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/. Embryol. exp. Morph. Vol. 23, 2, pp. 509-17, 1970
Printed in Great Britain
509
The effect of exogenous RNA extract on chick
embryo fibroblasts in vitro
By A. SANN 1 , D. S H A R P 1 AND J. MCKENZIE 1
From the Department of Embryology, Marischal College, Aberdeen
It is extremely difficult, if not impossible, to reconcile the conflicting claims
of those who have treated different cells and tissues with exogenous RNA. Some
authors (e.g. Niu, Cordova & Niu, 1961 ; Niu, Cordova & Radbill, 1962) maintain that RNA extracts alter the course of cell differentiation to conform in
morphological terms to the source of the RNA; in the same vein, Amos,
Askonas & Soeiro (1964) have shown that, under certain conditions, RNA from
mouse and bacterial cells can stimulate chick embryo fibroblasts to synthesize
protein related antigenically to the origin of the RNA. Shepley, Ambrose &
Kirby (1965), however, obtained stimulation of growth with permanent morphological changes in baby hamster kidney fibroblasts by the addition of RNA
from a variety of sources.
On the other hand, Holoubek, Fanshier & Crocker (1966) showed that nuclear
RNA synthesis in Ehrlich ascites cells was inhibited for 6-8 h by the addition
of homologous or rat liver RNA and our results are similar in that rat liver
RNA extract has been shown to have an adverse effect on the growth and
survival of chick embryo fibroblasts.
MATERIALS AND METHODS
Eggs of a Thornber hybrid x Light Sussex cross were incubated at 37-5 °C in
a commercial automatic incubator for 10| days. The embryos were removed
under aseptic conditions and placed in Hanks's Balanced Salt Solution. After
the head, limbs and viscera had been removed, the body wall was rinsed in
Hanks's BSS and chopped into fine fragments which were then incubated in
sterile 0-25 % trypsin solution (Hopkins and Williams) at 37-5 °C for 30 min.
Thereafter the mixture was filtered through several layers of absorbent gauze.
The resulting cell suspension was centrifuged at 1000 rev/min for 10 min on a
bench-type MSE centrifuge, washed and finally resuspended in single-strength
Eagle's medium (Burroughs Wellcome) and calf serum (Flow Laboratories) in
the proportion 9 : 1 respectively. When the concentration of cells had been
adjusted to give a final count of 5 x 105 cells/ml of growth medium, 1 ml was
1
Authors' address: Department of Embryology, Marischal College, Aberdeen, Scotland.
510
A. SANN, D. SHARP AND J. MCKENZIE
added to each of a series of pyrex test-tubes each containing a 22 mm x 4 mm
coverslip piece; a little C0 2 was introduced before the tubes were finally
stoppered with sterile silicon rubber stoppers. The cultures were incubated at
37-5 °C in an LSE incubator, the growth medium being changed every 3 days.
The RNA extract, obtained from rat livers by a modified Kirby (1956) phenol
method, was dried under vacuum, dissolved at a concentration of 3-6 mg/ml
chick Ringer saline (NaCl, 7-29 g; KCl, 0-37 g; CaCl2.6H.20, 017 g; deionized
water, 11.), stored at - 2 0 °C and, immediately before use, sterilized by passing
through an Oxoid membrane filter, grade 0-45.
At the first renewal of the growth medium, the required dose of RNA extract,
contained in 0-2 ml of saline, was added to each tube and thereafter the cell
growths were removed for staining (Jenner-Giemsa) and examination at intervals from 1 h onwards. Control cultures received no RNA extract.
The activity of the RNA extract was tested for its in vitro effect on the
fibroblasts after it had been treated in a variety of ways: (a) boiling for 15 min;
(b) dialysis against saline for 24 h using 24/32 Visking tubing; (c) digestion
with RNase (B.D.H.) for 24 h at a concentration of 4 mg RNase/4 mg RNA
extract; (d) digestion with RNase followed by dialysis for 24 h.
A simple extract of rat liver was also examined for its effect on the fibroblasts;
it was obtained by homogenizing the liver (1 mg liver/1 ml saline) in an M SE
homogenizer and, after the mixture had been centrifuged at 35000 g in an
MSE Superspeed 40 for 1 h, the supernatant was applied (0-2 ml/1 ml of growth
medium) to the chick embryo fibroblasts.
RESULTS
The appearance of normal chick embryo fibroblasts in monolayer culture is
shown in Fig. 1A: large irregular cells with several protoplasmic processes,
pink-staining, often vacuolated cytoplasm, and a round or oval nucleus containing usually two nucleoli. The density of cell growth is similar on all coverslips within a given culture series, but different series often display considerable
variation, i.e. in some the cells are few and sparsely scattered over the coverslip
surface while others have a profuse growth of densely packed cells. Many
coverslips with poor growths show a patchy distribution of dense colonies of
cells—a useful feature in judging the effect of the RNA extract.
Once the growth of the cells has been firmly established, i.e. by the third day,
the addition of RNA extract to the growth medium causes, first, a narrowing
and, later, a retraction of the cytoplasmic processes accompanied by a general
shrinkage of the cytoplasm (Fig. 1 B). This progresses to the stage where the
cells are dense and rounded, often clustered around strands of fibrillar material
on the surface of the coverslip (Fig. 1 C). The nucleus is, so far, still normal in
appearance but as the influence of the extract increases or continues the nucleus
becomes compact and densely stained; finally the cytoplasm assumes a pink-
RNA extract and fibroblasts
511
FIGURE 1
(A) Normal fibroblasts after 3 days in monolayer culture, x 400.
(B) Cells showing early effects of administered RNA extract 1 h after treatment.
Note the withdrawal of their cytoplasmic processes, x 400.
(C) Cells 3-5 h after RNA extract administration, clustering round strands of
fibrillar material, x 400.
(D) Advanced stages of RNA extract effect. Cells show dense nuclei and pinkstaining amorphous cytoplasm, x 400.
(E) Single colony surrounded by isolated cells in normal monolayer culture, x 100.
(F) Gross inhibition of isolated cells surrounding seemingly unaffected colony after
treatment with RNA extract, x 100.
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A. SANN, D. SHARP AND J. MCKENZIE
staining, amorphous appearance indicating cell death (Fig. ID). Perhaps the
most striking feature in the comparison of normal and severely affected cells is
the marked reduction in size of the latter; they appear no larger and sometimes
even smaller, than the nuclei of normal cells.
The degree of cellular damage and the rate of its progress through the
different stages are influenced by two factors: (a) the concentration of the RNA
extract in the growth medium and (b) the density of cell growth on the coverslip.
The influence of the first is best illustrated by the results obtained after
administering two different concentrations of the extract (0-36 and 0-72 mg/ml
of growth medium) within the same culture series. Each series has its own slight
modifications in timing but the basic pattern is always similar to that described
below :
3 h after treatment no effect can be seen with either dose.
At 5 h a considerable number of rounded cells are present in the culture
receiving the higher dose but none with the lower dose.
At 7 h all the cells treated with 0-72 mg of extract are affected while only a
few of those receiving 0-36 mg show any sign of cytoplasmic retraction.
At 1% h some of the heavily dosed cells are moribund while the cultures
receiving the lower dose exhibit only an occasional rounded cell.
9\ h after treatment with 0-72 mg there are many dead or dying cells but
only 30-40 % of those receiving 0-36 mg show even a mild effect.
24 h after receiving 0-72 mg only a few living but severely contracted cells
persist; the remainder are dead, but obviously the majority have floated off the
coverslip into the growth medium. However, cultures treated with the lower
dose show marked signs of recovery; only a few rounded cells can be found, the
others being apparently normal. Indeed, if they had not been examined earlier
these cultures would have given the appearance of not reacting at all to the
RNA extract.
At 48 h a few cells in the culture receiving the higher doses are clearly showing
signs of recovery; they have normal nuclei surrounded by darkly staining
cytoplasm, and some have achieved the normal fibroblastic shape.
The influence of cell density is illustrated in Fig. 1E and F, before and after
treatment with 0-36 mg of RNA extract/ml of growth medium. Wherever the
cells were densely aggregated before administering the extract there is little or
no effect but other fibroblasts which are scattered and growing separately on the
coverslip surface are greatly inhibited, damaged or dead. Similarly, comparing
cultures of different densities treated with the same concentration of extract
reveals that a dense culture may come through unscathed while sparse ones are
invariably severely damaged.
The boiled RNA extract produced exactly the same effect on the fibroblasts,
and dialysis for 24 h diminished the effect only slightly.
Digestion of the RNA extract introduced the complication of an RNase
effect on the fibroblast. Fortunately this was so different from that of the RNA
RNA extract and
fibroblasts
513
extract that it could be easily distinguished. It never appeared until at least 24 h
after the RNase was added and was usually best demonstrated at about 48 h ;
although the cells became more slender, they showed no signs of retracting their
processes as with RNA extract. The main feature, however, was the arrangement of the cells on the coverslip; they appeared to have aggregated to form a
network easily recognized under low magnification: strands of cells surrounding
rounded or almost square areas where cells were absent or scarce.
After digestion with RNase, the effect of the RNA extract was usually more
intense, appearing sooner and causing more damage to the cells with no
recovery within 72 h. Under these circumstances, of course, an RNase effect
was absent. If the digested extract were dialysed for 24 h before being applied
to the culture then the 'RNA extract' effect was greatly diminished or absent
but 24 h or more later the cells assumed the distribution pattern of the RNase
effect.
The supernatant from the liver homogenate had the same effect as the RNA
extract but prior dialysis of the supernatant did show some reduction in the
degree of cellular damage; the effect, however, was typically that of an RNA
extract.
DISCUSSION
These results lend support to previous claims that RNA extracts have an
adverse affect on cell growth and activity in vitro. According to Benitez, Murray
& Chargaff (1959), for example, inhibition of growth as well as heteromorphic
changes lasting several days occurred in subcutaneous areolar fibroblasts from
rats after 24 h treatment with microsomes from rat liver or kidney; apparently
permanent heteromorphic changes occurred after 2-3 days exposure. Holoubek
et al. (1966) treated intact Ehrlich ascites cells with homologous and heterologous RNA from rat liver and found that nuclear RNA synthesis in the cells was
inhibited for a period of 6-8 h.
But there is, as yet, no explanation of why other workers, e.g. Niu (1963) and
Niu et al. (1962) should claim evidence of specific differentiation according to
the source of the RNA, after using similar cell types (i.e. Ehrlich ascites),
administering RNA extracted by the same procedure, and employing experimental techniques which would be expected to provide similar results. Specific
differentiation in the form of cellular changes related to the RNA source has
been demonstrated in other types of cells; Sanyal & Niu (1966), for example,
found, in posterior segments of the chick blastoderm primitive streak, that
kidney and heart RNA stimulated the differentiation of tubular and vesicular
structures respectively, whilst chick brain RNA caused the differentiation of
neural tissue; Hillman & Niu (1963) initiated the development of brain and
notochord in explanted stage 5 chick embryos by the administration of brain
and notochord RNA. Although Malpoix (1964) could not elicit a specific
reaction from embryonic blood cells by externally supplied RNA, Esposito
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E M B 23
514
A. SANN, D. SHARP AND J. McKENZIE
(1964 A, b, c) was successful in obtaining specific responses from leukaemic cells
by exogenous RNA. Equally surprising are the findings of Shepley et al. (1965)
who have shown that exogenous RNA from various sources caused baby
hamster kidney fibroblasts to grow faster and more profusely in monolayer
cultures; the stimulatory effect seen and noted by these workers persisted in
subcultures of the treated cells and was expressed in the form of large, piled-up
colonies.
It might be claimed that the degree of differentiation in different target cells
is likely to give conflicting results and certainly chick embryo fibroblasts, no
matter how well they reproduce in vitro, must be regarded as well-differentiated
cells compared with the elements of the chick embryo blastodiscs. However, it
is unlikely to be a question of the degree of differentiation when two separate
groups of workers, both using Ehrlich ascites cells, claim, on the one hand
(Niu, 1963), that enzymes specific to the source of the RNA are synthesized by
these cells and, on the other hand (Holoubek et al. 1966), that there is inhibition
of RNA synthesis, a preliminary to protein synthesis.
In the present experiments, chick embryo fibroblasts during the first 24 h
after treatment assume morphological changes reflecting an inhibition of cell
growth and function, proceeding to degeneration and death of the cells: a far
more severe effect than reported by Holoubek et al. The explanation of what
appears to be merely a difference in the degree of the effect obtained may lie
with the dose or concentration of the RNA extract, but direct and accurate
comparison cannot be made of the doses of RNA and cell concentrations used
in our experiments and those of Holoubek et ah; the addition of 5 mg RNA to
cell numbers ranging from 2 x 106 to 2 x 108 includes the concentration we
provided for the chick embryo fibroblasts, viz. 0-36-0-72 mg/5x 105 cells.
Differences do, however, occur in the details of the techniques employed; the
numbers of cells given for the present experiments refer to the count when the
cultures were set up, i.e. 3 days before treatment, whereas Holoubek et al.
administered the RNA as soon as the cells were suspended in culture.
Variations in the density of our monolayer cultures provided an opportunity
to demonstrate the marked differences in vulnerability or resistance of the cells
in dense and sparse cultures to the RNA extract. This phenomenon suggests
that groups or colonies of cells can create in their immediate environment a
concentration of some lytic enzyme which is sufficient to inactivate the RNA
extract—or some part of it—at least in regard to its toxic effect on the fibroblast;
conversely, the isolated cells on the culture surface cannot, because of diffusion,
build up enough of this protective enzyme. There is no doubt that ribonuclease
would be the natural contender for this role but the reports in the literature on
the effect of RNase on exogenous RNA extracts when administered to cells
in vitro are as confusing as the results of administering the extracts themselves.
Some authors, e.g. Shepley et al. (1965) and Gazet & McKibbin (1965), claim
that RNase has no effect on the activity of the RNA; some, e.g. Sanyal & Niu
RNA extract and
fibroblasts
515
(1966), Niu et al. (1961) and Amos & Moore (1963), maintain that the RNA
activity is diminished or lost, while Benitez et al. (1959) report variable results
within their own series of experiments.
Our results show quite clearly that RNase digestion does not destroy the
effect of the RNA extract—indeed it may even increase it. The intact RNA
molecule is, therefore, not necessary in order to demonstrate cellular changes
and, furthermore, the active part of the extract is dialysable after RNase digestion. The parts of the RNA molecule most likely to be responsible are the
nucleotides but unfortunately Holoubek et al. (1966) have shown that none of
the nucleotides or higher products of RNase-digested RNA can penetrate cells
grown in vitro. Our results are, nevertheless, consistent with these authors' view
that the active element in the RNA extract is a protein usually found in combination with the RNA molecule, capable of combining with the chromosomal
DNA and thereby blocking normal synthesis of mRNA in the treated cells.
Several other authors, e.g. Allfrey, Littau & Mirsky (1963), Huang & Bonner
(1962) and Garen & Garen (1963), employing other experimental systems, have
already demonstrated that repression of RNA and of enzyme synthesis may be
effected by the combination of a protein with the nuclear DNA, and, since
Amos & Moore (1963) have shown that RNA extracts contain up to 10 %
protein, the aim, perhaps, should now be to determine the precise nature and
origin of the protein in such extracts as well as its mode of action.
SUMMARY
1. Chick embryo fibroblasts growing in monolayer culture and treated with
rat liver RNA extract show morphological changes indicating inhibition of
their growth and function.
2. With a high concentration of RNA extract administered to a sparse culture,
a rapid onset and a severe degree of cell inhibition, usually progressing to cell
death, are obtained. Only a few cells are found to survive and regain normal
characteristics 72 h after the extract is added to the culture.
3. With lower concentrations of RNA extract and dense cultures, the effects
are milder, few cells showing evidence of necrosis and most of them having a
normal appearance 24 h later.
4. RNase digestion of the RNA extract does not alter its effect on the chick
embryo fibroblast but much or all of its activity is lost after digested extract has
been dialysed for 24 h.
5. The findings are consistent with the view that RNA synthesis within the
fibroblasts has been repressed by a protein present in combination with RNA
in the administered extract.
33-2
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A. S A N N , D . S H A R P A N D J. M C K E N Z I E
RESUME
L'effet d'extrait de RNA exogène sur des fibroblastes
d'embryon de poulet in vitro
1. Des fibroblastes d'embryon de poulet en culture en couche monocellulaire
traités par du RNA extrait de foie de rat, présentent des changements morphologiques indiquant une inhibition de leur croissance et de leur survie.
2. L'administration de cet extrait de RNA en concentration élevée à une
culture dispersée produit un effet rapide et un degré sévère d'inhibition cellulaire,
conduisant généralement à la mort cellulaire. Quelques cellules seulement
peuvent survivre et récupérer leurs caractères normaux 72 h après l'adjonction
de l'extrait à la culture.
3. Avec des concentrations plus faibles d'extrait de RNA et des cultures
denses, les effets sont plus modérés; quelques cellules apparaissent nécrosées
mais la plupart ont une apparence normale après 24 h.
4. La digestion par la RNase de l'extrait de RNA ne modifie pas son action
sur les fibroblastes d'embryon de poulet mais la plus grande partie ou la
totalité de cette activité est perdue après une dialyse de 24 h de l'extrait digéré.
5. Ces résultats sont en accord avec l'idée que la synthèse de RNA à l'intérieur des fibroblastes a été réprimée par une protéine combinée avec le RNA
dans l'extrait administré.
REFERENCES
ALLFREY, V. G., LITTAU, V. C. & MIRSKY, A. E. (1963). On the role of histones in regulating
ribonucleic acid synthesis in the cell nucleus. Proc. natn. Acad. Sei. U.S.A. 49, 414-21.
AMOS, H., ASKONAS, JB. & SOEIRO, R. (1964). In: Metabolic Control Mechanisms in Animal
Cells. Natn. Cancer Inst. Monogr. 13, 155-65.
AMOS, H. & MOORE, M. O. (1963). Influence of bacterial ribonucleic acid on animal cells in
culture. Expl Cell Res. 32, 1-13.
BENITEZ, H. H., MURRAY, M. R. & CHARGAFF, E. (1959). Heteromorphic change of adult
fibroblasts by ribonucleoproteins. / . biophys. biochem. Cytol. 5, 25-34.
ESPOSITO, S. (1964a). Influence of exogenous RNA on the synthesis of RNA in leukaemic
cells. Medna exp. 11, 397-401.
ESPOSITO, S. (19646). Effect on leukaemic cells of ribonucleic acid extracted from calf's
spleen. Nature, Lond. 203, 1078.
ESPOSITO, S. (1964c). Formation of tryptophan pyrrolase in leukaemic cells incubated with
RNA from spleen of calf embryo. Acta haemat. 32, 265-70.
GAREN, A. & GAREN, S. (1963). Genetic evidence on the nature of the repressor for alkaline
phosphatase in E. Coli. J. molec. Biol. 6, 433-8.
GAZET, J. C. & MCKIBBIN, B. (1965). The influence of parenteral RNA and DNA on the
growth of sarcoma 180 and Ehrlich's ascites tumor in Swiss mice. 7. surg. Res. 5, 35-40.
HILLMAN, N. W. & Niu, M. C. (1963). Chick cephalogenesis. 1. The effect of RNA on early
cephalic development. Proc. natn. Acad. Sei. U.S.A. 50, 486-93.
HOLOUBEK, V., FANSHIER, L. & CROCKER, T. T. (1966). Inhibition of nuclear RNA synthesis
by added RNA. Expl Cell Res. 44, 362-74.
HUANG, R. C. & BONNER, J. (1962). Histone, a suppressor of chromosomal RNA synthesis.
Proc. natn. Acad. Sei. U.S.A. 48, 1216-22.
KIRBY, K. S. (1956). A new method for the isolation of RNA from mammalian tissues.
Biochem. J. 64, 405-8.
RNA extract and fibroblasts
517
MALPOIX, P. (1964). Influence of extraneous ribonucleic acid on the differentiation of
haemopoietic tissue in chick embryo. Nature, Lond. 203, 520-1.
Niu, M. C. (1963). The mode of action of ribonucleic acid. Devi Biol. 7, 379-93.
Niu, M. C , CORDOVA, C. C. & Niu, L. C. (1961). Ribonucleic acid-induced changes in
mammalian cells. Proc. natu. Acad. Sei. U.S.A. 47, 1689-700.
Niu, M. C , CORDOVA, C. C. & RADBILL, C. L. (1962). RNA-induced biosynthesis of
specific enzymes. Proc. natn. Acad. Sei. U.S.A. 48, 1964-9.
SANYAL, S. & Niu, M. C. (1966). Effects of RNA on the developmental potentiality of the
posterior primitive streak of the chick blastoderm. Proc. natn. Acad. Sei. U.S.A. 55,
743-50.
SHEPLEY, K., AMBROSE, E. J. & KIRBY, K. S. (1965). Alteration of hamster cells by nucleic
acid in vitro. Nature, Lond. 208, 1072-4.
(Manuscript received 8 April 1969, revised 15 May 1969)