The preparation and properties of cytoplasts from Ehrlich ascites tumor cells

Printed in Sweden Copyright 0 1979 by Academic Pnss, Inc. All rights of reproduction in any form reserved 0014-4627/79/080337-WW2.00/0

Experimental Cell Research 121 (1979) 337-345

THE PREPARATION

AND PROPERTIES

EHRLICH

ASCITES

OF CYTOPLASTS

TUMOR

FROM

CELLS

G. V. HENIUS, P. C. LARIS and J. D. WOODBURN Department of Biological Sciences, University of California, Santa Barbara, CA 93106, USA

SUMMARY A method is described in which cytochalasin B is used to fractionate Ehrlich ascites tumor cells into cytoplasts and (nucleated) karyoplasts. The plasma membrane and cytoplasm are selectively removed from these cells by this method such that the cytoplasts rarely contain membranous organelles (e.g., mitochondria) which are re&&ed in the katyoplast during fractionation. ATP concentrations similar to those found in v@le cells and glycolytic activity were measured in cytoplasts in the presence but not the absence of glycose. Cytoplasts also actively transport Na+, K+, and a-aminoisohutyric acid to steadystate distribution ratios similar to those found in whole cells. It was concluded that these cytoplasts are a simplified model system for the study of active transport in Ehrlich cells.

Cytochalasin B (CB) [l] is employed in a method of enucleation commonly used to fractionate cells into temporarily viable cytoplasts and karyoplasts [2]. Preliminary studies on the application of this method of enucleation to Ehrlich ascites tumor cells led to the development of the fractionation procedure reported here. An effect of CB on Ehrlich cells which is described below has made it possible to develop a method in which CB is used to fractionate these cells into cytoplasts and karyoplasts. A similar method has been used to fractionate rat liver cells [3]. A preliminary report on the fractionation of Ehrlich cells has already been presented [4]. This method of fractionating Ehrlich ascites tumor cells was developed in order to simplify investigations of the relationship between the Na+ electrochemical gradient and the active transport of amino acids in these cells. According to the Na+ gradient hypothesis [5], the energy in the Na+ elec-

trochemical gradient must be greater than or equal to that in the amino acid gradient. It is difficult to assessthe magnitude of the Na+ gradient in intact cells, however, because of reports that the nucleus of these cells sequesters Na+ [6, 7j. These investigations suggest that the cytoplasmic Na+ concentration is lower than that previously calculated from measurements of total cellular Na+ and water, because in these calculations Na+ was assumed to be equally distributed throughout the cellular water. Since determinations of both nuclear and cytoplasmic Na+ concentrations are indirect, the exact value of the Na+ gradient across the plasma membrane of intact cells is questionable. The Na+ gradient may be more directly estimated in the cytoplasts obtained by the methods described below, because these cytoplasts lack internal compartments. Investigations of the Na+ gradient hypothesis in these cytoplasts therefore preclude the problems associated with the Exp Cell Res 121 (1979)

338

Hen&s, Lark and Woodburn

nuclear seauestration of Na+ in whole Ehrlich cells. * MATERIALS

AND METHODS

The procedure for the preparation of Ehrlich ascites tumor cells has been described previously [8]. Nonhemorrhagic tumors from 3-4 mice were pooled for the nreparation of cvtoolasts. The cells were washed and iesuspended in -Na+ Ringer (154 mM NaCl, 6 mM KCI, 1.5 mM M&O,. and 3 mM Na+ phosphate buffer - _ at pH 7.4). Cytochalasin B (Sigma) was maintained in 10 mg/ ml stock solutions with absolute ethanol.

Cytoplast preparation Cells were incubated in 1: 5 dilutions at 37°C for l-2 min with 42 Frn CB. The cell suspension was then mildly homogenized in a loose-fitting teflon glass homogenizer for 3-5 min. (Note: The shear forces employed in this procedure were such that 10 min of this mild homogenization was required to increase the percentage of trypan stained cells in suspensions not treated with CB.) The homogenate was then diluted 3fold with Na+ Ringer containing 10 mM glucose at room temperature and centrifuged as follows: (1) The-nucleated fraction was sedimented by centrifugation at 250 g for 2 mm and the supematant was decanted. (2) The crude cytoplast fraction was collected by centrifuging this 250 g supematant at 3 500 g for 2 min. (3) The crude cytoplasrfraction was washed twice in Na+ Ringer containing 10 mM glucose, 1.2 mM CaClz, and 1% bovine serum albumin (BSA) in order to remove the CB from the preparation. This procedure was employed to prevent the occurrence of this drug’s most potent effect, the inhibition of glucose transport [9, 101 and hence glucose utilization. BSA assists in the removal of CB because it binds the drug

1111.

(4) The crude cytoplast fraction was purified by resuspending it at approx. 20 mg wet weight/ml in Na+ Ringer containing 10 mM glucose and centrifuging at 250 g for 2 min. The resultant pellets which contain nucleated contamination were washed repeatedly until the supematants, which consist of purified cytoplasts, were clear. (5) The purified cytoplasts were centrifuged at 3 500 g for 2 min and resuspended at 10-30 mg wet wt/ml in Na+ Ringer containina 10 mM glucose at room temperature- Purified cyioplasts w&e obtained approx. 1 h after the addition of CB to cells and used within 1 h of isolation.

Karyoplast

preparation

Karyoplasts were prepared by a method similar to that used for cytoplasts except that 1: 20 cell suspensions and 10 pm CB were used. Homogenates were immediately centrifuged at 150 g for 2 min and the resultant pellets were washed four times in Na+ Ringer Exp Cell Res 121 (1979)

in order to remove cvtonlasts and CB. The karvonlasts were then resuspended;n Na+ Ringer at 1 : 20.. Cvtoolasts. karvonlasts, and cells were incubated as described in the text before duplicate samples were centrifuged for analvsis. Karvoalasts and cells were centrifuged at 500 g-for 2 min and cytoplasts at 3500 g for 2 min. The wet (10-35 mg) and dry weight, and the Na+, K+, and ATP contents of the resultant pellets were determined as previously described for Ehrlich cells [12, 131. The internal water component of all pellets was determined from measurements of the wet weight, dry weight, and [3H]inulin (New England Nuclear) distribution 1141. The drv weight of cvtoplast pellets is approx. l&2$0.8% (nL5 experiments) dfthe wet weight, and 53+10% (n=8 experiments) of the wet weight is permeable to [3H]inulin. The uptake of [W]cY-aminoisobutyric acid (New England Nuclear) in cytoplasts was measured as previously described for Ehrlich cells [ 151.The concentration of lactic acid was determined in 5 ml aliquots of supematant by the method of Barker & Sumerson [16] using o,L-Li lactate (Siama) as a standard. DNA was extracted from wet &iets’by the method of Schmidt & Thannhauser as described by Leslie [17], and then assayed with diphenylamine (Mallinckrodt) by the procedure of Dische [ 181as modified by Burton [ 191. Suspensions were prepared for light microscopy by diluting them 1: 1 with a solution containing 0.85% NaCl and 0.5% trypan blue (Flow Laboratcries). A counting chamber was employed for cell counting. Samples were fixed for electron microscopy by resuspending pellets in a solution containing 2 % glutaraldehyde and 0.2 M sodium cacodylate at pH 7.4 for 25 min. Thev were then washed three times in cacodvlate buffer wiih 0.2 mM sucrose and post-fixed for 4dmin in cacodylate buffer with 1% 0~0,. The fixed material was then dehydrated with a series of graded ethanolwater solutions which included block-staining for 10 min with 2% uranyl acetate and 70% ethanol, transferred to propylene oxide, and embedded in Araldite 6005. Silver-&ay to gold sections (approx. 700-1000 A) were obtained with a Sorvall MT-2B ultramicrotome and collected onto 200-m&h copper grids. They were then stained for 20 min in 1% aqueous uranyl acetate [20] and 10 min in the lead citrate solution described by Reynolds [21]. Sections were examined with a Siemens IA electron microscope.

RESULTS

Effect ofcs men

Ehrli&

ascites

tumor

cells

are

treated with CB, ‘knobs’ [22] or ‘blebs’ [3, 23-271 visible with the light microscope form on the cellular surface (fig. 1). The blebs can develop completely on !W-100% of the cells within 1 min after the addition of CB, and they remain firmIy attached to the cell. Their formation is readily reversi-

Cytoplasts from Ehrlich ceils

Fig. 1. Light micrographs of cells (20-30 mg wet wtl ml) incubated in Na+ Ringer at room temperature. $zontrol; (b) treated for <5 min with 10 PM CB. Fig.

i. Electron micrographs of (a) control cells (2-3

339

mg wet wtlml) incubated at 37°C for 10 min in Na+ Ringer; (b) cells (20-30 mg wet wt/ml) incubated at room temperature for 1 min in Na+ Ringer containing 10 PM CB. (a) x5000; (b) x4500.

340

Henius, Lark and Woodburn

Fig. 3. Light micrograph of cytoplasts (2&30 mg wet wtlml) in Na+ Ringer at room temperature with 10 mM

glucose. S, cytoplast stained with trypan blue; U, unstained cytoplast. x500.

ble, for normal morphology is restored when cells treated with CB are washed three times in Na+ Ringer. Blebs were also observed to withdraw when cell suspensions containing 5 mg dry wt/ml and 4 ,uM CB were diluted lO-fold with Na+ Ringer containing 3 % BSA. The formation of blebs is dependent on dose of CB, the temperature, and the density of the cell suspension. The threshold and maximal responses of cells (5 mg dry wt/ml) at 37°C occurred at 2 and 10 PM CB, respectively. The effectiveness of a given dose of -CB decreased at higher suspension densities. The formation of blebs was maximal at 37°C and completely inhibited at 0°C. Evidence from electron micrographs in-

dicates that the blebs are bulbous protuberances of cytoplasm bound by plasma membrane (fig. 2). First, the surface membrane and contents of the blebs are continuous with the plasma membrane and cytoplasm of the nucleated portion of the cell. Second, the blebs contain ribosomes which are normally found in the cytoplasm. Membranous organelles, however, are rarely seen in blebs, and usually remain associated with the nucleated portion of the cell. Although CB induces the formation of blebs, it was generally found to be innocuous. It had no major effects on the morphology of subcellular components. Cellular concentration of Na+ and K+, and permeability to [3H]inulin and trypan blue were also unaffected by CB (data not shown).

Exp Cell Res I21 (1979)

Cytoplasts from Ehrlich cells

4. Electron micrographs of cytoplasts treated as in fig. 3. (a) X4500;@) ~24000; (c) ~35ooO.

Fig.

341

342

Henius, Laris and Woodburn

Table 1. Active transport

of Na+ and K+

in cytoplasts Cytoplasm (l-3 mg wet wt/ml were incubated for 1 h. Incubations were at 35°C in Na+ Ringer with 10 mM glucose except where indicated. NaCl replaced KC1 in K+ free incubations. K+ and ghrcose free incubations were preceded by two washes with the appropriate solution. Data is expressed as the mean + S.D. from the mean of duplicate samples Expt no.

Experimental conditions

17

Control 1 mM Ouabain 0°C Control K+ free Glucose free

19

Preparation karyoplasts

-[Nafl Ratio

&+I

0.11+0.01 0.55f0.02

1.18f0.10 0.16f0.01 0.85+0.00 0.57f0.05

of cytoplasts and

Mild homogenization of cell suspensions treated with CB was found to shear blebs free from the nucleated portion of the cell thus forming cytoplasts and karyoplasts. The blebs are sheared off without disrupting cells, such that cytoplasts and karyoplasts are the only cell fragments seen in homogenates. Cytoplasts were prepared from dense cell suspensions (20 mg dry wt/ ml) in order to maximize total yields. At this density, 42 PM CB resulted in maximal yields of 14% by dry weight, while 10 PM CB was ineffective. Mild homogenization of cells treated with CB also increased the percentage of nuclei which stained with trypan blue, but this value usually remained less than 10%. The separation of cytoplasts and karyoplasts by differential centrifugation was routinely monitored with a light microscope. The purified karyoplast fraction contained few if any cytoplasts in a field of several hundred karyoplasts. It is possible, however, that the karyoplast preparation is contaminated with whole cells since it is difExp Cell Res 121 (1979)

ficult to distinguish between cells and karyoplasts. The’cytoplast preparation contained less than lo5 nuclei (cells or karyoplasts)/mg dry weight (approx. 3% by weight). Usually, contamination was so slight that nuclei could not be detected by microscopic examination of 3 mg dry wtlml cytoplast suspensions. The purity of cytoplast preparations was also judged by measuring DNA content. This technique was not as sensitive as microscopic examination because DNA could only be detected in preparations determined by microscopic examination to be the most contaminated. When nuclear contamination was found, it consisted only of cells which stained with trypan blue. Since unstained nuclei were never observed in cytoplast preparations, it is unlikely that nucleated contamination contributes significantly to the activities attributed to cytoplasts in this report. Properties

of cytoplasts and karyoplasts

Light (fig. 3) and electron micrographs (fig. 4) show that purified cytoplast preparations are composed of structures which have dimensions and morphology which are similar to those of the original blebs. The preparation consists of membrane bounded structures which contain ribosomes, but are generally devoid of membranous organelles. When membranes are found within cytoplasts, they do not appear to be of nuclear or mitochondrial origin. Evidence indicates that purified cytoplast Table 2. ATP and lactic acid in cytoplasts Cytoplasts and data were treated as in table 1 Experimental conditions

[AmI

Internal (mM)

External [Lactate] W@

Control Glucose free

1.35_+0.07 0.03+0.01

0.01+o.oo

0.39+0.00

Cytoplasts from Ehrlich cells

0

20

40

Fig. 5. Abscissa:

60

60

100

time (min); ordinate:

343

120

[AIB] internal/

[AIB] external. Cytoplasts (l-3 mg wet wt/ml) were incubated in Ringer containing 10 mM glucose, [W]AIB, and the indicated concentrations of AIB. Incubations were in Na+ Ringer at 35°C except where indicated. In K+ Ringer, KC1 replaced NaCl. (I) 0.1 mM AIB, K+ Ringer; (2) 1 mM AIB, 22°C; (3) 1 mM AIB; (4) 0.1 mM AIB. 6

preparations contain more than one type of Fig. 6. Electron micrograph of karyoplast (20-30 mg cytoplast. First, light micrographs show wet wt/ml) incubated in Na+ Ringer at 37°C for 30 min. x6000. that cytoplasts which are smooth and spherical stain positively with trypan blue, while more irregularly shaped cytoplasts do not K+ (110-160 mM) concentrations similar to stain (fig. 3). This dichotomy is most readily whole cells. The maintenance of this low visualized by centrifuging cytoplast suspen- [Na+]/[K+] ratio (0.26+0.01, n=4 experisions which contain trypan blue. The upper ments) depends on the ATP concentration portion of the pellet stains blue, and is of the cytoplasts. ATP concentration and clearly demarcated from the lower, un- glycolytic activity as determined by the prostained portion of the pellet. Second, elec- duction of lactic acid both depend on the tron micrographs show that cytoplasts dif- presence of glucose (table 2). When freshly fer in their content of cytoplasm and ribo- isolated cytoplasts are incubated at 35°C somes (fig. 4b). Third, the percentage of in the presence of glucose, ATP concentracytoplast wet weights which is permeable to tions are similar to those of whole cells and [3H]inulin is variable (40-70%) and may in- [Na+]/[K+] ratios decrease by approx. 50%. clude cytoplasts which are leakier to [3H]- Incubations in the absence of glucose caused ATP concentrations to decrease and inulin than others. Active transport of Na+ and K+ in cyto- [Na+]/[K+] ratios to increase. Ratios were plasts was demonstrated by measuring Na+ also increased after cold storage, and inand K+ concentrations (table 1). Freshly cubations in the absence of K+ or the presisolated cytoplasts maintained in Na+ ence of ouabain, a specific inhibitor of the Ringer containing glucose at room tem- Na+/K+ ATPase. Since similar conditions perature have low Na+ (25-45 mM) and high cause [Na+]/[K+] ratios to increase in whole Exp Cell Res 121 (1979)

344 Henius, Laris and Woodburn cells by decreasing the active transport of chalasin was found to cause blebs in this Na+ and K+ [28, 291, low [Na+]/[K+] ratios and other reports [22, 26, 271, concentrain cytoplasts are presumably maintained by tions of CB in this range are usually associated with inhibition of cell motility [ 1, 303. the Na+/K+ pump. The active transport of the neutral non- Blebs also form under a variety of other metabolizable amino acid, o-amino iso- conditions [23-251. The procedure for fractionating Ehrlich butyric acid (AIB), was measured in cytoplasts (fig. 5). The properties of AIB trans- cells described in this report and the enuport in cytoplasts are comparable to those cleation method [2] are similar in that CB of intact Ehrlich cells. The uptake of AIB is used in both cases to fractionate cells into depends on the external concentrations of membrane bound fragments that temporariNa+ and AIB, and on temperature. AIB ly retain viability. The terms cytoplast and uptake reaches a steady state after approx. karyoplast have been used to describe such 1) h. Maximum distributions of 30: 1 were fragments obtained with the enucleation measured at 35°C with an external AIB con- method [31]. Since there are strong similarities between the products of both methcentration of 0.1 mM. Isolated karyoplasts are similar to intact ods, these terms were also applied to the cells in a number of ways. Electron micro- products of the fractionation of Ehrlich graphs (fig. 6) show that the subcellular cells. The cytoplasts derived from Ehrlich components found in whole cells are re- cells differ, however, from those obtained with the methods previously used [2, 31, in tained intact in the karyoplast. Karyoplasts, however, have a swollen endoplasmic reti- that they do not contain membranous orculum and are irregularly shaped with few ganelles. Unlike those derived from Ehrvilli on their surface. Residual blebs and lich cells, the cytoplasts described elsecondensed mitochondria are sometimes ob- where contained numerous mitochondria served. Freshly isolated karyoplasts are and an extensive endoplasmic reticulum [3, more permeable to r3H]inulin and have 321. The apparent absence of mitochondria higher [Na+]/[K+] ratios than intact cells. from these cytoplasts correlates with their The permeability to [3H]inulin and the requirement of exogenous glucose to main[Na+]/[K+] ratio approach values compara- tain ATP concentrations. It is tentatively ble to those measured in whole cells when concluded, therefore, that glycolysis is the the karyoplasts are incubated for 45 min at sole source of energy in the cytoplasts de37°C (data not shown). These results sug- rived from Ehrlich cells. gest that the structure and activity of EhrThe cytoplasts may be useful in a number lich cells is not irreversibly damaged by this of types of investigations. It has been a fractionation procedure. problem, for example, to distinguish between mitochondrial and plasma membrane activities of Ehrlich cells in a number .of DISCUSSION studies. First, the effects of respiration on The term bleb was applied to the structures amino acid transport has been the topic of which form on Ehrlich ascites tumor cells numerous reports [33-351. Second, it has treated with CB on the basis of previous been suggested that respiratory comporeports describing the structure of blebs on nents participate in the active transport of other cells [3,22-271. Although 10PM cyto- Na+ and K+ at the plasma membrane [28, Exp Cell Res 121 (1979)

Cytoplasts from Ehrlich

291. Third, mitochondria have been implicated as a source of error in the determination of plasma membrane potentials with lipid soluble ions and fluorescent probes [36]. Fourth, NADH dehydrogenase activity which is present in small amounts in purified plasma membranes [37, 381 has been recently implicated as a plasma membrane component responsible for the partial energization of amino acid transport [39]. Since cytoplasts lack mitochondria, these problems may be more readily solved with the cytoplast system than with whole cells. Cytoplasts may also simplify investigations of the energetic adequacy of the Na+ gradient hypothesis for amino acid transport. It is difficult to test this hypothesis in whole cells because the possibility of nuclear sequestration of Na+ makes estimates of the Na+ gradient unreliable [6]. This problem is circumvented in these cytoplasts because they do not have internal compartments. A forthcoming report will therefore be concerned with measuring the Na+ electrochemical and AIR gradients in cytoplasts. The authors wish to thank Dr S. K. Fisher for use of his microtome and darkroom facilities, and Ken Linburg and Janet Larsen for their excellent technical as&tance. This work was supported by grant number CA19234 awarded to P. C. L. by the NCI, DHEW.

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