Changes in nuclear RNA transport incident to carcinogenesis

Europ. J. Cancer Vol. 13, pp. 139-147. Pergamon Press 1977. Printed in Great Britain

Changes in Nuclear RNA Transport Incident to Carcinogenesis* D. E,. S C H U M M , M. HANAUSEK-WALASZEK,~ A. YANNAKELL and T. E. WEBB

Department of Physiological Chemistgy, The Ohio State University, College of Medicine, Columbus, Ohio, 43210, U.S.A.

Abstract--Rats were treated with the hepatocarcinogens thioacetamide and dimethylnitrosamine and the release (transport) of RNA from the isolated liver nuclei to homologous or heterologous liver cytosol was evaluated in a cell-free system at various times after treatment. Within 24 hr of treatment, cytosolfrom the carcinogen-treated animals enhanced the release of RNA from liver nuclei of untreated rats. This enhanced transport capacity of the cytosol persisted up to 4 months after treatment; furthermore, the RNA transport from the Ever nuclei of carcinogen-treated animals showed a partial loss of its A TP-dependence. Although the capacity ofcytosolfrom treated animals to support RNA transport dropped be.low control levels by 9 months after treatment, the RNA transport from nuclei of the carcinogen-treated animals remainedpartially A TP-independent. This A TP-independence, which is also a characteristic of nuclei from hepatomas, is not a characteristic of the age of the animal, nor is it due to differences in the pool size of nuclear A TP or the requirementfor polyadenylation of messenger RNA for transport.

or tumor cell nuclei, on macromolecules in the cytosol [7, 8, 121; some cytosol-dependence has been observed by other investigators [11, 12]. However, other cell-free systems release their nuclear RNA to incubation media of different composition in the absence of cytoplasmic macromolecules [6, 9]. Despite the apparent differences in cytosoldependence, both types of cell-free systems exhibit an ATP-dependence of RNA release when the nuclei are derived from normal liver [6-8, 13-15]. In contrast, liver nuclei from the liver of donor animals treated with the hepatocarcinogens dimethylaminoazobenzene, acetylaminofluorene, or thioacetamide was first reported by Smuckler and coworkers [15-171 to show a significant loss of their ATP-dependence. The release of RNA from myeloma cell nuclei was also reported [10] to be ATPindependent. In order to resolve the apparent discrepancies between ATP-dependence in normal liver nuclei and independence in myeloma cell nuclei (i.e. 2 cell types of different origin) a comparison was made of the ATPdependence of RNA release from the nuclei of normal rat liver, a differentiated rat hepatoma (Hepatoma 5123D) and an undifferentiated rat hepatoma (Novikoff hepatoma). The RNA release was found [14] to be totally dependent, partially dependent and independent of ATP, respectively, suggesting that ATP-dependence

INTRODUCTION

Accom~n~G to current theory both messenger and ribosomal RNA are derived from large nuclear precursors by non-conservative processing [1,21. There is also accumulating evidence that this nuclear processing and/or nucleocytoplasmic transport of the processed RNA are under post-transcriptional controls. For example, the proportion of potential messenger, or ribosomal RNA reaching the cytoplasm varies during development [31, with growth rate [4] and upon neoplastic transformation [5]. The limitations imposed on the study of the regulation of RNA processing and transport in the intact cell, has led to the development of ceU-free systems which support these processes [6-II I. One of these systems developed in thiis laboratory to study nucleocytoplasmic controls, shows a dependence of ribosomal and messenger RNA release as ribonucleoprotein particles from either normal,

Accepted 31 August 1976. *Supported by grant CA-12411 from the National Cancer Institute, U.S.P.H.S. and a Program Development Project Support Grant from the Ohio State University Cancer Research Center. tPresent address: Institute of Oncology, Department of Tumor Biology, ~I I01 Gilwice, Poland.

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D. E. Schumm, M. Hanausek-Walaszek, A. Tannarell and T. E. Webb

is related to the degree of differentiation of the tumor. The present study analyzes the loss of ATP-dependent release of RNA from liver nuclei incident to treatment of the host animals with hepatocarcinogens, using the cytosoldependent RNA transport system; special emphasis is placed on localizing the cellular site at which ATP-independence develops and the time-course of its development. MATERIAL A N D M E T H O D S

The donors of liver tissue were 200-250-g male rats of the Sprague-Dawley strain (Laboratory) Supply Co., Indianapolis, Indiana) before (controls) or after treatment with the well known hepatocarcinogens thioacetamide (Apache Chemicals, Seward, Illinois) or dimethylnitrosamine (freshly redistilled; Eastman Kodak, Rochester, New York). The carcinogenic regimens involved a course of nine daily injections of 50mg/kg of body weight of thioacetamide [18] or a single injection of dimethylnitrosamine (5.0 mg/kg of body weight) 24 hr after partial hepatectomy [19]. Although dimethylnitrosamine in particular may produce tumors at other sites (e.g. kidney tumors), when given to partially hepatectomized rats according to the indicated protocol, it induces hepatocellular carcinomas in at least 35% of the rats [19]. The protocols selected for dimethylnitrosamine and thioacetamide, besides inducing hepatomas, are particularly suited to the present study since the treatment is of short duration, allowing one to study the irreversible effects of the drugs after cessation of treatment. The nuclear RNA was prelabeled in vivo for 30 rain with [6-14C] orotic acid (40 #Ci/250 g) prior to removal of the liver for nuclear isolation. Non-specific toxicity of the carcinogens is a minor problem in the present study since most of the measurements were made weeks or months after cessation of treatment. All injections were via the intraperitoneal route and the animals were fasted for 17 hr (overnight) prior to use in order to deplete liver glycogen. The ascites form of the rapidly growing dedifferentiated Novikoff hepatoma was carried i.p. in 140 g female rats of the Sprague-Dawley strain. After washing with 0.9% saline, the nuclear RNA of the hepatoma cells was prelabeled by incubation (10 7 cells/ml) at 37°C for 20 min in Eagles minimal essential medium (Schwartz-Mann, Orangeburg, New York) containing 2/,Ci/ml of [5,6-3H] uridine (S.A. = 39.3 Ci/m-mole). Before homogenization the cells were removed from theincubation medium,

washed once with 0.9% saline and once with 2-0 vol of the homogenization buffer. Nuclei were isolated from liver tissue by homogenization in 15vol of 2-3 M sucrose, 3.3 mM calcium acetate as previously described [7, 20]. Novikoffhepatoma cells were disrupted by suspension in one volume of homogenizing medium composed of 30 mM sucrose, 2.0 mM MgC12, 3.0 mM CaC12, 10 mM Tris-HC1 (pHS.0), 0-1% Triton X-100, 0.5mM dithiothreitol [21] followed by 20 strokes of a tight-fitting Dounce homogenizer. The nuclei were purified by layering the homogenate over 20ml of 2.0 M sucrose, 3.3 mM calcium acetate and centrifuging for 60 min at 34,000 0. After washing in 1-0 M sucrose-1.0 mM calcium acetate (300 g for 5 min), the nuclei were resuspended in the same buffer for addition to the cell-free transport system. Liver cytosol (105,000 g supernatant) which contains RNA transport factors was prepared from a 1:2 homogenate and dialyzed at 4°(3 for 18 hr against T M K buffer (50 mM TrisHC1, pH 7.5, 2"5 mM MgC12, 25 mM KC1) as previously described [7, 8]. Cytosol was similarly prepared from the Novikoff hepatoma by swelling the washed cells for 10 min at 0°C in 2 volumes of T M K buffer, centrifuging out the cells, followed by a Dounce homogenization (20 strokes with a tight pestle) of the cell pellet. The resulting homogenate was centrifuged, dialyzed against T M K buffer and frozen until use. The protein concentration of the dialyzed cytosol was determined by the Biuret method [8]. The cell-free system which was used to study the release of labeled messenger-like RNA consisted [8] of approximately 5x106 prelabeled nuclei/m1 of medium containing 12 mg dialyzed cytosol protein/ml, 50 mM Tris-HC1 (pH 7.5), 25 mM KC1, 2"SmM MgC12, 0 . 5 m M CaC12, 0.3mM MnC12, 5 . 0 m M NaC1, 2 . 5 m M Na2HPO4, 5-0mM sperimidine, 2-0mM dithiothreitol, an energy source and 300/~g/ml of yeast RNA. Where indicated the energy source, composed of 2"0 mM ATP, 2-5 mM phosphoenolpyruvate and 35 units/ml of pyruvate kinase, was omitted from the reaction. The mixture was incubated at 30°C for 30min, or for the specified time interval. During the initial 30 rain of incubation over 80 % of the labeled nuclear RNA released to the medium is messenger-like [8, 20, 22]. The amount of RNA release from the nuclei was estimated as follows. After incubation, the assay mixtures were chilled on ice, and then centrifuged at 1000 g for 10 rain to remove

Changes in Nuclear R N A

Transport Incident to Carcinogenesis

the nuclei. RNA was precipitated by the addition of 1/10 volume of 50 % trichloroacetic acid. Following a wash with cold 95 % ethanol the precipitate was dissolved in solubilizer for radioassay in liquid scintillant [20]. Alternatively when a measure of the proportion of the labeled transported RNA containing poly (A) tracts was desired, the RNA was purified from the nuclei-freed incubation medium by phenol:chloroform (1:1) extraction as previously described [22]. The RNA was then passed through a column of oligo-dT-cellulose (Collaborative Research Inc., Waltham, Mass.) to separate the poly A containing RNA, which binds to the column in 0.5 M NaC1, from the RNA lacking poly A, which does not bind~under these conditions [23].

nitrosamine treatment (Fig. lb), there is a 230% increase in the capacity of cytosol to support RNA release from normal liver nuclei during the 30-min incubation. However, in contrast to thioacetamide treatment, nuclei from dimethylnitrosamine-treated rats release considerably more RNA to either homologous, or control liver cytosol than do control nuclei, the release at 30 rain being 250% for homologous cytosol and 175 % for control cytosol. This enhanced release is not observed with partial hepatectomy alone. Thus, the nuclear (b)'

(a) 12 I-

8

RESULTS

6

Effect o f hepatocarcinogens on RaVA transport

Preliminary to studying the prolonged (irreversible) effects of the hepatocarcinogens on the energy-dependence of RNA transport, the acute effect of these agents was investigated using the cell-free system described by Schumm and Webb [8]. i[n these experiments, the livers of the carcinogen-treated rats were tested 2 4 h r after a single injection of dimethylnitrosamine [19] or the last of 9 daily injections of thioacetamide [18]. The specific dosages are given under Material and Methods. Figure 1 shows the acute effect of (a) thioaeetamide or (b) dimethylnitrosamine treatment on RNA transport. The release of RNA from normal liver nuclei to normal liver cytosol (i.e. the control) is shown for comparison; note that the transport of labeled RNA essentially cease;~ after 30 rain of incubation. The release of [14C]-RNA from the nuclei to homologous cytosol prepared from liver 24 hr after cessation of treatment with thioacetamide was depressed to within 25% of the control value after 30 rain incubation. Thioacetamide-induced liver damage is observed despite the fact that the specific activity of the nuclear RNA is equivalent in the control and treated rat.';. In contrast, the release of messenger-like RNA from normal liver nuclei is markedly enhanced by incubation in medium containing cytosol from thioacetamide-treated rats. This enhancement, which amounted to 165 % of the control value at 30 min incubation, was not observed when cytosol was prepared from an animal 2 hr after a single injection of thioacetamide (M. Hanausek-Walaszek, unpublished observations). Similarly within 24, but not 2, hr of dimethyl-

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INCUBATION TIME (MINUTES)

Fig. 1. Acute effect of hepatocardnogens on the ability of liver nuclei and cytosol to support R N A transport in a cell-free system. The nuclei were prelabeled for 30 min in vlvo with [6-a46,] orotie add and the rats were examined 24 hr after the last dose of carcinogen. In all experiments the cytosol protein was 10 mg/ml of assay and the specific activities of the nuclei from normal, thioacetamide and dimethyl nitrosaminetreated rats were 10,800, 10,120 and 8850 counts/min 5 x I0 n nuclei, respectively. The amount of labeled R N A transported is expressed as percent of total nuclear counts. (a) Normal liver nuclei plus liver eytosol from normal ( ~ [[]~) or thioacetamide-treated rats ( - - A - - ) and nuclei from thioacetamide-treated rats plus cytosol from thioacetamidetreated ( - - 0 - - ) or normal ( - - 0 - - ) rats. (b) Normal liver nuclei plus liver cytosol from normal (--[[]--) or dimethylnitrosamine-treated ( ~ / x - - ) rats and nuclei from dimethylnitrosamine-treated rats and liver cytosol from dimethylnitrosamine-treated ( - - A - - ) or normal ( - - 0 - - ) rats.

damage, which may be due to the toxicity of the carcinogens, as estimated by RNA release, is either different from that produced by thioacetamide, or it is repaired within the 24 hr period. The molecular basis for this early enhanced transport is not clear; however, the possibility that it is due to increased nuclease activity was ruled out since there was no change in the soluble counts and the addition of exogenous ribonuclease inhibitor prepared from rat liver (Searle Diagnostic, Arlington Heights, Illinois) did not decrease the amount of RNA transported. Furthermore, none of the RNA

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D. E. Schumm, M. Hanausek-Walaszek, A. Yannarell and T. E. Webb

Table 1. Temporal changes in the capacity of cytosolfrom hepatocarinogen-treated rats to support messenger RNA releasefrom nuclei of normal (control) liver Cytosol derived from liver of Thioacetamide-treated (9 daily doses, 50 mg/kg) Dimethylnitrosaminetreated (one dose, 24 hr after partial hepatectomy, 50 mg/kg)

Percentage of normal liver homologous system at 5 days 1 month 2,5 months 4 months

2 hr

1 day

9 months

82*

175

155

157

200

162

81

83

236

248

248

234

200

60

*This rat received a single dose of thioacetamide. Duplicate experiments checked to within 10% in this and subsequent experiments.

release can be attributed to nuclear leakage of the RNA, since essentially all was dependent on the presence of cytosol proteins (M. Hanausek-Walaszek, unpublished observations). The temporal changes in the transport of messenger RNA in response to thioacetamide or dimethylnitrosamine treatment are summarized in Table 1. The enhanced capacity of cytosol from the carcinogen-treated rats to support [14C]-RNA release from normal liver nuclei is observed as early as one day and persists for at least 4 months (i.e. in Table 1 the transport of RNA from normal liver nuclei of untreated or control rats to cytosol derived from the liver of carcinogen-treated rats is compared to transport from normal liver nuclei to homologous cytosol). The enhancement than falls to below control levels at approximately 9 months after the final treatment. This decline is not attributable to aging, as transport in systems derived from normal rats of equivalent age were similar to those of young adults. Thus approximately 5 % of the total nuclear counts were being transported in

systems derived from the livers of 9-month-old rats during a 30-min incubation (A. Yannarell, unpublished observations). Note that these changes, observed from several days to months after cessation of treatment, must represent permanent or semi-permanent changes in regulatory mechanisms of the cell rather than to non-specific toxic effects. Also since the animals appeared healthy and any tumors present at time of assay, when present, were extremely small and were excised before processing of the liver, the presence of tumor tissue could not account for the effects observed. It should be emphasized that the main point of these experiments is that the enhanced capacity of the cytosol from carcinogen treated rats to support RNA release persists for at least 4 months but is lost by 9 months. The data in Table 2 describe the ATPdependence of RNA transport from the liver nuclei isolated from rats 4 months and 9 months after the cessation of treatment with hepatocarcinogens. As shown previously [14], the release of RNA from normal liver nuclei is essentially ATP-dependent. In contrast, the

Table 2. A TP-dependence of RNA release in homologous and heterologous cell-free systems derivedfrom the liver of normal and hepatocarcinogen-treated rats Pretreatment of donor liver Nuclear Cytosol source

none none none thioacetamide tImoacetamide dimethylnitrosamine dimethylnitrosamine

source

none thioacetamide dimethylnitrosamine none thioacetamide none dimethylnitrosamine

Percentage of [x 4C] RNA released from nuclei* 4 months post-treatment 9 months post-treatment

+ATP

-ATP

+ATP

-ATP

4.4 7.0 9.3 3.9 6.2 3.0 8.4

0.1 4.1 3.1 2"2 4"9 1.0 6"3

4.2 3"4 3.2 -3"4 -2"5

0" I 1.8 1"5 -1"4 0"7

Duplicate experiments checked to within 10%. *The columns labeled 4 months and 9 months post-treatment refer to the duration between the cessation ot treatment w i t h the carcinogen and the removal of the liver for the preparation ot the nuclei.

Changes in Nuclear R N A Transport Incident to Cardnogenesis

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release of RNA from normal liver nuclei to the liver cytosol derived from hepatocarcinogentreated rats was significantly ATP-independent. The ATP-independence varied from 30% in systems derived from dimethylamine-treated rats to 58% in comparable systems derived from thioacetamide-treated rats, both at 4 months after treatment. From an analysis of transport in the: homologous and heterologous systems, it can be concluded that both the nuclei and cytosol contribute to this ATPindependent component. Furthermore, the loss of ATP-dependence does not appear to be fully accounted for by the enhanced capacity of the cytosol to support RNA transport since significant ATP-independent transport exists even beyond 9 months after termination of treatment despite the fact that the amount of [14C] RNA released after 30 min of incubation is significantly lower than normal. (Note that the data in Fig. 1 show the acute affects of the carcinogens on RNA transport while the data in Table 2 show the effects observed 4 or 9 months after treatment.)

cytosol from normal liver. The data in Table 3 clearly show that, in the case of the hepatoma, modification from ATP-dependence to independence which accompanies transformation resides solely in the nucleus; i.e., transport in the liver (nuclei) : liver (cytosol) and hepatoma: liver systems is 0" 1 and 3.5 % respectively, and in liver: hepatoma and hepatoma: hepatoma 0-3 and 3.1%, respectively. The existence of a pool of ATP in the nucleus of the Novikoff hepatoma cell could produce an apparent loss of ATP-dependent KNA transport. In order to eliminate this possibility, cells were incubated with 3H-uridine for 10 min, followed by further incubation after the addition of 10 m M deoxyglucose, or 2 miV[ sodium cyanide for an additional 10 min prior to cellular disruption and nuclear isolation. As indicated in Table 4, this procedure did not alter the ATP-independence of the RNA release, although both the total counts in the

Effect of neoplastic transformation on R N A transport As shown earlier [14], RNA transport in homologous sys~ems derived from the moderately differentiated hepatoma 5123D and dedifferentiated Novikoff hepatoma is partially and completely ATP-dependent, respectively. Table 3 present,; data on crossover experiments

Percentage of nuclear counts released

Table 3. A TP-&#endence of RNA release in homologous and heterologous cell-free systems derived from normal liw'r and the Novikoff hepatoma Source of

Nuclei

Cytosol

liver hepatoma liver hepatoma

liver liver hepatoma hepatoma

Percentage of nuclear RNA transported + ATP

-- ATP

4.4 4"2 3.6 3.1

0.1 3.5 0.2 3.1

The data shown are the average of triplicate experimenu; the standard errors were less than 10%. between liver and the Novikoff hepatoma designed to identity, the site of the lesion leading to ATP-independent R N A release. The hepatoma system is simpler to study than the hepatocarcinogen-treated liver system since the tmcharacterized cytosol component responsible for enhanced RNA release is not present; in fact, normal liver nuclei release less RNA to cytosol from the Novikoff hepatoma than to

Table 4. Effectof cellular A TP-depletion on subsequent RNA releasefrom Novikoff hepatoma nuclei

Pretreatment with metabolic inhibitor*

none 2-deoxyglucose cyanide cordycepin

+ ATP

- ATP

3.1 2.3 2.5 1.3

3.1 2.2 2.4 1.1

*The intact cells were pretreated with the inhibitors before isolation of the nuclei. nucleus and the percent released after 30 re.in of incubation were decreased. This decrease was predictable since the reduction of the cellular ATP pools by cyanide and deoxyglucose would tend to inhibit RNA synthesis. Under conditions employed in the present experiments (i.e. a 30-min prelabel in vivo followed by a 30-min in vitro transport) over 80 % of the labelled RNA transported resembles messenger RNA since it is released as ribonucleoprotein particles with the density of informosomes [8, 20]. Because the nuclear processing of a significant fraction of the heterogeneous nuclear (pre-messenger) RNA includes the addition of a poly (A) tract to the 3'-end of the molecule [1], it was desirable to determine whether the proportion of transported messenger containing poly (A) tracts was affected by the elimination of ATP from the cell-free system. Therefore, the proportion of labeled poly (A)-containing RNA released

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D. E. Schumm, M. Hanausek-Walaszek, A. Yannarell and 7". E. Webb

from Novikoff hepatoma nuclei to homologous, or heterologous cytosol in the presence and absence of ATP, was estimated by determining the percentage of transported RNA which bound to an oligo-dT-cellulose column [23]. The results of these experiments are shown in Table 5. Surprisingly, the proportion of transported labeled RNA containing poly (A) tracts was the same in the absence and presence of ATP in both liver and Novikoff cytosols. The fact that there is no reduction in the poly (A) content is consistent with the earlier observation [26] that interference with polyadenylate formation in vivo leads to decreased RNA transport in vitro. In agreement with this finding, it was observed that treatment of Novikoff hepatoma cells with cordycepin (3'-deoxyadenosine) caused a reduction in the percent of RNA transported but no change in the ATP-independence of the transport (Table 4). The validity of the results of the cordycepin experiments rests on the premise that cordycepin does not interfere with ATP metabolism.

DISCUSSION Although the release of RNA from cell-free systems derived from the liver of hepatocarcinogen-treated rats clearly acquires a partial but significant ATP-independent component, the results also show an enhanced RNA transport during the first 4 months after treatment and a depressed transport after 9 months. However, when taken together the experimental evidence strongly suggests that a portion, if not all, of the ATP-independent transport observed after treatment with carcinogens is due to a specific lesion in RNA processing and, or transport. There is, however, one observation, not yet explicable, between the lesions(s) which characterizes the loss of ATP-dependence of RNA transport in the liver of hepatocarcinogen-treated rats (i.e. preneoplastic liver) and the Novikoff hepatoma (neoplastic liver). In the former, both the nuclei and the cytosol appear to contribute to the phenomenon, while in the latter, the lesion is clearly locallized to the nucleus. The observed loss of ATP-dependence of RNA transport following pretreatment with liver carcinogens are in general agreement with previous studies [15-17], the results of which are discussed below. The results of the present study rule out mere leakage of RNA from the nuclei since the transport in both the liver and hepatoma systems is completely dependent on macromolecules in the cytosol. As noted above the loss of ATP-dependence

of RNA transport, as exemplified by the Novikoff hepatoma, is attributable to an alteration in the nucleus. The fact that there was no decrease in the proportions of messenger RNA containing poly(A)-tracts transported from the Novikoff hepatoma nuclei when ATP (and phosphoenolpyruvate) was omitted from the incubation medium, suggests that in tumor cells the poly(A) tracts may be added to the nuclear pre-messenger RNA much sooner after synthesis than in the normal cell, although a number of other explanations are possible. It does not appear to be due to a large nuclear pool of ATP in the tumor cells. The elucidation of this difference awaits further study. On the other hand, ATP also appears to be necessary for RNA transport through the nuclear pores of normal liver tissue both in vivo and in vitro (i.e. in the cell-free system). Thus beryllium nitrate, an inhibitor of nuclear pore phosphatase inhibits messenger RNA release from normal and neoplastic cell nuclei in

Table 5. Proportion of labeled RNA containing poly (.4) tracts released from Novikoff hepatoma nuclei to Novikoff and liver cytosol in the presence and absence of ATP Percentage of labeled RNA with poly (A) tracts ATP in incubation - -

+

Novikoff cytosol

Liver cytosol

92

19

22

18

proportion to their ATP-dependence [14]. For example, RNA transport in the cell-free systems derived from normal liver, Hepatoma 5123D and the Novikoff hepatoma were inhibited approximately 80%, 35% and 2% by 30 mg/ml of beryllium nitrate; the corresponding energy-dependence of RNA transport in these systems are 100%, 25% and 0%, respectively. Together these results suggest that the loss of energy-dependence in the tumor is due to changes at the level of the nuclear pore complex. In this regard, it is of interest that in precancerous liver, cell foci induced by diethylnitrosamine can be identifed as adenosine triphosphatase-deficient islands [27, 28]. It is tempting to speculate that this enzyme deficiency and the loss of ATP-dependent transport are related. However, the fact that the adenosine-triphosphate-deficient islands in the livers of carcinogen-treated rats account for less than 1% of the tissue mass [27] makes it unlikely that such islands alone account for the ATP-

Changes in Nuclear R.)V'A Transport Incident to Carcinogenesis independent transport observed in the hepatocarcinogen-treated livers. One explanation is that the treatment with carcinogen may lead not only to a complete deletion of adenosine triphosphatase in a few cells which constitute the loci of precursor tumor cells, but rather to a partial reductie.n in the activity of this enzyme in most of the parenchymal cells and that the latter decrease is not observed by the histochemical test. It is clear that further studies are required to determine whether the loss of this enzyme and the ATP-dependence of RNA transport are related events. Aside from the locallization of the apparent lesion in tumor cells to nuclear RNA processing and/or transport the ATP-independent transport of RNA from nuclei of the Novikoff hepatoma provides further clues concerning the site of action of ATP. Thus the fact that RNA release from Novikoff nuclei is ATPindependent whether they are incubated in normal liver or hepatoma cytosol, and conversely the ATP-dependence of RNA transport from normal liver nuclei when incubated in either cytosol, rules out the possibility that the ATP-requirement in the normal cell is related to phosphorylation of the cytoplasmic proteins required for RNA transport. The possibility that energy donors other than ATP contribute to this ATP-independent-transport is doubtful. Since the cytosol is dialyzed, such pools, which are obviously absent from normal liver nuclei would have to be sufficient to support RNA transport for 30 min and this despite the loss of nucleotides during nuclear preparation. Furthermore, regeneration of such nucleotides would require ATP. It is clear that the enhanced RNA transport characteristic of cell-free systems derived from hepatocarcinogen-treated rats is due to an increase in the capacity of the cytosol to support RNA transport. Such a change may result from an increase in the positive feed-back, or a decrease in the negative feedback transport factors, both of which have been shown to regulate messenger RNA release in the cell-free system [13]. The modified RNA transport and/or loss of ATP-dependence may account for the defective regulation of hepatic tyrosine transaminase observed after treatment of the rats with dimethylnitrosamine or thioacetamide [29]. The results of the present study relative to the development of ATP-independence of RNA

release from liver nuclei following treatment of rats with hepatocarcinogens are in general agreement with the results of Smuckler and coworkers who studied the effect of acute intoxication [16] with thioacetamide; these studies were restricted to the period zero to 72 hr after a single dose of 50 or 200 mg/kg of body weight. These workers observed an initial drop in RNA (RNP) transport and a release of significant portion of RNA from the treated livers in the absence of ATP. In a further study utilizing this protocol, they reported [17] that the acute thioacetamide intoxication was associated with the appearance of more cytoplasmic RNA's (in vivo) with migrations of 9-16S, with increases in the proportion containing poly(A) and an enhanced leakiness of the nuclei toward 9S RNA in vitro. The in vitro studies utilized in modified form the RNA transport system originally developed by Ishikawa et al. [6], which in contrast to the system used in the present study does not show cytosol-dependence of RNA release. The RNA release in the Ishikawa system is also much higher than that of Schumm et al. (i.e. over 20% el. 5%). This difference may be partially accounted for by the longer (40 rain) in vivo labeling period employed [16] which would be expected to result in the transport of considerable labeled ribosomal RNA together with labeled messenger and 4S RNA. It should be noted in this regard that the degree of conversion of heterogeneous nuclear RNA to messenger RNA in some eukaryotic cells may vary from 4 to 20 % [30]; furthermore, a large fraction of this labeled nuclear RNA is ribosomal precursor which after a 30-rain in vivo label, is not transported in our system unless the in vitro incubation is extended beyond 30 min [8]. The reason for the difference in cytosol dependence of the two cell-free systems is less clear. Recent evidence from this laboratory indicates that the cytoplasmic transport factors are very specific and in low concentration in the cytosol [22, 31]; they can not be replaced by non-specific proteins such as dialyzed normal plasma [32], or 10 mg/ml of fl-maeroglobulin or serum albumin (D. E. Schumm, unpublished observations). The system of Schumm et al. [8] would appear to be useful for the study of nucleocytoplasmic controls in normal and neoplastic cells; however, both systems have detected changes in ATP-dependence of RNA transport incident to carcinogenesis.

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J. R. OREENBERG,Messenger RNA metabolism of animal cells. J. Cell Biol. 64, 269 (1975).

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D. E. Schumm, M. Hanausek-Walaszek, A. Tannarell and 7-. E. Webb 2. 3. 4. 5. 6.

7. 8. 9. I0. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

R. A. W~.INBm~G, Nuclear RNA metabolism. Ann. Rev. Biochem. ~2, 329 (1973). L . F . JOHNSOn, H. T. ABELSON,H. GREEN and S. PEN~AI% Changes in RNA in relation to growth of the fibroblast. Cell. 1, 95 (1974). J . M . HILL, Ribosomal R_NAmetabolism during renal hypertrophy. J. Cell Biol. 64, 260 (1975). R . W . SHEARER, Specificity of chemical modification of RNA transport by liver carcinogens in the rat. Biochemistry 13~ 1764 (1974). K. ISHIKAWA, C. KURODA, M. UEEI and K. OOATA, Messenger ribonucleoprotein complexes released from rat liver nuclei by ATP. Characterization of the RNA moiety of messenger ribonucleoprotein complexes. Biochim. biophys. Acta (Amst.) 213, 495 (1970). J. RAeEVSKISand T. E. WEBB, Processing and release of ribosomal RNA from isolated nuclei: Analysis of ATP and cytosol-dependenee. Europ. or. Biochem. 49, 93 (1974). D . E . SCHUMMand T. E. WEBB, Transport of informosomes from isolated nuclei of regenerating rat liver. Biochem. biophys. Res. Commun. 4B, 1259 (1972). N . K . CHATTERJEE and H. WEISSBACH, Release of RNA from HeLa cell nuclei. Arch. biochem. Biophys. (U.S.A.) 157, 160 (1973). S.E. STUART, F. M. ROTTMANand R . J . PATTERSON,Nuclear restriction of nucleic acids in the presence of ATP. Biochem. biophys. Res. Commun. 62~ 439 (1975). M. BRUNN~Rand H. RASKAS, Processing of adenovirns RNA before release from isolated nuclei. Proc. nat. Acad. Sci. 69, 3101 (1972). D . E . Scm~tM, D. J. McNAMARA and T. E. WEBS, Cytoplasmic proteim regulating messenger RNA release from nuclei. Nature New Biol. 24k~ 201 (1973). N. HAZANand R. McCAULEY, Effect ofphenobarbitone on nucleocytoplasmic transport of RNA in vitro. Biochem. J. 1 ~ , 665 (1976). D.E. SCHU~ and T. E. WEBS, Differential effect of ATP an RNA and DNA release from nuclei of normal and neoplastic liver. Biochem. biophys. Res. Commun. 67~ 706 (1975). E.A. SMUCKLERand M. KOPLITZ,Altered nuclear KNA transport associated with carcinogen intoxication in rats. Biochem. biophys. Res. Commun. 55~ 499 (1973). E . A . SMUCX~LERand M. KOPLITZ, Thioacetamide-induced alterations in nuclear RNA transport. CancerRes. 34, 827 (1974). E . A . SMUCI~LERand M. KoPLrrz, Polyadenylic acid content and electrophoretic behavior of in vitro released RNA's in chemical carcinogenesis. CancerRes. ~ 881 (1976). K. (~. KLEINFIELD,Altered patterns of RNA metabolism in liver cells'following thioacetamide treatment. Nat. CancerInst. Monogr. 23~ 369 (1966). V . M . C~DOCK, Induction of liver tumors in rats by a single treatment with nitroso compounds after partial hepatectomy. Nature (Lond.) 245~ 386 (1973). D . E . SCHUMM,H. P. MoRms and T. E. WE~B, Cytosol-modulated transport of messenger RNA from isolated nuclei. CancerRes. 33~ 1821 (1973). W.F. MAI~LUFF, E. C. MuRPI~ and R. C. C. HwANo, Transcription of RNA in isolated mouse myeloma nuclei. Biochemistry 12~ 3440 (1973). D.E. SCHUMUand T. E. WEBS, The in vivo equivalence of a cell-free system for R_NAprocessing and transport. Biochem.~biophys. Res. Commun. 5B~ 354 (1974). H. Avlv and P. LEDER, Purification of biologically active globin mRNA by chromatography on oligo-dT cellulose. Proc. nat. Acad. Sci. 69~ 1408 (1972). C . H . Lo, F. FARII~A,H. P. MORRISand S. WEmHOUSE,Glycolytic regulation in rat liver and hepatomas. Advanc. Enzyme Regul. 6~ 453 (1968). J . L . WEBS, Enzyme and Metabolic Inhibitors. Vol. 1, p. 782. Academic Press, New York (1963). D.E. SCHUMMand T. E. WEBB, Modified messenger RNA release from isolated hepatic nuclei after inhibition of polyadenylate formation. Biochem. d. 139~ 191 (1974). E. SCHEI~R and P. EmaELOT, Kinetics of induction and growth of precancerous liver-cell loci, and liver tumor formation by diethylnitrosamine in the rat. Europ. d. Cancer 115 689 (1975).

Changes in Nuclear RNA Transport Incident to Carcinogenesis 28. 29. 30. 31. 32.

E. SCHm~ERand P. EMMELOT,Foci of altered liver cells induced by a single dose of diethylnitrosamine and partial hepatectomy: Their contribution to hepatocarcinogenesis in the rat. EuroIJ..1".Cancer11, 145 (1975). M. HANAUSEK-WALASZ~K,D. E. S c ~ and T. E. WEBB, The repression and derepression of hepatic tyroslne transamlnase by carcinogens. Chem. biol. Interact. 12, 391 (1976). J. LENOYELand S. PENmA.N,HnRNA size and processing as related to different DNA content in two dipterans: Drosophila and Aedes. Cell 5, 281 (1975). A.Y.~NARELL, D. E. SCHUM~ and T. E. WEBB, Nature of facilitated messenger RNA transport from isolated nuclei. Biochem.J. 154, 379 (1976). D . E . SCHUM~tand T. E. W~BB, Differential effect of plasma fractions from normal and tumor-bearing rats on nuclear RNA restriction. Nature (Lond.) 256, 508 (1975).

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