Experimental Cell Research 94 (1975) 315-320

NUCLEAR OF NUCLEIC

PENETRATION ACIDS

AND

SYNTHESIS

IN MAMMALIAN E. D. FENSTER,

F. LACOUR

STIMULATION

BY POLY(A)-POLY(U) CELLS and J. HAREL

Institut Gustave-Roussy, Groupe de Recherche No 8 du CNRS, ViIlejuif F-WOO, France

SUMMARY The rapid penetration of poly(A)-poly(U) into cell nuclei is shown by radioautography, by recovery of acid-precipitable material from isolated nuclei and by sucrose gradient centrifugation of nuclear lysates. The majority of poly(A)-poly(U) remains intact in the nuclei for at least 24 h. This penetration is increased 20-fold by pretreatment of the cells with DEAE Dextran. In cells treated with DEAE Dextran, DNA and RNA syntheses are stimulated by poly(A)-poly(U) from the time the polymer complex is added and for at least 21 h.

The double-stranded synthetic polyribonucleotide complex poly(A)-poly(U) can stimulate both humoral antibody formation and cell-mediated immune responses [ 1, 21. Since these reactions are important in the control of turnours, the ability of this homopolymer duplex, which appears to be nontoxic and non-pyrogenic, to affect the growth of experimental tumours has been investigated. Some inhibition of the growth of mouse spontaneous mammary [3,4] and syngeneic transplantable tumours [5] and of a transplantable melanoma in hamster [4] has been reported. The mechanism of these effects is still not well understood. It is therefore of interest to study the early cellular events following treatment with poly(A)-poly(U). The uptake of a small percentage of radioactive poly(A)-poly(U) by Ehrlich ascites tumour cells has been reported [6]. The effects of this polyribonucleotide on DNA synthesis are contradictory [7, 81 and 21-751812

no elaborate study on the state of newly incorporated poly(A)-poly(U) has been published so far. We report here the uptake, in culture, of poly(A)-poly(U) by mouse ascites tumour cells and human lymphocytes. We show that a large proportion of the incorporated double-stranded complex rapidly migrates into the cell nucleus, and remains essentially intact for at least 2 h. In addition an early stimulation of both DNA and RNA syntheses is observed in human lymphocytes. MATERIALS

AND METHODS

Experiments were performed with normal human lymphocytes, LHN13, obtained from C. Rosenfeld [9] and maintained in suspension cultures in RPM1 1640 medium. or with FLS cells [IO] an ascitic tumor removed 6-g days after passage in Swiss mice.

Pa/y(A)-pa/y(U).

Poly(A) and poly(U) were synthesized with polynucleotide phosphorylase from the nucleoside diohosohates 3H-UDP. ‘“C-UDP or I%-ADP (The Radiochemical Centre. Amersham) by A. M. Michelson [I I] and each was mixed in an equimolar Exprl Cell Rcs 94 (1975)

3 16

Fenster, Lacour and Hare1

Table

1. Pf?netratiOn

Of

pOly@~pOly(l/)

FLS cells: 2 pglml (2~10~ cpm) of poly(IX’-A)+oly(U); 5 x106 cells/ml; 6 ml/ LHNl3 cells; 0.46 pglml(1.67~10~ cpm) of polyQI~poly(‘4c-U); 8 ~10~ cellslml, 5 ml AS, acid-soluble; AP, acid-precipitable The numbers in parentheses indicate the quantity AP poly(Atpoly(U) incorporated in IO’cells Cell type

Cytoplasm

Nuclei

cpm AS cpm AP

cpm AS cpm AP

of

Radioautography. Cells were exposed to poly(A)p01y(~H-U) in Eagle’s medium for 30 min, washed and resuspended in a drop of calf serum, spread on slides, fixed for 5 min with methanol and coated with Ilford K2 emulsion. After 15 days’ exposure, radioautographs were developed and stained with Giemsa. Nucleic acid synthesis. 3H-Thymidine and “C-uridine incorporation into TCA-precipitable material were taken as a measure of DNA and RNA synthesis.

RESULTS

Fig. 1 shows a typical radioautography of an FLS cell exposed to poly(A)-p01y(~H-U) (0.0097 pg) for 30 min. Most of the grains appear to be (0.0049 l-4 located in the nucleus. Table 1 demonstrates that in the absence of DEAE Dextran a small amount of polyratio with the non-radioactive comolementarv uolvmer. The activity of p01y(‘~C-A)-pdly(U) was 97000 mer was taken up by both FLS and LNH 13 cpm/pg; of poly(A)-poly(‘4C-U). 399 000 cpm/pg and of cells, and shows the distribution in the cytopoly(A)-pol~(~H-U), 60 000 cpm/pg. plasm and nuclei of acid-soluble and acidPenetration of poly(A)-poly(U). Cells were washed precipitable material. It is well known that with Eagle’s medium, resusnended in Eagle’s plus poly(A)$oly(U) and incubated for 30 min atj7”C with DEAE Dextran greatly facilitates cell penegentle swirling. The cells were washed twice with TBS buffer (8 g NaCl, 0.37 g KC], 0.25 g Na2HP04. 12 tration of nucleic acids. As shown in table H,O, 3.0 g Tris, 1.0 g glucose, 203 mg MgCI$l; pH 2.1, in the presence of DEAE Dextran a 7.2). resuspended in cold TBS-sucrose (0.2 M) convery large apparent increase of poly(A)taining 0.2% Nonidet P40, allowed to stand for 5 min in an ice bath and nuclei were pelleted by low speed poly(U) uptake was found. However, the centrifugation. Microscopic examination (phase congreater part of this augmentation could trast) and thereafter Giemsa staining showed an essentially pure nuclear preparation with occasional represent polymer, precipitated on the cell non-staining ‘halos’ of cytoplasmic matter. The nuclei surface in the presence of 100-200 pg were washed with TBS-sucrose and resuspended at room temperature in 0.7 ml TBS-sucrose, 0.2 ml (200 DEAE Dextran. Such precipitated comFLS

LHN

2906

I3 I2 032

1015

224

ILP) 16 079 8(0.0040 168

878

(“.oo35 18 563 /J&!)

pg) DNAse and 0.1 ml of 1% SDS. Lysis was rapid and the preparation was incubated for 10 min at 37°C. An additional 0.1-0.2 ml of SDS was then usually added to assure complete lysis. When DEAE Dextran was used to facilitate penetration, the cells were exposed to 50 pg/ml for 10 min at room temperature; then washed. After poly(A)poly(U) penetration, the cells were incubated for 5 min in 200 pg/ml Dextran sulphate and then treated as above.

Gradient centrifugcl?ion. For gradient centrifugation the lysate was diluted with an equal volume of TBS, layered on a 5-20% sucrose gradient (made in TBS) and centrifuged for I8 h at 23000 rpm in an SW 25.1 rotor at 15°C. One ml fractions were counted in Aquasol (New England Nuclear). To measure the distribution between acid-soluble and acid-precipitable material, the cytoplasmic contents released by Nonidet P40 and the nuclear lysate were precipitated with 8% TCA, centrifuged and the supematants and precipitates counted. Expfl Cell Res 94 (1975)

Table 2. Effect of DEAE Dextran on poly(A)-poly(U)penetration

of FLS cells

I, 22 pg/ml of poly(Y-A)-poly(IJ); II, IO *g/ml of poly(Y-A)-poly(U). Non-radioactive polymer was added to achieve these concentrations. The cell concentrations were lO’/ml DEAE Dextran

Dextran sulphate

Nuclear AP material, pg/lO’ cells

Expt I +

-

0.015 5.23

Expr II +

+

0.34

Poly(A)-poly(U):

cell penetration

Fig. I. Radioautography of incorporated poly(A& polyPH-U). Mouse ascites tumor cell (FLS tumour).

and nucleic acid synthesis

317

pared from cells exposed to poly(A)poly(U), treated with Dextran sulphate after removal of the polymer and incubated an additional 2 h shows that the peak is stable, although there is some increase in the amount of lower molecular weight material (fig. 2d). Poly(U) is lighter than poly(A)-poly(U) (fig. 2e) and is degraded in the presence of a nuclear lysate (fig. 2f). We conclude from these data that the material recovered from nuclei is double-stranded. In LNH 13 cells pretreated with DEAE Dextran DNA and RNA syntheses increased shortly after poly(A)-poly(U) was added (fig. 3). Similar results were obtained in three distinct experiments.

DISCUSSION plexes can be dissolved by addition of The uptake of poly(A jpoly(U) and of dextran sulphate in appropriate conditions [12]. FLS cells, exposed to DEAE Dextran, poly(A)-2 poly(U) has been demonstrated washed and incubated for 2 min at room by Schell [6] for Ehrlich ascites tumour temperature with poly(14C-A)-poly(U), re- cells. He recovered acid-precipitable matained 7660 cpm after two TRS washes. terial after nuclease treatment of these cells Treatment with Dextran sulphate removed and also re-isolated the triple-stmnded comall but 109 cpm. Finally the effect of DEAE plex by sucrose density gradient centrifugaDextran, including subsequent Dextran tion. Since poly(A)-poly(U) is thought to sulphate incubation, was to increase stimulate immune function and to affect poly(A)-poly(U) penetration into nuclei macromolecular synthesis, we wished to about 20-fold (table 211). The state of extend Schell’s observations to lymphopoly(A)-poly(U) in LHN 13 cells was de- cytes and to show that poly(A)-poly(U) termined by centrifugation of the nuclear reaches the cell nucleus. We confirm the entry, but not the state, lysate through a sucrose gradient. The cells were preincubated in DEAE Dextran of poly(A)-poly(U) by radioautography and, after penetration of poly(A)-poly(U), (fig. 1) and show that acid-precipitable matreated with Dextran sulfate. Fig. 2 shows terial is found in both cytoplasm and nuthat two controls-poly(A)-poly(U) alone clei (table 1). (fig. 2a) and poly(A)-poly(U) added to a We took advantage of the facilitation of poly(A)-poly(U) uptake by DEAE Dextran nuclear lysate (fig. 2b)-and poly(A)poly(U) which has penetrated the nuclei (table 2) to re-isolate the polymer from nu(fig. 2 c) all sediment at the same position in clei and to show that it sediments as a symthe gradient. A gradient of a lysate pre- metric peak at the same position as control Exptl Cell Res 94 (1975)

Fenster, Lacour and Hare1

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Exprl Cell Res 94 (1975)

Poly(A)-poly(U):

cell penetration

and nucleic acid synthesis

poly(A)-poly(U) in sucrose gradients (fig. 2). Poly(U) is significantly lighter than poly(A)-poly(U) (compare fig. 2a, e) and is degraded by a nuclear lysate (fig. 2f). This argues against both strand separation of penetrated poly(A)-poly(U) and its degradation and reincorporation into cellular RNA. Furthermore the greater part of poly(A)-poly(U) incorporated into nuclei is not destroyed for at least 2: h. Chess et al. [7] measured the ability of poly(A)-poly(U) to stimulate DNA synthesis in leucocytes and found that poly(A)poly(U) alone gave variable results, but that in combination with an antigenic stimulant, uniformly increased thymidine incorporation. In no case, however, did they report any effect before the fourth day of incubation of the cells with poly(A)POlYW).

Teng et al. [8], on the other hand, found that poly(Atpoly(U) depressed DNA synthesis in HeLa cells to 60 % of normal after just 4 h contact, but only when these cells were in late G 1 or S phase. We report here that lymphocytes, treated with DEAE Dextran show stimulated DNA and RNA syntheses shortly after the addition of

3 19

td

min; ordinate: (left) 3H; (right) ‘“C, cpm x 10m4. Nucleic acids synthesis. LNH 13 cells were washed with Eagle’s treated for 10 min with DEAE Dextran and resuspended at 1.2~ IO6 cells/ml with or without 100 @g/ml poly(Atpoly(U), and 3H-thymidine (3.5 pCi/ml) and 14C-uridine (0.44 &i/ml) were added to both samples. Cells were incubated at 37°C and incorporated into acid-precipitable material was followed. -, thymidine incorporation; - - -, uridine incorporation. Closed symbols, minus poly(A)-poly(U); open symbols, plus poly(A)-poly(U). Fig. 3. Abscissa:

pol~(At~ol~RJ).

We cannot say whether this represents an effect on the synthetic process or results from increased permeability of nucleosides

Fig. 2. Abscissa: min; ordinate: cpmx 10m3. Density gradient centrifugation of poly(A)-poly(U). (a) poly(A)-poly(“C-U) alone;(b)poly(A)-poly(r4C-U) added to the nuclear lysate prepared from lOrLHNl3 cells: (c) poly(A)-poly(“‘C-IJ) in the nuclear lysate obtained from 2x IO’ LHN13 cells after 30 min penetration. The polymer concentration was 4.3 pg/ml; (d) LHN13 cells (5~ 106/mI)were incubated for 40 min in 2.14 pg/ml poly(A)-p01y(‘~C-U), washed, treated with Dextran sulfate, rewashed and resuspended in Eagle’s for an additional 2 h. Thirty minutes were consumed by the several centrifugations, making the total chase equal to 21 h. (e) poly(‘4c-U); (f) poly(r4C-U) after a 10 min incubation at 37°C with a nuclear lysate.

in the presence of poly(A)-poly(U). While we have no direct evidence, the possibility is not excluded that this early increased nucleic acid synthesis is related to the immune stimulation by poly(A)-poly(U). We thank Dr E. Merlin-Nahon for the preparation of poly(Atpoly(U), Mr T. Huynh for maintenance of the cell cultures, and Dr A. Fourcade for indispensible counsel. This work was supported by Institut National de la Sante et de la Recherche Medicale (INSERM) and by Cancer Research Institute, Inc. New York. E. D. F. gratefully acknowledges the “Prix Annie Dalsace” and the cooperative hospitality of the personnel at Institut Gustave-Roussy. Expt/ Cell Res 94 (1975)

320

Fenster, Lacour and Hare1 REFERENCES

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Exprl

Cell

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