Uptake of polynucleotides by mammalian cells

323

Biochimica et Biophysica Acta, 340 (1974) 323--333 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 97954

UPTAKE OF POLYNUCLEOTIDES BY MAMMALIAN CELLS XIV. STIMULATION O F THE UPTAKE O F POLYNUCLEOTIDES BY POLY(L-LYSINE)

P.L. SCHELL

Medizinische Hochschule Hannover, Institut fiir Klinische Biochemie und Physiologische Chemic, Karl Wiechert Allee 9, 3 Hannover--Kleefeld (W. Germany) (Received October 8th, 1973)

Summary 1. The uptake of radioactively labelled synthetic homoribopolynucleotides by Ehrlich ascites t u m o r cells is stimulated by poly(L-lysine) (the term uptake includes irreversible binding to the cell surface and penetration into the cell). 2. This effect has been observed with poly(I), poly(C), poly(I) • poly(C}, poly(A), poly(U), poly(A) • poly(U) and poly(A) • 2poly(U). 3. Poly(L-lysine) can effect a stimulated uptake of polynucleotides by two different mechanisms: (i) formation of complexes between cell wall and poly(L-lysine) on the irttact cell, when the cells are treated with poly(L-lysine); (ii) formation of polynucleotide--poly(L-lysine) complexes which show a high affinity for the cellular membranes. 4. The modification of the cells caused by poly(L-lysine) (3(i)) and its effect on the polynucleotide uptake are both reversible. Pronase removes poly(L-lysine) from the cellular membrane. 5. The entry of ribopolymers into poly(L-lysine)-treated cells is n o t reversible. 6. The viability of the cells treated as indicated in 1--5 has been controlled by eosine staining and by the proliferation capacity of re-implanted cells. 7. The molecular mechanism of polynucleotide uptake appears to depend strongly on protonated groups which were introduced into the membrane and could thus contribute to the net charge of the cellular surface.

Introduction The uptake o f synthetic homoribopolymers into Ehrlich ascites t u m o r cells has been demonstrated previously [ 1 ]. In the earlier studies, we measured the uptake {adsorption and ingestion) using cells without a modified cell membrane. More recently it was demonstrated that H* ions [2] and deoxyribo-

324 nuclease [3] modify the outer cell membrane. These modifications increase both the rate of uptake and the a m o u n t of ingested homoribopolynucleotides. Both effects can be reversed by restoration of the original functional state of the cell membrane (i.e. by raising of pH or by treatment of the membrane with pronase [3], respectively). Polycationic substances can stimulate the entry of nucleic acids into cells [4]. The infectivity of purified virus RNA increases in the presence of these polymers and also of poly(L-lysine) [5,6]. Another biological effect of ribopolynucleotides, and particularly of certain double-stranded homoribopolynucleotides, is the stimulation of interferon production in mammalian cells. This effect has been reported to be increased considerably by the polycations mentioned above [7,8]. In this communication, experiments will be described which demonstrate that the uptake of poly(I), poly(C), poly(I) poly(C), poly(A), poly(U), poly(A) • poly(U) and poly(A) • 2poly(U) by Ehrlich ascites cells is stimulated when the cells are pretreated with poly(L-lysine). We believe that these results are caused by the interaction of poly(L-lysine) with the cell membrane. In a second type of experiment, the effect of polynucleotide--poly(L-lysine) complex formation on uptake was investigated. We measured the rapid adsorption of complex particles at the membrane surface. In our system ingestion of these particles into the cells, however, occurs only in some cases. Methods and Materials Ehrlich ascites t u m o r cells were harvested from NMRI mice. The incubation of such cells with radioactively labelled ribopolymers (Mr 10 s ) and the exposure of the cells to different enzymes correspond to procedures described previously [ 1--3]. These procedures and the washing and filtration technique [1] are also described in the legends of this communication.

Poly(L-lysine) treatment of the cells The cells were suspended in Hanks solution containing poly(L-lysine) {0--5 #g/ml) and were incubated at 0°C or 30°C for 15 min. The poly(L-lysine) solution was removed by sedimentation of the cells and washing with 0.9% NaC1. At this stage the cells are still viable, as can be demonstrated by re-implantation into mice (normal growth of tumor) and eosine staining (dye-incorporation test) [9]. Pronase treatment of the cells The cells were suspended in Hanks solution containing pronase (5 pg/ml) and incubated at 30°C for 15 min; the pronase was then removed by repeated sedimentation of the cells in 0.9% NaC1 solution (80 × g). Poly(L-lysine )- 3 H-labelled polynucleotide complexes The complexes were formed by mixing the polynucleotide and poly(L-lysine), b o t h dissolved in our incubation medium. The complex consisted of small insoluble particles which were stable in 1% sodium dodecylsulfate solu-

325

tion [10]. They could be retained by millipore filters from this detergent solution. The filters were subsequently washed with 0.1% sodium dodecylsulfate solution, dried, and then the radioactivity of the filters was measured in a liquid scintillation counter.

Treatment o f the cells with poly(L-lysine)--3H-labelled polynucleotide complexes The cells were incubated in 0.3 ml of polynucleotide--poly(L-lysine) and washed twice with 0.9% NaC1 solution. The cells were either filtered on glassfibre filters for measurement in a scintillation counter, or treated with enzyme solutions as described above or in previous papers [ 1--3 ]. The separation of the membrane fraction and the plasma fraction of homogenized cells follows the~procedure of Warren et al. [ 11 ]. Results and Discussion

Cells treated with poly(L-lysine) Ehrlich ascites t u m o r cells were incubated with a solution of poly(L-lysine) in Hanks solution and were exposed subsequently to poly(I) • poly(C) and other homoribopolymers; the uptake of polynucleotides was measured. Poly(Llysine) stimulates this uptake to an extent dependent on its concentration during the pretreatment procedure (Fig. 1). Temperature and pH variations of this pretreatment with poly(L-lysine), had little effect on the subsequent polynucleotide uptake. In Table I results are recorded which show the difference

u

poly ( E 3 ~

500-

'h

% o 8 E~

.(poly(C)

ZD e° 260-

g o 'b

0 0

Poly (L-iysine) (/ug/ml) Fig. 1. D e p e n d e n c e o f p o l y n u c l e o t i d e u p t a k e o n t h e c o n c e n t r a t i o n o f p o l y ( L - l y s i n e ) d u r i n g p o l y ( L - l y s i n e ) p r e t r e a t m e n t . T h e cells ( 1 . 4 • 1 0 7 / s a m p l e ) w e r e p r e t r e a t e d w i t h p o l y ( L - l y s i n e ) in H a n k s s o l u t i o n (0 ° C, 30 rain). P o l y ( L - l y s i n e ) was w a s h e d a w a y a n d t h e cells w e r e i n c u b a t e d in a m e d i u m c o n t a i n i n g 0 . 0 3 5 /~mole/ml, 105 d p m / m l [ 3 H I - l a b e l l e d p o l y n u c l e o t i d e (0 ° C, 30 rain). Excess m e d i u m was r e m o v e d b y t w o w a s h i n g s w i t h 0.9% NaCl s o l u t i o n a n d t h e cells w e r e t h e n p l a c e d o n glassfibre filters f o r scintillation c o u n t i n g . T h e u p t a k e is e x p r e s s e d in p m o l e s m o n o n u c l e o t i d e r e s i d u e s p e r 106 cells as d e s c r i b e d in T a b l e I.

* Poly(L-lysine) p r e t r e a t m e n t omitted.

P o l y ( I ) ' p o l y ( [ 3 H I C)

Poly([3H] l)-poly(C)

P o l y ( [ 3 H ] C)

P o l y ( [ 3 H ] I)

P o l y ( A ) . 2 p o l y ( [ 3 H ] U)

P o l y ( A ) - p o l y ( [ 3 H ] U)

P o l y ( [ 3 H ] A)

0 0 0 0 30 0 0 30 0 0 30 0 0 0 0 0 0 0 0 0 0

P o l y ( [ 3 H ] U)

1 30 1 30 30 1 30 30 1 30 30 6 30 1 30 1 6 30 1 6 30

Incubation time, incubation temper a t u r e (°C, r a i n )

Incubation medium

100 196 130 178 390 172 328 386 207 380 460 450 528 180 422 154 385 514 142 350 452

* 9.25 *20.0 "12.8 "21.7 *28.8 *10 *20.7 *42.5 "16.2 *25.0 *60.0 *27.5 *67.5 *23,0 *37.6 *38.0 *46.4 *29.5 '17.2 *42.3 *70.0

U p t a k e of the p o l y n u c l e o t i d e s (pmoles polynucleotide per 106 cells) 6.25 11.8 119 162 307 142 257 374 213 361 468 37.5 308 9.50 5.75 21.2 30.0 35.0 27.0 32.5 27.5

* 0.45 * 6.00 * 13.2 * 18.8 * 24.5 * 10.5 * 18.8 * 32.5 * 15.0 * 21.3 * 65.0 "149 * 27.0 * 6.25 * 3.00 * 20.8 * 22.5 * 17.5 * 4.62 * 9.25 * 5.75

pancreas ribonuclease pancreas ribonuclease phosphodiesterase phosphodiesterase phosphodiesterase phosphodiesterase phosphodiesterase phosphodiesterase phosphodiesterase phosphocliesterase phosphodiesterase phosphodiesterase phosphodiesterase pancreas ribonucleasc pancreas ribonuclease phosphodiesterase phosphodiesterase phosphodiesterase phosphodiesterase phosphodiestcrase p h o s p h o diesterase

P o l y n u c l e o t i d e s r e m a i n i n g in cells a f t e r s u b s e q u e n t t r e a t m e n t with n u c l e a s e ( p m o l e s p o l y n u c l e o t i d e p e r 105 cells)

The cells (1,4 - 1 0 7 / s a m p l e ) w e r e p r e i n c u b a t e d in H a n k s m e d i u m , p H 7.2, c o n t a i n i n g 5 P g / m l p o l y ( L - l y s i n e ) (0¢'C, 30 rain). This m e d i u m w a s r e m o v e d b y c e n t r i f u g a t i o n in 0.9% NaCI solution. A s u b s e q u e n t i n c u b a t i o n of the cells was carried o u t in 0.3 m l of a m e d i u m w h i c h c o n t a i n e d r i b o p o l y m e r s at t h e f o l l o w i n g r a d i o a c t i v i t y ( 3 H ) a n d c o n c e n t r a t i o n : 0 . 0 3 5 p m o l e / m l a n d 105 d p m / m l . P o l y n u c l e o t i d e s r e m a i n i n g in t h e cells w e r e d e t e r m i n a t e d by i n c u b a t i o n of the cells w i t h e i t h e r r i b o n u c l e a s e (EC 2 . 7 . 7 . 1 6 ) or p h o s p h o d i e s t e r a s e (EC 3 . 1 . 4 . 4 ) w h i c h r e m o v e a d s o r b e d p o l y m e r s . C o n t r o l : u n d e r o u r c o n d i t i o n s t h e p o l y m e r s in s o l u t i o n w e r e digested c o m p l e t e l y b y p a n c r e a s r i b o n u c l e a s c ( p o l y ( U ) a n d p o l y ( C ) ) o r s n a k e - v e n o m d i e s t e r a s e ( p o l y ( A ) , p o l y ( A ) • p o l y ( U ) , p o l y ( A ) • 2 p o l y ( U ) , poly(1) a n d p o l y ( l ) • p o l y ( C ) ) . T h e u p t a k e is e x p r e s s e d in p m o l e s m o n o n u c l e o t i d e r e s i d u e s p e r 106 cells or in p m o l e s m o n o n u c l e o t i d e pairs or t r i p l e t s p e r 106 cells, r e s p e c t i v e l y .

UPTAKE OF POLY(U), POLY(A), POLY(A)- POLY(U), POLY(A) • 2POLY(U), POLY(1), POLY(C) and POLY(1) - POLY(C) BY EHRLICHASCITESTUMOR CELLS PRETRATEDWITHPOLY-(L-LYSINE)

TABLEI

b~

327 500-

s

S

%

o_ o250-

/ 0 0 Time (rain) Fig. 2. T i m e course of the uptake of poly([3H] I) and poly([3H] I) • poly(C) into poly(L-lysine)-treated Ehrlich ascites t u m o r cells. T h e incubations with poly(L-lysine) and subsequently with the polynucleotides have already been described in Table I. T h e incubation temperature was 30 ° C.

between adsorption of the polynucleotides to the cellular surface and their entry into the cells. The corresponding control data with poly(L-lysine) omitted, are also included in Table I. The enzymatic degradation and removal of polynucleotides from cellular membranes have been described and discussed previously [1,3]. The values for poly(U) in this table show that enzymatic degradation occurs and, therefore, indicate adsorption to the membrane. Thus a control is provided which demonstrates that this enzymatic test is possible with poly(L-lysine)-treated cells. Poly(I) enters the cells only partially and poly(C) is absorbed to the membrane. Hardly any poly(I) • poly(C) penetrates into the cells. From Table I it can be seen that generally poly(L-lysine) treatment of the cells amplifies the uptake but does not alter the mode of this uptake. Time-course experiments (Fig. 2) confirm the result of Table I that the uptake proceeds very rapidly even at 0°C, and that the 30-min incubations in Table I correspond to the plateau states. The possibility of stand separation during the interaction of poly(I) • poly(C) with the cellular surface of poly(L-lysine)-treated cells can be excluded. Cells were exposed to a mixture of double-labelled poly(I) • poly(C) (0.012 gmole/ml) and non-labelled poly(C) (0.025 pmole/ml). Any strand separation of poly(I) • poly(C) would n o w have resulted in isotope dilution of the corresponding labelled strand and in a shift in the ratio of the two labelled partners taken up by the cells; this did not occur. The concentration dependence of the relative efficiency of [3 H]-labelled polynucleotide uptake [1,12] into poly(L-lysine)-treated cells was measured, the samples being of identical radioactivity and varying concentration (Fig. 3).

328 7, "cr

-~ o 150- I Q. c

~00-

~ 50

I

),poly(C)

L poly(I).poly {[3H]C)

o~

0.0

0.0£

I I '/ r .v 0.06 0,14 0.28 PoLy (I). poly (C) (//moles/ml)

Fig. 3. D e p e n d e n c e o f t h e relative e f f i c i e n c y o f p o l y n u c l e o t i d e u p t a k e o n p o l y n u c l e o t i d e c o n c e n t r a t i o n . T h e cells w e r e p r e t r e a t e d w i t h p o l y ( L - l y s i n e ) , 0 . 0 5 ~ g / m l at 3 0 ~ C for 3 0 rain. P o l y ( [ 3 H ] I) - p o l y ( C ) : t h e different media had identical radioactivities and different concentrations. They were prepared by mixing an i n c u b a t i o n m e d i u m c o n t a i n i n g 0 . 0 0 3 5 p m o l e p o l y ( [ 3 H ] I ) • poly(C), 3-104 dpm/ml with various a m o u n t s o f c o n c e n t r a t e d n o n - l a b e l l e d p o l y ( I ) • p o l y ( C ) . T h e c o n c e n t r a t i o n ( 0 . 0 0 1 7 # m o l e / m l ) w a s prep a r e d b y d i l u t i o n a n d t h e c o r r e s p o n d i n g d a t a w e r e c o r r e c t e d b y a f a c t o r o f t w o . P o l y ( I ) • p o l y ( [ 3 H ] C): A n a n a l o g o u s r a n g e w a s p r e p a r e d f r o m p o l y ( I ) • p o l y ( [ 3 H ] C), 1 . 5 • 1 0 4 d p m / m l . T h e p r e p a r a t i o n o f t h e cells, t h e p r e i n c u b a t i o n , i n c u b a t i o n a n d a n a l y s i s p r o c e d u r e s w e r e i d e n t i c a l t o T a b l e I. T h e i n c u b a t i o n w a s carried o u t at 0 ° C for 6 rain. E a c h p o i n t is a n average o f c o r r e s p o n d i n g v a l u e s o f a t r i p l i c a t e e x p e r i m e n t . T h e c u r v e s are r e p r e s e n t a t i v e for f o u r i n d i v i d u a l e x p e r i m e n t s . In t h e s e i s o t o p e - d i l u t i o n e x p e r i m e n t s t h e relative e f f i c i e n c y o f u p t a k e ( p o l y n u c l e o t i d e t a k e n u p f r o m t h e m e d i u m d i v i d e d b y t h e c o n c e n t r a t i o n o f p o l y n u c l e o t i d e in t h e m e d i u m [ 1 2 ] ) h a s b e e n e x p r e s s e d d i r e c t l y b y t h e d p m v a l u e s o f [ 3 H ] - l a b e l l e d p o l y n u c l e o t i d e s t a k e n u p b y t h e cells. T h e 2 : 1 ratio o f t h e d p m v a l u e s b e t w e e n t h e t w o curves, o b t a i n e d b y i n c u b a t i o n in t h e t w o r e s p e c t i v e m e d i a is m a i n t a i n e d , e x c e p t at v e r y l o w c o n c e n t r a t i o n s . • • p o l y ( [ 3 H ] I) • p o l y ( C ) ; - - e - - ~ - - , p o l y ( I ) • p o l y ( [ 3 H ] C).

Along this range of isotope dilutions, relative efficiency of uptake can be expressed directly by the values of radioactivity taken up by the cells. Owing to the generally increased efficiency of uptake of poly(L-lysine)treated cells, polynucleotide uptake from media of much lower polynucleotide concentration can n o w be measured. Identical maxima of the relative efficiency were found at a concentration of 0.003 pmole/ml polynucleotide in the incubation medium for poly([ 3 HI I) • poly(C) and poly(I) • poly([ 3 H] C). This identical behaviour of the two species indicates that they may be identical, i.e. that the double-strand character may be maintained. The uptake of labelled poly(U), poly(A) • poly(U) and poly(A) • 2poly(U) by poly(L-lysine)-treated cells has also been studied (Table I). Poly(L-lysine) increases the total uptake of these ribopolymers. The effect depends on the poly(L-lysine) concentration during the pretreatment procedure, as was already described for poly(I) and poly(I) • poly(C) (Fig. 1). The stimulation of uptake is particularly enhanced in the case of poly(U) which is known [1] to become attached to the cellular surface. Poly(A), poly(A) • poly(U) and poly(A) • 2 poly(U) enter the cells. Non-labelled poly(U) was added to poly( [' 4 C] A) • 2poly([3 H] U). There was no indication of strand separation between poly(A) and poly(U) of this triple strand during the interaction with poly(L-lysine)-treated cells.

329 TABLE II

REVERSIBILITY UPTAKE

OF

THE

POLY(L-LYSINE)-INDUCED

INCREMENT

OF

POLYNUCLEOTIDE

T h e e x p e r i m e n t a l c o n d i t i o n s c o r r e s p o n d w i t h t h o s e o f T a b l e I. T h e cells w e r e p r e t r e a t e d w i t h p o l y ( L - l y -

sine), 5 ~ug/ml at 0 ° C f o r 3 0 rain. O n e p o r t i o n o f t h e s e cells w a s t h e n e x p o s e d t o p o l y ( [ 3 H ] U) ( 0 . 0 3 5 ~ t m o l e l m l , 105 d p m / m l a t 0 ° C f o r 3 0 rain), a n d a n o t h e r p o r t i o n t r e a t e d w i t h p r o n a s e d i s s o l v e d in H a n k s s o l u t i o n (2 ~ g / m l a t 30~C f o r 1 5 rain). Still a n o t h e r p o r t i o n w a s t r e a t e d w i t h p r o n a s e (5 /zg/ml) u n d e r i d e n t i c a l c o n d i t i o n s . T h e s e p r o n a s e - t r e a t e d cells w e r e t h e n w a s h e d o n c e a n d e x p o s e d t o p o l y ( [ 3 H ] U) ( 0 . 0 3 5 / ~ m o l e / m l , 1 0 S d p m / m l ) . T h e s a m e p r o c e d u r e s w e r e carried o u t w i t h cells w h i c h w e r e p r e t r e a t e d a n a l o g o u s l y in m e d i u m w i t h o u t p o l y ( L - l y s i n e ) . Pronase treatment o f t h e cells (min}

0 2 5

P o l y n u c l e o t i d e u p t a k e o f cells Pretreatment with poly(L-lysine) ( p m o l e s )

Control, poly(L-lysine) omitted (pmoles)

175 42 29

26 20 23

Time-course experiments were carried o u t with all polynucleotides and ascites cells pretreated with poly(L-lysine) solutions (5 pg/ml). The results closely correspond to the situation in Fig. 2. Poly(L-lysine) can be removed from the cellular surface by exposing the cells to pronase (5 #g/ml, 15 min, 30°C). This removal can be followed by measuring the corresponding reduction of the poly(L-lysine)-induced increment of polynucleotide uptake (Table II). Control data show that pronase alone does n o t alter polynucleotide uptake. On the other hand, poly(L-lysine) is completely degraded by pronase under our conditions. From the data of Table II, it can be deduced that the presence of poly(L-lysine) at the cellular membrane is responsible for the observed increase in polynucleotide uptake.

Polynucleotides treated with poly(L-lysine) In Hanks solution, polynucleotide--poly(L-lysine) complexes are easily formed when the two components are mixed. These complexes aggregate and the resulting particles can be retained on millipore filters. Sedimentation analysis also confirms particle formation. The particles are stable in sodium dodecylsulfate solution [ 10 ]. Complexes of poly(L-lysine) and poly(A), poly(U), poly(A) • poly(U) or poly(A) • 2poly(U) are formed in the same way. All these particles easily associate with the cells. The following experiments were performed to determine whether polynucleotide--poly(L-lysine) complexes enter the cells or remain adsorbed to the membrane surface. The problem was attacked by two different methods. The cells were exposed to the polynucleotide--poly(Llysine) complex and excess medium was washed away. Afterwards the cells were treated with pronase, which was completely removed by washing, and by autodigestion (incubation of the cells: 30°C, 10 rain) to remove traces adsorbed to the cells. Thus any nucleoprotein particle adsorbed to the membrane surface was "opened u p " to allow nucleolytic degradation by a nuclease applied subsequently.

330 ' F A B L E III A D S O R P T I O N A N D I N C L U S I O N OF EHRLICH ASCITES TUMOR CELLS

POLYNUCELOTIDE--POLY(L-LYSINE)

COMPLEXES

BY

T h e cells ( 1 . 4 • 1 0 V / s a m p l e ) w e r e i n c u b a t e d in m e d i a (0.3 ml) c o n t a i n i n g 0 , 0 3 5 p m o l e / m l [ 3 H I - l a b e l l e d p o l y n u c l e o t i d e s (105 d p m / m l ) w h i c h w e r e m i x e d w i t h e q u i m o l a r a m o u n t s of p o l y ( L - l y s i n e ) b e f o r e ( b a s e d on t h e m o l a r r a t i o of the r e s p e c t i v e p o l y p e p t i d e a n d p o l y n u c l e o t i d e s u b u n i t s ) . T h e u p t a k e is e x p r e s s e d in ~tmoles p o l y n u c l e o t i d e r e s i d u e s p e r 106 ceils. (A) T h e ceils w e r e w a s h e d a n d i n c u b a t e d w i t h the f o l l o w i n g e n z y m e s o l u t i o n s for 15 rain at 30~C a c c o r d i n g to t h e s c h e m e s b e l o w . (1) S n a k e - v e n o m diesterase, 0.1 m g / m l ; p r o n a s e , 2 ~lg/ml; H a n k s s o l u t i o n , 20 m i n ; s n a k e - v e n o m diesterase, 0.1 r n g / m l ; (2) p r o n a s e , 5 p g / m l ; H a n k s s o l u t i o n , 20 m i n ; s n a k e - v e n o m d i e s t e r a s e , 0.1 m g / m l ; s n a k e - v e n o m diesterase, 0.1 m g / m l ; (3) p r o n a s e , 5 p g / m l ; H a n k s s o l u t i o n , 20 rain; p a n c r e a s r i b o n u c l e a s e , 0.5 m g / m l ; s n a k e - v e n o m dies(erase, 0.1 m g / m l . This m e t h o d of m a x i m a l d e g r a d a t i o n was e m p l o y e d in all cases. C o n t r o l : u n d e r these cond i t i o n s t h e p o l y n u c l e o t i d e - - p o l y ( L - l y s i n e ) c o m p l e x e s in s o l u t i o n were digested c o m p l e t e l y . T h e t o t a l a m o u n t of cells p e r p r o b e a n d t h e p e r c e n t a g e of d e a d cells were d e t e r m i n e d b y d y e i n c o r p o r a t i o n ( c o s i n e ) a n d c o u n t i n g : overall losses d u r i n g the p r o c e d u r e s 4 15%, d e a d cells E 8%. (B) T h e ceils w e r e w a s h e d a n d h o m o g e n i z e d by an u l t r a t u r r a x b l e n d e r a n d s u s p e n d e d in 0 . 2 5 M sucrose solution. This s o l u t i o n was p l a c e d on t o p of a 50% ( w / w ) s u c r o s e s o l u t i o n a n d c e n t r i f u g e d . T h e t o p p h a s e was c e n t r i f u g e d to s e p a r a t e soluble p l a s m a c o m p o n e n t s a n d t h e m e m b r a n e s . T h e soluble f r a c t i o n was p r e c i p i t a t e d w i t h t r i c h l o r o a c e t i c acid 5% at 0"C, a n d f i l t e r e d on glassfibre filters. T h e m e m b r a n e s w e r e s u s p e n d e d in e t h a n o l a n d also filtered. B o t h filters w e r e w a s h e d , d r i e d a n d m e a s u r e d in a scintillation c o u n t e r . Incubation medium

Incubation t i m e , incubation temperature (°C, m i n )

U p t a k e of polynucleotide-poly(L-lysine) complexes

(A) R i b o p o l y m e r s r e m a i n i n g at t h e cells a f t e r e n z y m e treatment (1,2,3)

(B) P o l y n u c l e o t i d e s in m e m b r a n e fract i o n ( p m o l e s ) divided by polynucleotides in p l a s m a fraction (pmoles)

( p m o l e s p o l y n u c l e o t i d e / l O 6 cells) P o l y ( [ 3 H ] I) P o l y ( [ 3 H] C) P o l y ( [ 3 HI I) • p o l y ( C ) P o l y ( I ) ' p o l y ( [ 3 H ] C) P o l y ( [ 3 H ] U) P o l y ( A ) ' p o l y ( [ 3 H ] U) P o l y ( A ) . 2 p o l y ( [ 3 H ] U)

0 30 0 30 0 30 0 30 0 0 30 0 30

6 30 1 30 6 30 6 30 30 l 30 1 30

645 400 272 210 480 308 550 412 712 276 667 255 787

107 125 27.5 17.5 145 112 77.5 95.0 45.0 230 575 218 657

(1)

(1) (2) (3) (1) (3) (3)

0.22 0.31 0.15 0.17 0.32 0.35 0.15 0.20 0.07 0.88 1.00 0.78 0.82

The data o f Table Ill indicate that poly(I) • poly(C)--poly(L-lysine) particles were n o t taken up into the cells. Different nucleolytic degradation patterns for poly(I) and poly(C) might even suggest a separation of the two c o m p o n e n t s followed by a partial ingestion of poly(I). On the other hand we f o u n d an uptake of poly(L-lysine) complexes of p o l y ( [ 3 H ] U ) , p o l y ( A ) , poly([3 H] U) and poly(A) • 2poly([ 3 H] U) into the ceils. The cellular m e m b r a n e appeared to be unimpaired after these c o m b i n e d treatments, as was shown by a dye-inc o r p o r a t i o n test (cosine). A second experimental approach confirmes the data of these e n z y m e treatments. The cells were disrupted and a separation of cell-wall fraction, plasma fraction and nucleus fraction, according to Warren et al. [ 1 1 ] , was carried out. The resulting ratios of cell-wall fraction:plasma fraction are recorded in Table III (B).

331

375-

~ © 25O-

~2 0

~ 125L

~~ 0

j/

©

o:i

o'.2

o:3

Poly(L-lysine) (~ moles/ml ) Fig. 4. U p t a k e o f poly(1) • p o l y ( C ) ~ p o l y ( L - l y s i n e ) m i x t u r e s b y Ehrlich ascites t u m o r cells: variations of p o l y ( L - l y s i n e ) c o n t e n t . The e x p e r i m e n t a l procedure f o l l o w s closely Table III (30°C, 30 rain). The m e d i a c o n t a i n e d p o l y ( [ 3 H ] I) • p o l y ( C ) 0 . 0 3 5 # m o l e , 10 s d p m / m l and various a m o u n t s of poly(L-lysine).

The uptake of poly(I) • poly(C) at constant concentration was studied as a function of varying concentrations of poly(L-lysine) (Fig. 4). The uptake is favoured at a polynucleotide/poly(L-lysine) ratio of 1/1. Such maximum polynucleotide uptake could not be demonstrated in experiments with poly(L-lysine)-treated cells (Fig. 1). This discrepancy (and the data of Table III) infers that the uptake of polynucleotide--poly(L,lysine) particles and, on the other hand, the uptake of polynucleotides by poly(L-lysine)-treated cells are different phenomena. The two types of molecular interaction do not even seem to be related since poly(L-lysine) pretreatment of the membranes did not give rise to any further modification of the uptake of polynucleotide--poly(L-lysine) complexes (Table IV). T A B L E IV U P T A K E O F P O L Y N U C L E O T I D E - - P O L Y ( L - L Y S I N E ) C O M P L E X E S BY C E L L S P R E T R E A T E D WITH POLY(L-LYSINE) The e x p e r i m e n t a l c o n d i t i o n s are t h o s e o f Table I and Table III. Incubation medium: polynucleotide-p o l y (L-lysine) c o m p l e x

I n c u b a t i o n time, i n c u b a t i o n ternperature (°C, rain)

Uptake into pretreated cells

Uptake into untreated cells

( p m o l e s p o l y n u c l e o t i d e / l O 6 cells)

P o l y ( [ 3 H ] I) P o l y ( [ 3 H ] C) Poly([3H] I)-poly(C) P o l y ( [ 3 H ] U)

0 1 3 0 30 0 1 3 0 30 0 1 3 0 30 0 1 0 30

575 430 227 243 435 328 263 630

645 400 272 210 490 308 278 712

332 Conclusion The uptake of synthetic homoribopolynucleotides by Ehrlich ascites t u m o r cells is stimulated either by pretreatment of the cells with poly(L-lysine) or by complex formation of polynucleotides and poly(L-lysine), followed by subsequent incubation of the cells with this complex. An a t t a c h m e n t of such nucleoprotein-like particles at the surface of the cellular membranes of Ehrlich ascites t u m o r cells can be demonstrated with particles composed of poly(I) • poly(C) and poly(L-lysine). These complexes do n o t enter the cells and the polynucleotides can be removed by nucleases. Particles composed of poly(A) • poly(U) and poly(L-lysine) or poly(A) • 2poly(U) and poly(L-lysine), however, enter the cells and the polynucleotides are no longer attacked by nucleases. This might occur via an intermediate surface position like the one proposed for poly(I) • poly(C)--poly(L-lysine). Owing to the decreased net negative charge in the complex this first contact of polynucleotide and cellular membrane is less restricted. A subsequent entry could occur from this favourable starting point. The incorporation of poly(L-lysine) into the surface of artificial membranes can create a net positive charge on one side of the membrane [13]. The insertion of basic molecules, like poly(L-lysine), into the membrane surface of ascites t u m o r cells might similarly introduce groups into these membranes which are already protonated at pH 7. The arrangement, therefore, imitates an ionic situation around the cells in which protonation of naturally occurring basic groups of the membrane must have taken place. Protonation of the cellular membrane of intact cells is, however, most likely to have occurred at pH 5, particularly when the reversibility of the effect can be easily demonstrated. Under those conditions of reduced pH, the uptake of polynucleotides is greatly stimulated [2] and this process is reversible. The data of this paper and of the previous one [2] imply an analogy in the principal uptake mechanism for the three differently modified cells (normal cells, normal protonated cells and poly(L-lysine)-treated cells). In each case, poly(U) is adsorbed to the cellular surface, and other polymers like poly(A) • poly(U) enter the cells. The three cell types show analogous kinetics such as time and concentration dependence of polynucleotide uptake. From this identity, one may conclude that analogous molecular mechanisms of uptake exist under those three conditions and that protonated groups contribute to the net positive charge of the outer membrane and the enhanced uptake of polynucleotides. Acknowledgement This work was supported by the Deutsche Forschungsgemeinschaft. References 1 2 3 4

Schell, P.L. ( 1 9 7 1 ) B i o c h i m . B i o p h y s . A c t a 240, 4 7 2 - - 4 8 4 Schell, P.L. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 262, 4 6 7 - - 4 7 5 Schcll, P.L. a n d Muller, W.tf. (19"/1) B i o c h i m . B i o p h y s . A c t a 247, 5 0 2 - - 5 0 5 K o c h , G. a n d Bishop, J.M. ( 1 9 6 8 ) V i r o l o g y 35, 9 - - 1 7

333 5 Bishop, J.M. and Koch, G. (1969) F u n d a m e n t a l Techniques in Virology, p. 143, Academic Press, New York 6 Wensky, P. and Koch, G. (1971) J. Virol. 6, 35--40 7 Carter, W.A. and Pitha, P.M. (1971) in Biological Effects of Polynucleotides (Beers, R.F, and Braun, W., eds), pp. 89--105, Springer-Verlag, Berlin 8 Bausek, H.G. and Merrigan, T.C. (1969) Virology 39, 4 9 1 - - 4 9 8 9 Eaton, M.D., Scala, A.R. and Jewell, M. (1959) Cancer Res. 19, 945--951 10 Havllza, D. and Koch, G. (1971) Arch. Biochem. Biophys. 147, 85--91 11 Warren, L., Glick, M.C. and Nass, M.K. (1966) J. Cell. Comp. Physiol. 68, 269--276 12 Schell, P.L. (1968) Biochim. Biophys. Acta 166, 156--161 13 Montal, M. (1972) J. Membrane Biol. 7, 245--253