Intervirology 12: 47-56 (1979)
Lysosome Stability during Lytic Infection by Simian Virus 40 Katie H. Einck and Leonard C. Norkin Department of Microbiology, University of Massachusetts, Amherst, Mass.
Key Words. SV40 • Cytopathology • Lysosomes
The mechanisms by which viruses damage and kill cells remain largely unknown. One important hypothesis is that lysosomal rupture and the destructive release of lysosomal hydro lases into the surrounding cytoplasm might play an important role in the chain of events leading to cell death [1], Histochemical and cell fractionation procedures indicate that lysoso mal changes do occur during infection by a number of cytopathic viruses, including vac cinia virus, Newcastle disease virus, mouse hepatitis virus [1], adenovirus type 5, fowl plague virus [2], simian virus 40 (SV40), herpes simplex virus [3], and poliovirus [4]. Address imjuiries to: Dr. Leonard C. Norkin, De partment of Microbiology, University of Massachu setts, Amherst, MA 01003 (USA) Received: December 11, 1978
It has not been possible to answer with certainty whether these lysosomal changes play an important role in cell killing by viruses, in part because a useful definition of cell death has not been applied to this problem. Thus, the possibility that lysosomal changes occur postmortem rather than antemortem has not been excluded. Furthermore, biochemical studies of cell fractions from infected cells can not distinguish between actual enzyme release and changes in lysosomes (such as increased size) causing them to rupture more easily dur ing fractionation. Lysosomal rupture, as the critical event in lethal cell injury resulting from most causes, was once considered an attractive hypothesis. However, it is no longer accepted widely or without reservation. This is largely because of studies which showed that in cells damaged by
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Summary. By 48 h postinfection, 40-80% of SV40-infected CV-1 cells have undergone irre versible injury as indicated by trypan blue staining. Nevertheless, at this time the lysosomes of these cells appear as discrete structures after vital staining with either acridine orange or neutral red. Lysosomes, vitally stained with neutral red at 24 h postinfection, were still intact in cells stained with trypan blue at 48 h. Acid phosphatase activity is localized in discrete cytoplasmic particles at 48 h, as indicated by histochemical staining of both fixed and unfixed cells.
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Materials and Methods Cell Cultures The CV-1 line of green monkey kidney (GMK) cells, the LLC-MK 2 line of rhesus monkey kidney cells, and BA LB 3T3 cells were obtained from the American Type Culture Collection, Rockville, Md. All cells were cultivated in Dulbecco modified Eagle medium (DME; GIBCO, Grand Island, N.Y.) containing 10% fetal calf serum and gentamicin (50 ug/ml) in a humidified 5% COa atmosphere. Cell cultures did not contain myco plasma as indicated by electron microscopy [7]. Virus Small-plaque SV40 (strain 777) was prepared for infection as previously described [8], Virus was titered by plaque assay on CV-1 monolayers. Unless otherwise
indicated, all experiments were at an input multiplicity of 100 PFU/cell. Fluorescence Microscopy Acridine orange vital staining was carried out by the method of Robbins and Marcus [9], The working solutions, prepared immediately before use, were made from a 3 x 10~8 M stock solution by diluting in DME to final concentrations of 3 x 10~8 M and 3 x 10~9 M. Cells grown on 11 mm diameter coverslips were stained by immersion in 2-3 ml of working solution at 37° for 5-10 min. These were then wet-mounted, observed, and photographed with Professional Ektachrome ASA 200 (Kodak) under dark-field UV illumination. The frac tion of T-antigen-containing nuclei was determined by indirect immunofluorescence as described previously [8], Histochemistry Acid phosphatase activity was localized histochemically by the Burstone method [10]. Cells were cultured on 11 mm diameter coverslips or in Lab-tek chambers and were fixed briefly in buffered 4% formol-calcium or left unfixed. Samples were briefly washed in 0.1 M acetate buffer, pH 5.2, prior to staining with the azo dye, fast blue RR (Sigma Chemical Co., St. Louis, Mo.), which was coupled to the substrate, naphthol AS-MX phosphate (Sigma), as described by Burstone [10]. Samples were incubated with the stain in 0.1 M acetate buffer, pH 5.2, for 2 h at 37°. Trypan Blue Staining The fraction of nonviable trypan blue-stainable cells was measured as previously described [8]. Neutral Red - Trypan Blue Staining Cells cultured on coverslips in 35-mm dishes were stained with neutral red at 24 h postinfection by adding 0.05 ml of 0.1 % stock solution to 2 ml of media in each dish. At 48 h postinfection the coverslips were stained briefly with trypan blue, wet-mounted, and photo graphed within 15 min, using high-speed Ektachrome (Tungsten) film, ASA 160 (Kodak), and phase optics.
Results Vital Staining with Acridine Orange The lysosomes of unfixed cells have the unique ability to concentrate basic dyes such
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a variety of treatments (e.g., metabolic in hibitors, the cytolytic action of complement, oxygen deprivation), the lysosomes remained as intact structures after the cells acquired many characteristics typical of lethal cell dam age [5,6], For this reason, and others stated above, we have sought to re-evaluate the role of lysosomes in viral cytopathology. In the studies reported here we have tested in a direct way whether the lysosomes of SV40infected cells rupture prior to cell death. Cell death is considered to occur at the time that the damage becomes irreversible [5], Although we cannot operationally apply this definition of cell death, it is known from studies of lethal cell injury resulting from a variety of other causes that cells become trypan blue-stainable only after they have undergone irreversible damage [6], Experiments reported here, in which lysosomes were labeled with acridine orange dye, neutral red dye, or by histochemical staining for acid phosphatase, all indicate that lysosomes remain intact well into the post mortem stage of cell injury. Thus, lysosomal rupture per se does not appear to be respon sible for cell killing in this system.
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Fig. 1. Vital staining of CV-I cells with acridine orange dye. a Uninfected cells, b Infected cells at 48 h. c Infected cells at 48 h containing grossly swollen AOP. All plates were photographed at the same magnifica tion.
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as acridine orange [11], The initial segregation of acridine orange into lysosomes occurs pas sively, but the rapid and continued accumula tion of large quantities of dye is an energyrequiring process [9,12]. It is likely that acri dine orange enters lysosomes in an unprotonated form and is trapped in the acidic interior by protonation [11]. Cytoplasmic red dening occurs under conditions in which the dye cannot be segregated fast enough [9], The vital staining of lysosomes by acridine orange is, therefore, quite different from the use of this dye as a differential lluorochrome for nucleic acids in fixed cells [13]. Some dye is taken up by nuclei and nucleoli in unfixed cells, resulting in a bright green fluorescence. However, much greater concentrations of dye are achieved in lysosomes, resulting in a bril liant orange or red fluorescence. The shift in the fluorescence to a longer wavelength proba bly results from a concentration-dependent association of single dye molecules, which fluoresce green, into dimers which fluoresce red [14], The lysosomes of uninfected CV-I, LLCMK-, and BALB 3T3 cells, vitally stained with 3x10 9 M acridine orange, appear as discrete bright orange particles, frequently clustered on one side of the nucleus (fig. la). These will hereafter be referred to as AOP (acridine orange particles) [9], Increasing the acridine orange concentration to 3 * 10 8 M resulted in a similar pattern with more intense staining of the AOP. Cytoplasmic reddening was ob served in these cultures when the acridine
orange concentration was increased to 3 * 10-7 M.
CV-1 green monkey kidney cells, which are killed more quickly by SV40 than some other simian cell lines [8], were stained with 3 x 10 !l M acridine orange at 24 and 48 h after infec tion. At these times, all cells contained intact AOP. In most of these cells the AOP were normal or slightly swollen (fig. lb). However, grossly swollen AOP were observed in about 10-15% of the infected cells by 48 h (fig. Ic). Swollen vacuoles, which did not contain acri dine orange, were also occasionally seen. De spite the fact that the AOP of the infected CV-1 cells were still intact at 48 h, 40-80% of these cells were stainable with trypan blue at this time. All cells of the infected CV-I cultures showed cytoplasmic reddening when exposed to 3 x 10 8 M acridine orange for 5 min at 24 and 48 h after infection. Cytoplasmic redden ing was never seen in parallel control cells under these conditions. The relative diameters of the bright green fluorescing nuclei of the infected and control CV-1 cultures were measured at 24 and 48 h postinfection. The average diameter of the infected cell nuclei was 27% larger than that of the control nuclei by 24 h and 43% larger by 48 h. For comparison, the acridine orange stain ing patterns of the LLC-MKc line of rhesus monkey kidney cells and of BALB 3T3 cells were also examined. SV40 infection of LLCMK -2 cells results in productive infection ac companied by relatively slow cell killing [8], while infection of 3T3 cells is abortive and the cells are spared. The AOP of the infected LLC-MK> cells were normal at 24 h postinfection and slightly swollen at 48 h postinfection. The AOP of the 3T3 cells were normal at both of these times.
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Cytoplasmic reddening and nuclear swelling were not observed in either of these cell types. Distribution of Acid Phosphatase Histochemical staining for acid phospha tase in fixed control CV-1 cells was localized in discrete cytoplasmic particles (fig. 2a). Sim ilar acid phosphatase-positive particles were also seen in fixed infected CV-i cells at 24 and 48 h (fig. 2b, c). Diffuse acid phosphatase activ ity in the cytoplasm was not detected even as late as 48 h postinfection. Because histochemical staining of unfixed cells might reveal changes in the permeability of lysosomal membranes to substrate as well as enzymes [2], we also observed the staining pattern of unfixed cells. In these studies, one determines whether treated (infected) cells can withstand the same controlled amount of dam age (exposure to substrate under staining con ditions) as control cells before becoming per meable to the substrate [15]. Unfixed control and infected cells at 24 and 48 h postinfection were exposed to the substrate, as described in ‘Materials and Methods’, for 15, 30, and 45 min. After 15 min there was no staining of either infected or control cells. After 30 min about 1% of the infected and control cells showed discrete acid phosphatase-containing particles. By 45 min, acid phosphatase-posi tive particles were seen in all of the control and infected cells (fig. 2d— f). We found no evidence of diffuse acid phosphatase staining in the cytoplasm of unfixed control cells or in fected cells at either 24 or 48 h after infection.
Fig. 2. Fixed and unfixed CV-1 cells stained for acid phosphatase, a Fixed uninfected cells, b Fixed infected cells at 24 h. c Fixed infected cells at 48 h. d Unfixed uninfected cells, e Unfixed infected cells at 24 h. f Unfixed infected cells at 48 h. All plates were photographed at the same magnification.
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SV40 Cytolysis and Lysosomes 51
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Fare o f Pres rained Lysosomes In the studies described above, lysosomes were found to be intact at 24 and 48 h after infection. However, it is possible that new lysosomes continue to be produced during the course of the infection. Consequently, it is possible that the intact lysosomes represent those most recently produced and that ‘older’ lysosomes might have ruptured.
To determine whether old lysosomes re main intact, cultures were stained with neutral red at 24 h postinfection and examined at subsequent times. Neutral red is another basic dye that is concentrated in lysosomes by an energy-dependent process similar to that de scribed above for acridine orange [11], Neutral red was used here because when the neutral red-stained cultures are subsequently stained
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Fig.3. CV-I cellsstained with neutral red and trypan blue, a Uninfected cells, b-d Infected cells at 48 h. All plates were photographed at the same magnification.
with trypan blue, both dyes can be seen using bright-field microscopy. We found that lysosomes stained with neu tral red at 24 h may retain the dye as late as 48 h, even in trypan blue-stainabie cells. Some of these trypan blue-stainable cells appear to have a full complement of stained lysosomes (fig. 3b), while others have many fewer lyso somes (fig.3c). In some of the trypan bluestained cells the neutral red was diffuse, and others appeared to have lost the dye entirely. It might be noted, however, that some of the uninfected control cells, which were unstainable with trypan blue, also failed to retain the neutral red dye. In some of the infected cells the neutral red-stained lysosomes appeared to be grossly swollen (fig. 3d). Surprisingly, all of the cells containing grossly swollen lysosomes were able to exclude trypan blue.
Discussion In studies of lethal cell injury, it is useful to consider cell death to have occurred when the cell is no longer able to recover if the injurious agent is removed. Thus, the moment of cell death is when the injury becomes irre versible. In studies of virus-induced cell injury, this definition of cell death is not directly appli cable in an operational sense. Nevertheless, stainability by trypan blue can be used as a first approximation of the time of cell death because cells undergoing lethal injuries resulting from a variety of causes become stainable with try pan blue at the time that the damage becomes irreversible [6]. Previously, we found that the release of cytoplasmic lactic dehydrogenase virus from cells lytically infected with SV40 somewhat precedes and closely parallels the accumulation of trypan blue-stainable cells
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[15a], Thus, in the arguments that follow, stainability with trypan blue is our criterion for cell death. Events that occur prior to the acquisition of stainability are antemortem and those occurring subsequently are post mortem. Our studies with the vital lysosomotropic dyes, acridine orange and neutral red. indicate that lysosomes in SV40-infected CV-I cells remain as intact structures as late as 48 h after infection when between 40 and 80% of these cells are trypan blue-stainable. These findings are incompatible with the most extreme model for lysosomal involvement in lethal cell injury in which lysosomes rupture and quickly re lease their hydrolytic enzymes into the cyto plasm. Our histochemical studies on the distribu tion of acid phosphatase activity in fixed and unfixed infected cells also fail to support a model of cell killing involving antemortem lysosomal rupture. Histochemical results alone would be inconclusive because apparent enzyme localizations could result from diffu sion of enzyme or reaction product within cells. Furthermore, a certain amount of the enzyme activity is inactivated by fixation, possibly ob scuring released enzyme. Nevertheless, the acid phosphatase and vital dye staining pat terns together provide strong evidence against lysosomal rupture prior to the irreversible stage of cell injury. In an earlier study [3], lysosomal changes in SV40-infected cells were followed utilizing the Gomori metal precipitation technique to stain unfixed cells for acid phosphatase. It was reported that lytic SV40 infection of BSC-1 green monkey kidney cells resulted in a ‘first stage activation' in which the lysosomal mem branes of infected cells at 24 h were more per meable than those of control cells to the sub strate. [3-glycerophosphate. By 72 h there was
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SV40 Cytolysis and Lysosomes
a ‘second stage activation' in which acid phos phatase appeared to escape into the cytoplasm and nucleus, as suggested by a diffuse staining pattern. Using the Burstone method, we saw no difference in the acid phosphatase staining patterns between unfixed control and infected cells during the first 48 h of infection. The reason for the dissimilarity between our results and those of the earlier study is not clear. Regardless, it is now recognized that for light microscopy the use of azo dye-naphthol AS phosphates to demonstrate acid phosphatase is preferred over the Gomori metal precipita tion techniques in which some precipitate for mation is not directly related to sites of enzyme activity [16,17]. The diffuse cytoplasmic and nuclear acid phosphatase staining seen at 72 h in unfixed cells using the Gomori technique [3], if not an artifact induced by the staining con ditions, probably represents a postmortem change, since most cells become trypan bluestainable by 48 h. Previously, we found that SV40 infection of CV-I cells resulted in a redistribution of lysosomal N-acetyl-^-glucosaminidase from a particle bound to a soluble fraction [8]. En zyme redistribution was thought to indicate release into the cytoplasm. Because SV40-induced enzyme redistribution followed the same time course and occurred to the same extent in LLC-MKi cells, which became trypan bluestainable at a much slower rate than the CV-I cells, we suggested that lysosomal enzyme re lease per se is not primarily responsible for cell killing by SV40. While the results reported above support the main conclusion of our earlier study, they also cause us to reconsider whether enzyme release into the cytoplasm actually occurs dur ing the antemortem phase of SV40-induced cell injury. Biochemical studies of cellular homogenates cannot, unfortunately, distin
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guish between actual enzyme release from or ganelles and enzyme release resulting from a change in the resistance of lysosomes to rup ture during homogenization. Changes in lysosome resistance to breakage could reflect a change in lysosome size, a change in the fre quencies of different kinds of lysosomes, or a change in the stability of the lysosomal mem brane. Enzyme redistribution might also reflect the portion of the cell population which has entered the postmortem stages of cell injury. It is clear that uninfected cells secrete lyso somal hydrolases into the media and that extra cellular enzyme accumulation increases as a result of infection [8], The histochemical results reported here suggest that there is no prior accumulation of activity in the cytoplasm. However, enzymes released into the cytoplasm might rapidly diffuse throughout the cell [18] and go undetected by histochemical proce dures. Thus, it is possible that during the ante mortem stage of infection there is a small in crease in lysosomal permeability that cannot be detected by the procedures reported here. If this were so, it would remain unresolved whether small differences in lysosomal leak iness could be responsible for irreversible in jury. Yet it is difficult to envision how lyso somes could concentrate acridine orange, which has a molecular weight of only about 300 daltons, while releasing very much larger enzymes into the cytoplasm. As described above, about 10-15% of the SV40-infected CV-I cells have grossly swollen acridine orange- or neutral red-stained struc tures at 48 h. The cells with the grossly swollen neutral red-stained structures, and presumably those with the swollen AOP, are all able to exclude trypan blue at this time. These grossly swollen stained bodies may be autophagic structures (which functionally may be regarded as secondary lysosomes) [19], as suggested by
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SV40 Cytolysis and Lysosomes
cytoplasmic enzymes and by the shutdown of membrane-associated electron transport [8. 15a]. Enzyme release, which does not appear to be related to virus release, was coincident with declining levels of membrane phospholipid synthesis and preceded the appearance of try pan blue-stainable cells by several hours. These cytopathic effects occur prior to any measur able decline in ATP levels and protein synthe sis. Therefore, we suggest that impaired mem brane metabolism plays an important role in cell killing by SV40. It has been suggested that altered membrane permeability, resulting in impaired cellular and intracellular compart ment volume regulation, is a major determi nant of lethal cell injury in systems involving both viral and nonviral damage [6,21], The antemortem nuclear swelling seen in SV40infected CV-I cells described here, as well as our earlier results, are consistent with this hypothesis. Acknowledgments This investigation was supported in part by Public Health Service grant I ROI Al 14049 from the National Institute of Allergy and Infectious Diseases, by Bio medical Research Support grant RR 07048, and by a grant from the American Cancer Society, Massachu setts Division. Inc. We are gratefid to Cheryl Coguen for her excellent technical assistance.
References 1 Allison, A.C. and Sandclin, K.: Activation of lysosomal enzymes in virus-infected cells and its possible relationship to cytopathic effects. J. exp. Med. 117: 879-887 (1965). 2 Allison, A.C. and Mullucci, L.: Histochemical studies of lysosomes and lysosomal enzymes in virus-infected cell cultures. J. exp. Med. 121: 463476(1965). 3 Allison, A.C. and Black, P. H .: Lysosomal changes in lytic and nonlytic infections with the simian
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an ultrastructure analysis in progress. A strong case has been made for the concept that autophagy represents a cellular survival mecha nism in response to pathologic influences [20], Increased autophagic activity might help to isolate an injured area and prevent the spread of degenerative changes. The accumulation and retention of acridine orange and neutral red by lysosomes is an energy-requiring process [5.9], Energy is prob ably needed to drive a lysosomal proton pump which is necessary for the intralysosomal trap ping of the dye [II]. Therefore, the ability of SV40-infected CV-I cells to concentrate acri dine orange and retain neutral red, after they have become trypan blue-stainable, indicates that irreversible cell damage does not result from a shutdown of the cells’ capacity to pro duce energy. This is in agreement with our previous finding that SV40-infected CV-I cells retain normal levels of ATP well after they become trypan blue-stainable [15a]. Because SV40-infected CV-1 cells show cytoplasmic reddening when exposed to acri dine orange, under conditions in which cyto plasmic reddening is not observed in control cells, the infected cells probably cannot segre gate the dye as rapidly as can the control cells. This might reflect damage to lysosomal mem branes. damage to the lysosomal proton pump, or decreased availability of ATP to drive the pump in infected cells. We do not yet know the sequence of cytopathic events leading to irreversible injury nor do we know the specific action of the virus which sets this sequence in motion. The results reported here suggest that lysosomes do not play an important role in the cell-killing pro cess. Earlier, we reported that lytic SV40 infec tion is characterized by severe dysfunction of cellular membranes as indicated by release into the medium of lysosomal, mitochondrial, and
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vacuolating virus (SV40). J. natn. Cancer Inst. 39: 775-787 (1967). Guskey, L. E .; Smith, P.C., and Wolff, D.A.: Pat terns of cytopathology and lysosomal enzyme re lease in poliovirus-infected HEp-2 cells treated with either 2-(a-hydroxybenzyl)-benzimidazole or gua nidine HCI. J. gen. Virol. 6: 151 —161 (1970). Hawkins, H .K .; Ericsson, J.L .E .: Bibcrficld, P., and Trump, B. F .: Lysosome and phagosome sta bility in lethal cell injury: morphologic tracer stud ies in cell injury due to inhibition of energy metab olism, immune cytolysis and photosensitization. Am. J. Path. 68: 255-288 (1972). Trump, B .F.; Croker, B.P., and Mergner, W .J.: The role of energy metabolism, ion, and water shifts in the pathogenesis of cell injury: in Richter and Scarpelli, Cell membranes: biological and patho logical aspects, pp. 84-128 (Williams & Wilkins, Baltimore 1971). De Harven, E.: Identification of tissue culture contaminants by electron microscopy; in Fogh, Contamination in tissue culture, pp. 205-231 (Aca demic Press, New York 1973). Norkin, L.C. and Ouellette, J.: Cell killing by simian virus 40: variation in the pattern of lysoso mal enzyme release, cellular enzyme release, and cell death during productive infection of normal and simian virus 40-transformed simian cell lines. J. V irol./«: 48-57 (1976). Robbins, E. and Marcus, P. I.: Dynamics of acri dine orange-cell interaction. I. Interrelationships of acridine orange particles and cytoplasmic redden ing. J. Cell Biol. 18: 237-250(1963). Burstone, M.S.: Histochemical comparison of naphthol AS-phosphates for the demonstration of phosphatases. J. natn. Cancer Inst. 20: 601-610 (1958). Dc Duve, C.; de Barsy, T.; Poole, B.; Trouet, A.; Tulkens, P., and van Hoof, F .: Lysosomotropic agents. Biochem. Pharmacol. 23:2495-2531 (1974).
12 Robbins, E.; Marcus, P.I., and Gonatas, N .K .: Dynamics of acridine orange-cell interaction. II. Dye-induced ultrastructural changes in multivesicular bodies (acridine orange particles). J. Cell Biol. 21 .-49-62(1964). 13 Armstrong, J.A .: Histochemical differentiation of nucleic acids by means of induced fluorescence. Expl Cell Res. 11: 640-643 (1957). 14 Bradley, D .F .: Molecular biophysics of dye poly mer complexes. Trans. N.Y. Acad. Sci. 24 : 64-71 (1961). 15 Bitensky, L.: The reversible activation of lysosomes in normal cells and the effects of pathological con ditions; in DeRouck and Cameron, CIBA Sympo sium on Lysosomes, pp.362-384 (Little, Brown, Boston 1963). 15a Norkin, L .C .: Cell killing by simian virus 40: im pairment of membrane formation and function. J. Virol. 2 /: 872-879 (1977). 16 Barka, T. and Anderson, P.J.: Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. J. Histochem. Cytochem. 10: 741-753 (1962). 17 Pearse, A .G .E.: Histochemistry: theoretical and applied, vol. I, chapt. 16 (Churchill, London 1968). 18 Danielli, J .F .: Discussion; in DeRouck and Came ron, Cl BA Symposium on Lysosomes, p. 353 (Little. Brown, Boston 1963). 19 Kerr, J.F .R .: Some lysosome functions in liver cells reacting to sublethal injury: in Dingle and Fell, Lysosomes in biology and pathology, vol. 3, pp. 365-394 (North-Holland, Amsterdam 1969). 20 Ericsson. J. L. E .: Mechanism of cellular autophagy: in Dingle and Fell, Lysosomes in biology and pathology, vol.2, pp. 345-394 (North-Holland, Amsterdam 1969). 21 Vainio, T.; Judah, J.D ., and Bjotvedt, G.: Mech anism of cellular damage by virus: a study of anti histamine drugs. I. Murine hepatitis virus and liver explant cultures. Expl Molec. Path. 1 :15-26 (1962).
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Lysosome Stability during Lytic Infection by Simian Virus 40 Katie H. Einck and Leonard C. Norkin Department of Microbiology, University of Massachusetts, Amherst, Mass.
Key Words. SV40 • Cytopathology • Lysosomes
The mechanisms by which viruses damage and kill cells remain largely unknown. One important hypothesis is that lysosomal rupture and the destructive release of lysosomal hydro lases into the surrounding cytoplasm might play an important role in the chain of events leading to cell death [1], Histochemical and cell fractionation procedures indicate that lysoso mal changes do occur during infection by a number of cytopathic viruses, including vac cinia virus, Newcastle disease virus, mouse hepatitis virus [1], adenovirus type 5, fowl plague virus [2], simian virus 40 (SV40), herpes simplex virus [3], and poliovirus [4]. Address imjuiries to: Dr. Leonard C. Norkin, De partment of Microbiology, University of Massachu setts, Amherst, MA 01003 (USA) Received: December 11, 1978
It has not been possible to answer with certainty whether these lysosomal changes play an important role in cell killing by viruses, in part because a useful definition of cell death has not been applied to this problem. Thus, the possibility that lysosomal changes occur postmortem rather than antemortem has not been excluded. Furthermore, biochemical studies of cell fractions from infected cells can not distinguish between actual enzyme release and changes in lysosomes (such as increased size) causing them to rupture more easily dur ing fractionation. Lysosomal rupture, as the critical event in lethal cell injury resulting from most causes, was once considered an attractive hypothesis. However, it is no longer accepted widely or without reservation. This is largely because of studies which showed that in cells damaged by
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Summary. By 48 h postinfection, 40-80% of SV40-infected CV-1 cells have undergone irre versible injury as indicated by trypan blue staining. Nevertheless, at this time the lysosomes of these cells appear as discrete structures after vital staining with either acridine orange or neutral red. Lysosomes, vitally stained with neutral red at 24 h postinfection, were still intact in cells stained with trypan blue at 48 h. Acid phosphatase activity is localized in discrete cytoplasmic particles at 48 h, as indicated by histochemical staining of both fixed and unfixed cells.
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Materials and Methods Cell Cultures The CV-1 line of green monkey kidney (GMK) cells, the LLC-MK 2 line of rhesus monkey kidney cells, and BA LB 3T3 cells were obtained from the American Type Culture Collection, Rockville, Md. All cells were cultivated in Dulbecco modified Eagle medium (DME; GIBCO, Grand Island, N.Y.) containing 10% fetal calf serum and gentamicin (50 ug/ml) in a humidified 5% COa atmosphere. Cell cultures did not contain myco plasma as indicated by electron microscopy [7]. Virus Small-plaque SV40 (strain 777) was prepared for infection as previously described [8], Virus was titered by plaque assay on CV-1 monolayers. Unless otherwise
indicated, all experiments were at an input multiplicity of 100 PFU/cell. Fluorescence Microscopy Acridine orange vital staining was carried out by the method of Robbins and Marcus [9], The working solutions, prepared immediately before use, were made from a 3 x 10~8 M stock solution by diluting in DME to final concentrations of 3 x 10~8 M and 3 x 10~9 M. Cells grown on 11 mm diameter coverslips were stained by immersion in 2-3 ml of working solution at 37° for 5-10 min. These were then wet-mounted, observed, and photographed with Professional Ektachrome ASA 200 (Kodak) under dark-field UV illumination. The frac tion of T-antigen-containing nuclei was determined by indirect immunofluorescence as described previously [8], Histochemistry Acid phosphatase activity was localized histochemically by the Burstone method [10]. Cells were cultured on 11 mm diameter coverslips or in Lab-tek chambers and were fixed briefly in buffered 4% formol-calcium or left unfixed. Samples were briefly washed in 0.1 M acetate buffer, pH 5.2, prior to staining with the azo dye, fast blue RR (Sigma Chemical Co., St. Louis, Mo.), which was coupled to the substrate, naphthol AS-MX phosphate (Sigma), as described by Burstone [10]. Samples were incubated with the stain in 0.1 M acetate buffer, pH 5.2, for 2 h at 37°. Trypan Blue Staining The fraction of nonviable trypan blue-stainable cells was measured as previously described [8]. Neutral Red - Trypan Blue Staining Cells cultured on coverslips in 35-mm dishes were stained with neutral red at 24 h postinfection by adding 0.05 ml of 0.1 % stock solution to 2 ml of media in each dish. At 48 h postinfection the coverslips were stained briefly with trypan blue, wet-mounted, and photo graphed within 15 min, using high-speed Ektachrome (Tungsten) film, ASA 160 (Kodak), and phase optics.
Results Vital Staining with Acridine Orange The lysosomes of unfixed cells have the unique ability to concentrate basic dyes such
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a variety of treatments (e.g., metabolic in hibitors, the cytolytic action of complement, oxygen deprivation), the lysosomes remained as intact structures after the cells acquired many characteristics typical of lethal cell dam age [5,6], For this reason, and others stated above, we have sought to re-evaluate the role of lysosomes in viral cytopathology. In the studies reported here we have tested in a direct way whether the lysosomes of SV40infected cells rupture prior to cell death. Cell death is considered to occur at the time that the damage becomes irreversible [5], Although we cannot operationally apply this definition of cell death, it is known from studies of lethal cell injury resulting from a variety of other causes that cells become trypan blue-stainable only after they have undergone irreversible damage [6], Experiments reported here, in which lysosomes were labeled with acridine orange dye, neutral red dye, or by histochemical staining for acid phosphatase, all indicate that lysosomes remain intact well into the post mortem stage of cell injury. Thus, lysosomal rupture per se does not appear to be respon sible for cell killing in this system.
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Fig. 1. Vital staining of CV-I cells with acridine orange dye. a Uninfected cells, b Infected cells at 48 h. c Infected cells at 48 h containing grossly swollen AOP. All plates were photographed at the same magnifica tion.
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as acridine orange [11], The initial segregation of acridine orange into lysosomes occurs pas sively, but the rapid and continued accumula tion of large quantities of dye is an energyrequiring process [9,12]. It is likely that acri dine orange enters lysosomes in an unprotonated form and is trapped in the acidic interior by protonation [11]. Cytoplasmic red dening occurs under conditions in which the dye cannot be segregated fast enough [9], The vital staining of lysosomes by acridine orange is, therefore, quite different from the use of this dye as a differential lluorochrome for nucleic acids in fixed cells [13]. Some dye is taken up by nuclei and nucleoli in unfixed cells, resulting in a bright green fluorescence. However, much greater concentrations of dye are achieved in lysosomes, resulting in a bril liant orange or red fluorescence. The shift in the fluorescence to a longer wavelength proba bly results from a concentration-dependent association of single dye molecules, which fluoresce green, into dimers which fluoresce red [14], The lysosomes of uninfected CV-I, LLCMK-, and BALB 3T3 cells, vitally stained with 3x10 9 M acridine orange, appear as discrete bright orange particles, frequently clustered on one side of the nucleus (fig. la). These will hereafter be referred to as AOP (acridine orange particles) [9], Increasing the acridine orange concentration to 3 * 10 8 M resulted in a similar pattern with more intense staining of the AOP. Cytoplasmic reddening was ob served in these cultures when the acridine
orange concentration was increased to 3 * 10-7 M.
CV-1 green monkey kidney cells, which are killed more quickly by SV40 than some other simian cell lines [8], were stained with 3 x 10 !l M acridine orange at 24 and 48 h after infec tion. At these times, all cells contained intact AOP. In most of these cells the AOP were normal or slightly swollen (fig. lb). However, grossly swollen AOP were observed in about 10-15% of the infected cells by 48 h (fig. Ic). Swollen vacuoles, which did not contain acri dine orange, were also occasionally seen. De spite the fact that the AOP of the infected CV-1 cells were still intact at 48 h, 40-80% of these cells were stainable with trypan blue at this time. All cells of the infected CV-I cultures showed cytoplasmic reddening when exposed to 3 x 10 8 M acridine orange for 5 min at 24 and 48 h after infection. Cytoplasmic redden ing was never seen in parallel control cells under these conditions. The relative diameters of the bright green fluorescing nuclei of the infected and control CV-1 cultures were measured at 24 and 48 h postinfection. The average diameter of the infected cell nuclei was 27% larger than that of the control nuclei by 24 h and 43% larger by 48 h. For comparison, the acridine orange stain ing patterns of the LLC-MKc line of rhesus monkey kidney cells and of BALB 3T3 cells were also examined. SV40 infection of LLCMK -2 cells results in productive infection ac companied by relatively slow cell killing [8], while infection of 3T3 cells is abortive and the cells are spared. The AOP of the infected LLC-MK> cells were normal at 24 h postinfection and slightly swollen at 48 h postinfection. The AOP of the 3T3 cells were normal at both of these times.
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Cytoplasmic reddening and nuclear swelling were not observed in either of these cell types. Distribution of Acid Phosphatase Histochemical staining for acid phospha tase in fixed control CV-1 cells was localized in discrete cytoplasmic particles (fig. 2a). Sim ilar acid phosphatase-positive particles were also seen in fixed infected CV-i cells at 24 and 48 h (fig. 2b, c). Diffuse acid phosphatase activ ity in the cytoplasm was not detected even as late as 48 h postinfection. Because histochemical staining of unfixed cells might reveal changes in the permeability of lysosomal membranes to substrate as well as enzymes [2], we also observed the staining pattern of unfixed cells. In these studies, one determines whether treated (infected) cells can withstand the same controlled amount of dam age (exposure to substrate under staining con ditions) as control cells before becoming per meable to the substrate [15]. Unfixed control and infected cells at 24 and 48 h postinfection were exposed to the substrate, as described in ‘Materials and Methods’, for 15, 30, and 45 min. After 15 min there was no staining of either infected or control cells. After 30 min about 1% of the infected and control cells showed discrete acid phosphatase-containing particles. By 45 min, acid phosphatase-posi tive particles were seen in all of the control and infected cells (fig. 2d— f). We found no evidence of diffuse acid phosphatase staining in the cytoplasm of unfixed control cells or in fected cells at either 24 or 48 h after infection.
Fig. 2. Fixed and unfixed CV-1 cells stained for acid phosphatase, a Fixed uninfected cells, b Fixed infected cells at 24 h. c Fixed infected cells at 48 h. d Unfixed uninfected cells, e Unfixed infected cells at 24 h. f Unfixed infected cells at 48 h. All plates were photographed at the same magnification.
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SV40 Cytolysis and Lysosomes 51
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Fare o f Pres rained Lysosomes In the studies described above, lysosomes were found to be intact at 24 and 48 h after infection. However, it is possible that new lysosomes continue to be produced during the course of the infection. Consequently, it is possible that the intact lysosomes represent those most recently produced and that ‘older’ lysosomes might have ruptured.
To determine whether old lysosomes re main intact, cultures were stained with neutral red at 24 h postinfection and examined at subsequent times. Neutral red is another basic dye that is concentrated in lysosomes by an energy-dependent process similar to that de scribed above for acridine orange [11], Neutral red was used here because when the neutral red-stained cultures are subsequently stained
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Fig.3. CV-I cellsstained with neutral red and trypan blue, a Uninfected cells, b-d Infected cells at 48 h. All plates were photographed at the same magnification.
with trypan blue, both dyes can be seen using bright-field microscopy. We found that lysosomes stained with neu tral red at 24 h may retain the dye as late as 48 h, even in trypan blue-stainabie cells. Some of these trypan blue-stainable cells appear to have a full complement of stained lysosomes (fig. 3b), while others have many fewer lyso somes (fig.3c). In some of the trypan bluestained cells the neutral red was diffuse, and others appeared to have lost the dye entirely. It might be noted, however, that some of the uninfected control cells, which were unstainable with trypan blue, also failed to retain the neutral red dye. In some of the infected cells the neutral red-stained lysosomes appeared to be grossly swollen (fig. 3d). Surprisingly, all of the cells containing grossly swollen lysosomes were able to exclude trypan blue.
Discussion In studies of lethal cell injury, it is useful to consider cell death to have occurred when the cell is no longer able to recover if the injurious agent is removed. Thus, the moment of cell death is when the injury becomes irre versible. In studies of virus-induced cell injury, this definition of cell death is not directly appli cable in an operational sense. Nevertheless, stainability by trypan blue can be used as a first approximation of the time of cell death because cells undergoing lethal injuries resulting from a variety of causes become stainable with try pan blue at the time that the damage becomes irreversible [6]. Previously, we found that the release of cytoplasmic lactic dehydrogenase virus from cells lytically infected with SV40 somewhat precedes and closely parallels the accumulation of trypan blue-stainable cells
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[15a], Thus, in the arguments that follow, stainability with trypan blue is our criterion for cell death. Events that occur prior to the acquisition of stainability are antemortem and those occurring subsequently are post mortem. Our studies with the vital lysosomotropic dyes, acridine orange and neutral red. indicate that lysosomes in SV40-infected CV-I cells remain as intact structures as late as 48 h after infection when between 40 and 80% of these cells are trypan blue-stainable. These findings are incompatible with the most extreme model for lysosomal involvement in lethal cell injury in which lysosomes rupture and quickly re lease their hydrolytic enzymes into the cyto plasm. Our histochemical studies on the distribu tion of acid phosphatase activity in fixed and unfixed infected cells also fail to support a model of cell killing involving antemortem lysosomal rupture. Histochemical results alone would be inconclusive because apparent enzyme localizations could result from diffu sion of enzyme or reaction product within cells. Furthermore, a certain amount of the enzyme activity is inactivated by fixation, possibly ob scuring released enzyme. Nevertheless, the acid phosphatase and vital dye staining pat terns together provide strong evidence against lysosomal rupture prior to the irreversible stage of cell injury. In an earlier study [3], lysosomal changes in SV40-infected cells were followed utilizing the Gomori metal precipitation technique to stain unfixed cells for acid phosphatase. It was reported that lytic SV40 infection of BSC-1 green monkey kidney cells resulted in a ‘first stage activation' in which the lysosomal mem branes of infected cells at 24 h were more per meable than those of control cells to the sub strate. [3-glycerophosphate. By 72 h there was
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SV40 Cytolysis and Lysosomes
a ‘second stage activation' in which acid phos phatase appeared to escape into the cytoplasm and nucleus, as suggested by a diffuse staining pattern. Using the Burstone method, we saw no difference in the acid phosphatase staining patterns between unfixed control and infected cells during the first 48 h of infection. The reason for the dissimilarity between our results and those of the earlier study is not clear. Regardless, it is now recognized that for light microscopy the use of azo dye-naphthol AS phosphates to demonstrate acid phosphatase is preferred over the Gomori metal precipita tion techniques in which some precipitate for mation is not directly related to sites of enzyme activity [16,17]. The diffuse cytoplasmic and nuclear acid phosphatase staining seen at 72 h in unfixed cells using the Gomori technique [3], if not an artifact induced by the staining con ditions, probably represents a postmortem change, since most cells become trypan bluestainable by 48 h. Previously, we found that SV40 infection of CV-I cells resulted in a redistribution of lysosomal N-acetyl-^-glucosaminidase from a particle bound to a soluble fraction [8]. En zyme redistribution was thought to indicate release into the cytoplasm. Because SV40-induced enzyme redistribution followed the same time course and occurred to the same extent in LLC-MKi cells, which became trypan bluestainable at a much slower rate than the CV-I cells, we suggested that lysosomal enzyme re lease per se is not primarily responsible for cell killing by SV40. While the results reported above support the main conclusion of our earlier study, they also cause us to reconsider whether enzyme release into the cytoplasm actually occurs dur ing the antemortem phase of SV40-induced cell injury. Biochemical studies of cellular homogenates cannot, unfortunately, distin
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guish between actual enzyme release from or ganelles and enzyme release resulting from a change in the resistance of lysosomes to rup ture during homogenization. Changes in lysosome resistance to breakage could reflect a change in lysosome size, a change in the fre quencies of different kinds of lysosomes, or a change in the stability of the lysosomal mem brane. Enzyme redistribution might also reflect the portion of the cell population which has entered the postmortem stages of cell injury. It is clear that uninfected cells secrete lyso somal hydrolases into the media and that extra cellular enzyme accumulation increases as a result of infection [8], The histochemical results reported here suggest that there is no prior accumulation of activity in the cytoplasm. However, enzymes released into the cytoplasm might rapidly diffuse throughout the cell [18] and go undetected by histochemical proce dures. Thus, it is possible that during the ante mortem stage of infection there is a small in crease in lysosomal permeability that cannot be detected by the procedures reported here. If this were so, it would remain unresolved whether small differences in lysosomal leak iness could be responsible for irreversible in jury. Yet it is difficult to envision how lyso somes could concentrate acridine orange, which has a molecular weight of only about 300 daltons, while releasing very much larger enzymes into the cytoplasm. As described above, about 10-15% of the SV40-infected CV-I cells have grossly swollen acridine orange- or neutral red-stained struc tures at 48 h. The cells with the grossly swollen neutral red-stained structures, and presumably those with the swollen AOP, are all able to exclude trypan blue at this time. These grossly swollen stained bodies may be autophagic structures (which functionally may be regarded as secondary lysosomes) [19], as suggested by
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SV40 Cytolysis and Lysosomes
cytoplasmic enzymes and by the shutdown of membrane-associated electron transport [8. 15a]. Enzyme release, which does not appear to be related to virus release, was coincident with declining levels of membrane phospholipid synthesis and preceded the appearance of try pan blue-stainable cells by several hours. These cytopathic effects occur prior to any measur able decline in ATP levels and protein synthe sis. Therefore, we suggest that impaired mem brane metabolism plays an important role in cell killing by SV40. It has been suggested that altered membrane permeability, resulting in impaired cellular and intracellular compart ment volume regulation, is a major determi nant of lethal cell injury in systems involving both viral and nonviral damage [6,21], The antemortem nuclear swelling seen in SV40infected CV-I cells described here, as well as our earlier results, are consistent with this hypothesis. Acknowledgments This investigation was supported in part by Public Health Service grant I ROI Al 14049 from the National Institute of Allergy and Infectious Diseases, by Bio medical Research Support grant RR 07048, and by a grant from the American Cancer Society, Massachu setts Division. Inc. We are gratefid to Cheryl Coguen for her excellent technical assistance.
References 1 Allison, A.C. and Sandclin, K.: Activation of lysosomal enzymes in virus-infected cells and its possible relationship to cytopathic effects. J. exp. Med. 117: 879-887 (1965). 2 Allison, A.C. and Mullucci, L.: Histochemical studies of lysosomes and lysosomal enzymes in virus-infected cell cultures. J. exp. Med. 121: 463476(1965). 3 Allison, A.C. and Black, P. H .: Lysosomal changes in lytic and nonlytic infections with the simian
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an ultrastructure analysis in progress. A strong case has been made for the concept that autophagy represents a cellular survival mecha nism in response to pathologic influences [20], Increased autophagic activity might help to isolate an injured area and prevent the spread of degenerative changes. The accumulation and retention of acridine orange and neutral red by lysosomes is an energy-requiring process [5.9], Energy is prob ably needed to drive a lysosomal proton pump which is necessary for the intralysosomal trap ping of the dye [II]. Therefore, the ability of SV40-infected CV-I cells to concentrate acri dine orange and retain neutral red, after they have become trypan blue-stainable, indicates that irreversible cell damage does not result from a shutdown of the cells’ capacity to pro duce energy. This is in agreement with our previous finding that SV40-infected CV-I cells retain normal levels of ATP well after they become trypan blue-stainable [15a]. Because SV40-infected CV-1 cells show cytoplasmic reddening when exposed to acri dine orange, under conditions in which cyto plasmic reddening is not observed in control cells, the infected cells probably cannot segre gate the dye as rapidly as can the control cells. This might reflect damage to lysosomal mem branes. damage to the lysosomal proton pump, or decreased availability of ATP to drive the pump in infected cells. We do not yet know the sequence of cytopathic events leading to irreversible injury nor do we know the specific action of the virus which sets this sequence in motion. The results reported here suggest that lysosomes do not play an important role in the cell-killing pro cess. Earlier, we reported that lytic SV40 infec tion is characterized by severe dysfunction of cellular membranes as indicated by release into the medium of lysosomal, mitochondrial, and
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5
6
7
8
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vacuolating virus (SV40). J. natn. Cancer Inst. 39: 775-787 (1967). Guskey, L. E .; Smith, P.C., and Wolff, D.A.: Pat terns of cytopathology and lysosomal enzyme re lease in poliovirus-infected HEp-2 cells treated with either 2-(a-hydroxybenzyl)-benzimidazole or gua nidine HCI. J. gen. Virol. 6: 151 —161 (1970). Hawkins, H .K .; Ericsson, J.L .E .: Bibcrficld, P., and Trump, B. F .: Lysosome and phagosome sta bility in lethal cell injury: morphologic tracer stud ies in cell injury due to inhibition of energy metab olism, immune cytolysis and photosensitization. Am. J. Path. 68: 255-288 (1972). Trump, B .F.; Croker, B.P., and Mergner, W .J.: The role of energy metabolism, ion, and water shifts in the pathogenesis of cell injury: in Richter and Scarpelli, Cell membranes: biological and patho logical aspects, pp. 84-128 (Williams & Wilkins, Baltimore 1971). De Harven, E.: Identification of tissue culture contaminants by electron microscopy; in Fogh, Contamination in tissue culture, pp. 205-231 (Aca demic Press, New York 1973). Norkin, L.C. and Ouellette, J.: Cell killing by simian virus 40: variation in the pattern of lysoso mal enzyme release, cellular enzyme release, and cell death during productive infection of normal and simian virus 40-transformed simian cell lines. J. V irol./«: 48-57 (1976). Robbins, E. and Marcus, P. I.: Dynamics of acri dine orange-cell interaction. I. Interrelationships of acridine orange particles and cytoplasmic redden ing. J. Cell Biol. 18: 237-250(1963). Burstone, M.S.: Histochemical comparison of naphthol AS-phosphates for the demonstration of phosphatases. J. natn. Cancer Inst. 20: 601-610 (1958). Dc Duve, C.; de Barsy, T.; Poole, B.; Trouet, A.; Tulkens, P., and van Hoof, F .: Lysosomotropic agents. Biochem. Pharmacol. 23:2495-2531 (1974).
12 Robbins, E.; Marcus, P.I., and Gonatas, N .K .: Dynamics of acridine orange-cell interaction. II. Dye-induced ultrastructural changes in multivesicular bodies (acridine orange particles). J. Cell Biol. 21 .-49-62(1964). 13 Armstrong, J.A .: Histochemical differentiation of nucleic acids by means of induced fluorescence. Expl Cell Res. 11: 640-643 (1957). 14 Bradley, D .F .: Molecular biophysics of dye poly mer complexes. Trans. N.Y. Acad. Sci. 24 : 64-71 (1961). 15 Bitensky, L.: The reversible activation of lysosomes in normal cells and the effects of pathological con ditions; in DeRouck and Cameron, CIBA Sympo sium on Lysosomes, pp.362-384 (Little, Brown, Boston 1963). 15a Norkin, L .C .: Cell killing by simian virus 40: im pairment of membrane formation and function. J. Virol. 2 /: 872-879 (1977). 16 Barka, T. and Anderson, P.J.: Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. J. Histochem. Cytochem. 10: 741-753 (1962). 17 Pearse, A .G .E.: Histochemistry: theoretical and applied, vol. I, chapt. 16 (Churchill, London 1968). 18 Danielli, J .F .: Discussion; in DeRouck and Came ron, Cl BA Symposium on Lysosomes, p. 353 (Little. Brown, Boston 1963). 19 Kerr, J.F .R .: Some lysosome functions in liver cells reacting to sublethal injury: in Dingle and Fell, Lysosomes in biology and pathology, vol. 3, pp. 365-394 (North-Holland, Amsterdam 1969). 20 Ericsson. J. L. E .: Mechanism of cellular autophagy: in Dingle and Fell, Lysosomes in biology and pathology, vol.2, pp. 345-394 (North-Holland, Amsterdam 1969). 21 Vainio, T.; Judah, J.D ., and Bjotvedt, G.: Mech anism of cellular damage by virus: a study of anti histamine drugs. I. Murine hepatitis virus and liver explant cultures. Expl Molec. Path. 1 :15-26 (1962).
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