP. Radiolabeling RNA Efflux from Isolated Nuclei. Nuclei were isolated from and isolation of L5178Y cell nuclei and determination of RNA release were per mouse L-cells prelabeled with [3H]uridine as described in "Ma formed as described in "Materials and Methods." Appearance of tnchloroacetic terials and Methods." Starting with 4 x 105 cells/ml, cultures acid-precipitable radioactivity in the external medium after incubation of nuclei at 37Â°in the presence of 2.5 mM ATP (â€¢),2.5mM ATP plus 100 <Â¿Mara-ATP(O), or reached, after a labeling period of 2 hr in the presence of 2 u.C\/ 2.5 mM EDTA (H). Points, mean of 3 independent experimental series; bars, S.D. ml, a density of 5 x 105 cells/ml; about 7x10 dpm were found to be incorporated into 106 nuclei. Transport measurements were performed within 0.5 hr after the preparation of nuclei was finished. The appearance of acid-precipitable radioactivity was 300 monitored after a 10-min preincubation period of the nuclei in the presence of varying ara-ATP concentrations (Chart 2). The ex periments revealed that 100 U.Mara-ATP decreases the amount : of RNA left in the postnuclear supernatant after an incubation period of 20 min (30 min) by 27.0% (30.2%). If the amount of = 5 200 RNA released due to leakage of nuclei (measured in the presence o o a 3 of EDTA in the transport medium instead of ATP) is subtracted, o e the drop of RNA efflux in the presence of 100 U.Mara-ATP Is amounts to 29.0% (32.4%). The concentration for half-maximal inhibition was estimated to be approximately 120 UM (Chart 3). lÃ¬o 2 ATP and Poly(A) Binding of Pore Laminae. In our scheme o 100- of the nucleoside triphosphatase cycle, binding of ATP to the carrier structure for mRNA transport induces a conformational change resulting in the release of the bound mRNA (10). In order 'C to examine whether also ara-ATP is able to stimulate the release of bound poly(A) from pore laminae, pore laminae were prepared from nuclear ghosts of rat liver and preincubated in the presence of poly([3H]A) as outlined in legend to Table 3; then the dissocia 0.5 1.0 1.5 tion of the pore laminae poly(A) complex formed in the presence of ATP or ara-ATP was determined. The results summarized in ara-ATP [mM] Table 3 show that addition of 5 UMATP to the incubation mixture Charts. Effects of increasing ara-ATP concentrations on RNA release from isolated nuclei. The acid-precipitable radioactivity was determined after an incuba results in the release of 31.6% of the bound poly(A) from the tion period of 15 min; for further details, see legend to Chart 2. The concentration pore laminae. In the presence of the same concentration of ara- for half-maximal inhibition was obtained as indicated (broken line).

SEPTEMBER 1984 3815

Tabte3 Tabte4 Effect of ara-ATP on the release of bound poly(A) from pore laminae Induction of his* revenants in S. typhimurium 14700 by adenine derivatives Pore laminae from rat liver were prepared (13) and washed 3 times with 50 mM The assays were performed in the absence or the presence of postmitochondnal Tris-HCI (pH 8; 1 mu MgCi,) by resuspension and centrifugation (20,000 x g; 10 fraction (S-9) as described under "Materials and Methods." The controls included min; 4Â°).Binding of poly(A) to the pore laminae was performed for 10 min at 20Â° were dimethyl sulfoxide, benzo(a)pyrene. and methyl methanesulfonate. m 1-ml reaction mixtures containing 230 M9 of pore laminae protein and 10 nmol of poiyd'H |A) in the Tns- MgCi; buffer described above. After incubation. 75-Ml aliquots ofrevertants/plateMinus were layered over 100M! 30% (w/v) sucrose cushions [containing 50 mM Tris-HCI tration (pH 8.0) and 1 mM MgCI..| and centrifugea in an A-100/18 rotor of a Beckmann CompoundsAdenosineDeoxyadenosineara-Aara-AMPControls(MM)15 S9114,123,121,116 Airfuge (12 psi; 30 min; 6Â°).The pellets were resuspended in 100 M' of the Tris- 132, 103, 128 MgCI2 buffer and incubated then in the absence or the presence of ATP or ara- 4415 101.101.127,114145.125.122,104120,115115,128,120,101 104, 127, ATP for 10 min at 20Â°as indicated. Thereafter, the pore laminae were pelleted again by centrifugaron through the sucrose cushion as described, and the radio activity of the supematants and the pellets resuspended in 100 M!of 0.9% NaCI 4415 120,92,123,11796,98,116,129102,123,105,108138,102,116,122 solution was counted in Aquasol. Poly(A) bound 4415 104,119,124,9592,102,111,126 127,115,103,119150,118,108,117 AfterwithNo treatment protein0.65 Â±0.04' treatment 44No. 113,77.102,98126,145,131,104piussg135,136,116.97,125144, 5 MMATP 0.44 Â±0.02 68.4 5 MMara-ATPpmol/Mg 0.42 Â±0.03%100 66.1 100 /Â»IdimethylsulfoxideConcen 162, 148, 106 Mean Â±S.D. of 3 independent experiments. Benzo(a)pyrene 4.9 101,109,113,122 598,608,621,607 9.8 126,124, 127,141 1002,1111,1207, liver nuclei as well as with poly(A) polymerase found to be 1014 associated with nuclear matrix preparations from the same source. Methyl methane 1666.0 1256, 1197,1341, 1324,1254, 1537, sulfonate 1156 1249 The experiments revealed that all 3 poly(A) polymerase activ ities were inhibited by ara-ATP in a competitive way with respect to ATP. The kinetic constants were determined as follows. bules are regulated by a series of nucleotide-dependent enzymes Terminal riboadenylate transferase: Km, 29.1 ^M; Kâ€ž164.3/Â¿M. (63). As possible targets for ara-ATP, the following 2 ATP- Nuclear poly(A) polymerase (intact rat liver nuclei): Km, 102.1 Â¿Â¿M;dependent microtubule-associated enzymes were investigated: KI, 47.1 tiM. Nuclear matrix-bound poly(A) polymerase: Km, 51.2 ATPase (55,71) associated with microtubule-associated Protein A/M;Kâ€ž10.8UM.In the case of terminal riboadenylate transferase, 1 (28); and cAMP-dependent protein kinase (55, 60) associated the K,:Kâ€ž,ratioamounted to 5.65, while the corresponding values with microtubule-associated Protein 2 (65). for both nuclear poly(A) polymerase activities were significantly Only a small effect was observed with ara-ATP on microtubule- lower: enzyme activity in intact rat liver nuclei, 0.46; nuclear associated protein kinase (Table 2). Using the standard assay matrix-bound poly(A) polymerase, 0.21. These results indicate mixture without exogenous substrate (autophosphorylation of that cytoplasmic poly(A) polymerase, in contrast to the nuclear microtubule protein), the protein kinase activity was determined enzyme(s), is inhibited by ara-ATP only to a small extent. to be 0.53 pmol MP incorporated per /*g microtubule protein in Cell Membrane-associated 5'-Nucleotidase Activity. Al the absence of cAMP and 0.72 pmol ^P incorporated per fig in though evidence exists that ara-AMP is able to enter cells directly the presence of cAMP. After addition of 100 ^M ara-ATP, the (49), dephosphorylation of ara-AMP to ara-A, which is then enzyme activities in the assays without or with 5 /Â¿McAMPwere actively transported across the cell membrane, is assumed to be inhibited by 14.1% (0.46 pmol 32P incorporated per Â¿Â»9)and the usual way of the cellular uptake of this antibiotic (39). A 5'- 16.8% (0.60 pmol 32P incorporated per ^g), respectively (Table nucleotidase, isolated from rat liver microsomes (59), which 2). In contrast to nuclear envelope-associated nucleoside tri- catalyzes the cleavage of phosphate from both ribonucleoside phosphatase, microtubule-associated adenosine triphosphatase 5'-monophosphates and deoxyribonucleoside 5'-monophos- was found to be insensitive towards ara-ATP even in the pres phates might be involved in transport of adenosine (18) and ence of a drug concentration in the standard reaction mixture as perhaps also of ara-A. Therefore, the ability of 5'-nucleotidase high as 200 UM. in mouse liver cell membranes to hydrolyze ara-AMP has been Mutagenicity. As shown in Table 4, neither the naturally investigated. Under the assay conditions used, the enzyme prep occurring adenine nucleosides nor their arabinosyl derivatives aration showed linear time kinetics for up to 30 min. The Km of have mutagenic potential as measured by the TA100 S. typhi the enzyme for 5'-AMP was determined to be 9.1 /Â¿M.However, murium strain of the Ames test. The addition of S-9 (post if 5'-AMP was replaced by the identical amount of ara-AMP in mitochondnal fraction) does not change the number of revert- the 5'-nucleotidase assay (see "Materials and Methods"), no ants, indicating that ara-A as well as ara-AMP are neither direct degradation of this nucleotide to ara-Ado by the enzyme prepa mutagens nor premutagens under the assay conditions used. ration could be detected (data not shown). Moreover, it could be demonstrated that hydrolysis of AMP by 5'-nucleotidase is not DISCUSSION inhibited in the presence of ara-AMP concentrations as high as 200 MM.Therefore, it must be concluded that ara-AMP does not In the present report, it could be demonstrated that poly(A)- affect intracellular polynucleotide synthesis as a consequence of containing mRNA transport from the nucleus to the cytoplasm is an impaired cellular uptake of the needed nucleotide constitu significantly inhibited by ara-ATP. In the presence of 120 Â¿tMara- ents. ATP, the RNA efflux rate was found to be suppressed half- ATP-dependent Enzyme Activities Associated with Micro- maximally. tubule Protein. The dynamics and the organization of microtu- Most probably, inhibition of nucleocytoplasmic RNA translo-

3816 CANCER RESEARCH VOL. 44

cation is caused by inhibition of nuclear envelope nucleoside Our results revealed that ara-AMP does not influence cellular triphosphatase by ara-ATP. This enzyme has been shown to membrane-bound 5'-nucleotidase; neither inhibition of the en deliver the energy required for RNA transport by hydrolysis of zyme activity nor degradation of the nucleotide analogue by the the terminal high-energy phosphodiester bond of 5'-ATP (4). The enzyme could be detected. Especially the latter finding is sur nucleoside triphosphatase displays a very broad substrate spec prising, since 5'-nucleotidase was proposed to facilitate the ificity. ATP, 3'-dATP, 2'-dATP, and GTP are accepted as sub cellular uptake of ara-AMP via its dephosphorylation to the strate by the enzyme (2). The inhibition of the nucleoside tri nucleoside stage in a way similar to that observed in the case of phosphatase by ara-ATP is of the competitive type with respect 5'-AMP (18). Once taken up by the cell, ara-A is completely to 5'-ATP. The KÂ¡:Kmratiowas determined to be 0.27. In com phosphorylated to ara-ATP or deaminated to ara,-Hx which is parison, the inhibition of DMA polymerase a from quail oviduct is subsequently phosphorylated to ara-ITP as shown previously in characterized by a K,:Â«â„¢ratioof 1.20 (43). the L-cell system (40). ara-ATP was found to be degraded by nucleoside triphospha In in vitro assays, nuclear ghost-associated nucleoside tri tase under the liberation of phosphate. Therefore, this antibiotic phosphatase activity has been shown to be inhibited by micro- behaves like cordycepin S'-triphosphate which is also hydrolyzed tubule protein (12). ATP-dependent reactions are attributed to by the enzyme (5), but the latter drug did not inhibit nucleoside regulate microtubule formation and dynamics (36); thereby ATP triphosphatase activity (5). However, cordycepin 5'-triphosphate, can serve either as an allosteric effector (72) or as substrate for a potent inhibitor of poly(A) polymerase (46), was found to inhibit certain microtubule-associated enzymes (71). The microtubule- predominantly the efflux of mRNA from isolated nuclei by a associated cAMP-dependent protein kinase which is associated mechanism not yet understood (5). with the projection portion of high-molecular-weight microtubule- The inhibition of nuclear envelope nucleoside triphosphatase associated Protein 2 (HMW-MAP2) (65) has been shown to be by ara-ATP probably provides a useful tool for studies of the involved in controlling microtubule steady-state treadmilling (29) mechanism of action of nucleoside triphosphatase-mediated nu- and in modulating microtubule-polynucleotide interaction (56) via cleocytoplasmic mRNA transport. Until now, only one nucleotide phosphorylation of HMW-MAP2 and of r proteins (55, 60). In our analogue inhibiting nucleoside triphosphatase markedly, adeno- experiments, only little effect of ara-ATP on microtubule-associ sine 5'-[7-thio]triphosphate (2), was known which possesses a ated protein kinase could be detected. Microtubule-associated nonhydrolyzable 7-phosphodiester linkage and acts, therefore, ATPase (71), which is apparently associated with HMW-MAP1 rather unspecifically. (28), was not affected by ara-ATP. Besides the inhibition of nuclear envelope nucleoside triphos It is remarkable that ara-A as well as ara-AMP possess neither phatase, the reduced release of RNA in the presence of ara-ATP direct mutagenic nor premutagenic potencies when tested by could be caused in part also by inhibition of nuclear envelope- the TA100 S. fyp/7/mur/um-mammalian microsome variant of the associated protein phosphokinase activity. This enzyme activity Ames test. which is not part of the nucleoside triphosphatase cycle (10) is As a consequence of the results presented in this paper, the thought to be involved in modulating the affinity of the "carrier conclusion must be drawn that the antiviral and antitumor mode structure" for mRNA transport via phosphorylation of 2 distinct of action of ara-ATP is not restricted to the level of DNA repli proteins [P106 and P64 (10)]. Phosphorylation of isolated pore cation but also involves not negligible points of attack on the laminae has been shown to result in a reduced affinity for poly(A) levels of posttranscription (polyadenylation of mRNA) and of binding (13). nucleocytoplasmic mRNA transport (nuclear envelope-associ A second, more indirect way by which ara-ATP can affect the ated nucleoside triphosphatase and protein phosphokinase). appearance of poly(A)-containing mRNA in the cytoplasm may These findings may help to explain some phenomena observed result from inhibition of mRNA polyadenylation within the nu under ara-A treatment until now inexplicable such as weight loss cleus. From experiments with the poly(A) polymerase inhibitor induced by ara-A possibly resulting from lower protein synthesis cordycepin 5'-triphosphate, it is known that the polyadenylation (32, 50) or musculoskeletal pain syndrome (50). step occurring during nuclear hnRNA processing is essential for nucleocytoplasmic RNA transport (58). Previously, it was re ACKNOWLEDGMENTS ported by Rose and Jacob (52) that ara-ATP inhibits the initial polyadenylation of mRNA by chromatin-associated poly(A) po We thank Dr. P. S. Agutter (Napier College, Edinburgh) for invaluable discus sions. We are indebted to R. Steifen for technical assistance. lymerase, while the rate of the poly(A) elongation reaction cata lyzed by a nuclear sap poly(A) polymerase fraction was less affected by the antibiotic. In our experiments, we found both REFERENCES poly(A) synthesis occurring in isolated, intact nuclei and poly 1. Agutter, P. S. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1984 American Association for Cancer Research. Inhibition of Nuclear Envelope Nucleoside Triphosphatase-regulated Nucleocytoplasmic Messenger RNA Translocation by 9-β-d-Arabinofuranosyladenine 5′ -Triphosphate in Rodent Cells

Heinz C. Schröder, Doris E. Nitzgen, August Bernd, et al.

Cancer Res 1984;44:3812-3819.

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