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(CANCER RESEARCH 33, 1796-1803, July 1973] Ribosomes of Mouse Liver following Administration of Dimethylnitrosamine1 Gary M. Williams2 and Tore Hultin Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden \T. //.], and Department of Pathologv and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 [G. M. W.\ SUMMARY Administration of dimethylnitrosamine (DMNA), 50 mg/kg, to mice for 2 hr resulted in a 60% inhibition of hepatic protein synthesis. This effect was accompanied by a disaggregation of both free and membrane-bound polysomes. The increased monomers in the membrane-bound fraction remained associated with the membranes. Isolated total monomers from DMNA-treated mice were equivalent to control monomers in their utilization of the synthetic template polyuridylic acid. Also, isolated polysomes from both groups had the same endogenous amino acid-incor porating activity. The lack of a direct action of DMNA on the state of aggregation of polysomes was demonstrated by the finding that cycloheximide, which arrests the readout of polysomes, largely protected the polysomes from disaggre gation by DMNA. damage is apparent from the finding that microsomal RNA (41) and rRNA (22, 28) is highly methylated by DMNA. Nevertheless, the functional activity of the generated mon omers has not been tested. This paper reports that isolated ribosome monomers from DMNA-treated mouse liver are normal as regards their activity in translating a synthetic messenger. Further more, the ribosomes that are membrane bound remain attached after disaggregation to monomers. Isolated poly somes were also found to be functionally normal following treatment, and the possibility of a direct destruction of mRNA on polysomes was further rendered unlikely by the finding that arrest of polysome readout by cycloheximide partially protected against the effect of DMNA. MATERIALS AND METHODS Materials INTRODUCTION DMNA3 will induce liver tumors in a variety of species and, as with many other hepatocarcinogens, toxic doses cause a marked inhibition of hepatic protein synthesis (18). Although a number of disturbances in the protein synthetic apparatus have been found following DMNA treatment, the primary critical lesion has not been identified (19). Adminis tration of DMNA results in a rapid disorganization of the endoplasmic reticulum (3, 9, 11, 26) and a disaggregation of polysomes (25), which parallels the disruption of protein synthesis (41). The in vitro amino acid-incorporating activ ity of microsomes isolated from treated liver is depressed (8, 12) and unfractionated ribosomes (25) and microsomes (20) have been found to be more responsive to stimulation with synthetic messenger. These latter results were interpreted as reflecting a loss of mRNA from polysomes due to attack on the mRNA (20, 25). However, the potential for ribosomal 1This work was supported by the research grants from the Swedish Cancer Society and the Swedish Natural Science Research Council. 2The work was performed during the tenure of a Research Training Fellowship awarded by the International Agency for Research on Cancer. 3The abbreviations used are: DMNA, dimethylnitrosamine; poly U, polyuridylic acid; HM, homogenizing medium; LM, light medium; TCA, trichloroacetic acid; PMS, postmitochondrial supernatant; DOC, sodium deoxycholate. Received February 28, 1973; accepted April 16, 1973. 1796 DMNA was obtained from Eastman Kodak Co., Roches ter, N. Y. It was purified by distillation and dissolved in 0.9% NaCl solution on the day of use. The sources of other chemicals were: cycloheximide, Sigma Chemical Co., St. Louis, Mo.; poly U, Miles Chemical Co., Kankakee, 111.; Soluene 100, Packard Instrument Co., Downers Grove, 111.; DL-leucine-I4C (34 Ci/mole), DL-phenylalanine-14C (48 Ci/ mole), and 14C-labeled orotic acid (61 Ci/mole), Radiochemical Centre, Amersham, England. Buffer A: 25 mM KC1:5 mM MgCl2:35 mM Tris-HCl (pH 7.7 at 25°).Buffer B: 50 mM KC1:5 mM MgCl2:35 mM Tris-HCl. HM: 0.25 M sucrose in Buffer A. LM: 0.15 Msucrose in Buffer A. Animals Female white mice of the Naval Medical Institute strain, weighing 25 to 30 g, were fasted overnight before treatment. In the early morning, experimental animals were given i.p. injections of DMNA, 50 mg/kg body weight, and controls were treated with an equal volume of 0.9% NaCl solution. Sacrifice was by decapitation and the gallbladders were removed before the livers were extirpated and chilled in LM. Except in the study of in vivo leucine incorporation, the livers of 5 animals were pooled for each experimental point. CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1973 American Association for Cancer Research. VOL. 33 DMNA-treated Mouse Liver Ribosomes sedimented through layers of 1.38 M and 2.0 M sucrose, while membranous material is trapped at the interface of the sucrose layers. It has been shown (31) and confirmed in this work (Table 4) that following the 1st centrifugation for sedimentation of free ribosomes some free monomers remain in the overlying 2.0 M sucrose cushion. Therefore, this cushion was removed, diluted, and layered over a new cushion of 2.0 Msucrose in Buffer A placed on the pellet of free ribosomes. The tube was oriented in the centrifuge with the pellet outward and centrifuged for 20 hr at increased centrifugal force (50,000 rpm, Spinco rotor 50 Ti). The membrane-bound ribosomes were liberated by homogenization in 1% DOC and centrifuged along with the free ribosomes through a 2.0 Msucrose cushion. The efficiency of the separation was tested in the following way, using isolated, l4C-labeled free monomers as markers. Mice were given i.p. injections of 4 ¿tCi14Clabeled orotic acid. After 2 days, the food was removed, and they were fasted for an additional 2 days to increase the monomer content in the liver. Monomers were collected and pelleted as described. The pellet was suspended in LM and Preparation and Fractionation of Ribosomes added as part of the medium used in the homogenization of normal liver. Free and membrane-bound ribosomes were Total Ribosomes. Minced livers were homogenized in 2.5 separated according to the procedure for obtaining sedimen volumes of HM in a glass homogenizer fitted with a tation profiles. After the initial 24-hr centrifugation, the motor-drived Teflon pestle. The PMS was prepared by distribution of radioactivity in the gradient was determined centrifuging the homogenate for 7 min at 15,000 x g. It was by pipetting alternate 0.1-ml samples of the gradient as well added to one-ninth volume of a 10% DOC solution and as aliquots of the PMS and suspended pellet onto filters. gently mixed on ice. In a modification of the method of The filters were placed in ice-cold 10% TCA; washed twice Munro et al. (27), the ribosomes were sedimented through a 5.0-ml cushion of 1.0 M sucrose in Buffer A by centrifuga- in cold 5% TCA; and dehydrated in cold and then warm (2:1:1), and tion for 2.5 hr at 50,000 rpm in a Spinco 50 Ti rotor at 4°. absolute ethanol, ethanol:ether:chloroform ether. Radioactivity was counted as above. The A260:A280 ratio of the ribosomes was always around 1.4. Monomers. DOC-treated PMS, 1.7 ml, was applied to a Sedimentation Analysis 35-ml 15 to 40% sucrose gradient in Buffer A. This was centrifuged for 4 hr at 27,000 rpm in a Spinco SW27 rotor. Samples were suspended in Buffer A containing 5% The gradient was monitored by displacement with heavy mouse liver cell sap and applied to a 5-ml 15 to 55% linear sucrose solution through a Beckman Model DK-2 recording sucrose gradient in Buffer A. Gradients were centrifuged in spectrophotometer provided with a LKB 4712A-4 flow cell, a Spinco SW 50.1 rotor for 65 min at 47,500 rpm (4°). and the monomer peak was collected. The monomer Sedimentation profiles were recorded at 260 nm as de fraction was layered on 3.5 ml of 1.5 Msucrose Buffer B and scribed. centrifuged for 16 hr at 40,000 rpm in a Spinco 40 rotor. The A260:A280ratio was constantly around 1.4. Polysomes. The sample and gradient were identical to Isolation of Free and Total Ribosomes that used for monosomes, but the gradient was centrifuged Isolation was performed by the method of Blobel and for only 2 hr. The lighter portion of the polysome region was composed mainly of smaller aggregates with low amino Potter (5) in which minced livers were homogenized in H M acid-incorporating activity due, at least in part, to a with 15 strokes to produce full liberation of free ribosomes. substantial content of monomers. Therefore, only the These were isolated from the PMS by sedimentation medium and heavy fractions, which had comparable activi through layers of 0.5 M and 2.0 M sucrose in Buffer A, and ties, as Wettstein et al. (45) have found, were studied. These total ribosomes were pelleted in the same manner from fractions were sedimented as for the monomers. The PMS made to 1% DOC. A2io:A280 ratio was always about 1.4. In vivo Incorporation of Leucine Mice were given i.p. injections of 8 /¿CiDi.-leucine-14C per 100 g body weight and were sacrificed 15 min later. Livers were homogenized in 30 volumes of LM and replicate 1.0-ml aliquots were made to 10% TCA; Celile, 10 mg/ml; and nonradioactive leucine, 1.5 mg/ml. After heating at 90° for 30 min, the precipitated protein was collected by filtration on Munktell 20/150 paper discs overlayered with 10 mg Celite. The precipitates were washed with 15 ml 5% TCA and dehydrated by successive 10-ml washes with 2-propanol, 2-propanol: ether (1:1), and ether. The filters were placed in counting vials, and the precipitates were allowed to dissolve in 0.5 ml Soluene for 2 hr. Counting was performed in a Packard scintillation counter at 55% effi ciency in 10 ml of scintillation fluid composed of 0.5% PRO and 0.015% POPOP in toluene. The protein content of the homogenate was measured by the method of Lowry et al. (17) for the calculation of specific activity of proteins. Replicates agreed within 10%. RNA Analyses Separation of Free and Membrane-bound Ribosomes for Sedimentation Analysis In studies in which some samples might contain DNA, the method of Scott et al. (34) was used throughout. In the Separation was performed by a modification of the case of ribosomes in which no other fractions were analyzed, method of Blobel and Potter (7) in which free ribosomes are RNA was measured by a modification of the method of JULY 1973 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1973 American Association for Cancer Research. 1797 Gary M. Williams and Tore HuitÃ-n Ogur and Rosen (29) as previously described (13). The values obtained by the 2 methods were closely similar. Table I In vivo incorporation of leucine- "C into protein Treatment In Vitro Amino Acid Incorporation cpm/mg protein Control (3)"DM hr(2)DMNA, N A, 50 mg/kg, l 50 mg/kg, 2 hr (4)697 inhibition 145*388 ± 119275 ± ±1344461 Cell sap was prepared by homogenizing in 2 volumes of LM the livers of mice fasted overnight and centrifuging the PMS for 90 min at 50,000 rpm in a Spinco 50 Ti rotor. The " Numbers in parentheses, number of animals. supernatant was freed of amino acids by passage through a ' Mean ±S.E. column of Sephadex G-25 (42) and stored frozen at -20° until use. Table 2 The incubation mixture contained, in 0.15 ml: 20 ^1 In vitro amino acid-incorporating activity of total ribosomes ribosome suspension in LM (15 A260 units; 10 to 20 /ig RNA); 75 n\ cell sap (about 1.5 mg protein); 1 mM ATP; cpm/IO/ig 0.25 mM GTP; 10 mM phosphoenolpyruvate; pyruvate RNA° % inhibition kinase, 40 ¿tg/ml;90 mM KC1; 19 amino acids excluding phenylalanine (42); and DL-phenylalanine-14C, 0.67 ¿uCi/ml. Control 71 DMNA, 50 mg/kg, 2 hr 32 55 MgCl2 was added in concentrations ranging from 6 to 18 mM. Incubation was performed in a shaking water bath for 5 'At 7 mM MgCl2. min at 35°and was stopped by placing the samples on ice. Aliquots of 0.1 ml were immediately pipetted onto filter papers and processed by the method of Mans and Novelli (21). Radioactivity was counted as above. One incubation 500 was performed in each experiment without ribosomes added. This blank had a constantly low isotope content that was identical to nonincubated complete mixtures and was subtracted from the counts of the experimental samples. Incorporation was linear for 8 to 10 min and proportional to 400 added ribosomes up to 3 times the amount normally used. The assay conditions were established to allow maximal incorporation in the presence of poly U. The ratio of mg protein cell sap to mg ribosomal RNA was greater than 100:1 (27). Poly U stimulation was maximal at a concentra g 300 tion of 900 Mg/m'- Higher concentrations did not depress ir incorporation and so 1200 ng/m\ was rountinely used to S. assure saturation. i o 200 RESULTS Effect of DMNA on Protein Synthesis. Treatment with DMNA at a dose of 50 mg/kg produced a large depression of in vivo hepatic leucine incorporation after 1to 2 hr (Table 1). In order to study the functional activity of ribosomes at a point where protein synthesis was severely inhibited, we performed the experiments reported below using a 2-hr interval of treatment at this dosage, except where noted. This period of treatment was felt to be sufficiently short to consider our observations to reflect the status of the elements studied during the evolution of inhibition. Total ribosomes isolated from the DMNA-treated mice had a correspondingly reduced activity of endogenous in vitro amino acid incorporation (Table 2). Optimal incorpo ration for both types of ribosomes was found at 6 to 8 mM MgCl2. Similar to what others have found (20, 25), the treated ribosomes responded to the addition of poly U with a greater increment in incorporation than control ribosomes (Chart 1). In addition, the Mg2+ optimum was shifted from about 10 mM for the controls to about 11 to 12 mM for the 1798 CL O 100 9 10 11 Mg2+ CONCENTRATION (mM) 12 13 Chart I. In vitro poly U-stimulated polyphenylalanine synthesis of total ribosomes from control and DMNA-treated mouse liver. treated animals. Dialyzed cell sap or pH 5 enzymes (14) from DMNA-treated animals yielded the same or slightly better incorporation per mg protein as control material. Effects of DMNA on Free and Membrane-bound Ribosomes. DMNA is activated by a microsomal enzyme system (19), and it might therefore be suspected that CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1973 American Association for Cancer Research. DMNA-treated Mouse Liver Ribosomes membrane-associated polysomes would be more severely affected by reactive metabolites than their free counter parts. This possibility was not substantiated by the finding that the sedimentation profiles of both free and membranebound ribosomes contained greatly increased peaks of monomers following DMNA treatment (Chart 2). DMNA treatment has been reported to cause a detachment of ribosomes from the endoplasmic reticulum (3, 9). If this were to occur under the present conditions, then this effect should be reflected by an increase in RNA in the free fraction and a corresponding reduction in the content of the membrane-bound fraction. Analysis of the RNA in these fractions as separated for sedimentation profiles revealed no such alteration following treatment (Table 3, Method 1). Since the total recovery by this method seemed low, an alternate method for isolation of free ribosomes (5) was also tested. In this experiment, the recovery of free ribosomes as a percentage of the PMS was slightly greater, and again there was no increase in RNA in the free fraction after DMNA (Table 3, Method 2). In the 2 above methods for the separation of free ribosomes, these particles pass through the rapidly ac cumulating membrane fraction during sedimentation. Some evidence has been presented in support of the view that monomers recovered from the membrane fraction represent essentially free particles that have not been completely separated during centrifugation (40). Since no shift of RNA from the membrane-bound to the free ribosome fraction was observed during the DMNA treatment (Table 3), the identity of the monomers in the membrane-bound fraction was further investigated. For this purpose the distribution of added free monomers, prelabeled with I4C-labeled orotic acid, was studied in the gradient used for separating free and membrane-bound ribosomes. Less than 5% of the added radioactive monomers were retained in the membranebound fraction even when material below the interface was included to assure recovery of all membranous material (Table 4). This small proportion, which may include heavy subunits, is insufficient to account for the amount of monomers found in the membrane-bound fraction and indicates that these particles do not represent entirely an artifact of separation. An interesting incidental finding was the extensive trapping of monomers in the 2 M sucrose cushion overlying the free pellet. Because of this observation BOUND the procedure for separating free and membrane-bound ribosomes was modified to give more complete sedimenta tion of the free particles (cf. Chart 2 and Table 3). The distribution experiment was also performed with labeled total free ribosomes in which 7% of the counts were as monomers. In this case, only 0.6% of the counts were retained in the membrane-bound fraction. It was felt that, if monomers were attached to endoplasmic membranes as it seemed, these monomers should be released after lysis of the membranes by DOC, thereby resulting in a larger monomer peak than would be present in the same material not treated with DOC (43). Therefore, an aliquot of PMS was diluted with the same volume of LM as that of DOC used on another aliquot. Sedimentation profiles revealed in contrast to the finding of Webb (43) that DOC treatment resulted in a considerable increase in the monomer peak size (Chart 3), Chart 2. Sedimentation analysis of separated free and membrane- consistent with the release of monomers from membranes. bound ribosomes from control and DMNA-treated mice. The PMS from DMNA-treated mice contained a larger peak of free monomers than the control, and the magnitude Table 3 of the increment following DOC treatment was greater than RNA in fractions of rat liver that observed with control PMS. This latter result could Table 4 inPMS Distribution of "C-labeled free monomers on gradient after separation of brane-bound0.620.700.58'0.81'Totalrecovered2.32C2.25e1.841.93 sepa ongradient4.233.962.382.54Totalfree1.771.551.261.12Free as%PMS42.039.253.044.0Totalmem free and bound ribosomes ration"1"yTreatmentControlDMNAControlDMNATotal Methodof total cpm inPMS1004.844.243.194.0 A.B.C.D.E.F.G.Total gradientAbove in PMS placed on sucroselayersBetween interface of 2 M " Average of 2 experiments of each kind. * Analyses performed on fractions obtained during separation for sedimentation profiles (cf. Chart 2). "Sum of free plus membrane bound. " Isolation of free and total ribosomes in PMS according to the method of Blobel and Potter (5). ' Calculated as difference between total recovered and free. JULY visibleband interface and last ml)Totalof material (0.8 fraction(B in membrane-bound C)Remainder + sucroseFree of 2 M pelletTotal recovered (D + E + F)cpm534016888256246123005017% 1973 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1973 American Association for Cancer Research. 1799 Gary M. Williams and Tore Hull in 80S TOP BOTTOM Chart 3. Size of 80 S monomer peak in control (A) and DMNAtreated (B) PMS with and without DOC treatment. In the samples not treated with DOC, the absorbance curve in the polysome region indicates light scattering due to the presence of membranes. somes (16, 44) and the polysome profile remains fixed (10). This effect has been used to probe the susceptibility of such arrested polysomes to direct attack by other inhibitors of protein synthesis (1, 10, 31, 37, 38). The mouse is less sensitive than the rat to cycloheximide (38), but administra tion of 100 mg/kg body weight i.p. for 1 hr followed by a 2nd injection of 25 mg/kg resulted in an 86% inhibition of in vivo incorporation of leucine. The incomplete inhibition of protein synthesis was reflected by an increase in the monomer peak after cycloheximide alone (Chart 5, cf. Chart 2). Under these conditions, when DMNA, 50 mg/kg, was given with the 2nd dose of cycloheximide, 25 mg/kg, for 1 hr there was a definite reduction in the amount of polysomes disaggregated to monomers by DMNA (Chart 5). In the Sprague-Dawley rat, cycloheximide, 2 mg/kg, given for 30 min produced a 76% inhibition in in vivo leucine incorporation. The effect of a subsequent injection of DMNA, 200 mg/kg, for 40 min on polysome disaggregation was markedly diminished (Chart 5). In both the mouse 600 reflect a larger component of monomers attached to membranes after DMNA treatment and strengthens the < interpretation that monomers are indeed bound to mem tr branes. Fractions corresponding to the monomer peaks 400 were collected, and the A320was obtained to determine the content of ferritin (27). This was minor and did not differ for O any of the monomer peaks. Therefore, the A26owas not OÕT> 00 corrected. Functional Activity of Isolated Monomers and Polysomes. Isolated monomer fractions from livers of control and DMNA-treated mice were only slightly contaminated by o ce heavy subunits and dimers, as indicated by the sedimenta 200 tion profiles (not illustrated). They had very little or no endogenous amino acid-incorporating activity. Poly U was CL utilized equally well by both types of monomers as a O template for the synthesis of polyphenylalanine (Chart 4), and the stimulated activity of the monomers was greater than that of total ribosomes (cf. Chart 1). The Mg2+ dependence of the reaction was the same for both types of monomers with the optimum incorporation occurring at a Mg2+ concentration of 11 to 12 mM. 1214 16 10 18 The phenylalanine-incorporating activity of isolated polyMg2+ CONCENTRATION (mM) somes was measured over a MgCl2 concentration range of 6 Chart 4. ¡nvitro poly U-stimulated polyphenylalanine synthesis of to 12 mM. Optimum endogenous incorporation for both isolated monomers from control (O) and DMNA-treated (•)mouse liver. control and DMNA polysomes occurred at 9 to 10 mM Mg2+. The peak activity of the DMNA polysomes did not Table 5 differ from that of controls (Table 5). The addition of poly In vitro amino acid-incorporating activity of isolated polysomes at ¡0mM U did not result in very much increase in incorporation i and average of valuesfor medium and heavy fractions of polysomes apparently because of the absence of monomers for utiliza tion of the template. Consequently, the Mg2+ optimum was cpm/lOfigRNA not raised in the presence of poly U. Effect of DMNA after Cycloheximide Treatment. Cypoly U95 cloheximide reduces the rate of polysome readout (44) Control through inhibition of elongation (2, 15, 16). This results in DMNAEndogenous54 51+ 86 an equilibrium shift toward a high concentration of poly 1800 CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1973 American Association for Cancer Research. DMNA-treated MOUSE Chart 5. Sedimentation analysis of mouse and rat ribosomes after treatment with DMNA, cycloheximide (cyclo) plus DMNA, and cycloheximide. and the rat, cycloheximide produced a shift to heavier polysomes. This is regularly observed with cycloheximide (15). DISCUSSION In the above experiments the interval of DMNA treat ment was kept short in the hope that alterations giving rise to the inhibition of protein synthesis could be detected. It was found that disaggregation of polysomes occurred without detachment of the monomers from endoplasmic membranes, as determined in 2 different ways. In DMNAtreated animals, the association of ribosomes with endoplas mic membranes has been previously studied only with the electron microscope. Some observers have not described a decrease in the ribosome content of the endoplasmic reticulum (26) while others have observed degranulation (3, 9). The latter studies (3, 9) were performed after longer intervals of treatment than the present and it may be that degranulation is a subsequent event to disaggregation. There is considerable evidence that monomers remain adherent to membranes after in vitro runoff (6) and treatment with a variety of agents that result in polysome disaggregation (4, 6, 32, 33). Thus, the present results are in agreement with these other observations, indicating that there is a binding of monomers to endoplasmic membranes that is not dependent upon polysome integrity. The inhibition of protein synthesis by DMNA was quantitatively reproduced in vitro by total ribosomes from treated animals. Previous reports that the cell sap factors involved in protein synthesis are normal after DMNA (8, 12, 20) were confirmed. The reduced activity of treated ribosomes was accompanied by an increased responsiveness to stimulation with poly U as others have described (20, 25). In addition, we further documented that the maximal stimulated incorporation with poly U occurred at a higher Mouse Liver Ribosomes Mg2+ concentration for the DMNA-treated ribosomes (11 to 12 mM) than for control ribosomes (9 to 10 mM). In view of the fact that both control and DMNA-treated isolated monomers had a Mg2+ optimum of 11 to 12 mM for poly U-stimulated incorporation, we believe that the shift in Mg2+ optimum of DMNA-treated total ribosomes may be due to more effective utilization of poly U at the higher Mg2+ concentration by the greater proportion of monomers present. A similar increase in the Mg2+ optimum for poly U-stimulated incorporation has been previously reported for ribosomes from livers of animals exposed to hepatotoxins (36) and the interpretation was offered that this effect may be due to an altered capacity of the treated ribosomes to bind bivalent cations (35, 36). The monomers generated under these conditions have not been directly tested as in the present study. Vernie et al. (39) have recently shown that membrane-bound but not free polyribosomes from DMNAtreated liver have an altered Mg2+ plus Ca2+ dependence for endogenous incorporation. The significance of this effect has not yet been determined. The isolated monomers from DMNA-treated livers were functionally as active in utilizing poly U as control mono mers. Bearing in mind that utilization of a synthetic tem plate is not entirely analogous to translation of an endog enous messenger, it is concluded that the generated mono mers are normal as regards this function. This finding com plements the observations that the stability of that of rRNA is not decreased after methylation in vivo by DMNA (22) and that DMNA-generated monomers and those run off during in vitro protein synthesis are similar in their sedimentation characteristics in low Mg2+ concen tration (40). Isolated polysomes were contaminated by monomers to a very slight degree as reflected by the small increment in incorporation in the presence of poly U. This indicates that the 16 hr of centrifugation did not lead to extensive runoff of monomers. The polysomes from DMNA-treated livers repeatedly showed activity comparable to controls, which is in contrast to the finding of Mizrahi and DeVries (25) that treated polysomes are less active. This reduced activity was related by them to polysome instability (25). The same laboratory has subsequently suggested from less direct studies that reduced activity might be due to smaller aggregate size of polysomes from DMNA-treated liver (40). This seems unlikely from our result, in agreement with Wettstein et al. (45), that the in vitro activity of aggregates is constant for fractions from which monomers have been excluded. Our finding indicates, therefore, no impairment of the activity of the ribosomes on engaged mRN A at the time when protein synthesis was markedly inhibited. This conclu sion is strengthened by the further finding of a partial protection by cycloheximide on the DMNA-induced break down of polysomes. This observation is similar to that recently reported by Stewart (37) and implies that the effect of DMNA does not occur entirely through a direct attack on the connecting mRN A strand. The duration of cyclohex imide inhibition of protein synthesis was too short to attribute its protective effect to a reduction of DMNA metabolism and indeed Stewart (37) has shown that there is JULY 1973 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1973 American Association for Cancer Research. 1801 Gary M. Williams and Tore HuitÃ-n Liver Cells after Dimethylnitrosamine, 2-Fluorenamine, or Prednisono change even after longer intervals of cycloheximide lone Treatment Studied by Electron Microscopy. J. Nail. Cancer treatment. The lack of complete protection is undoubtedly Inst., 30: 1045 1075, 1963. due, at least in part, to the fact that in vivo protein synthesis 12. HuitÃ-n,T., Arrhenius, E., Low, H., and Magee, P. N. Toxic Liver was not entirely inhibited by cycloheximide. These results Injury. Inhibition by Dimethylnitrosamine of Incorporation of La with polysomes do not support the earlier suggestions of a belled Amino Acids into Proteins of Rat-Liver Preparations in Vitro. DMNA-induced lesion in polysomal mRNA (20, 24, 25). Biochem. J. 76: 109 116, 1960. Since the above experiments revealed no defect in the 13. HuitÃ-n,T., Naslund, P. H., and Nilsson, M. O. Dissociation of translational capacity of DMNA-treated monomers or Ribosomes from Dormant Cysts of Anemia salina in Potassium-free polysomes, we are led to suggest that the critical lesion may Media. Exptl. Cell Res., 55: 269 274, 1969. be in the process of initiation of protein synthesis. A 14. Keller, E. B., and Zamecnik, P. C. The Effect of Guanosine Diphosphate and Triphosphate on the Incorporation of Labelled possibility not precluded by the present studies of isolated Amino Acids into Proteins. J. Biol. Chem., 221: 45 49, 1956. monomers is a defect of these particles in reacting with the normal initiation codon, AUG. Furthermore, since the 15. Kisilevsky, R. The Regulatory Parameter of Protein Synthesis Most Affected by Ethionine and Cycloheximide. A Comparison of Compu utilization of poly U as a template at high Mg2+ concentra ter and in VivoStudies. Biochim. Biophys. Acta, 272:463-472, 1972. tion does not require the ribosome-associated protein 16. Lodish, H. F., Housman, D., and Jacobsen, M. Initiation of Hemo synthesis initiation factors (23, 30), another possibility is globin Synthesis. Specific Inhibition by Antibiotics and Bacteriophage that these factors could be impaired or deficient. On the Ribonucleic Acid. Biochemistry, 10: 2348-2356, 1971. other hand, evidence has been found that DMNA methy- 17. Lowry. O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. lates nRNA and it has been suggested, but not demon Protein Measurements with the Folin Phenol Reagent. J. Biol. Chem., 193: 265 275, 1951. strated, that inactivation of mRNA may be involved (41). The present findings of no direct effect of DMNA on 18. Magee, P. H. Toxic Liver Injury. Inhibition of Protein Synthesis in Rat Liver by Dimethylnitrosamine in Vivo. Biochem. J., 71: 606 611, engaged mRNA indicate that the defect in mRNA would 1958. have to be an impairment of stable or newly synthesized 19. Magee, P. N., and Swann, P. F. Nitroso Compounds. Brit. Med. Bull. mRNA to initiate new peptide chains. ACKNOWLEDGMENTS The authors gratefully acknowledge the excellent technical assistance of Birgit Lundberg. REFERENCES 1. Alpers, D. H., and Isselbacher, K. J. Biochemical Effects of CC14on Rat Intestinal Mucosa. Biochim. Biophys. Acta, 158:4\4 424, 1968. 2. Baliga. B. S., Pronczuk, A. W., and Munro, H. N. Mechanism of Cycloheximide Inhibition of Protein Synthesis in a Cell Free System Prepared from Rat Liver. J. Biol. Chem., 244: 4480 4489, 1969. 3. Benedetti. E. L., and Emmelot. P. 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Nature. 197. 430 435, 1963. 1973 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1973 American Association for Cancer Research. 1803 Ribosomes of Mouse Liver following Administration of Dimethylnitrosamine Gary M. Williams and Tore Hultin Cancer Res 1973;33:1796-1803. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/33/7/1796 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1973 American Association for Cancer Research.