The Extraction of Intracisternal A-Particles from a Mouse Plasma-Cell Tumor1

(CANCER RESEARCH 28, 2137-2148,October1968]

Edward 1.Kuff, Nelson A. Wivel, and Kira K. Lueders Laboratory of Biochemistry and Viral Leukemia and Lymphoma Branch, National Cancer lnstitute, NIH, Department of Health, Education, and Welfare, USPHS, Bethesda, Maryland ?A%114

SUMMARY 29, A. J. Dalton, M. Potter, and H. B. Andervont, personal communication). Plasma-cell tumors in BALB/c mice contain numerous in The other type, with which the present study deals, has been tracistemnal A-particles which remain localized within micro found in myeloma lines arising both in C3H mice (carrying somal vesicles when the tumor cells are disrupted by homoge the mammary tumor agent) and in mice of the agent-free nization. Liberation of the particles has been achieved by sub BALB/c strain. Measuring between 70 and 100 m@iin diameter, jecting microsome suspensions to mechanical shear in the pres these particles consist of two concentric shells surrounding a ence of an optimal concentration of Triton X-100. The particles relatively electron-lucent core and are characterized by an cx were concentrated by two cycles of sedimentation in sucrose clusive localization within the cistemnae of the endoplasmic potassium citrate solutions, pH 72, and finally banded iso reticulum of the tumor cells. They appear to form by budding pycnically in a sucrose density gradient containing dilute at the reticulum membranes and may be very numerous in th,@ potassium citrate. Most of the particles were recovered in a neoplastic plasma cells. Particles of similar morphology and density range of 1.20.-i .24 gm/cu cm. intracellular localization have been described in a number of A-particles extracted by this procedure retained their char other malignant cell types (4, 6, 7, 11, 14, 17, 35) . They are acteristic inner and outer shells. Electron microscopy of the referred to as â€œintmacisternal A-particlesâ€• in a recently pro gradient-isolated fractions revealed some residual contamina posed classification of oncogenic RNA viruses (30) ; however, tion of the A-particles with microsomal membranes. The iso their inclusion in this scheme rests at present on morphologic lated material consisted of about 80% protein, 14% phospho grounds rather than upon any direct evidence that the particles lipid (other lipids not studied) and 5 to 6% RNA. No DNA contain nucleic acid and represent viruses. There is, in fact, a was detected. Deoxycholate treatment, which lysed the con complete lack of information regarding both their biochemical taminating membranes and simultaneously stripped the A-parti properties and their functional significance. des of their outer shells, sharply reduced the phospholipid In the present study, we have undertaken the extraction of content of the fraction but did not remove RNA. Evidence intracistemnal A-particles from a BALB/c plasma-cell tumor is presented that 40 to 50% of the total RNA in the isolated line. The results demonstrate the feasibility of isolating the fractions was contributed by other cytoplasmic components. particles in quantities sufficient for biochemical analysis. The remainder may have represented RNA intrinsic to the A-particles themselves ; however, further studies are required MATERIALSAND METHODS to establish this point. The MOPC-104E plasma-cell tumor line (23) was used cx INThODUCTION clusively in this work. Subcutaneous tumors, generally weighing between 1 and 2.5 gm each, were harvested 16 to 18 days Electron microscopy has revealed two types of â€œvirus-likeâ€• following transplantation in female BALB/c mice. The excised particles in murine plasma-cell tumors (5) . One is morpho tissue was quickly chilled to 0Â°C, minced, and homogenized logically indistinguishable from the intracytoplasmic A-particle in 4 volumes of ice-cold Solution A consisting of 0.25 si sucrose, classically associated with mouse mammary tumors (1 ) . These 0.05 M Tris Cl, pH 7.6, 0.025M KC1,and 0.005 @rMgCl2 particles are found dispersed singly or in clusters within the (19). All subsequent procedures were carried out between O@ cytoplasmic matrix of the tumor cells and are not obviously and 5Â°C. Nuclei and whole cells were removed by centrifuga associated with any intracellular organelle. They are apparently tion of the homogenates for 10 minutes at 700 x g. The nuclear confined to plasma-cell tumors that have arisen or been trans supernatant fractions were then centrifuged either for 10 mm planted in mice carrying the mammary tumor agent (5, 15, utes at 11,500 rpm (10,000 X g) in the No. 296 angle rotor of the International PR2 centrifuge or, in the case of large

1 A preliminary report of this work was presented at the 51st scale preparations, for 20 mm at 10,500 rpm (13,000 x g) in Annual Meeting of the American Society for Experimental Pa the No. 856 rotor. The pellets from this centrifugation were thology (20). resuspended with vigorous hand homogenization in fresh So Received March 14, 1968; accepted June 20, 1968. lution A to a final volume equivalent to one half the original

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1968 American Association for Cancer Research. Edward L. Kuff, Nelson A. Wivel, and Kira K. Lueders wet weight of tissue. These preparations, referred to as â€œmem in a mixture of 0.5% aqueous uranyl acetate and 0.54% su brane fractions,â€•contained the bulk of the mitochondria and crose, pH 5.0. The specimens were dehydrated in graded alco microsomes, and, as will be illustrated later, the A-particles hols and embedded in an Epon-Araidite mixture (25). Ultrathin localized within cisternae of the microsomal vesicles. sections were cut on an LKB ultratome with a diamond knife. To a given volume of membrane fraction, 02 volume of a The sections were placed on Formvar-coated, carbon-stabilized 10% solution of Triton X-100 in water was added with constant copper grids, double stained with uranyl acetate (13) and lead mixing. The still turbid mixture was rapidly expressed 5 times citrate (31), and examined in a Siemens Elmiskop I electron through a 23-gauge hypodermic needle attached to a syringe microscope, using an accelerating voltage of 80 kv and a 50- of appropriate size. The sheared preparation, now containing micron objective aperture. the A-particles in free form (see Results), was immediately Two percent phosphotungstic acid, adjusted to pH 6.3 with diluted with 7â€”8volumes of 0.15 M potassium citrate, pH 7.2, 5 N KOH, was used for negative staining. A drop of the sample and centrifuged for 30 minutes at 30,000 rpm (105,000 x g) and a drop of the negative stain were applied in succession to using polycarhonate tubes in the Spinco No. 30 rotor. The a coated grid, each drop being allowed to remain on the grid slightly turbid supernatant fraction was discarded, and the about 30 seconds before the bulk of the fluid was removed. pellets were resuspended by homogenization in Solution B (25% The remaining thin layer of the mixture was allowed to dry sucrose containing 0.05 @ipotassium citrate, pH 72) . The frac on the grid. tion was filtered through a thin layer of pyrex wool to remove some gelatinous material which became apparent at this stage RESULTS and then sheared 3 times as before. A small amount of incom pletely dispersed material was removed by centrifugation for Comments on the Isolation Procedure 10 mm at 10,000 X g, and the preparation was then layered Upon disruption of the tumor cells, the A-particles retained over a cushion of 48% sucrose containing 0.05 @ipotassium their original relationships to the endoplasmic reticulum, i.e., citrate, pH 7.2, and centrifuged in either of 2 ways depending they were localized within the cavities of the microsomal vesi on the scale of the isolation and/or the demands of time: des (compare Figs. 1 and 2) . A similar observation has been (a) 4-mi aliquots over 1.3-mi cushions were centrifuged for reported by Parsons (28) . Very rarely were free A-particles 2 hr at 65,000 rpm (420,000 x g) in the Spinco SW-65L rotor; seen either in the usual membrane fractions or in fractions pre or (b) 25-mi aliquots over 5-mi cushions were centrifuged for pared at higher gravitational forces. It was therefore necessary 15 hr at 25,000 rpm (90,000 X g) in the Spinco SW-25.1 rotor. to devise a technic for liberating the A-particles prior to any Following centrifugation, the tube contents, including a very further fractionation steps. heavy band of material overlying the cushion, were removed In preliminary experiments isolated membrane fractions were as completely as possible from above the small pellets and subjected to mechanical shear under a variety of conditions discarded. The pellets were resuspended in a small volume of with the aim of disrupting the microsomal vesicles without 0.05 M potassium citrate and dispersed by shearing (3 to 5 severely damaging the contained A-particles. The treated frac cycles through a 25-gauge needle) . At this point, the material tions were then centrifuged for 30 mm at 105,000 x g and from as much as 40 gm of tumor has been concentrated in a the pellets examined by electron microscopy. Prolonged ho volume of 1 ml. mogenization with a tight-fitting pestle of the Potter-Elvehjem For isopycnic banding of the A-particles, 0.3-mi aliquots of type or repeated vigorous expulsion of the fraction through the above suspension were layered over precooled 5-mi linear 23- or 25-gauge hypodermic needles appeared to be ineffective gradients formed from 33 and 68% (w/v at 20Â°C) sucrose in fragmenting the microsomal vesicles, and only a few A- solutions containing 0.05 i@ipotassium citrate, pH 72. The particles were released. Fractions were also examined after hay tubes were centrifuged for 3 hours at 65,000 rpm (420,000 x g) ing been expressed from a French pressure cell under pressures in a Spinco SW-65L rotor to achieve equilibrium (see Results), ranging from 5,000 to 20,000 psi. Although treatment at pro after which the contents were sampled and the absorbancies gressively higher pressures resulted in marked, and ultimately monitored at 260 m@ and 280 mj@as described elsewhere (19). profound, disruption of the vesicular structures, there appeared Solution densities (gm/cu cm at 20Â°C) were calculated from to be a parallel destruction of the intracisternal particles. Some the refractive indices of the gradient fractions. Particulate com liberated and apparently intact A-particles were seen in ma ponents were routinely recovered from the gradient fractions terial treated at 12,000 to 15,000 psi, but they were judged to by centrifugation for 1 hour at 125,000 x g (Spinco SW-39 L be too few in number to warrant further attempts at isolation. rotor), after the fractions had first been diluted with 3 to 5 Detergent action presented an alternative means of disrupt volumes of Solution B. The pellets were either fixed in situ for ing the microsomes and releasing the A-particles. However, electron microscopy (see below) or resuspended in sterile 0.145 when either sodium deoxycholate or the nonionic detergent M sodium chloride for biochemical analysis and bioassay. Triton X-100 was added to membrane fractions in amounts Quantitative determinations of nucleic acid and lipid phos sufficient to lyse the microsomal membranes, the liberated A- phorus were carried out as previously described (21 ) . Protein particles were found to have been stripped of their outer mem was assayed by the Folin phenol method (22) using crystallized branes. bovine serum albumin (Armour and Co.) as a standard. In the procedure finally adopted, the function of the detergent The pellets for electron microscopy were fixed for 1 hour in was not to lyse the microsomal vesicles but, instead, to sensi a chrome-osmium solution (3) and were pre-fixed for 90 minutes tize them to disruption during the subsequent shearing step.

2138 CANCER RESEARCH VOL. 28

Exploratory studies revealed that the relative amounts of de tergent and membrane material, rather than the absolute con 30i centration of detergent, were critical in determining the degree of preservation of A-particle morphology. Thus, when each ml 120

of mixture contained the membrane material. from approxi I 15 @ mately 2 gm of tissue (standard conditions), Triton X-lOO at S a 2% level had no discernible effects on the morphology of particles (Fig. 3) . However, when the concentration of mem brane material was reduced 10-fold (02-gm equivalents of tis sue per ml), as little as a 0.5% Triton sufficed to remove the FRACTION NUMBER outer envelopes of the particles (Fig. 4). We have found it useful to express both the tissue equiva Chart 1. Sucrose density-gradient banding of A-particle fraction. lence of the membrane fraction and the amount of Triton on A pellet enriched in A-particles was prepared by centrifugation a weight basis and to consider the ratio between these quan through 48% (w/v) sucrose (see Methods). An aliquot (02 ml) representing material from 0.95 gm of tissue was applied to each tities in any given mixture. All ratios of tissue:Triton equal to of 3 replicate gradients (5.0 ml volume, 33% to 68% (w/v) sucrose or greater than 100:1 were found to be compatible with preser containing 0.05 M potassium citrate pH 72) and centrifuged for vation of the A-particles on subsequent shearing. However, at the indicated times at 65,000 rpm in a Spinco SW-65L rotor at ratios above 200 : 1, the microsomal vesicles became less sus 5Â°C. The tube contents were sampled and monitored for absorb ceptible to disruption, and A-particles were liberated in reduced ancy and solution density as described in the text. The direction numbers. These data may be useful in adapting the procedure of sedimentation is towards the right. to situations where tissue is less abundant than in the present instance. The sheared membrane fractions were diluted with 0.15 @z equilibration of the A-particle (major) band while maintaining potassium citrate in order to dissociate ribosomal components an adequate separation of the ribosomal components. from the microsome membranes through chelation of divalent In a control experiment, the isolation technic was applied to cations (33) . As a result, more than two-thirds of the total normal BALB/c mouse liver, which is devoid of the A-particles. RNA in the membrane fraction remained in the supernatant Membrane fractions from equal weights of liver and tumor fluid after centrifugation at 30,000 rpm for 30 minutes. Po were carried in parallel through the entire procedure. In the tassium citrate was chosen as the chelating agent since it has case of liver, no light-scattering band was observed in the been used extensively in the isolation of RNA-containing murine gradient tubes following centrifugation, and very little absorb leukemia viruses (26). ancy was recorded in the gradient position corresponding to The pellet from the 30,000 rpm centrifugation contained the the A-particle peak (Chart 2). A-particles in liberated form, but still consisted primarily of Electron Microscopy of Gradient-isolated A-Particle Fractions extraneous membrane components. The vast majority of these membranes failed to penetrate the 48% sucrose cushion at the Fractions representing the major band (p = 1.22 gm/cu cm) next centrifugation (probably as a result of reduction in their were diluted and centrifuged as described under Methods in ribosome content, since in the absence of potassium citrate order to sediment the particulate components. Thin sections treatment, rough-surfaced tumor microsomes readily sediment of the fixed pellets presented the appearance shown in Fig. 6. through 48% sucrose) . The pellets obtained by centrifugation The isolated A-particles consisted of two concentric shells through 48% sucrose represented a marked concentration of surrounding a relatively electron-lucent core. Most of the single A-particles at the expense of other components of the mem particles had a circular or slightly ovoid outline and averaged brane fraction, as illustrated in Fig. 5. about 95 millimicrons in diameter. The outer shell was approxi In order to evaluate the time required for isopycnic band mately 5 millimicrons thick and was separated from the inner ing of A-particles in sucrose density gradients, aliquots of a shell by an electron-lucent space about 5 millimicrons in width. resuspended 48% sucrose pellet were layered over 3 replicate The inner shell was approximately 18 millimicrons in width gradients and centrifuged for various periods before sampling. and delineated a central relatively electron-lucent core about The resultant optical density profiles are shown in Chart 1. 35 millimicrons in diameter. In some instances the core appeared The major band of optical density (visible as a light-scattering to contain fine particulate material (Fig. 6, P) . Complex forms zone before sampling) appeared to have reached an equilibrium consisting of two, three, or even five A-particle buds were also distribution centered around a density of 1.22 gm/cu cm by observed (Fig. 6, arrows) . In addition, the fraction contained 180 minutes of centrfugation. The ratio of absorbancies at 260 membrane structures which were probably remnants of micro and 280 mj.@(A260:A280) varied between 1.28 and 1.31 in the somal vesicles, as well as some finely granular material of peak fraction of this band. In contrast, a complex of sharp moderate electron density, the nature of which is not clear. peaks, with A260:A280 ratios of about 1.8, moved steadily down The gradient-isolated preparations were also plated directly ward in the gradients with the passage of time. Electron micros on grids and examined after negative staining with phospho copy and chemical analysis indicated that these minor peaks tungstic acid (Fig. 7) . The disposition of material on the grids represented ribosomal material, probably in the form of dis suggested a fairly extensive aggregation of A-particles within sociated subumts. A 3-hour centrifugation appeared to permit the fraction. We think it likely that this aggregation was in

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Table 1 Original fractioniiz% membrane fraction of 00 originalProtein20,6001720.83RNA2,890153034Phospholipid5,82030.4052Total29,3102180.74(liz)A-particie

>- U z

0 U) Yields of protein, RNA, and phospholipid in isolated A-particle 4 fraction. A-particles were isolated by isopycnic banding in a sucrose density gradient. Values are expressed per gin wet weight of tissue.

20 25 FRACTION NUMBER pared with the amounts in the original membrane fraction. The isolated fraction represented less than 1% of the starting ma Chart 2. Comparison of isolation procedure as applied to liver terial ; however, we have no means at present of evaluating the and plasma-cell tumor. Membrane fractions from equal weights efficiency of recovery of A-particles in the isolation procedure. (2.4 gm) of normal BALB/c mouse liver and of MOPC-104E tu Table 2 presents analytic data obtained for the A-particle mor were carried in parallel through the extraction procedure. The fractions in several isolation experiments. Similar results were entire yield (48% sucrose pellet) from each tissue was subjected obtained whether the entire A-particle band (Experiments II to sucrose density-gradient analysis as in Chart 1 (centrifugation and III) or only the center portion (Experiment I) were taken time 3 hours). The small spikes seen in fractions 18 and 20 of the MOPC-lO4E pattern probably resulted from discontinuities in the for analysis. Protein constituted 80% and phospholipid about sucrose gradient ; they were not seen when gradients were allowed 14% of the recovered material (polysaccharide and other types to age for 2 or more hours before use (e.g., Chart 1). of lipid were not studied) . The RNA content varied between 4.8% and 6.8% in different experiments. Analysis by a sensi tive modification (2) of the diphenylamine reaction indicated duced by the pelletization steps prior to density-gradient frac that DNA was either entirely absent or constituted less than tionation. It is apparent that occlusion of extraneous compo 1% of the total nucleic acid. In all instances, RNA as measured nents within clumps of A-particles could reduce the efficiency of purification during isopycnic banding. 2Deoxycbolate-treated@IIIIIIIivProteinTable

Effects of Deoxycholate on Isolated A-Particles Gradient-isolated fractions were treated with deoxycholate in order to remove as much of the contaminating membrane RXA 6.8 4.8 5.4 72 6.4 material as possible and hopefully to provide a more purified Phospholipid79.1 14.181.1 14.180.4 14288.4 4.4912 2.5 preparation of the inner shells of the A-particles. The fractions were first dialyzed for 8 hours at 5Â°C against Solution B to Composition of isolated A-particle fractions in various experi reduce the sucrose concentration, then treated at 5Â°C with 0.1 ments. A-particles were isolated by isopycnic banding in sucrose density gradients. Roman numerals indicate separate experiments. volume of a 10% solution of deoxycholate and immediately Values are expressed as percents of the total material (protein + reapplied to the usual 33â€”68%sucrose gradient. Centrifugation RNA + phospholipid) recovered in each instance. Per gm of for as short a time as 1 hour at 80,000 x g now resulted in original tissue, the total material recovered in the A-particle frac complete sedimentation of the particulate material through the tions in the various experiments were : I, 161 pg; II, 266 jig; gradient. The appearance of the pellet upon electron micros III,3SSjzg; IV, 264 jig. copy is shown in Fig. 9. Much of the background material aGradientisolatedfractionsweretreatedwith 1% sodium had been removed as expected if the deoxycholate were to have deoxycholate and the particulate components recovered by centrif lvsed the contaminating membrane components. The outer shell ugation as described in the text. In Experiment I aliquots were of the A-particles was also removed by the detergent (compare analyzed both before and after deoxycholate treatment. Figs. 8 and 9) . The remaining inner portions of the particles must have had a density in excess of 1.25 gm/cu cm to have by the orcinol reaction for ribose accounted fully for the UV sedimented through the 68% sucrose at the bottom of the absorption of hot perchloric extracts of the fractions. gradient. Deoxycholate treatment caused a marked reduction in the phospholipid content of the fractions (Table 2) concomitant Gross Chemical Composition of Isolated A-Particle Fractions with its lytic action on the contaminating microsomal mem Following density-gradient centrifugation, the A-particle bands branes and on the outer envelopes of the A-particles them (corresponding to fractions 13â€”18in the patterns shown in selves. On the other hand, the RNA content was not reduced Charts 1 and 2) were analyzed for their content of protein, by deoxycholate. This observation was consistent with the lo RNA, and phospholipid as described in Methods. Table 1 shows calization of some RNA within the inner shell or core of the the yields of these components in a typical experiment com A-particles. However, the variation in RNA content observed

2140 CANCER RESEARCH VOL.28

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1968 American Association for Cancer Research. Extraction of Intracisternal A-Particles in different experiments suggested that a proportion of the no evidence for RNA components of this size in the plasma R.NA in the isolated fractions might represent contamination cell tumor A-particles. In several instances, RNA was prepared by exogenous ribonucleoprotein. For example, ribosomal sub from isolated A-particle fractions through the use of phenol units or degradation products could be carried into the fractions and sodium dodecyl sulfate, essentially according to the pro adsorbed to the surface of the A-particles or occluded in small cedure of Duesberg and Robinson (9) . The total recovery of aggregates. RNA was nearly quantitative. No discrete components larger than 30 S were detected either by sucrose density-gradient The Partition of RNA during A-Particle Isolation Evaluated analysis or by examination in the Spinco Model E analytical by Isotope Dilution ultracentrifuge using the UV absorption system. A typical su Liver membrane fractions, although devoid of A-particles, crose gradient pattern is presented in Chart 3A . The absorb contain cytoplasmic components (notably microsome-associated ancy scan shows a very heterogeneous distribution superim ribosomes) which might be expected to contribute exogenous posed upon which is a single discrete peak (arrow) , estimated RNA to isolated A-particle fractions. In the experiment sum by planimetry of the pattern to represent about 15% of the marized in Table 3, membrane fractions were prepared from total A260. The sedimentation rate of this component was found the livers of normal BALB/c mice following the administration on further analysis to correspond precisely to that of the large of uridine-5-3H ; the liver membranes containing labeled ribo ribosomal RNA (Chart 3B), i.e., 29 S (18). nucleoprotein components were mixed with unlabeled tumor membranes, and the combined fraction was then carried through DISCUSSION the standard isolation procedure. It is seen from Table 3 that more than 90% of the total radioactivity in RNA had been The most important result of the present study is the demon eliminated prior to density-gradient fractionation. The latter stration that intracisternal A-particles may be isolated in rela step provided a further marked reduction in radioactive (exog tively large numbers and in an apparently good state of mor enous) RNA, without, however, eliminating it completely. The phologic preservation. It is reasonable to suppose that avail RNA in the final A-particle preparation had a reduced specific ability of the isolated particles may contribute ultimately to activity compared to that in the original combined membrane an understanding of their biochemical properties and functional fraction. If it is assumed that the cytoplasmic components of significance. However, there are two aspects of the present liver and tumor reacted similarly during the isolation procedure, isolation technic which call for special comment in this context. it can be calculated that 43% of the RNA in the A-particle First, the use of a detergent to liberate the A-particles seems fraction represented contamination by other elements, the re undesirable in principle, since several membrane-active agents, mainder being intrinsic to the A-particles themselves. For ob including Triton X-100 itself, have been found either to com vious reasons, this type of calculation is uncertain ; the experi pletely inactivate the RNA-contaiing Rauscher virus (36) or ment is important, however, in demonstrating the need for to greatly reduce its infectivity (34) . In the procedure as pres improvement in the isolation technic before RNA content of ently constituted, we have attempted to balance the relative the particles can be finally evalulated. amounts of Triton and membrane material in such a way that the detergent would be largely bound to the exposed micro Ultracentrifugai Behavior of RNA Extracted from A-Particle somal and mitochondrial surfaces. It was hoped that the A- Fractions particles, when subsequently released by shearing, would thus High molecular weight RNA's (sedimentation rates near 70 5) encounter a medium containing little or no free detergent. Al have been extracted from several murine and avian oncogenic though the isolated particles did retain their external coats as viruses (8, 10, 12, 27, 32) . Our results thus far have provided judged by electron microscopy, there is of course no assurance

Table 3 RadioactivityRNAin RNASpecific % of Total of radioactivity Fraction original50,000880006,67512137,50010020.656,675100137,500RNATotal@igS original. cpxn% cpm/jig Tumor membranes Liver membranes Combined membranes 30,000 rpm pellet 40,800 30 48% sucrose pellet 7.92.433690.654100.301.11 10,900100 A-particle band from sucrose density gradient Distribution of RNA during A-particle isolation in the presence of added uridine-@H-labeled liver membrane fraction. A membrane fraction was isolated from the livers of 4 normal BALB/c female mice, each of which had received 100 jic of uridine-5-3H (specific activity 9 c/mmole) intraperitoneally 15 hours before sacrifice. A second membrane fraction was pre pared from 27.5 gm of MOPC-104E tumor (nonradioactive) . The fractions were combined and A-particles were isolated from the mixture in the usual fashion.

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significance of the 29 S component found in the phenol-extracted RNA preparations cannot be evaluated at present, although the sedimentation rate is indicative of ribosomal RNA. Demon stration of a discrete RNA component differing from the usual cytoplasmic RNA's in size, base composition, and/or sensitivity 0 to added ribonuclease may resolve this critical question even if it proves impossible to eliminate all contamination of the isolated A-particle fractions. Previous bioassays of cell-free preparations from plasma-cell tumors have yielded consistently negative results (5, 24, 29). Since, as we have seen, the A-particles remain within the micro somal vesicles when the tumor cells are disrupted by the usual means, it might be supposed that the surrounding membranes FRACTION NUMBER could have constituted a barrier to the expression of biologic activity. However, during the course of the present work, we Chart 3. Sucrose density-gradient analysis of RNA extracted have injected concentrated suspensions of free A-particles from isolated A-particle fraction. A, RNA was extracted by a both subcutaneously and intraperitoneally into BALB/c and phenol-sodium dodecyl sulfate procedure (9) and applied to a C3Hf/He infant mice (48 hours old) without any obvious linear 5â€”25%sucrose gradient (28 ml) containing 0.15 M sodium effects over a subsequent 10-month observation period. Simi chloride and 0.015 M sodium citrate pH 7.0. The tube was cen larly, no differences in growth properties or cell morphology trifuged for 15.5 hr at 25,000 rpm (Spinco SW-25 rotor) and 5Â°C. have been detected between control cultures of mouse embryo Tube contents were sampled and monitored for absorbancy as cells and parallel cultures to which isolated A-particles had described in the text. B, fractions representing the shaded area of been added, even though in the latter case the A-particles could A were pooled and dialyzed overnight at 5Â°C against 0.05 M be shown by electron microscopy to have persisted in the cul sodium chloride-0.1 M Tris chloride, pH 72. Half of the dialyzed ture medium and to have been present in vacuoles within some sample was applied to a 5-25% sucrose gradient containing the same buffer. A sample of ribosomal RNA isolated (18) from of the cells. Possible â€œhelper-virusâ€•activity, assayed by rescue MOPC-104E tumor was applied to a second gradient. Both tubes of Moloney sarcoma virus activity in vitro ( 16) , is currently were centrifuged and sampled as above, and the resultant absorb under study. ancy distributions superimposed. The solid line in B represents In addition to continued efforts towards a direct demonstra the ribosomal RNA. tion of biologic activity, other lines of investigation related to the functional significance of the intracisternal A-particles now seem feasible. Chief among these are the search for nucleic acid that these were not altered in a way that might block biologic components unique to the particles and the characterization of activity. An important technical improvement, then, would be the protein components by immunologic and chemical technics. a purely mechanical means of efficiently disrupting the micro Of interest in the latter respect are the serologic relationships somal vesicles without damaging the A-particles. It might be between A-particles and known murine viruses, as well as the recalled here that a few apparently intact particles were ob possible occurrence of marker proteins which might reflect the served in membrane preparations that had been expressed from function of viral genome within the tumor cells. a French pressure cell. Although not suited for large-scale iso lation of the particles, such preparations may be useful for ACKNOWLEDGMENTS assay of biologic activity. Second, the present isolation procedure, while greatly con The authors are grateful to Mrs. Marian V. A. Smith and Mr. centrating the A-particles, failed to achieve their complete Raymond Steinberg for excellent technical assistance. separation from certain other cytoplasmic components. To some extent, the apparent â€œcontaminationâ€•of the isolated fractions REFERENCES may reflect the heterogeneity of the original A-particle popu lation rather than a deficiency in technics. Thus, incompletely 1. Bernhard, W. The Detection and Study of Tumor Viruses with developed particles (Fig. 1) could carry fragments of associated the Electron Microscope. Cancer Res., 50: 712â€”727,1960. endoplasmic reticulum into the fraction if the density of the 2. Burton, K. Study of the Conditions and Mechanism of the complex were not sufficiently different from that of the free Diphenylamine Reaction for the Colorimetric Estimation of A-particles themselves. Of more concern is the possibility of Deoxyribonucleic Acid. Biochem. J., 62: 315â€”323,1956. artifactitious association between the A-particles and other dc 3. Dalton, A. J. Chrome-Osmium Fixative for Electron Micros copy. Anat. Record, 151: 281, 1955. ments (ribosomes and membrane fragments) during the pre 4. Dalton, A. J., and Felix, M. D. The Electron Microscopy of parative procedures. We are currently trying to develop an Normal and Malignant Cells. Ann. N.Y. Acad. Sci., 63: 1117â€” isolation technic which would avoid repeated packing of the 1140, 1956. particles and the attendant likelihood of induced aggregation 5. Dalton, A. J., Potter, M., and Merwin, R. M. Some Ultra prior to isopycnic banding. structural Characteristics of a Series of Primary and Trans.. The biochemical data thus far obtained are consistent with planted Plasma-Cell Tumors of the Mouse. J. Natl. Cancer the presence of some RNA intrinsic to the A-particles. The Inst., 56: 1221â€”1267,1961.

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6. de Harven, E., and Friend, C. Electron Microscope Study of a 21. Kuff, E. L., and Zeigel, R. F. Cytoplasmic Ribonucleoprotein Cell-Free Induced Leukemia of the Mouse. J. Biophys. Components of the Novikoff Hepatoma. J. Biophys. Biochem. Biochem. Cytol., 4: 151â€”156,1958. Cytol., 7 : 465â€”478,1960. 7. Dmochowski, L., and Grey, C. E. Electron Microscopy of Tu 22. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. moss of Known and Suspected Viral Etiojogy. Texas Rept. J. Protein Measurement with the Folin Phenol Reagent. J. Biol. Med., 15: 704â€”756,1957. Biol. Chem., 193: 265â€”275,1951. 8. Duesberg, P. H., and Blair, P. B. Isolation of the Nucleic Acid 23. Mclntire, K. R., Asofsky, R. M., Potter, M., and Kuff, E. L. of Mouse Mammary Tumor Virus (MTV). Proc. Natl. Acad. Macroglobulin-Producing Plasma-Cell Tumor in Mice : Iden Sci. U. S., 55: 1490â€”1497,1966. tification of a New Light Chain. Science, 150: 361â€”363,1965. 9. Duesberg, P. H., and Robinson, W. S. Isolation of the Nucleic 24. Merwin, R. M., and Redmon, L. W. Induction of Plasma Cell Acid of Newcastle Disease Virus (NDV) . Proc. Natl. Acad. Tumors and Sarcomas in Mice by Diffusion Chambers Placed Sci. U. S., 64: 794â€”800,1965. in the Peritoneal Cavity. J. Natl. Cancer Inst., 31 : 997â€”1018, 10. Duesberg, P. H., and Robinson, W. S. Nucleic Acid and Pro 1963. teins Isolated from the Rauscher Mouse Leukemia Virus 25. Mollenhauer, H. H. Plastic Embedding Mixtures for Use in (MLV). Proc. Natl. Acad. Sci. U. S., 55: 219â€”227,1966. Electron Microscopy. Stain Technol., 39: 111â€”114,1964. 11. Friedlaender, M., and Moore, D. H. Occurrence of Bodies 26. Moloney, J. B. Biological Studies on a Lymphoid-Leukemia within Endopiasmic Reticulum of Ehrlich Ascites Tumor Cells. Virus Extracted from Sarcoma 37. I. Origin and Introductory Proc. Soc. Exptl. Biol. Med., 9@: 828â€”831,1956. Investigations. J. Natl. Cancer Inst., 24: 933-951, 1960. 12. 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OCTOBER 1968 2143

Fig. 1. Portion of an intact plasma cell in MOPC-104E tumor. There are fairly numerous A-particles located in the cisternae of the endoplasmic reticulum. An area of thickened membrane in association with budding particles is indicated (arrow). X 45,000. Fig. 2. Membrane fraction of plasma-cell tumor. The A-particles remain in the cavities of the microsomal vesicles. X 53,000. Fig. 3. A gloup of A-particles with Triton X-lOO (2.0% Triton added to the membrane material from 2 gm of tumor tissue). The outer shells of the Particles are visible and the single particles measure about 95 millimicrons in diameter. X 53,000. Fig. 4. A-particles treated with Triton X-100 (0.5% Triton added to the membrane material from 02 gm of tumor tissue) . The par tides have been stripped of their outer shells, and the single particles measure about 85 millimicrons in diameter. Note the decreased density of the inner shells. X 53,000. Fig. 5. Thin section of a pellet centrifuged through a 48% sucrose cushion showing a marked concentration of A-particles. Numer ous membrane fragments and aggregates of granular material are apparent. x 53,000. Fig. 6. A-particle fraction from the sucrose density gradient. The outer shells of the particles are readily visible. There are several elongated forms which consist of two to five buds (arrows) . Compare with structures in intact plasma cell (Fig. 1) . A few particles (P) have centers which are relatively electron dense. There are remnants of miciosomal vcsicles and foci of particulate material which may represent ribosomal degradation products. x 53,000. Fig. 7. A-particle fraction from the sucrose density gradient which is negatively stained with phosphotungstic acid. The stain pene trates the cores of the particles and the outer shell is clearly defined. Most of the particles remain in large aggregates; a small aggregate is indicated (arrow) . X 31,000. Fig. 8. Untreated A-particle fraction essentially the same as seen in Fig. 6. Note that all the particles have a distinct outer shell. x 100,000. Fig. 9. Treatment of A-particle fraction with 1.0% deoxycholate. The outer shell has been stripped from the particles. Note the absence of contaminant membrane material. X 100,000.

2144 CANCER RESEARCH VOL. 28

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Edward L. Kuff, Nelson A. Wivel and Kira K. Lueders

Cancer Res 1968;28:2137-2148.

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