Transformation of Cells by Rous Sarcoma Virus: Cytoplasmic Vacuol ization ARTRICE V. BADER' AND J O H N P. BADER' 1 Office of Coordinator f o r Ultmstructiiral S t u d i e s a n d 2 C k c m i s t r y B r a n c h , N a t i o l Z ~ ~C l( Z ~ C ~Institute, T N(Zttona1 r71strt2ctf:sof H ( , ( l l t h , B e t k e s d o , M a r y l n n d 2001 4
ABSTRACT Chick embryo cells transformed by the Bryan "high titer" strain of Rous sarcoma virus (RSV-BH) are heavily vacuolated. A variety of microscopic techniques have been used i n demonstrating that the vacuoles are cytoplasmic, bounded by membrane, and are composed largely of water. Proteins, lipids, glycoproteins, glycolipids, glycosaminoglycans, glycogen, and nucleic acids were undetectable i n the vacuoles. Physiological requirements for development of the vacuoles, and reversal of vacuolization, were examined i n cells infected with a virus mutant, RSV-BH-Ta, which induces reversible temperature-dependent transformation. Na+ was the only component of the cell culture medium found essential for both the development and reversal of vacuoles. Glucose depletion or dinitrophenol treatment inhibited vacuolization, suggesting a possible energy requirement i n the vacuolization process. Ouabain, an inhibitor of Na+-K+ATPase, enhanced vacuolization, but a variety of other substances affecting cell surface components were inactive. Two sugars, glucosamine and mannosamine, prevented the disappearance of vacuoles. The observations suggest that cellular vacuolization may be a normal physiological response to a n increase i n water and Na+, and, i n the specific case of transformation by RSV-BH, may be relevant to the physiological basis for malignancy
The Bryan "high titer" strain of Rous accompanying malignant transformation. sarcoma virus (RSV-BH)induces in infected In this light, a study of the vacuoles, and cells a characteristic morphological change the vacuolization process, could reveal spewhich accompanies the transformation of cifk physiological processes involved in these cells to malignant forms. These mor- transformation. phological changes include rounding, an Mutants of RSV-BH have been isolated increase in size, (Golde, '62; Bader et al., which induce temperature dependent trans'74) and intense cytoplasmic vacuolization formation (Bader and Brown, '71). Infected (Haguenau and Beard, '62; Distefano and cells appear nontransformed when grown Dougherty, '65). The increased size has at 41", but are transformed at 37", albeen attributed to an initial increase in though virus production occurs at both the accumulation of intracellular water, temperatures. The morphological changes, followed by a generalized increase in cel- including vacuolization, begin to appear lular mass (Bader et al.,'74). within ten minutes after shifting infected Cytoplasmic vacuolization of cells in cells from 41" to 37" and these changes culture commonly has been attributed to occur without a requirement for DNA, cellular degenerative processes, foreboding RNA, or protein synthesis (Bader, '72). cell death. Contrary to this view, RSV-BH Vacuolization, therefore, precedes and is transformed cells containing numerous independent of several metabolic changes cytoplasmic vacuoles can grow and divide known to accompany transformation, inthrough many generations, with no obvious cluding increased rates of hexose uptake degenerative effects ascribable to the vacu- and hyaluronate synthesis, and may be a oles. Vacuolization, therefore, could be associated with the physiological changes Received April 14, '75. Accepted J u n e 13, '75. J. CELL. PHYSIOL.,8 7 : 3 3 4 6 .
33
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ARTRICE V. BADER AND JOHN P. BADER
direct consequence of a functioning viral gene product. Resolution of factors affecting the development of vacuoles could point to the physiological function of this viral gene product, and perhaps lead to the identification of the primary physiological molecule responsible for malignancy in this system. An examination of the vacuoles of RSVBH infected cells by microscopic and histological techniques is described. By shifting mutant-infected cells from 41 to 37”, and the reverse, an examination of factors affecting the vacuolization process was possible. Components of the cell culture medium have been assessed in the vacuolization process, and several exogenously added substances have been examined for their effects on vacuolization. The results demonstrate that the intracytoplasmic vacuoles of RSV-BH transformed cells are membrane bound organelles composed largely of water, and suggest that vacuoles occur as a cellular response to the increased accumulation of water and Naf. O
MATERIALS AND METHODS
Virus The Bryan “high titer” strain of Rous sarcoma virus (RSV-BH), a transforming virus, is mixed with Rous-associated virus (RAVl), a subgroup A nontransforming avian leukosis virus, and probably a second nontransforming virus, RAVo. The mutant RSV-BH-Ta, also called tdBElBH (Vogt et al., ’74) transforms infected cells at 37 but when placed at 41”, these infected cells revert to normal phenotype. RSV-BHTa stocks also contain RAVl and RAVo, but the presence of these viruses has no effect on the transformation process. O,
Cells and media Chick embryo cells were prepared from 10-day-old embryos, and replated at 2- to 3-day intervals. Cultures were grown in Eagle’s Minimal Essential Medium supplemented with dextrose (2 g/l final concentration), sodium pyruvate (5 mM), 10% tryptose phosphate broth (Difco), 5% fetal bovine serum, penicillin (50 U/ml), streptomycin (50 pglml), tylosine (50 pglml), and gentamycin (20 pglml). Cultures were maintained in humidified, C02- atmosphere incubators at 41” or 37”, as dictated by individual experiments.
Cells were infected (2 to 10 focus-forming units per cell) as secondary cultures within 24 hours after the transfer, and transferred at 2- to 3-day intervals thereafter. Transformation was evident in RSVBH or RSV-BH-Ta infected cells within a day after infection, and practically all cells were transformed within six days after infection. Infected cultures were used for experiments on the second day after replating before cells became confluent. Scanning electron microscopy Cells were grown on 9 X 9 mm2 glass coverslips (Bellco Glass), seeded in 60 mm petri dishes and processed through dehydration in the plates. Prior to fixation, cells were washed twice with phosphate buffered or Tris buffered saline (pH 7.4). Cells were fixed in 3% glutaraldehyde in Sorenson’s buffer (pH 7.4) then washed in phosphate buffered saline or distilled water. Cells were dehydrated in a graded series of ethyl alcohol (70, 90, 95 and 100%) followed by amyl acetate. Samples were then processed by the “critical point” drying method (Anderson, ’51) in a Denton DCP.l apparatus. Coverslips were removed from the dishes and mounted on specimen stubs to be vacuum coated with carbon and palladium on a rotating, tilting stage. Micrographs were taken on a JEOL scanning microscope, model JSM-U3 at 25” tilt and 15 kv. Transmission electron microscopy Cells were washed twice with Tris-buffered saline prior to fixation. Several fixatives were used: 1.5% glutaraldehyde i n cacodylate buffer, 3% glutaraldehyde in Sorenson’s buffer, 3 % glutaraldehyde i n Dulbecco’s phosphate buffered saline, 1% osmium prepared in each of the above buffers, and Dalton’s chrome osmium. Glutaraldehyde as the primary fixative was always followed by post fixation in osmic acid in the corresponding buffer. Cells which were used for sectioning perpendicular to the plane of growth were processed on the plates as cell monolayers. Other cells were scraped from the dishes and pelleted at 3,400 revlmin (International Centrifuge Model CL for 5 min). Cells were stained with a 0.5% aqueous solution of uranyl acetate with sucrose buffered to pH 4.9. Pellets of cells were further processed as previously described
CYTOPLASMIC VACUOLIZATION
Figs. 1
35
Phase contrast micrographs of transformed and non-transformed chick embryo
(CE) cells. ( A ) Cells infected with RSV-BH and grown a t 39O. The cells have no obvious directional orientation and are heavily vacuolated. (B) Non-infected C E cells grown a t 39". Note the difference between these spindle shaped fibroblasts a n d the larger, rounded transformed cells in figure A. (C) Cells infected with RSV-BH-Ta a n d grown a t 37O. The general morphology of these cells is similar to those infected with wild type RSV-BH pictured in figure A. (D) RSVBH-Ta infected cells grown a t 41". Note the absence of vacuoles and general resemblance of these cells to the non-infected CE cells shown i n B. Magnification, approximately x 380.
(Valentine and Bader, '68). Grids were examined in an AEI-801 electron microscope.
Histology Formalin (10% ), ethanol (95% ), or glutaraldehyde (1.5%) was added directly to cells in petri dishes. Selection of the fixative was determined by the staining procedure to be used. The cells were processed further by the Pathological Technology Section of the National Cancer Institute.
The following staining procedures were employed (Sanders, '72) to test for the indicated macromolecules: PAS, McManus: glycogen, mucin, fibrin and collagen. PAS, Alcian Blue: acid and neutral mucopolysaccharides, mucin. Oil Red 0: lipids. Sudan Black B: lipids. Giemsa: nucleic acids, proteins. Stains-all, pH 7.0: nucleic acids, proteins, glycosaminoglycans.
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ARTRICE V. BADER AND JOHN P. BADER
Pyronin Y and Azure B: nucleic acids, proteins. After staining, the cells were immersed in either water or ethanol and observed under light and phase contrast optics. RESULTS
Phase contrast microscopy Cells transformed by RSV-BH contained refractile vacuoles throughout the cytoplasm under conditions in which growth and division of cells fluorished (fig. 1A). Noninfected CE cells (fig. 1B) contained virtually no vacuoles under the same growth conditions. Cells infected with the mutant virus, RSV-BH-Ta, contained vacuoles at 37" (fig. 1C) but not at 41" (fig. 1D). Development of vacuoles could be detected within ten minutes after shifting RSV-BH-Ta infected cells from 41 " to 37" and vacuoles arose in the perinuclear region of the cytoplasm.
Scanning microscopy Transformed and nontransformed cells were viewed with a scanning electron microscope in order to investigate possible differences in surface structure. During the scanning of RSV-BH cells areas of the surface were observed to collapse, leaving craters in the cell surface (fig. 2). This phenomenon suggested that the vacuoles, as viewed in phase contrast microscopy, were composed of a volatile substance which vaporized during the scanning process. The transition from smooth to cratered surface also indicated that the vacuoles were something other than mere invaginations of cell surface into the cytoplasm. Such cratering was not observed in noninfected CE cells. Transmission electron microscopy Vacuolated cells after fixation, staining and sectioning were examined by trans-
I Figs. 2 Scanning electron micrographs of non-infected CE cells a n d cells transformed by RSV-BH. (A) Non-infected cells showing cell processes a n d smooth surfaces. (B) Cells infected with RSV-BH. Numerous "craters" appear in the cell surface during viewing of the cells in the microscope, indicating evaporation from the vacuoles of a volatile substance. Magnification, approximately x 2,000 a n d x 1,500 respectively.
CYTOPLASMIC VACUOLIZATION
mission electron microscopy. Sections cut perpendicularly to the plane of the plastic substratum revealed intracytoplasmic vacuoles (fig. 3 ) , eliminating the possibility that entrapment of some substance between the cell and the substratum was responsible for the noted refractility and craterization. In addition, these perpendicular sections, as well as random sections through cells fixed while attached to the substratum, or cells suspended before fixing, all revealed vacuoles bounded by membrane (figs. 4, 5). Vacuoles were rarely found in noninfected CE cells, and sections from these cells were easily distinguishable, because of the absence of vacuoles, from those of transformed cells in the electron microscope. The cell section seen in figure 6 is typical of non-infected CE cells. The development of vacuoles was examined after shifting RSV-BH-Ta infected cells from 41 to 3 7 " . Within minutes numerous small membrane-bound vacuoles could be detected, and these increased in size and number with time, consistent with observations using phase microscopy (Bader, '72). Resolution of the bounding membrane was more difficult in larger vacuoles. Vacuoles may increase in size by accumulation of intravacuolar substance, a suggestion supported by phase microscopy viewing of RSV-BH-Ta cells undergoing the vacuolization process. However, the occasional development of large vacuoles in cells which earlier contained numerous smaller vacuoles, suggests that coalescence of vacuoles may occur, and increase in the size of vacuoles may occur by this mechanism as well. The origin of the vacuoles could not be resolved by direct viewing in the electron O
Fig. 3 This figure a n d figures 4-8 are micrographs obtained from transmission electron microscopy. Cells were fixed with glutaraldehyde, followed by post fixation i n buffered osmium tetroxide. Sections were doubly stained with alcoholic uranyl acetate and lead citrate. In this figure a section from a RSV-BH transformed cell cut perpendicul a r l y to the plane of the plastic substratum is shown. Vacuoles typical of cells transformed by this virus (vac) a r e seen throughout the length of the cell. One vacuole appears to be causing a n identation of the nucleus ( N a n d arrow). Virus particles (VP) are found in the extracellular space adjacent to the surface membrane, upper left corner of micrograph. Bar, 1 p m . Approximately X 6,000.
37
microscope. In heavily vacuolated cells, most mitochondria remained intact with no obvious aberrations, although occasional mitochondria were found disrupted (fig.
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ARTRICE V. BADER AND JOHN P. BADER
Fig. 4 Section from chick embryo cells transformed by RSV-BH-Ta and maintained at 37’. Vacuoles of variable size, typical of RSV-BH transformed cells, are shown (VAC). Most mitochondria (M) seem unaffected. although a few appear to be disintegrating (D). Vacuoles in close proximity to one another are found (arrows) a n d intervacuolar membranes a r e apparent (arrow a). A phagocytic vacuole (PV) also is present. Virus particles are seen i n extracellular spaces (VP) and a n occasional particle is seen within a vacuole. Bar, 1 p m . Approximately X 14,000.
4). A suggestion that membranes of the Golgi apparatus were involved in vacuolization of neurons (Whetsell and Bunge, ’69) led us to examine such membranes. No obvious changes in Golgi membranes were found in heavily vacuolated cells (fig. 7). The vacuoles contained no obvious substance, being devoid of any electron-dense material. This lack of substance distinguished the vacuoles from phagocytic vacuoles, which often contained virus particles as well as other unidentifiable matter (fig. 4).
Histological staining A variety of histological techniques was used in attempts to reveal molecular con-
stituents of vacuoles. These included fixing cells in formaldehyde, glutaraldehyde, or ethanol, and staining with oil Red 0, sudan black, PAS (McManus), PAS (Alcian Blue), Giesma, pyronin Y plus Azure B, or “Stains-all.” In all cases, the vacuoles remained unstained, indicating the absence of lipids, glycolipids, proteins, glycoproteins, glycosaminoglycans, glycogen, and nucleic acids. Fig. 5 Section from RSV-BH-Ta transformed cells ( 3 7 ” ) at higher magnification ( X 56,000) showing the membrane of the vacuole. The tripartite structure (Arrow A) typical of biological membranes is seen. Arrow B illustrates a small vacuole i n close proximity to a larger one where the increased thickness indicates apposed membranes. Bar, 0.1 p m .
CYTOPLASMIC VACUOLIZATION
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ARTRICE V. BADER AND JOHN P. BADER
Fig. 6 Electron micrograph of non-infected CE cells. Cellular organelles: Nucleus (N), mitochondria (MI, and endoplasmic reticulum (ER). This cell section differs from those seen in figures 3 a n d 4 by the absence of vacuoles which are typical of transformation by RSV-BH or RSV-BH-Ta. Bar, 1 p m . Approximately x 15,000.
Uptake of neutral red When neutral red (0.01%) was added to the cell culture medium, the dye was rapidly taken into cells and within ten minutes could be found in the vacuoles of transformed cells. Two kinds of vacuoles could be distinguished on the basis of the color of the neutral red. The predominant type was reddish-brown, the color of the dye in the medium at pH 7.4. Only this type was found within the first few hours after shifting RSV-BH-Ta cells from 41 to 37", or after shifting for longer intervals in the presence of cycloheximide or Actinomycin D. RSV-BH transformed cells, or RSV-BHTa cells incubated for extended times at 37" both contained other vacuoles staining purple with neutral red. These probably represent lysosomes, or some other acidic organelle. In any case, the incorporation of neutral red into the vacuoles demonstrates that, whatever else the vacuoles O
may contain, water is a major component. The rapid uptake of neutral red into preexisting intracellular vacuoles, and the perinuclear location of developing vacuoles, suggested that vacuoles developed de novo in the cytoplasm. Nonetheless, it was possible that rapid pinocytosis, and accumulation and coalescence of miniscule vesicles, could result in vacuolization. To examine this possibility, inulin-*4C, which cannot penetrate the membrane barrier into the cytoplasm, was added to RSV-BH-Ta cells during the development of vacuolization after shifting from 41 to 37". No difference in bound radioactivity was found between these cells and those maintained at 4 1 suggesting that possible differences in pinocytotic properties are not responsible for vacuolization. O
O ,
Effects of temperature and p H Earlier studies had shown that vacuoli-
41
CYTOPLASMIC VACUOLIZATION
vacuolization developed below pH 6.8, and vacuolization increased with increasing pH. Also, RSV-BH-Ta cells maintained at 41" became vacuolated if exposed to alkaline medium (pH 7.8) for several hours, and the degree of vacuolization increased with extended incubation.
Fig. 7 Portion of a transformed cell showing numerous vacuoles of different sizes in t h e Golgi region of the cell. No obvious changes in the Golgi membranes a r e apparent. Bar 1 p m . Approximatel y x 24,000.
zation was detectable within ten minutes after shifting RSV-BH-Ta infected cells from 40.5 O to 36 O . Greater consistency was found using 41" as the transformation nonpermissive temperature, and an optimum lower temperature for transformation was examined. Also, cells in which the medium had become acidic appeared less vacuolated than cells in alkaline medium, and the effect of pH over the physiological range was examined. The combined results show that vacuolization is both a temperature-dependent and pH-dependent process (fig. 8). Vacuolization was found earlier, and by six hours had progressed to a greater extent, in cells incubated at 37" than at 39 36", 34 ', or 22 , demonstrating an optimal temperature for vacuolization in this system. Both the rate and extent of vacuolization was dependent on pH over the pH range 6.6 to 7.6 (fig. 8). Little O ,
Components of the medium The components of the cell culture medium were examined for their essentiality in the vacuolization process. Removal of serum enhanced vacuolization slightly, but deletion of tryptose phosphate broth, antibiotics, amino acids, or vitamins had no obvious effect on the development of vacuoles in RSV-BH-Ta cells (Bader and Bader, '74). Glucose could also be removed without any immediate effect on vacuolization. Thus, vacuoles could develop in the b d anced salt solution (BSS) containing Na+, K+, Mg++,Ca++,C1-, SO;, HCO,, and PO;. Salt solutions deficient in various anions and cations were then examined. None of the anions were found essential for vacuolization, although C1- could not be examined satisfactorily. Of the cations (table l),Ca++could be eliminated without effect, but in the absence of Mg++,less vacuolization was observed. Deletion of K+ had a minor enhancing effect which could be augmented by preincubating cells at 41" in K+-deficient medium and changing the medium again before shifting cultures to 37". TABLE 1
Eflects ofdeletion of ion5 f r o m B S S on development or reverrul Of7NtC710~22t~tton
Temperature shifts 4lo+37O
Earle's BSS complete Minus SO4 Minus HC09Minus PO,+ Minus N a + plus sucrose Minus Na+ plus Li+ Minus Na+ plus choline+ Minus K+ Minus Mg++ Minus C a t +
O
1
Degree of vacuolization
+ + + I
37'-41'
0
+++ +++ +++ ++++
++++
0
0
+++ ++++ ++ +++
0
0 0
+++ + 0 0
42
++++
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ARTRICE V. BADER AND JOHN P. BADER
+++
++
+
I
- +-&--& -4v 6.6
7.0
/
7.4
I 7.8
pH OF MEDIUM Fig. 8 Effects of pH a n d temperature on vacuolization. Growth medium buffered with phosphate a n d bicarbonate w a s adjusted with HCl or NaOH a n d equilibrated to the appropriate p H in carbon-dioxide incubators. The medium was applied to RSV-BH-Ta infected cells grown a t 41° and containing no vacuoles. Cultures were then placed i n incubators at indicated temperatures. Six hours later cultures were examined for degree of vacuolization ( , r a r e vacuolated cells; , 1 to 10% of the cells vacuolated; 10 to 50% vacuolated; greater t h a n 50% vacuolated; intermediate values were given to cultures where vacuolization appeared greater t h a n in cultures at lower pH and less t h a n i n cultures of higher pH, incubated at the s a m e temperature). The pH varied by less t h a n 0.1 unit during the course of the experiment
+ + + +,
++
Eventually, it was found that a solution containing Na+ as the only cation (0.15 M NaCl 0.027 M, NaHC03) supported the development of vacuoles. Replacement of the Na+ in BSS with isoosmotic sucrose resulted in the appearance of vacuoles at 41 O as well as 3 7 " , and to a lesser extent induced vacuoles in noninfected CE cells as well. However, addition of sucrose (0.1 M) to the Na+-containing salt solution also induced vacuolization in noninfected CE cells a s well as in RSV-BH-Ta infected cells maintained a t 41". The relation of these sucrose-induced vacuoles to the vacuolization occurring during transformation is unknown. Replacement of Na+ with Li+ had an o p posite effect to that of sucrose; no vacuoles
+
+ + +,
+
appeared either at 41" or 37". When choline chloride was used as a cationic substitute for Na+, vacuolization equivalent to Na+-containing medium was found. In contrast to sucrose, cholinef did not induce vacuolization in noninfected CE cells or RSV-BH-Ta cells at 41 O. The effects of ion-deficient media on the reversibility of vacuolization upon shifting RSV-BH-Ta infected cells from 37" to 41 O also was examined. Reversal of vacuolization occurred in the simple NaC1-NaHC03 solution. Although choline+ could substitute for Naf in the development of vacuoles, vacuoles which developed in the presence of choline+ were retained after shifting cells to 41 O . These results suggested that the development and loss of vacu-
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Temperature shifts Medium Additives
BSS Minus glucose Minus glucose Minus glucose BSS BSS BSS BSS BSS BSS BSS BSS BSS BSS BSS BSS
4I0+37O
I
+ + + 2
++ + ++ ++ +++ +++ +++ ++ +++ +++ +++ ++++ +++ +++ +++
deoxyglycose glucosamine deoxyglucose glucosamine added glucose glutamine fructose glutamine fructose N-acetylglucosamine mannose mannosamine galactose galactosamine fucose
+
37O-41'
0 0 0
++ ++++ 0 ++ 0
0 0
0 0
++++ 0 0 0
1 All additives at 1 0 - 2 M . Culture medium was replaced with medium containing additive one hour before shifting temperatures. Additional cultures containing additives were kept at the original temperature. None of these differed from nontreated controls maintained at the same temperature. 2 Degree of vacuolization.
oles occurring under the usual culture conditions was specifically a Na--dependent process.
Brief incubation of RSV-BH-Ta cells at 37 with mannosamine or glucosamine, and to a lesser extent glutamine, prevented the reversal of vacuolization (table 2) upon Glucose requirement shifting to 41". Although the RSV-BH-Ta Although Na+ was the only medium cells changed from rounded to elongated component immediately required for vacu- shapes, and appeared to become smaller olization, a delayed requirement for glucose after shifting temperatures, the vacuoles could also be shown (table 2). RSV-BH-Ta persisted. None of the other sugars affected cells preincubated at 41 in glucose-defi- the reversal of vacuolization. cient medium for two hours or longer beDinitrophenol, an uncoupler of oxidative came less vacuolated than controls after phosphorylation, was examined for possible shifting to 37". Slight inhibition by inclu- effects on vacuolization, or loss of vacuoles sion of deoxyglucose (10 mm) in glucose- after shifting vacuolated cells to the higher containing medium was also found; deoxy- temperature. A minor inhibitory effect glucose is known to interfere with glucose on vacuolization was observed with 2, 4metabolism in addition to affecting other dinitrophenol at concentrations of 1 mM metabolic properties. The addition of de- or greater. No effect on the reversal of vacoxyglucose to glucose-deficient medium uolization was seen. was even more effective in preventing vacuAdenine and derivatives olization, although within a few hours toxic effects were clearly evident. Several A suggestion that adenosine triphosphate other sugars in addition to 2-deoxyglucose (ATP) when added to erythrocytes could were examined for possible effect on the affect the rigidity of the surface membrane development of vacuolization (table 2). No (Weed et al., '69; Paleck et al., '71) led us marked effects were found with glucosa- to examine the effect of this substance on mine, fructose, N-acetyl-glucosamine, man- transformation. After observing a slight nose, galactose, galactosamine, fucose, inhibitory effect of exogenous ATP on vacor increased amounts of glucose. Although uolization, other adenine derivatives were neither fructose nor glutamine alone af- examined (table 3). The nucleoside derivafected vacuolization the two together in- tives of adenine (adenosine, B'deoxyadenohibited slightly the development of vacuoles. sine, 3'deoxyadenosine), and the adenosine Mannosamine enhanced vacuolization. analogue 2, 6-diamino-purine riboside, O
O
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ARTRICE V. BADER AND JOHN P. BADER
teristic of Rous-transformed cells by many investigators (Bader et al., '74; Tenenbaum and Doljanski, '43; Haguenau and Beard, '62; DiStefano and Dougherty, '65). Until Temperature shifts now, no serious attempts to characterize Additives 41O-37' 37O+4I0 these vacuoles, or to examine factors affecting vacuolization, have been reported. + + + Z 0 None We feel that an understanding of the na+ 0 Adenosine ture of vacuolization will be invaluable to +++ 0 Adenine +++ 0 AMP a determination of physiological events +++ 0 ADP associated with transformation to malig+ + + 0 ATP nancy by Rous sarcoma virus. These phys+ + + 0 3'5' cyclic AMP iological events may not be involved in +++ 0 dibutyryl cyclic AMP + 0 2'deoxyadenosine malignant transformation induced by other + 0 3'deoxyadenosine means, or by other viruses, perhaps not 2,6 diaminopurine even other strains of Rous sarcoma virus. ++ 0 riboside As an example, perhaps, cells infected with 1 All Additives at lo-:! M. the Schmidt-Ruppin strain of Rous sarcoma 2 Degree of vacuolization. virus usually are nonvacuolated, and easily were the most potent inhibitors of vacuoli- can be distinguished from RSV-BH transformed cells on the basis of morphology zation; ATP, ADP, AMP, and adenine were alone (Bader et al., '74). Several other much less effective. Guanosine, uridine, cytidine, and their deoxy-derivatives failed biophysical properties of Schmidt-Ruppin transformed cells differ from those of RSVto inhibit or enhance vacuolization or affect BH transformed cells (Bader et al., '74). the reversal of vacuolization (table 3). Their These biophysical differences may be a phosphorylated derivatives were equally reflection of basic physiological differences ineffective. The preference of cells for in- induced by the viruses, although the outcorporation of nucleosides over incorpora- come in both cases is transformation to tion of phosphorylated derivatives or free malignancy. bases suggests that the effect of adenosine An increased accumulation of water is is intracellular and may be unrelated to found in RSV-BH transformed cells (Bader the effect of exogenous ATP on erythro- et al., '74), and vacuolization may be a cytes. cellular response to this increase. Cells commonly take in Na+ with water from Effects of other substances physiological solutions. The failure of Li+ Ouabain (10-5 M and higher concen- to replace Na+ in the formation of vacuoles trations) an inhibitor of Na+-K+-ATPase, suggest that this process, and its reversal enhanced the rate of vacuolization of RSV- may be Na+-specific. Although choline' BH-Ta cells after shifting from 41 to 37" could substitute for Na+ in the developand induced vacuoles in cells held at 41 ment of vacuoles, these choline+-vacuoles Several other substances reported to have failed to revert. Slight enhancement of the effects on surfaces or other structural com- vacuolization process was noted using soluponents of cells also were examined for tions deficient in K+. This result differs effects on vacuolization. These include di- from results of earlier experiments where methyl sulfoxide, dextran sulfate, aqueous no attempt was made to deplete cells of agar extract, vinblastine sulfate, colchi- residual K+ (Bader and Bader, '74). In adcine, colcemid, hyaluronic acid and hyal- dition, the cardiac glycoside, ouabain, inuronidase. None of these significantly creased the rate of the development of affected either the development or the re- vacuoles, and induced vacuolization in versal of the vacuolization process. non-vacuolated cells. Ouabain is an effective inhibitor of Na+-K+ATPase, the enDISCUSSION Vacuolization of cells transformed by zyme responsible for the cellular incorpoRous sarcoma virus was observed some ration of K+. The incorporation of K+ into fifty years ago by Carrel ('25), and since the cell is coupled to the expulsion of Na+. that time has been reported as a charac- In K+-deficient medium, or in ouabain1
O
O .
CYTOPLASMIC VACUOLIZATION
containing medium, the transport of Na+ from the cell would be delayed, and increased vacuolization could result from the relative increase in Na+. The specificity for Na+ in vacuolization and its reversal, and the induction of vacuolization by ouabain, suggest that (Na+-K+)-ATPasemay be involved in this process. This enzyme plays such a key role in cation and osmotic regulation that i t is hard to ignore in studies where specificities for monovalent cations and differences in accumulation of intracellular water have been demonstrated. The effects of glucosamine and mannosamine in preventing the reversal of vacuolization are difficult to interpret. Possibly these substances inhibit enzymes involved in the dissolution of vacuolar membranes, or are otherwise involved in glycoprotein or glycolipid metabolism related to the vacuoles. Vacuoles have been described in many kinds of cells under a variety of circumstances. Of particular interest, are those induced in rodent neurons after exposure to ouabain, where an involvement of the Golgi region was implicated in the vacuolization process (Whetsell and Bunge, ’69). The early appearance of vacuoles in the perinuclear region of RSV-BH-Ta transformed cells indicated that the Golgi region may be affected, but an electron microscopic examination of this area revealed no obvious connection. Whether the vacuoles represent specialized organelles involved in maintaining ionic or hydrostatic equilibria, or are organelles whose primary function is in some other area of metabolism, has not been resolved. Mitochondria were not grossly affected during the vacuolization process, and nothing unusual is noted among mitochrondria of vacuolated cells. The failure to detect increased uptake of inulin 14C into vacuolating cells, and the perinuclear location of developing vacuoles suggests that pinocytosis at the cell surface membrane is not responsible for vacuolization, although more definitive experiments to resolve this possibility are in progress. Resolution of the origin of vacuoles may require separation and isolation of vacuoles away from other cellular organelles, and characterization of their molecular constituents. Preliminary attempts to isolate
45
vacuoles in this laboratory have been unsuccessful, due mainly to an extraordinary instability of vacuoles in physiological buffers. ACKNOWLEDGMENTS
The authors acknowledge the excellent technical contributions of Monica Bigelow, Nancy R. Brown and Joan Kondratick in the performance of these experiments, and the cooperation of Dr. Bruce Wetzel in the processing of samples for scanning microsCOPY. LITERATURE CITED Anderson, T. F. 1951 Techniques for the preservation of 3 dimensional structure in preparing specimens for the electron microscope. Transac. N. Y. Acad. Sci., 13: 130-134. Bader, J. P. 1972 Temperature-dependent transformation of cells infected with a mutant of Bryan Rous sarcoma virus. J . Viral., 10- 267276. Bader, J. P., and A. V. Bader 1974 Characteristics of cells transformed by Bryan Rous sarcoma virus, i n Mechanisms of Virus Desease. W. S . Robinson and C. F. Fox, eds. W. A. Benjamin, Inc. pp. 3 0 L 3 1 3 . Bader, J. P., and N. R. Brown 1971 Induction of mutations in an RNA tumour virus by a n analogue of a DNA precursor. Nature N. Biol., 234; 11-12. Bader, J. P., D. A. Ray and N. R. Brown 1974 Accumulation of water during transformation of cells by a n avian sarcoma virus. Cell, 3: 309-31 5. Carrel, A . 1925 Effets de l’extract de sarcomes fusocellularies sur des cultures pures de fibroblasts. Comptes Rendus Acad. Sci. Paris, 92: 477. DiStefano, H. S., and R. M. Dougherty 1965 Cytological observations of “nonproducer” R o u s sarcoma cells. Virology, 27. 360-377. Golde, A. 1962 Chemical changes in chick embryo cells infected with Rous sarcoma virus i n vitro. Virology, 1 6 : 9-20. Haguenau, F., and J. W. Beard 1962 The avian sarcoma-leukosis complex; its biology and ultrastructure. I n : Ultrastructure i n biological systems. I. Tumors Induced by Viruses; Ultrastruct u r d Studies. A. J. Dalton and F. Haguenau, eds. Academic Press, New York, pp. 1-59. Paleck, J., W. A. Curby a n d F. G. Lionetti 1971 Effects of calcium and adenosine triphosphate on volume of human red cell ghosts. Amer. J. Physiol., 220: 19-26. Sanders, B. J . 1972 Animal histology procedures of the Pathological Technology Section of the National Cancer Institute, DHEW Public No. (NIH)72-275, U.S. Dept. of HEW, PHS, NIH. Tenenbaum, E., and L. Doljanski 1943 Studies of Rous sarcoma cells cultivated in vitro. I. Cellular composition of pure cultures of Rous sarcoma cells. Cancer Res., 3: 5 8 S 6 0 3 . Valentine, A. F., and J. P. Bader 1968 Produc-
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ARTRICE V . BADER AND JOHN P. BADER
tion of virus by mammalian cells transformed by Rous sarcoma and murine sarcoma viruses. J . Virol., 2; 224-237. Vogt, P. K., R. A. Weiss and H . Hanafusa 1974 Proposal for numbering mutants of avian leukosis and sarcoma viruses. J. Virol., 13: 551-554. Weed, R. I., P. L. LaCelle, E. W. Merrill, G . Craib,
A. Gregory, F. Karch and F. Pickens 1969 Metabolic dependence of red cell deformability. J. Clin. Invest., 48: 7 9 S 8 0 9 . Whetsell, W. O., and R. P. Bunge 1969 Reversible alterations in the Golgi complex of cultured neurons treated with an inhibitor of active N a and K transport. J. Cell Biol., 42: 490-500.
ABSTRACT Chick embryo cells transformed by the Bryan "high titer" strain of Rous sarcoma virus (RSV-BH) are heavily vacuolated. A variety of microscopic techniques have been used i n demonstrating that the vacuoles are cytoplasmic, bounded by membrane, and are composed largely of water. Proteins, lipids, glycoproteins, glycolipids, glycosaminoglycans, glycogen, and nucleic acids were undetectable i n the vacuoles. Physiological requirements for development of the vacuoles, and reversal of vacuolization, were examined i n cells infected with a virus mutant, RSV-BH-Ta, which induces reversible temperature-dependent transformation. Na+ was the only component of the cell culture medium found essential for both the development and reversal of vacuoles. Glucose depletion or dinitrophenol treatment inhibited vacuolization, suggesting a possible energy requirement i n the vacuolization process. Ouabain, an inhibitor of Na+-K+ATPase, enhanced vacuolization, but a variety of other substances affecting cell surface components were inactive. Two sugars, glucosamine and mannosamine, prevented the disappearance of vacuoles. The observations suggest that cellular vacuolization may be a normal physiological response to a n increase i n water and Na+, and, i n the specific case of transformation by RSV-BH, may be relevant to the physiological basis for malignancy
The Bryan "high titer" strain of Rous accompanying malignant transformation. sarcoma virus (RSV-BH)induces in infected In this light, a study of the vacuoles, and cells a characteristic morphological change the vacuolization process, could reveal spewhich accompanies the transformation of cifk physiological processes involved in these cells to malignant forms. These mor- transformation. phological changes include rounding, an Mutants of RSV-BH have been isolated increase in size, (Golde, '62; Bader et al., which induce temperature dependent trans'74) and intense cytoplasmic vacuolization formation (Bader and Brown, '71). Infected (Haguenau and Beard, '62; Distefano and cells appear nontransformed when grown Dougherty, '65). The increased size has at 41", but are transformed at 37", albeen attributed to an initial increase in though virus production occurs at both the accumulation of intracellular water, temperatures. The morphological changes, followed by a generalized increase in cel- including vacuolization, begin to appear lular mass (Bader et al.,'74). within ten minutes after shifting infected Cytoplasmic vacuolization of cells in cells from 41" to 37" and these changes culture commonly has been attributed to occur without a requirement for DNA, cellular degenerative processes, foreboding RNA, or protein synthesis (Bader, '72). cell death. Contrary to this view, RSV-BH Vacuolization, therefore, precedes and is transformed cells containing numerous independent of several metabolic changes cytoplasmic vacuoles can grow and divide known to accompany transformation, inthrough many generations, with no obvious cluding increased rates of hexose uptake degenerative effects ascribable to the vacu- and hyaluronate synthesis, and may be a oles. Vacuolization, therefore, could be associated with the physiological changes Received April 14, '75. Accepted J u n e 13, '75. J. CELL. PHYSIOL.,8 7 : 3 3 4 6 .
33
34
ARTRICE V. BADER AND JOHN P. BADER
direct consequence of a functioning viral gene product. Resolution of factors affecting the development of vacuoles could point to the physiological function of this viral gene product, and perhaps lead to the identification of the primary physiological molecule responsible for malignancy in this system. An examination of the vacuoles of RSVBH infected cells by microscopic and histological techniques is described. By shifting mutant-infected cells from 41 to 37”, and the reverse, an examination of factors affecting the vacuolization process was possible. Components of the cell culture medium have been assessed in the vacuolization process, and several exogenously added substances have been examined for their effects on vacuolization. The results demonstrate that the intracytoplasmic vacuoles of RSV-BH transformed cells are membrane bound organelles composed largely of water, and suggest that vacuoles occur as a cellular response to the increased accumulation of water and Naf. O
MATERIALS AND METHODS
Virus The Bryan “high titer” strain of Rous sarcoma virus (RSV-BH), a transforming virus, is mixed with Rous-associated virus (RAVl), a subgroup A nontransforming avian leukosis virus, and probably a second nontransforming virus, RAVo. The mutant RSV-BH-Ta, also called tdBElBH (Vogt et al., ’74) transforms infected cells at 37 but when placed at 41”, these infected cells revert to normal phenotype. RSV-BHTa stocks also contain RAVl and RAVo, but the presence of these viruses has no effect on the transformation process. O,
Cells and media Chick embryo cells were prepared from 10-day-old embryos, and replated at 2- to 3-day intervals. Cultures were grown in Eagle’s Minimal Essential Medium supplemented with dextrose (2 g/l final concentration), sodium pyruvate (5 mM), 10% tryptose phosphate broth (Difco), 5% fetal bovine serum, penicillin (50 U/ml), streptomycin (50 pglml), tylosine (50 pglml), and gentamycin (20 pglml). Cultures were maintained in humidified, C02- atmosphere incubators at 41” or 37”, as dictated by individual experiments.
Cells were infected (2 to 10 focus-forming units per cell) as secondary cultures within 24 hours after the transfer, and transferred at 2- to 3-day intervals thereafter. Transformation was evident in RSVBH or RSV-BH-Ta infected cells within a day after infection, and practically all cells were transformed within six days after infection. Infected cultures were used for experiments on the second day after replating before cells became confluent. Scanning electron microscopy Cells were grown on 9 X 9 mm2 glass coverslips (Bellco Glass), seeded in 60 mm petri dishes and processed through dehydration in the plates. Prior to fixation, cells were washed twice with phosphate buffered or Tris buffered saline (pH 7.4). Cells were fixed in 3% glutaraldehyde in Sorenson’s buffer (pH 7.4) then washed in phosphate buffered saline or distilled water. Cells were dehydrated in a graded series of ethyl alcohol (70, 90, 95 and 100%) followed by amyl acetate. Samples were then processed by the “critical point” drying method (Anderson, ’51) in a Denton DCP.l apparatus. Coverslips were removed from the dishes and mounted on specimen stubs to be vacuum coated with carbon and palladium on a rotating, tilting stage. Micrographs were taken on a JEOL scanning microscope, model JSM-U3 at 25” tilt and 15 kv. Transmission electron microscopy Cells were washed twice with Tris-buffered saline prior to fixation. Several fixatives were used: 1.5% glutaraldehyde i n cacodylate buffer, 3% glutaraldehyde in Sorenson’s buffer, 3 % glutaraldehyde i n Dulbecco’s phosphate buffered saline, 1% osmium prepared in each of the above buffers, and Dalton’s chrome osmium. Glutaraldehyde as the primary fixative was always followed by post fixation in osmic acid in the corresponding buffer. Cells which were used for sectioning perpendicular to the plane of growth were processed on the plates as cell monolayers. Other cells were scraped from the dishes and pelleted at 3,400 revlmin (International Centrifuge Model CL for 5 min). Cells were stained with a 0.5% aqueous solution of uranyl acetate with sucrose buffered to pH 4.9. Pellets of cells were further processed as previously described
CYTOPLASMIC VACUOLIZATION
Figs. 1
35
Phase contrast micrographs of transformed and non-transformed chick embryo
(CE) cells. ( A ) Cells infected with RSV-BH and grown a t 39O. The cells have no obvious directional orientation and are heavily vacuolated. (B) Non-infected C E cells grown a t 39". Note the difference between these spindle shaped fibroblasts a n d the larger, rounded transformed cells in figure A. (C) Cells infected with RSV-BH-Ta a n d grown a t 37O. The general morphology of these cells is similar to those infected with wild type RSV-BH pictured in figure A. (D) RSVBH-Ta infected cells grown a t 41". Note the absence of vacuoles and general resemblance of these cells to the non-infected CE cells shown i n B. Magnification, approximately x 380.
(Valentine and Bader, '68). Grids were examined in an AEI-801 electron microscope.
Histology Formalin (10% ), ethanol (95% ), or glutaraldehyde (1.5%) was added directly to cells in petri dishes. Selection of the fixative was determined by the staining procedure to be used. The cells were processed further by the Pathological Technology Section of the National Cancer Institute.
The following staining procedures were employed (Sanders, '72) to test for the indicated macromolecules: PAS, McManus: glycogen, mucin, fibrin and collagen. PAS, Alcian Blue: acid and neutral mucopolysaccharides, mucin. Oil Red 0: lipids. Sudan Black B: lipids. Giemsa: nucleic acids, proteins. Stains-all, pH 7.0: nucleic acids, proteins, glycosaminoglycans.
36
ARTRICE V. BADER AND JOHN P. BADER
Pyronin Y and Azure B: nucleic acids, proteins. After staining, the cells were immersed in either water or ethanol and observed under light and phase contrast optics. RESULTS
Phase contrast microscopy Cells transformed by RSV-BH contained refractile vacuoles throughout the cytoplasm under conditions in which growth and division of cells fluorished (fig. 1A). Noninfected CE cells (fig. 1B) contained virtually no vacuoles under the same growth conditions. Cells infected with the mutant virus, RSV-BH-Ta, contained vacuoles at 37" (fig. 1C) but not at 41" (fig. 1D). Development of vacuoles could be detected within ten minutes after shifting RSV-BH-Ta infected cells from 41 " to 37" and vacuoles arose in the perinuclear region of the cytoplasm.
Scanning microscopy Transformed and nontransformed cells were viewed with a scanning electron microscope in order to investigate possible differences in surface structure. During the scanning of RSV-BH cells areas of the surface were observed to collapse, leaving craters in the cell surface (fig. 2). This phenomenon suggested that the vacuoles, as viewed in phase contrast microscopy, were composed of a volatile substance which vaporized during the scanning process. The transition from smooth to cratered surface also indicated that the vacuoles were something other than mere invaginations of cell surface into the cytoplasm. Such cratering was not observed in noninfected CE cells. Transmission electron microscopy Vacuolated cells after fixation, staining and sectioning were examined by trans-
I Figs. 2 Scanning electron micrographs of non-infected CE cells a n d cells transformed by RSV-BH. (A) Non-infected cells showing cell processes a n d smooth surfaces. (B) Cells infected with RSV-BH. Numerous "craters" appear in the cell surface during viewing of the cells in the microscope, indicating evaporation from the vacuoles of a volatile substance. Magnification, approximately x 2,000 a n d x 1,500 respectively.
CYTOPLASMIC VACUOLIZATION
mission electron microscopy. Sections cut perpendicularly to the plane of the plastic substratum revealed intracytoplasmic vacuoles (fig. 3 ) , eliminating the possibility that entrapment of some substance between the cell and the substratum was responsible for the noted refractility and craterization. In addition, these perpendicular sections, as well as random sections through cells fixed while attached to the substratum, or cells suspended before fixing, all revealed vacuoles bounded by membrane (figs. 4, 5). Vacuoles were rarely found in noninfected CE cells, and sections from these cells were easily distinguishable, because of the absence of vacuoles, from those of transformed cells in the electron microscope. The cell section seen in figure 6 is typical of non-infected CE cells. The development of vacuoles was examined after shifting RSV-BH-Ta infected cells from 41 to 3 7 " . Within minutes numerous small membrane-bound vacuoles could be detected, and these increased in size and number with time, consistent with observations using phase microscopy (Bader, '72). Resolution of the bounding membrane was more difficult in larger vacuoles. Vacuoles may increase in size by accumulation of intravacuolar substance, a suggestion supported by phase microscopy viewing of RSV-BH-Ta cells undergoing the vacuolization process. However, the occasional development of large vacuoles in cells which earlier contained numerous smaller vacuoles, suggests that coalescence of vacuoles may occur, and increase in the size of vacuoles may occur by this mechanism as well. The origin of the vacuoles could not be resolved by direct viewing in the electron O
Fig. 3 This figure a n d figures 4-8 are micrographs obtained from transmission electron microscopy. Cells were fixed with glutaraldehyde, followed by post fixation i n buffered osmium tetroxide. Sections were doubly stained with alcoholic uranyl acetate and lead citrate. In this figure a section from a RSV-BH transformed cell cut perpendicul a r l y to the plane of the plastic substratum is shown. Vacuoles typical of cells transformed by this virus (vac) a r e seen throughout the length of the cell. One vacuole appears to be causing a n identation of the nucleus ( N a n d arrow). Virus particles (VP) are found in the extracellular space adjacent to the surface membrane, upper left corner of micrograph. Bar, 1 p m . Approximately X 6,000.
37
microscope. In heavily vacuolated cells, most mitochondria remained intact with no obvious aberrations, although occasional mitochondria were found disrupted (fig.
38
ARTRICE V. BADER AND JOHN P. BADER
Fig. 4 Section from chick embryo cells transformed by RSV-BH-Ta and maintained at 37’. Vacuoles of variable size, typical of RSV-BH transformed cells, are shown (VAC). Most mitochondria (M) seem unaffected. although a few appear to be disintegrating (D). Vacuoles in close proximity to one another are found (arrows) a n d intervacuolar membranes a r e apparent (arrow a). A phagocytic vacuole (PV) also is present. Virus particles are seen i n extracellular spaces (VP) and a n occasional particle is seen within a vacuole. Bar, 1 p m . Approximately X 14,000.
4). A suggestion that membranes of the Golgi apparatus were involved in vacuolization of neurons (Whetsell and Bunge, ’69) led us to examine such membranes. No obvious changes in Golgi membranes were found in heavily vacuolated cells (fig. 7). The vacuoles contained no obvious substance, being devoid of any electron-dense material. This lack of substance distinguished the vacuoles from phagocytic vacuoles, which often contained virus particles as well as other unidentifiable matter (fig. 4).
Histological staining A variety of histological techniques was used in attempts to reveal molecular con-
stituents of vacuoles. These included fixing cells in formaldehyde, glutaraldehyde, or ethanol, and staining with oil Red 0, sudan black, PAS (McManus), PAS (Alcian Blue), Giesma, pyronin Y plus Azure B, or “Stains-all.” In all cases, the vacuoles remained unstained, indicating the absence of lipids, glycolipids, proteins, glycoproteins, glycosaminoglycans, glycogen, and nucleic acids. Fig. 5 Section from RSV-BH-Ta transformed cells ( 3 7 ” ) at higher magnification ( X 56,000) showing the membrane of the vacuole. The tripartite structure (Arrow A) typical of biological membranes is seen. Arrow B illustrates a small vacuole i n close proximity to a larger one where the increased thickness indicates apposed membranes. Bar, 0.1 p m .
CYTOPLASMIC VACUOLIZATION
39
40
ARTRICE V. BADER AND JOHN P. BADER
Fig. 6 Electron micrograph of non-infected CE cells. Cellular organelles: Nucleus (N), mitochondria (MI, and endoplasmic reticulum (ER). This cell section differs from those seen in figures 3 a n d 4 by the absence of vacuoles which are typical of transformation by RSV-BH or RSV-BH-Ta. Bar, 1 p m . Approximately x 15,000.
Uptake of neutral red When neutral red (0.01%) was added to the cell culture medium, the dye was rapidly taken into cells and within ten minutes could be found in the vacuoles of transformed cells. Two kinds of vacuoles could be distinguished on the basis of the color of the neutral red. The predominant type was reddish-brown, the color of the dye in the medium at pH 7.4. Only this type was found within the first few hours after shifting RSV-BH-Ta cells from 41 to 37", or after shifting for longer intervals in the presence of cycloheximide or Actinomycin D. RSV-BH transformed cells, or RSV-BHTa cells incubated for extended times at 37" both contained other vacuoles staining purple with neutral red. These probably represent lysosomes, or some other acidic organelle. In any case, the incorporation of neutral red into the vacuoles demonstrates that, whatever else the vacuoles O
may contain, water is a major component. The rapid uptake of neutral red into preexisting intracellular vacuoles, and the perinuclear location of developing vacuoles, suggested that vacuoles developed de novo in the cytoplasm. Nonetheless, it was possible that rapid pinocytosis, and accumulation and coalescence of miniscule vesicles, could result in vacuolization. To examine this possibility, inulin-*4C, which cannot penetrate the membrane barrier into the cytoplasm, was added to RSV-BH-Ta cells during the development of vacuolization after shifting from 41 to 37". No difference in bound radioactivity was found between these cells and those maintained at 4 1 suggesting that possible differences in pinocytotic properties are not responsible for vacuolization. O
O ,
Effects of temperature and p H Earlier studies had shown that vacuoli-
41
CYTOPLASMIC VACUOLIZATION
vacuolization developed below pH 6.8, and vacuolization increased with increasing pH. Also, RSV-BH-Ta cells maintained at 41" became vacuolated if exposed to alkaline medium (pH 7.8) for several hours, and the degree of vacuolization increased with extended incubation.
Fig. 7 Portion of a transformed cell showing numerous vacuoles of different sizes in t h e Golgi region of the cell. No obvious changes in the Golgi membranes a r e apparent. Bar 1 p m . Approximatel y x 24,000.
zation was detectable within ten minutes after shifting RSV-BH-Ta infected cells from 40.5 O to 36 O . Greater consistency was found using 41" as the transformation nonpermissive temperature, and an optimum lower temperature for transformation was examined. Also, cells in which the medium had become acidic appeared less vacuolated than cells in alkaline medium, and the effect of pH over the physiological range was examined. The combined results show that vacuolization is both a temperature-dependent and pH-dependent process (fig. 8). Vacuolization was found earlier, and by six hours had progressed to a greater extent, in cells incubated at 37" than at 39 36", 34 ', or 22 , demonstrating an optimal temperature for vacuolization in this system. Both the rate and extent of vacuolization was dependent on pH over the pH range 6.6 to 7.6 (fig. 8). Little O ,
Components of the medium The components of the cell culture medium were examined for their essentiality in the vacuolization process. Removal of serum enhanced vacuolization slightly, but deletion of tryptose phosphate broth, antibiotics, amino acids, or vitamins had no obvious effect on the development of vacuoles in RSV-BH-Ta cells (Bader and Bader, '74). Glucose could also be removed without any immediate effect on vacuolization. Thus, vacuoles could develop in the b d anced salt solution (BSS) containing Na+, K+, Mg++,Ca++,C1-, SO;, HCO,, and PO;. Salt solutions deficient in various anions and cations were then examined. None of the anions were found essential for vacuolization, although C1- could not be examined satisfactorily. Of the cations (table l),Ca++could be eliminated without effect, but in the absence of Mg++,less vacuolization was observed. Deletion of K+ had a minor enhancing effect which could be augmented by preincubating cells at 41" in K+-deficient medium and changing the medium again before shifting cultures to 37". TABLE 1
Eflects ofdeletion of ion5 f r o m B S S on development or reverrul Of7NtC710~22t~tton
Temperature shifts 4lo+37O
Earle's BSS complete Minus SO4 Minus HC09Minus PO,+ Minus N a + plus sucrose Minus Na+ plus Li+ Minus Na+ plus choline+ Minus K+ Minus Mg++ Minus C a t +
O
1
Degree of vacuolization
+ + + I
37'-41'
0
+++ +++ +++ ++++
++++
0
0
+++ ++++ ++ +++
0
0 0
+++ + 0 0
42
++++
1
ARTRICE V. BADER AND JOHN P. BADER
+++
++
+
I
- +-&--& -4v 6.6
7.0
/
7.4
I 7.8
pH OF MEDIUM Fig. 8 Effects of pH a n d temperature on vacuolization. Growth medium buffered with phosphate a n d bicarbonate w a s adjusted with HCl or NaOH a n d equilibrated to the appropriate p H in carbon-dioxide incubators. The medium was applied to RSV-BH-Ta infected cells grown a t 41° and containing no vacuoles. Cultures were then placed i n incubators at indicated temperatures. Six hours later cultures were examined for degree of vacuolization ( , r a r e vacuolated cells; , 1 to 10% of the cells vacuolated; 10 to 50% vacuolated; greater t h a n 50% vacuolated; intermediate values were given to cultures where vacuolization appeared greater t h a n in cultures at lower pH and less t h a n i n cultures of higher pH, incubated at the s a m e temperature). The pH varied by less t h a n 0.1 unit during the course of the experiment
+ + + +,
++
Eventually, it was found that a solution containing Na+ as the only cation (0.15 M NaCl 0.027 M, NaHC03) supported the development of vacuoles. Replacement of the Na+ in BSS with isoosmotic sucrose resulted in the appearance of vacuoles at 41 O as well as 3 7 " , and to a lesser extent induced vacuoles in noninfected CE cells as well. However, addition of sucrose (0.1 M) to the Na+-containing salt solution also induced vacuolization in noninfected CE cells a s well as in RSV-BH-Ta infected cells maintained a t 41". The relation of these sucrose-induced vacuoles to the vacuolization occurring during transformation is unknown. Replacement of Na+ with Li+ had an o p posite effect to that of sucrose; no vacuoles
+
+ + +,
+
appeared either at 41" or 37". When choline chloride was used as a cationic substitute for Na+, vacuolization equivalent to Na+-containing medium was found. In contrast to sucrose, cholinef did not induce vacuolization in noninfected CE cells or RSV-BH-Ta cells at 41 O. The effects of ion-deficient media on the reversibility of vacuolization upon shifting RSV-BH-Ta infected cells from 37" to 41 O also was examined. Reversal of vacuolization occurred in the simple NaC1-NaHC03 solution. Although choline+ could substitute for Naf in the development of vacuoles, vacuoles which developed in the presence of choline+ were retained after shifting cells to 41 O . These results suggested that the development and loss of vacu-
43
Temperature shifts Medium Additives
BSS Minus glucose Minus glucose Minus glucose BSS BSS BSS BSS BSS BSS BSS BSS BSS BSS BSS BSS
4I0+37O
I
+ + + 2
++ + ++ ++ +++ +++ +++ ++ +++ +++ +++ ++++ +++ +++ +++
deoxyglycose glucosamine deoxyglucose glucosamine added glucose glutamine fructose glutamine fructose N-acetylglucosamine mannose mannosamine galactose galactosamine fucose
+
37O-41'
0 0 0
++ ++++ 0 ++ 0
0 0
0 0
++++ 0 0 0
1 All additives at 1 0 - 2 M . Culture medium was replaced with medium containing additive one hour before shifting temperatures. Additional cultures containing additives were kept at the original temperature. None of these differed from nontreated controls maintained at the same temperature. 2 Degree of vacuolization.
oles occurring under the usual culture conditions was specifically a Na--dependent process.
Brief incubation of RSV-BH-Ta cells at 37 with mannosamine or glucosamine, and to a lesser extent glutamine, prevented the reversal of vacuolization (table 2) upon Glucose requirement shifting to 41". Although the RSV-BH-Ta Although Na+ was the only medium cells changed from rounded to elongated component immediately required for vacu- shapes, and appeared to become smaller olization, a delayed requirement for glucose after shifting temperatures, the vacuoles could also be shown (table 2). RSV-BH-Ta persisted. None of the other sugars affected cells preincubated at 41 in glucose-defi- the reversal of vacuolization. cient medium for two hours or longer beDinitrophenol, an uncoupler of oxidative came less vacuolated than controls after phosphorylation, was examined for possible shifting to 37". Slight inhibition by inclu- effects on vacuolization, or loss of vacuoles sion of deoxyglucose (10 mm) in glucose- after shifting vacuolated cells to the higher containing medium was also found; deoxy- temperature. A minor inhibitory effect glucose is known to interfere with glucose on vacuolization was observed with 2, 4metabolism in addition to affecting other dinitrophenol at concentrations of 1 mM metabolic properties. The addition of de- or greater. No effect on the reversal of vacoxyglucose to glucose-deficient medium uolization was seen. was even more effective in preventing vacuAdenine and derivatives olization, although within a few hours toxic effects were clearly evident. Several A suggestion that adenosine triphosphate other sugars in addition to 2-deoxyglucose (ATP) when added to erythrocytes could were examined for possible effect on the affect the rigidity of the surface membrane development of vacuolization (table 2). No (Weed et al., '69; Paleck et al., '71) led us marked effects were found with glucosa- to examine the effect of this substance on mine, fructose, N-acetyl-glucosamine, man- transformation. After observing a slight nose, galactose, galactosamine, fucose, inhibitory effect of exogenous ATP on vacor increased amounts of glucose. Although uolization, other adenine derivatives were neither fructose nor glutamine alone af- examined (table 3). The nucleoside derivafected vacuolization the two together in- tives of adenine (adenosine, B'deoxyadenohibited slightly the development of vacuoles. sine, 3'deoxyadenosine), and the adenosine Mannosamine enhanced vacuolization. analogue 2, 6-diamino-purine riboside, O
O
44
ARTRICE V. BADER AND JOHN P. BADER
teristic of Rous-transformed cells by many investigators (Bader et al., '74; Tenenbaum and Doljanski, '43; Haguenau and Beard, '62; DiStefano and Dougherty, '65). Until Temperature shifts now, no serious attempts to characterize Additives 41O-37' 37O+4I0 these vacuoles, or to examine factors affecting vacuolization, have been reported. + + + Z 0 None We feel that an understanding of the na+ 0 Adenosine ture of vacuolization will be invaluable to +++ 0 Adenine +++ 0 AMP a determination of physiological events +++ 0 ADP associated with transformation to malig+ + + 0 ATP nancy by Rous sarcoma virus. These phys+ + + 0 3'5' cyclic AMP iological events may not be involved in +++ 0 dibutyryl cyclic AMP + 0 2'deoxyadenosine malignant transformation induced by other + 0 3'deoxyadenosine means, or by other viruses, perhaps not 2,6 diaminopurine even other strains of Rous sarcoma virus. ++ 0 riboside As an example, perhaps, cells infected with 1 All Additives at lo-:! M. the Schmidt-Ruppin strain of Rous sarcoma 2 Degree of vacuolization. virus usually are nonvacuolated, and easily were the most potent inhibitors of vacuoli- can be distinguished from RSV-BH transformed cells on the basis of morphology zation; ATP, ADP, AMP, and adenine were alone (Bader et al., '74). Several other much less effective. Guanosine, uridine, cytidine, and their deoxy-derivatives failed biophysical properties of Schmidt-Ruppin transformed cells differ from those of RSVto inhibit or enhance vacuolization or affect BH transformed cells (Bader et al., '74). the reversal of vacuolization (table 3). Their These biophysical differences may be a phosphorylated derivatives were equally reflection of basic physiological differences ineffective. The preference of cells for in- induced by the viruses, although the outcorporation of nucleosides over incorpora- come in both cases is transformation to tion of phosphorylated derivatives or free malignancy. bases suggests that the effect of adenosine An increased accumulation of water is is intracellular and may be unrelated to found in RSV-BH transformed cells (Bader the effect of exogenous ATP on erythro- et al., '74), and vacuolization may be a cytes. cellular response to this increase. Cells commonly take in Na+ with water from Effects of other substances physiological solutions. The failure of Li+ Ouabain (10-5 M and higher concen- to replace Na+ in the formation of vacuoles trations) an inhibitor of Na+-K+-ATPase, suggest that this process, and its reversal enhanced the rate of vacuolization of RSV- may be Na+-specific. Although choline' BH-Ta cells after shifting from 41 to 37" could substitute for Na+ in the developand induced vacuoles in cells held at 41 ment of vacuoles, these choline+-vacuoles Several other substances reported to have failed to revert. Slight enhancement of the effects on surfaces or other structural com- vacuolization process was noted using soluponents of cells also were examined for tions deficient in K+. This result differs effects on vacuolization. These include di- from results of earlier experiments where methyl sulfoxide, dextran sulfate, aqueous no attempt was made to deplete cells of agar extract, vinblastine sulfate, colchi- residual K+ (Bader and Bader, '74). In adcine, colcemid, hyaluronic acid and hyal- dition, the cardiac glycoside, ouabain, inuronidase. None of these significantly creased the rate of the development of affected either the development or the re- vacuoles, and induced vacuolization in versal of the vacuolization process. non-vacuolated cells. Ouabain is an effective inhibitor of Na+-K+ATPase, the enDISCUSSION Vacuolization of cells transformed by zyme responsible for the cellular incorpoRous sarcoma virus was observed some ration of K+. The incorporation of K+ into fifty years ago by Carrel ('25), and since the cell is coupled to the expulsion of Na+. that time has been reported as a charac- In K+-deficient medium, or in ouabain1
O
O .
CYTOPLASMIC VACUOLIZATION
containing medium, the transport of Na+ from the cell would be delayed, and increased vacuolization could result from the relative increase in Na+. The specificity for Na+ in vacuolization and its reversal, and the induction of vacuolization by ouabain, suggest that (Na+-K+)-ATPasemay be involved in this process. This enzyme plays such a key role in cation and osmotic regulation that i t is hard to ignore in studies where specificities for monovalent cations and differences in accumulation of intracellular water have been demonstrated. The effects of glucosamine and mannosamine in preventing the reversal of vacuolization are difficult to interpret. Possibly these substances inhibit enzymes involved in the dissolution of vacuolar membranes, or are otherwise involved in glycoprotein or glycolipid metabolism related to the vacuoles. Vacuoles have been described in many kinds of cells under a variety of circumstances. Of particular interest, are those induced in rodent neurons after exposure to ouabain, where an involvement of the Golgi region was implicated in the vacuolization process (Whetsell and Bunge, ’69). The early appearance of vacuoles in the perinuclear region of RSV-BH-Ta transformed cells indicated that the Golgi region may be affected, but an electron microscopic examination of this area revealed no obvious connection. Whether the vacuoles represent specialized organelles involved in maintaining ionic or hydrostatic equilibria, or are organelles whose primary function is in some other area of metabolism, has not been resolved. Mitochondria were not grossly affected during the vacuolization process, and nothing unusual is noted among mitochrondria of vacuolated cells. The failure to detect increased uptake of inulin 14C into vacuolating cells, and the perinuclear location of developing vacuoles suggests that pinocytosis at the cell surface membrane is not responsible for vacuolization, although more definitive experiments to resolve this possibility are in progress. Resolution of the origin of vacuoles may require separation and isolation of vacuoles away from other cellular organelles, and characterization of their molecular constituents. Preliminary attempts to isolate
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vacuoles in this laboratory have been unsuccessful, due mainly to an extraordinary instability of vacuoles in physiological buffers. ACKNOWLEDGMENTS
The authors acknowledge the excellent technical contributions of Monica Bigelow, Nancy R. Brown and Joan Kondratick in the performance of these experiments, and the cooperation of Dr. Bruce Wetzel in the processing of samples for scanning microsCOPY. LITERATURE CITED Anderson, T. F. 1951 Techniques for the preservation of 3 dimensional structure in preparing specimens for the electron microscope. Transac. N. Y. Acad. Sci., 13: 130-134. Bader, J. P. 1972 Temperature-dependent transformation of cells infected with a mutant of Bryan Rous sarcoma virus. J . Viral., 10- 267276. Bader, J. P., and A. V. Bader 1974 Characteristics of cells transformed by Bryan Rous sarcoma virus, i n Mechanisms of Virus Desease. W. S . Robinson and C. F. Fox, eds. W. A. Benjamin, Inc. pp. 3 0 L 3 1 3 . Bader, J. P., and N. R. Brown 1971 Induction of mutations in an RNA tumour virus by a n analogue of a DNA precursor. Nature N. Biol., 234; 11-12. Bader, J. P., D. A. Ray and N. R. Brown 1974 Accumulation of water during transformation of cells by a n avian sarcoma virus. Cell, 3: 309-31 5. Carrel, A . 1925 Effets de l’extract de sarcomes fusocellularies sur des cultures pures de fibroblasts. Comptes Rendus Acad. Sci. Paris, 92: 477. DiStefano, H. S., and R. M. Dougherty 1965 Cytological observations of “nonproducer” R o u s sarcoma cells. Virology, 27. 360-377. Golde, A. 1962 Chemical changes in chick embryo cells infected with Rous sarcoma virus i n vitro. Virology, 1 6 : 9-20. Haguenau, F., and J. W. Beard 1962 The avian sarcoma-leukosis complex; its biology and ultrastructure. I n : Ultrastructure i n biological systems. I. Tumors Induced by Viruses; Ultrastruct u r d Studies. A. J. Dalton and F. Haguenau, eds. Academic Press, New York, pp. 1-59. Paleck, J., W. A. Curby a n d F. G. Lionetti 1971 Effects of calcium and adenosine triphosphate on volume of human red cell ghosts. Amer. J. Physiol., 220: 19-26. Sanders, B. J . 1972 Animal histology procedures of the Pathological Technology Section of the National Cancer Institute, DHEW Public No. (NIH)72-275, U.S. Dept. of HEW, PHS, NIH. Tenenbaum, E., and L. Doljanski 1943 Studies of Rous sarcoma cells cultivated in vitro. I. Cellular composition of pure cultures of Rous sarcoma cells. Cancer Res., 3: 5 8 S 6 0 3 . Valentine, A. F., and J. P. Bader 1968 Produc-
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tion of virus by mammalian cells transformed by Rous sarcoma and murine sarcoma viruses. J . Virol., 2; 224-237. Vogt, P. K., R. A. Weiss and H . Hanafusa 1974 Proposal for numbering mutants of avian leukosis and sarcoma viruses. J. Virol., 13: 551-554. Weed, R. I., P. L. LaCelle, E. W. Merrill, G . Craib,
A. Gregory, F. Karch and F. Pickens 1969 Metabolic dependence of red cell deformability. J. Clin. Invest., 48: 7 9 S 8 0 9 . Whetsell, W. O., and R. P. Bunge 1969 Reversible alterations in the Golgi complex of cultured neurons treated with an inhibitor of active N a and K transport. J. Cell Biol., 42: 490-500.
