The Extraction of Intracisternal A-Particles from a Mouse Plasma-Cell Tumor1
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(CANCER RESEARCH 28, 2137-2148,October1968] The Extraction of Intracisternal A-Particles from a Mouse Plasma-Cell Tumor1 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 “intmacisternal A-particles― 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 “virus-like― following transplantation in female BALB/c mice. The excised particles in murine plasma-cell tumors (5) . One is morpho tissue was quickly chilled to 0°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 5°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 OCTOBER 1968 2137 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 “mem in a mixture of 0.5% aqueous uranyl acetate and 0.54% su brane fractions,―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 7—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 20°C) sucrose in fragmenting the microsomal vesicles, and only a few A- solutions containing 0.05 i@ipotassium citrate, pH 72.