- Email: [email protected]
Uptake of exogenous ribonucleic acid by ascites tumor cells
Experimental
Cell Research
UPTAKE
I.
43, 381-390
381
(1966)
OF EXOGENOUS RIBONUCLEIC ASCITES TUMOR CELLS
AUTORADIOGRAPHIC
AND
P. GALAND,’
J.
ACID BY
CHROMATOGRAPHIC
REMY
and
L.
STUDIES
LEDOUX
Section de Biochimie Cellulaire, Ddpartement de Radiobiologie, Centre d’Etude de 1’Energie Nuclkaire, Mol-Donk, Belgium Received
February
15, 1966
WE
have previously reported results of experiments leading to the conclusion that ascites tumor cells (Landschiitz) were able to take up heterologous ribonucleic acid (RNA) [ 13, 151. Similar results have been obtained by Hare1 et al. [3] and Amos [I ] and have, since then, been confirmed by several authors working with different organisms [12, 181. It was, however, necessary to obtain more detailed evidence that the utilization of an exogenous RNA did not involve its degradation by the cells, followed by de novo synthetic processes. It was also interesting to know whether the RNA taken up by the cells retains its initial characteristics of structure and composition. This study is the object of the present paper.
MATERIAL
AND
METHODS
Cells
The transplantable ascitic Landschiitz carcinoma (grown intraperitoneally into C+ mice) was used. The median survival time of the hostmice was 16 days. The cells, harvested after neck dislocation and dissection of the mice, are washed free from red cells by slow centrifugation (500 rev/min) in 9 % NaCl. They are then suspended in an adequate medium and incubated at 37°C in a Bekso agitating thermostate. The final cells number was about 106/ml. Media
The medium of Krebs-Ringer, modified according to King et al. [20] was used, buffered with 0.1 M phosphate (pH 7.4). Glucose is present at a final concentration of 1.4 mmoles/L. 1 Present 1, Belgium.
address:
Laboratoire
Central
de MBdecine
NuclBaire,
115, bd. de Waterloo,
Experimental
Bruxelles
Cell Research
43
382
P. Galand,
J. Remy and L. Ledoux
Similar results were obtained with the medium of Pileri et al. [34] consisting of 8.5 %.NaCI: 15 ml, glucose (36 mg/ml of 0.1 M phosphate buffer at pH 7.2): 5 ml. To one volume of this mixture, an equal volume of ascitic plasma is added. Labeled
RNA’s
(3H) or (%) RNA is prepared from yeast grown in presence of (3H) uridine or (14C) adenine (Amersham products). The method of Crestfield et al. [9] is used. TABLE
,-
I.
Distribution of the activity in % of total activity
Guanine (1)
Subsfrafe
of incubafion
(*‘C)
Substrate (a) (b)
of incubation
(sH)
25k3
75*5
-
-
26f3
73&S
-
-
25k3
74&S
-
-
19t2
26+3
34a4
21+2
22f3
29+4
28f4
2112
21$2
27k4
32t5
20&Z
RNA
yeast RNA before incubation RNA from cells incubated for 40
min with 350 pg/ml of labeled RNA (c) id. but 3.5 mg of equimolecular proportionsof the 4 nucleotides added
Uridylic acid
RNA
(a) yeast RNA before incubation (b) RNA from cells incubated for 40 min with 100 pg/ml of labeled RNA (c) id. but 250 ,ug of adenine added per ml during incubation (2)
Adenine
Cytidylic acid
The product obtained has a maximum/minimum U.V. absorption ratio of 2.7 and is free of measurable amounts of DNA and proteins. It has an activity of 15,000 dpm/,ug in the caseof (%) RNA and of 2600 dpm/pg in the caseof (3H) RNA. We also prepared bacterial RNA, labeled with tritium, by cultivating Escherichia coli, strain M226-8 Davis (kindly supplied by Professor Wiame), in presence of (3H) adenosine (Amersham) and extracting the RNA following the technique of Kirby
WI.
The final purified product has an activity of 25,000 dpm/yg. In the experiments summarized in Table I, we used a commercial preparation of yeast RNA (Mann product) labeled by tritium exchange (this was achieved by Dr M. Winand, in the Department of the Radioisotopes, C.E.N., Mol). The tritiated product contained 15 per cent of acid soluble products at the time of the experiment. It had an activity of 12,740 dpm/pg, and a min/max U.V. absorption ratio of 2.08. Experimental
Cell Research
43
RNA uptake Biochemical
by ascites
tumor
383
cells. I
determinations
The cells are washed by three repeated centrifugations at 1000 rpm in presence of 9 L NaCl ascitic plasma (1:l). They are then submitted to the extracting procedure of Schmidt-Thannhauser [37]. The concentration in nucleic acid-soluble products is measured by spectrophotometry, using the Beckman DKl recording spectrophotometer. The RNA and DNA contents are determined, respectively by the orcinol and by the diphenylamine procedures. In someexperiments the nucleotides concentration of those fractions was estimated from the U.V. spectrum recorded by use of the Beckman DKI recording spectrophotometer. In this case the difference E,,, rnp - Ezoornp is taken as a measure of this concentration. Protein determinations were made by the calorimetric method of Lowry et al. [28] using the Folin-Ciocalteu reagent. Radioactivity
measurements
They are made in liquid scintillation, using a Packard Tri-Carb automatic scintillation counter. Autoradiography
The cells are included in a 1.3 per cent agar block, which is then dehydrated and, after paraffin embedding, is cut at 34 p. Ilford emulsion in gel form, type L4 is used according to Ficq [Ill. Before the autoradiographic process, the slices are treated with cold 5 per cent perchloric acid, in order to eliminate acid soluble compounds. Ribonucleasetests were made following Brachet [5]. Chromatographic
procedures
The basecomposition of the RNA hydrolysate, obtained as describedby Markham and Smith [29], was determined after ascendingchromatography on Whatman No. 3 paper, using a mixture of methanol-HCl-water (7:2:1), Kirby [21]. The four spots were eluted with HCl I N, and their activity was measuredby scintillation counting. The chromatographic analysis of the RNA preparations (yeast-bacterial or tumorRNA) was performed with the centrifuged DEAE-cellulose paper pulp technique of Ledoux et al. [26], Davila et al. [9], reviewed by Ledoux [23]. RESULTS Kinetics
of RNA
uptake
Cells from a tumor implanted for 8 days mere incubated in vitro with 350 &ml of yeast-(3H) RNA. The radioactivity of the nucleic acid and acidsoluble fractions of the cells was measured and expressed as dpm/O.D. units. 25-661807
Experimental
Cell Research
43
384
P. Galand,
J. Remy and L. Ledoux
Fig. 1 a and b shows the results so obtained for incubation times varying from 0 to 40 min. In 1 a the RNA preparation used was contaminated by 10 per cent labeled acid-soluble products. In 1 b no labeled acid-soluble compounds were present in the RNA preparation used.
0 Duration
of incubatron
Fig. 1 o.
(mtn.)
510
20 Duration
PO of incubation
(min.)
Fig. 1 b.
Fig. l.-Specific activity of the nucleic acid (N.A.) and acid-soluble (A.S.) fractions from cells incubated with tritiated yeast RNA (350 pg/ml). (a) Yeast RNA preparation contained 10 % acid-soluble labeled components. Standard deviation of the mean is indicated. * . . . * ( l ), A.S.; ___ (o), N.A. (b) Yeast RNA preparation free from labeled acid-soluble components. Same values for the axes as in Fig. 1 a. * . . . . , A.S.; -, N.A.
This curve is representative of the shape of the curves obtained in similar experiments in which the external concentration in RNA varied from 0.1 to 1 mg/ml (using bacterial or yeast RNA). It can be seen that an initial rapid uptake is followed by a slowing-down of the phenomenon. The autoradiographs made at corresponding times (see Fig. 4), show that the label is actually intracellular. The curve obtained by using the grain counts instead of the radioactivity measurements, tits with the curve illustrated in Fig. 1. An important feature of those experiments is that, when we used a freshly prepared dialysed RNA, devoid of acid soluble components, there appears no label in the cellular acid-soluble fraction. For an external concentration of 500 pg RNA/ml, calculation shows that the amount taken up by the cells after 10 min incubation represents 2-4 per cent of the normal cellular RNA content. Experimental
Cell
Research
43
RNA
385
uptake by ascites tumor cells. I
Autoradiography Fig. 2 shows the results obtained in experiments in which the cells were incubated for different times with 375 &ml of (3H) RNA, then submitted to the autoradiographic process.
b
a
L d
Y
2
1
I
15
2s
I
45 rime
(min.)
-dm e
Fig. 2.
Fig. 3.
Fig. 2.-Autoradiography: grains counts over the different cellular regions as a function of incubation time in presence of (SH) RNA (see text), (A), mean grains count over nucleolus; * . . ( l ), mean grains count over non-nucleolus part of the nucleus; - ( l ), mean grains count over cytoplasm. Fig. 3.-Distribution of the chromatographic fractions (chromatography on DEAE cellulose) of the RNA extracted from cells incubated with labeled yeast RNA as compared with the RNA used and with the normal ascites cells RNA. The numbers in abscissa refer to the order of elution of the fractions in the stepwise chromatography. The ordinate values are proportional to the relative concentration of each fraction. (a) Radioactivity distribution in the RNA fractions from cells incubated with the yeast (14C) RNA (chromatographic profile in Fig. 3b); (-), after 20 min of incubation; - - -, after 40 min of incubation. (b) Chromatographic profile of the (14C) RNA used in Fig. 3~. (c) Radioactivity distribution in the RNA fractions from cells incubated with the yeast (sH) RNA (see Fig. 3d); (- - - -), after 20 min of incubation; (+-), after 40 min of incubation. (d) Chromatographic profile of the (8H) RNA used in Fig. 3~. (e) Chromatographic profile of normal ascites cells RNA (spectrophotometric measurements).
The number of developed grains over the different cellular regions (cytoplasm, nucleolus and non-nucleolar nucleus) was counted at different incubation times. It can be seen that the cytoplasm and the nucleus become nearly equally labeled and that they present a similar rate of uptake of the labeled RNA. Experimental
Cell Research
43
386
P. Galand,
J. Remy and L. Ledoux
The nucleolus, however, remains poorly labeled during the time considered. The addition, in the incubation medium, of a non-radioactive mixture of equimolecular amounts of the four 5’-nucleotides, has no effect, quantitatively nor qualitatively, on the resulting RNA uptake. The kinetics of labeling of the different parts of the cell is, in fact, completely different from what is generally observed for the incorporation of precursors in ribonucleic acids [33, 401. Chroma tographic
study
The cells were incubated with 100 &ml of yeast (14C) RNA. Chromatographic analysis showed that 75 per cent of the label was bound to adenine and 25 per cent to guanine. After 20 and 40 min of incubation in presence of this (l”C) RNA, the cells are washed and their RNA is extracted by the phenol procedure of Kirby [22]. This fraction is then submitted to the base composition analysis in parallel with chromatographic separation on DEAE-cellulose paper [9, 23, 251. The distribution of radioactivity among the bases and nucleotides or among the six frac,tions separated by chromatography was then compared with the results obtained with the (1°C) RNA, treated by the same way. Table I shows the data obtained for the base composition analysis. This table also shows results obtained with a yeast (3H) RNA used in similar experiments. In parallel assays, we added an excess of unlabeled adenine or nucleosides and nucleotides to the labeled RNA during incubation. It can be seen from Table I, that the distribution of activity between the four bases or nucleotides is the same in the RNA extracted from the incubated cells and in the RNA preparation used. Addition of unlabeled precursors does not modify the results. The fact that the RNA taken up by the cells retains its base composition clearly supports the conclusion that this uptake does not lead to a great alteration of its properties, even after 40 min incubation. Fig. 3 shows the results obtained by measuring the distribution of the radioactivity among the six fractions separated by chromatography on DEAE-cellulose paper. It can be seen that the radioactivity is distributed in a similar manner in the case of the (‘“C) RNA (Fig. 3b) used and in the case of the RNA prepared from cells incubated for 20 and 40 min with the labeled RNA (Fig. 3 a). Figs. 3 c and 3 d show the results of a comparative experiment in which (3H) RNA hydrolysate was used. They show that in this case no label appears Experimental
Cell
Research
43
RNA uptake
Fig. 4.-Ascites cells treated for 20 min 20 min with 5 y0 perchloric acid. Emulsion:
by ascites tumor
387
cells. I
with (3H) yeast RSA 3 EL sections Ilford in gel form, type L 4.
of cells,
treated
for
in the fractions corresponding to polymerized RNA (of the six fractions, separated by this chromatographic technique, two, the lirst and second ones, correspond to acid soluble products) [23, 251. The chromatographic profile of the RNA constitutive of the ascites cells, as established by spectrophotometric measurements (Fig. 3c), is very different from the profile of the yeast RNA used in these experiments.
DISCUSSION
Apparently, ascites tumor cells (Landschtitz) are able to utilize exogenous RNA. That this utilization does not involve prior degradation of the RNA is suggested by the fact that no label appears in the acid-soluble fraction of the cells when the RNA preparation used is itself free of acid-soluble products. This is in total agreement with the results obtained with a similar material by Hare1 et al. [17] using homologous RNA. It also agrees with the results Experimental
Cell
Research
43
388
P. Galand,
J. Remy and L. Ledoux
of Amos [I] who showed that the pretreatment of the exogenous RNA by ribonuclease decreases the rate of its uptake by embryonic cells in tissue culture. The keeping of the distribution of the radioactivity among the bases and nucleotides of the labeled RNA after its reextraction from the cells has also been observed by Niu et al. [32] in the case of uptake of liver RNA by ascites tumor cells. Those results support the hypothesis of a direct penetration of intact exogenous RNA into the cells. This is also confirmed in our experiments by the chromatographic assays on DEAE-cellulose paper, which indicates that the RNA taken up by the cells retains its chromatographic properties, even in the cells incubated during 40 min. Our results seem to exclude a degradationsynthesis process for the incorporation of the exogenous RNA by the ascites cells. In addition to this, the autoradiography gives results showing that the labeling of the different parts of the cell occurs with kinetics quite different from what is generally found in the case of cellular RNA synthesis [33, 401, the cytoplasm being here labeled at the same time as the nucleus and always more heavily than the nucleolus. The question of the mechanism of penetration of RNA into living cells is not yet clear. However, the known existence of pinocytotic activity in ascites tumor cells [lo] provides a hypothetic possibility for the mechanism of uptake of the exogenous macromolecules by living cells. It seems, indeed, that the penetration of a macromolecule like RN,4 does not represent an isolated case. Similar observations were made in the case of ribonuclease, which was shown to enter into vegetal or animal cells [B, 24, 27j. More recent work indicates the possibility of DNA uptake by several living materials [l, 4, 18, 35, 38, 391. Gartler [16], Bensch and King [3] and Hill and Jakubikova [ 191 have provided more direct evidence that utilization by animal cells of a homologous DNA might not be due to a combined degradation-resynthesis process. A similar conclusion, in the case of a heterologous DNA, appears from the works of Schimizu et al. [36], Ledoux and Charles [25] and Ledoux [23]. A general mechanism of the “pinocytosis” type can thus be involved in the uptake of macromolecules and it is not necessary to imagine a special process for this uptake. Our results show that there is the possibility of an eventual biological action of the absorbed RNA. This has been studied by several authors Experimental
Cell Research
43
RNA uptake
by ascites tumor cells. I
389
[2, 30, 31, 351. Let us remember that we have shown that in the case of ascites cells, yeast RNA activates the incorporation of amino acids in the proteins [14]. Detailed results on this work will be published elsewhere. SUMMARY
Ascites tumor cells are able to ingest a labeled heterologous RNA which is dissolved in their incubation medium. The maximum quantity taken up after 40 min of incubation represents 2-4 per cent of the normal RNA content of those cells. Autoradiographic study shows that the RNA taken up is mainly located :n the nucleus and the cytoplasm of the cells. Far less is fixed by the nucleolus. The base composition as well as the chromatographic behaviour on DEAEcellulose paper of the exogenous RNA (yeast RNA) seem to be maintained after ingestion by the cells. The results suggest that ascites tumor cells can take up intact RNA, without greatly modifying its structure and composition. The mechanism of this RNA uptake has not yet been clarified. We wish to thank Prof. M. Errera for his interest and advice during the course of this work. This work has been performed under the contract Euratom-C.E.N. 014-62-I-BIAB. REFERENCES 1. AMOS,
H.,
2. AMOS,
H. and KEARNS,
Biochem.
Res. Comm. 5, 1 (1961). K. E., Nafure 195,806(1962).
Biophys.
3. BENSCH, K. G. and KING, D. W., Science 33, 381 (1961). 4. BORENFREUND, E. and BENDICH, A., J. Biophys. Biochem. 5. BRACHET, J., Quart. J. Microscop. Sci. 94, 1 (1953).
Cytol.
9, 81 (1961).
6. -7. __
Nature 175, 879 (1954). Biochim. Biophys. Acta 19, 583 (1956). 8. CRESTFIELD, A. M., SMITH, K. C. and ALLEN, F. W., J. Biol. Chem. 216, 185 (1955). 9. DAVILA, C., CHARLES, P. and LEDOUX, L., In press. 10. EASTY, D. M., LEDOUX, L. and AMBROSE, E. J., Biochim. Biophys. Acfa 20, 528 (1956). 11. FICQ, A., in J. BRACHET and A. E. MIRSKY (eds.), The Cell, Vol. 1, chap. 3. Academic Press, 12.
13. 14. 15. 16. 17. 18. 19. 20.
New York, 1959. FISHMAN, M., HAMMERSTR~M, GALAND, P., Arch. Intern.
__
ibid.
R. A. and
PhysioI.
V. P., Nature 71, 816 (1963).
BOND,
Biochem.
198, 549 (1963).
72, 319 (1964).
GALAND, P. and LEDOUX, L., ibid. 69, 383 (1961). GARTLER, S. M., Biochem. Biophys. Res. Comm. 3, 127 (1960). HAREL, C., HAREL, J. and LACOUR, F., Compf. Rend. Acad. Sci. HILL, M. and DRASIL, V., Expff CeZI Res. 21, 569 (1960). HILL, M. and JAKUBIKOVA, J., ibid. 26, 541 (1952). KING, D. W., PAULSON, S. R., HANNAFORD, N. D. and PUCKETT, Report 1710, Fort Knox, Kentucky, 1955.
253, 2000 (1960).
N. L., Army
Experimental
Med.
Res.
Cell Research
Lab.
43
P. Galand,
J. Remy and L. Ledoux
21. KIRBY, K. S., Biochim. Biophys. Acta 18, 575 (1955). 22. __ Biochem. J. 64, 405 (1956). 23. LEDOUX, L:, in Progress in Nucleic Acid Research. Vol. 4. Acad. Press, New York, 1966. 24. LEDOUX L. and BALTUS, E., Experienfia 10, 401 (1954). 25. LEDOUX, L. and CHARLES, P., Arch. Intern. Physiol. Biochem. 70, 158 (1962). 26. LEDOUX, L., CHARLES, P. and DAVILA, C., Arch. Intern. Physiol. Biochem. 71, 820 (1963). 27. LEDOUX, L., LECLERC, J. and VANDERHAEGHE, F., Nature 174, 793 (1954). 28. LOWRY, 0. H., ROSEBROUGH, S. I., FARR, A. L. and RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 29. MARKHAM, R. and SMITH, J. D., Biochem. J. 49, 401 (1951). 30. NIU, M. C., Fed. Proc. 22, part 1, 354 (1962). 31. __ Deuetop. Biol. 7, 379 (1963). 32. NIU, M. C., CORDOVA, C. C. and NIU, L. C., Proc. Nat1 Acad. Sci. 47, 1681 (1961). 33. PERRY, R. P., HELL, A. and ERRERA, M., Biochim. Biophys. Acta 49, 47 (1961). 34. PILERI, A., LEDOUX, L. and VANDERHAEGHE, F., Exptl Cell Res. 17, 218 (1959). 35. RIEKE, W. O., .I. Cell Biol. 13, 205 (1962). 36. SCHIMIZU, T., KOYAMA, S. and I~A~UCHIN, M., Biochim. Biophys. Acta 55, 795 (1962). 37. SCHMIDT,‘G. and THAN.NHAUSER, S. T., J. Biochem. Chem. 161, 83 (1945). 38. SCHWARZ, M. R. and RIEKE, \I:. O., Science 136, 151 (1962). 39. WILCZOK, T., Nature 196, 1314 (1962). 40. ZALOKAR, M., Exptl Cell Res. 19, 559 (1960).
Experimental
Cell Research
43
Cell Research
UPTAKE
I.
43, 381-390
381
(1966)
OF EXOGENOUS RIBONUCLEIC ASCITES TUMOR CELLS
AUTORADIOGRAPHIC
AND
P. GALAND,’
J.
ACID BY
CHROMATOGRAPHIC
REMY
and
L.
STUDIES
LEDOUX
Section de Biochimie Cellulaire, Ddpartement de Radiobiologie, Centre d’Etude de 1’Energie Nuclkaire, Mol-Donk, Belgium Received
February
15, 1966
WE
have previously reported results of experiments leading to the conclusion that ascites tumor cells (Landschiitz) were able to take up heterologous ribonucleic acid (RNA) [ 13, 151. Similar results have been obtained by Hare1 et al. [3] and Amos [I ] and have, since then, been confirmed by several authors working with different organisms [12, 181. It was, however, necessary to obtain more detailed evidence that the utilization of an exogenous RNA did not involve its degradation by the cells, followed by de novo synthetic processes. It was also interesting to know whether the RNA taken up by the cells retains its initial characteristics of structure and composition. This study is the object of the present paper.
MATERIAL
AND
METHODS
Cells
The transplantable ascitic Landschiitz carcinoma (grown intraperitoneally into C+ mice) was used. The median survival time of the hostmice was 16 days. The cells, harvested after neck dislocation and dissection of the mice, are washed free from red cells by slow centrifugation (500 rev/min) in 9 % NaCl. They are then suspended in an adequate medium and incubated at 37°C in a Bekso agitating thermostate. The final cells number was about 106/ml. Media
The medium of Krebs-Ringer, modified according to King et al. [20] was used, buffered with 0.1 M phosphate (pH 7.4). Glucose is present at a final concentration of 1.4 mmoles/L. 1 Present 1, Belgium.
address:
Laboratoire
Central
de MBdecine
NuclBaire,
115, bd. de Waterloo,
Experimental
Bruxelles
Cell Research
43
382
P. Galand,
J. Remy and L. Ledoux
Similar results were obtained with the medium of Pileri et al. [34] consisting of 8.5 %.NaCI: 15 ml, glucose (36 mg/ml of 0.1 M phosphate buffer at pH 7.2): 5 ml. To one volume of this mixture, an equal volume of ascitic plasma is added. Labeled
RNA’s
(3H) or (%) RNA is prepared from yeast grown in presence of (3H) uridine or (14C) adenine (Amersham products). The method of Crestfield et al. [9] is used. TABLE
,-
I.
Distribution of the activity in % of total activity
Guanine (1)
Subsfrafe
of incubafion
(*‘C)
Substrate (a) (b)
of incubation
(sH)
25k3
75*5
-
-
26f3
73&S
-
-
25k3
74&S
-
-
19t2
26+3
34a4
21+2
22f3
29+4
28f4
2112
21$2
27k4
32t5
20&Z
RNA
yeast RNA before incubation RNA from cells incubated for 40
min with 350 pg/ml of labeled RNA (c) id. but 3.5 mg of equimolecular proportionsof the 4 nucleotides added
Uridylic acid
RNA
(a) yeast RNA before incubation (b) RNA from cells incubated for 40 min with 100 pg/ml of labeled RNA (c) id. but 250 ,ug of adenine added per ml during incubation (2)
Adenine
Cytidylic acid
The product obtained has a maximum/minimum U.V. absorption ratio of 2.7 and is free of measurable amounts of DNA and proteins. It has an activity of 15,000 dpm/,ug in the caseof (%) RNA and of 2600 dpm/pg in the caseof (3H) RNA. We also prepared bacterial RNA, labeled with tritium, by cultivating Escherichia coli, strain M226-8 Davis (kindly supplied by Professor Wiame), in presence of (3H) adenosine (Amersham) and extracting the RNA following the technique of Kirby
WI.
The final purified product has an activity of 25,000 dpm/yg. In the experiments summarized in Table I, we used a commercial preparation of yeast RNA (Mann product) labeled by tritium exchange (this was achieved by Dr M. Winand, in the Department of the Radioisotopes, C.E.N., Mol). The tritiated product contained 15 per cent of acid soluble products at the time of the experiment. It had an activity of 12,740 dpm/pg, and a min/max U.V. absorption ratio of 2.08. Experimental
Cell Research
43
RNA uptake Biochemical
by ascites
tumor
383
cells. I
determinations
The cells are washed by three repeated centrifugations at 1000 rpm in presence of 9 L NaCl ascitic plasma (1:l). They are then submitted to the extracting procedure of Schmidt-Thannhauser [37]. The concentration in nucleic acid-soluble products is measured by spectrophotometry, using the Beckman DKl recording spectrophotometer. The RNA and DNA contents are determined, respectively by the orcinol and by the diphenylamine procedures. In someexperiments the nucleotides concentration of those fractions was estimated from the U.V. spectrum recorded by use of the Beckman DKI recording spectrophotometer. In this case the difference E,,, rnp - Ezoornp is taken as a measure of this concentration. Protein determinations were made by the calorimetric method of Lowry et al. [28] using the Folin-Ciocalteu reagent. Radioactivity
measurements
They are made in liquid scintillation, using a Packard Tri-Carb automatic scintillation counter. Autoradiography
The cells are included in a 1.3 per cent agar block, which is then dehydrated and, after paraffin embedding, is cut at 34 p. Ilford emulsion in gel form, type L4 is used according to Ficq [Ill. Before the autoradiographic process, the slices are treated with cold 5 per cent perchloric acid, in order to eliminate acid soluble compounds. Ribonucleasetests were made following Brachet [5]. Chromatographic
procedures
The basecomposition of the RNA hydrolysate, obtained as describedby Markham and Smith [29], was determined after ascendingchromatography on Whatman No. 3 paper, using a mixture of methanol-HCl-water (7:2:1), Kirby [21]. The four spots were eluted with HCl I N, and their activity was measuredby scintillation counting. The chromatographic analysis of the RNA preparations (yeast-bacterial or tumorRNA) was performed with the centrifuged DEAE-cellulose paper pulp technique of Ledoux et al. [26], Davila et al. [9], reviewed by Ledoux [23]. RESULTS Kinetics
of RNA
uptake
Cells from a tumor implanted for 8 days mere incubated in vitro with 350 &ml of yeast-(3H) RNA. The radioactivity of the nucleic acid and acidsoluble fractions of the cells was measured and expressed as dpm/O.D. units. 25-661807
Experimental
Cell Research
43
384
P. Galand,
J. Remy and L. Ledoux
Fig. 1 a and b shows the results so obtained for incubation times varying from 0 to 40 min. In 1 a the RNA preparation used was contaminated by 10 per cent labeled acid-soluble products. In 1 b no labeled acid-soluble compounds were present in the RNA preparation used.
0 Duration
of incubatron
Fig. 1 o.
(mtn.)
510
20 Duration
PO of incubation
(min.)
Fig. 1 b.
Fig. l.-Specific activity of the nucleic acid (N.A.) and acid-soluble (A.S.) fractions from cells incubated with tritiated yeast RNA (350 pg/ml). (a) Yeast RNA preparation contained 10 % acid-soluble labeled components. Standard deviation of the mean is indicated. * . . . * ( l ), A.S.; ___ (o), N.A. (b) Yeast RNA preparation free from labeled acid-soluble components. Same values for the axes as in Fig. 1 a. * . . . . , A.S.; -, N.A.
This curve is representative of the shape of the curves obtained in similar experiments in which the external concentration in RNA varied from 0.1 to 1 mg/ml (using bacterial or yeast RNA). It can be seen that an initial rapid uptake is followed by a slowing-down of the phenomenon. The autoradiographs made at corresponding times (see Fig. 4), show that the label is actually intracellular. The curve obtained by using the grain counts instead of the radioactivity measurements, tits with the curve illustrated in Fig. 1. An important feature of those experiments is that, when we used a freshly prepared dialysed RNA, devoid of acid soluble components, there appears no label in the cellular acid-soluble fraction. For an external concentration of 500 pg RNA/ml, calculation shows that the amount taken up by the cells after 10 min incubation represents 2-4 per cent of the normal cellular RNA content. Experimental
Cell
Research
43
RNA
385
uptake by ascites tumor cells. I
Autoradiography Fig. 2 shows the results obtained in experiments in which the cells were incubated for different times with 375 &ml of (3H) RNA, then submitted to the autoradiographic process.
b
a
L d
Y
2
1
I
15
2s
I
45 rime
(min.)
-dm e
Fig. 2.
Fig. 3.
Fig. 2.-Autoradiography: grains counts over the different cellular regions as a function of incubation time in presence of (SH) RNA (see text), (A), mean grains count over nucleolus; * . . ( l ), mean grains count over non-nucleolus part of the nucleus; - ( l ), mean grains count over cytoplasm. Fig. 3.-Distribution of the chromatographic fractions (chromatography on DEAE cellulose) of the RNA extracted from cells incubated with labeled yeast RNA as compared with the RNA used and with the normal ascites cells RNA. The numbers in abscissa refer to the order of elution of the fractions in the stepwise chromatography. The ordinate values are proportional to the relative concentration of each fraction. (a) Radioactivity distribution in the RNA fractions from cells incubated with the yeast (14C) RNA (chromatographic profile in Fig. 3b); (-), after 20 min of incubation; - - -, after 40 min of incubation. (b) Chromatographic profile of the (14C) RNA used in Fig. 3~. (c) Radioactivity distribution in the RNA fractions from cells incubated with the yeast (sH) RNA (see Fig. 3d); (- - - -), after 20 min of incubation; (+-), after 40 min of incubation. (d) Chromatographic profile of the (8H) RNA used in Fig. 3~. (e) Chromatographic profile of normal ascites cells RNA (spectrophotometric measurements).
The number of developed grains over the different cellular regions (cytoplasm, nucleolus and non-nucleolar nucleus) was counted at different incubation times. It can be seen that the cytoplasm and the nucleus become nearly equally labeled and that they present a similar rate of uptake of the labeled RNA. Experimental
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The nucleolus, however, remains poorly labeled during the time considered. The addition, in the incubation medium, of a non-radioactive mixture of equimolecular amounts of the four 5’-nucleotides, has no effect, quantitatively nor qualitatively, on the resulting RNA uptake. The kinetics of labeling of the different parts of the cell is, in fact, completely different from what is generally observed for the incorporation of precursors in ribonucleic acids [33, 401. Chroma tographic
study
The cells were incubated with 100 &ml of yeast (14C) RNA. Chromatographic analysis showed that 75 per cent of the label was bound to adenine and 25 per cent to guanine. After 20 and 40 min of incubation in presence of this (l”C) RNA, the cells are washed and their RNA is extracted by the phenol procedure of Kirby [22]. This fraction is then submitted to the base composition analysis in parallel with chromatographic separation on DEAE-cellulose paper [9, 23, 251. The distribution of radioactivity among the bases and nucleotides or among the six frac,tions separated by chromatography was then compared with the results obtained with the (1°C) RNA, treated by the same way. Table I shows the data obtained for the base composition analysis. This table also shows results obtained with a yeast (3H) RNA used in similar experiments. In parallel assays, we added an excess of unlabeled adenine or nucleosides and nucleotides to the labeled RNA during incubation. It can be seen from Table I, that the distribution of activity between the four bases or nucleotides is the same in the RNA extracted from the incubated cells and in the RNA preparation used. Addition of unlabeled precursors does not modify the results. The fact that the RNA taken up by the cells retains its base composition clearly supports the conclusion that this uptake does not lead to a great alteration of its properties, even after 40 min incubation. Fig. 3 shows the results obtained by measuring the distribution of the radioactivity among the six fractions separated by chromatography on DEAE-cellulose paper. It can be seen that the radioactivity is distributed in a similar manner in the case of the (‘“C) RNA (Fig. 3b) used and in the case of the RNA prepared from cells incubated for 20 and 40 min with the labeled RNA (Fig. 3 a). Figs. 3 c and 3 d show the results of a comparative experiment in which (3H) RNA hydrolysate was used. They show that in this case no label appears Experimental
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RNA uptake
Fig. 4.-Ascites cells treated for 20 min 20 min with 5 y0 perchloric acid. Emulsion:
by ascites tumor
387
cells. I
with (3H) yeast RSA 3 EL sections Ilford in gel form, type L 4.
of cells,
treated
for
in the fractions corresponding to polymerized RNA (of the six fractions, separated by this chromatographic technique, two, the lirst and second ones, correspond to acid soluble products) [23, 251. The chromatographic profile of the RNA constitutive of the ascites cells, as established by spectrophotometric measurements (Fig. 3c), is very different from the profile of the yeast RNA used in these experiments.
DISCUSSION
Apparently, ascites tumor cells (Landschtitz) are able to utilize exogenous RNA. That this utilization does not involve prior degradation of the RNA is suggested by the fact that no label appears in the acid-soluble fraction of the cells when the RNA preparation used is itself free of acid-soluble products. This is in total agreement with the results obtained with a similar material by Hare1 et al. [17] using homologous RNA. It also agrees with the results Experimental
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of Amos [I] who showed that the pretreatment of the exogenous RNA by ribonuclease decreases the rate of its uptake by embryonic cells in tissue culture. The keeping of the distribution of the radioactivity among the bases and nucleotides of the labeled RNA after its reextraction from the cells has also been observed by Niu et al. [32] in the case of uptake of liver RNA by ascites tumor cells. Those results support the hypothesis of a direct penetration of intact exogenous RNA into the cells. This is also confirmed in our experiments by the chromatographic assays on DEAE-cellulose paper, which indicates that the RNA taken up by the cells retains its chromatographic properties, even in the cells incubated during 40 min. Our results seem to exclude a degradationsynthesis process for the incorporation of the exogenous RNA by the ascites cells. In addition to this, the autoradiography gives results showing that the labeling of the different parts of the cell occurs with kinetics quite different from what is generally found in the case of cellular RNA synthesis [33, 401, the cytoplasm being here labeled at the same time as the nucleus and always more heavily than the nucleolus. The question of the mechanism of penetration of RNA into living cells is not yet clear. However, the known existence of pinocytotic activity in ascites tumor cells [lo] provides a hypothetic possibility for the mechanism of uptake of the exogenous macromolecules by living cells. It seems, indeed, that the penetration of a macromolecule like RN,4 does not represent an isolated case. Similar observations were made in the case of ribonuclease, which was shown to enter into vegetal or animal cells [B, 24, 27j. More recent work indicates the possibility of DNA uptake by several living materials [l, 4, 18, 35, 38, 391. Gartler [16], Bensch and King [3] and Hill and Jakubikova [ 191 have provided more direct evidence that utilization by animal cells of a homologous DNA might not be due to a combined degradation-resynthesis process. A similar conclusion, in the case of a heterologous DNA, appears from the works of Schimizu et al. [36], Ledoux and Charles [25] and Ledoux [23]. A general mechanism of the “pinocytosis” type can thus be involved in the uptake of macromolecules and it is not necessary to imagine a special process for this uptake. Our results show that there is the possibility of an eventual biological action of the absorbed RNA. This has been studied by several authors Experimental
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RNA uptake
by ascites tumor cells. I
389
[2, 30, 31, 351. Let us remember that we have shown that in the case of ascites cells, yeast RNA activates the incorporation of amino acids in the proteins [14]. Detailed results on this work will be published elsewhere. SUMMARY
Ascites tumor cells are able to ingest a labeled heterologous RNA which is dissolved in their incubation medium. The maximum quantity taken up after 40 min of incubation represents 2-4 per cent of the normal RNA content of those cells. Autoradiographic study shows that the RNA taken up is mainly located :n the nucleus and the cytoplasm of the cells. Far less is fixed by the nucleolus. The base composition as well as the chromatographic behaviour on DEAEcellulose paper of the exogenous RNA (yeast RNA) seem to be maintained after ingestion by the cells. The results suggest that ascites tumor cells can take up intact RNA, without greatly modifying its structure and composition. The mechanism of this RNA uptake has not yet been clarified. We wish to thank Prof. M. Errera for his interest and advice during the course of this work. This work has been performed under the contract Euratom-C.E.N. 014-62-I-BIAB. REFERENCES 1. AMOS,
H.,
2. AMOS,
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