Previous experiments have shown that under proper conditions ribonucleic acid (RNA) can indeed initiate differentiation [1, 2]. Of particular interest in this work is the development of highly specific structure. That the type of structure developed correlates with the tissue source of RNA indicates a response of the reacting tissue to a message carried directly by RNA or RNA-containing substances of the inducing tissue. Thus, on the cellular level, when a piece of the epidermal ectoderm of urodele gastrula is transplanted into the presumptive
kidney area, the ectoderm contributes to the formation of extra kidney tubules [3] and develops into muscle in the presumptive muscle area [4]. At the molecular level, ribonucleoproteins (RNP) from different tissues were found to be capable of inducing the formation of muscle[ 5, 6], blood [7], thymus [1], and other tissues [2].

Some two years ago, an examination of the effects of normal RNA on mouse ascites cells was initiated. After incubation with liver RNA, only 10 per cent of the transplanted ascites cells gave rise to tumors. The loss of the tumor-forming ability was correlated with induced cellular changes rather than with regression caused by either cell death or immunological response [8]. The cellular changes were shown in vivo by the lack of mitotic division and invasiveness. In vitro, they were demonstrated by the acquired capacity of synthesizing the liver protein, serum albumin [9].

In this communication, observations which indicated an inhibitory effect of RNA on tumor growth [8, 9] are presented in detail. These studies and comparable studies with the Novikoff hepatoma [10] appear to support the concept that RNA induces cellular transformation in mammalian cells.

Materials and Methods.

Mice and ascites cells: During 1959 and 1960, white Swiss mice of the
Rockefeller Institute, and in 1961, Swiss Webster mice supplied by Pied Piper Farms were used in
this study. The ascites tumor cells were developed and maintained through successive transfers
in the peritoneal cavity of the Princeton strain by Dr. John B. Nelson of the Rockefeller Institute,
whose generosity in supplying ascites fluid biweekly during 1959 and 1960 the authors are pleased
to acknowledge. Upon receipt of the cell suspension, our customary procedure was to wash the
ascites cells with 0.9% saline. Intramuscular injection of the washed cells give rise to solid tumors [11],
and the mice usually died within a month. For the preparation of tumor RNA, solid tumors without
necrosis (8 to 10 days) were used as source material. Mice from the same litter were used for
control and experimental series. The number in each series was limited to 5 to 8 mice, weighing
10 to 15 grams each.

Isolation of RNA: RNA was prepared from mouse and calf liver (L-RNA) and solid tumor
(T-RNA) by a modified Kirby procedure involving low-speed centrifugation [2]. Beginning in
November 1960, the isolated RNA was centrifuged at 40,000 RPM (Spinco Model-L Rotor #40)
for 30 min. The supernatant fluid was subsequently treated with ether and the ether was expelled
by nitrogen. The preparations were used immediately or frozen in dry ice-acetone and stored
at -2^oC for 7 to 10 days. No apparent loss of activity was detected.

Ultraviolet absorption spectra of both preparations of RNA were similar, with the lowest
absorption at 230 m/u and the highest at 258 miu. In each case, the ratio, absorption at 260 mm/absorption
at 230 mm, exceeded 2.2. The amount of RNA calculated from the orcinol determination,
the phosphorus content, and the absorption at 260 mm agreed, thus indicating that the substances present in solution were RNA. In addition, routine laboratory tests for protein, DNA,
and polysaccharides were negative. However, the RNA hydrolysate gave a ninhydrin color
reaction, indicating the presence of some amino acids.

Preparation of ascites cells for study: The ascites cells were washed 5 to 6 times with 30 to 50
volumes of 0.9% saline and divided into aliquots. One served as control, the second and third
were mixed well with 10 volumes of L-RNA with an optical density (O.D.) at 260 mu of 25/ml and
O.D. 100/ml respectively, and the fourth was mixed with T-RNA with O.D. 100/ml. They were
incubated for 12 to 20 hr in the cold (5 to 8^oC). To insure contact of RNA with the cells, the
mixture was stirred at intervals, especially during the first few hours of incubation. Next morning,
each sample was centrifuged and resuspended in fresh samples of the incubation medium.

Before and after each incubation, one volume of the ascites cells and 1/2 volume of 1 % eosin were
mixed thoroughly. Within 5 min, the number of stained and unstained cells were counted.
The unstained cells are viable and tumor-producing [12].

Each aliquot of cells was subdivided for (1) Intramuscular injection in the thigh with 0.05 ml
saline containing 5 X 10⁶ cells. The thigh was subsequently fixed daily up to the 10th day to
trace the fate of the injected cells. (2) Quantitative determination of nucleic acid. Both RNA-treated
and control series were centrifuged and cells quickly washed twice with saline. The cell
suspension was treated with an equal amount of cold 4% perchloric acid (PCA), mixed well, and
held at 2^oC for 5 min. The acid-soluble substances were then removed by centrifugation. The
sediment was washed once with 2% PCA and extracted with 10% PCA at 90^oC for 15 min.
Nucleic acids were estimated by the ultraviolet absorption at 260 mu. (3) Amino acid incorporation.
The samples were centrifuged and cells resuspended in 5 to 8 volumes of the incubation
mixture [13]. They were placed on crushed ice and 0.1 ml containing 1 uc of C¹⁴-DL-leucine was
then added for each ml of cell suspension. The samples were then transferred to a shaker at
37^oC, and two minutes were allowed for temperature equilibration. The shaking speed was 100
cycles per min. Incorporation was stopped at intervals by addition of equal volumes of ice-cold
10% trichloroacetic acid (TCA).

Incorporation was also followed on cells cultivated in milk dilution bottles [14] for various lengths
of time.

Fractionation of protein: The TCA protein was centrifuged in the cold and washed twice with
cold 5% TCA. The sediment was extracted with 10 volumes of absolute alcohol and then designated
as general protein (G.P.). The alcohol was removed by dialysis against distilled water
containing some DL-leucine. On removal of alcohol, precipitates appeared, which were collected
by centrifugation and called insoluble protein (I.P.) The substance in solution with serum
albumin added as carrier (30 ug/ml) was precipitated by addition of the antiserum against bovine
serum albumin. Due to its solubility in absolute alcohol [15], especially after treatment with 5%
TCA, and precipitability by the antiserum, the substance in question appeared to be related to
serum albumin (S.A.) and was so designated.

The nucleic acids of G.P. were removed by heating the 5% TCA suspension at 90^oC for 15 min.
Afterwards, this sample was treated in the same manner as I.P. and S.A. The lipids were removed
by three extractions with alcohol-ether (3:1) at 62^oC for 5 min each, followed by washing with
alcohol-ether-chloroform (2:2:1) and ether. The dried samples were weighed and counted in an
automatic gas flow counter.

Methods of treatment with RNAse and DNAse: One ml of the L-RNA (O.D. 200/ml) was mixed
separately with 1 ml saline containing 1 mg RNAse or DNAse (Worthington Co.). Together
with samples of RNAse and DNAse in saline (control series) they were incubated at 37^oC for 20
min. These four samples were separately mixed with 0.2 ml of the ascites cells, kept in the
refrigerator for 15 to 20 hr with frequent stirring, and each then divided into two equal aliquots.
One aliquot was used immediately for a test of its tumor-producing capability. The other was
centrifuged and the cells were resuspended respectively in the same fresh enzyme solution. They
were kept in 5 to 8^oC for another 15 to 20 hr before centrifugation, resuspension, and intramuscular
injection.

Preparation of C¹⁴-liver RNA and determination of C¹⁴ distribution in RNA: Mice weighing 25 to
30 grams were starved 12 hr and then given 0.5 ml of C¹⁴-orotic acid (5 uc) and 0.5 ml of C¹⁴-
adenosine (5 uc) intraperitoneally. Five hr later, livers from 15 animals were pooled and the gall
bladders removed. RNA was prepared and found radioactive, presumably due to C¹⁴-labeled
bases (adenine, cytosine, and uracil) of the L-RNA.

C¹⁴-L-RNA was hydrolyzed by 1N HC1 for 1 hr at 100^oC. The hydrolysates were subjected to
paper chromatography according to Smith and Markham [16]. The four spots representing guanine,
adenine, cytidylic acid, and uridylic acid were dried and counted for radioactivity. Samples were
eluted with 0.1N HCl, evaporated until dry, and counted.

C¹⁴-L-RNA was employed to treat the washed ascites cells. After 20 hr of incubation, the
sample was centrifuged. For the removal of free C¹⁴-L-RNA, the cells were quickly washed twice.
RNA isolated from these cells was radioactive and thus designated as C¹⁴-T-RNA. Radioactivity
of its hydrolysates was determined in the same manner as that of C¹⁴-L-RNA.

Results:

Viability of the ascites cells: Of prime importance in the present
investigation was the viability of the ascites cells, particularly after incubation
with RNA. Comparing the two groups of cells, blebs appeared more frequently
in the RNA-treated cells than in the untreated. The latter packed more firmly
when centrifuged. Neither phase nor electron microscopy could detect any additional
difference between them. Staining with eosin or trypan blue revealed
that 60 to 95 per cent of the washed ascites cells and 40 to 80 per cent of the RNA-treated
counterpart resisted staining. It is pertinent to add that the presence of
only 19 per cent viable cells in the RNAse-treated ascites population was sufficient
to produce solid tumors in a frequency of approximately 60 per cent at the site of
injection (see below). Accordingly, one would expect tumor formation by the
RNA-treated cells. The apparent reduction in tumor formation (see below)
would therefore suggest that other factors are operating.
The presence of metabolically active cells in the RNA-treated sample was further
indicated by the study of amino acid incorporation into proteins (Fig. 1).

FIG. 1.-Time course of incorporation of DLleucine-C'4 into proteins of un-treated and L-RNA-treated ascites cells.

It can be seen that all three series were capable of incorporating DL-leucine-C¹⁴
into TCA proteins. The rate of incorporation by those cells exposed to higher
concentrations of RNA was reduced to approximately 1/4 Of the control. This
reduction could result from either cell death or cell change, or both. Since the
difference in viability between the L-RNA-treated and untreated cells, as estimated
by staining with eosin, was only 15 to 20 per cent, it seems unlikely that cell death
was responsible for the decrease of 3/4 in metabolic activity. Besides, cancer
cells ordinarily are metabolically more active than normal cells [17].

Rate of tumor formation: Intramuscular injection of the ascites cells and the
RNA-treated counterpart resulted in the development of solid tumor at the site of
injection. The frequency varied, however, according to the concentration and the
tissue source of the RNA employed. As a rule, if L-RNA-treated cells developed
into solid tumor, the appearance of the tumor was frequently delayed. Table 1

summarizes the data obtained with the Rockefeller Swiss mice (up to the summer
of '60). It can be seen that under our experimental conditions a proper concentration
of L-RNA (O.D. 100/ml) indeed affected the development of the ascites
tumor. In contrast, treatment with T-RNA had no significant effect. Our protocol
revealed further that the inhibition was permanent, because those mice that
had developed no tumors within a month gave none in six months or longer. In
addition, they did not do so even on subsequent inoculation with untreated ascites
cells (25 Swiss Webster mice).

The effect of nucleases on L-RNA: Subsequent to the finding that L-RNA could
inhibit tumor growth, enzyme studies were carried out to see if RNA macromolecules
were required for this inhibition. Both RNAse and DNAae were used, one
serving as control for the other. Since RNAse is reportedly tumor-inhibitory [18, 19],
our cells were tested separately after the first and the second treatments. This
was to insure doubly the adequacy of the enzymatic treatment. Table 2 provides

data obtained with the Swiss Webster mice. RNAse treatment of L-RNA led
to its inactivity in lowering tumor frequency, whereas treatment with DNAse
had no effect. The sensitivity to RNAse and indifference to DNAse provides a
strong point in favor of the necessity of macromolecular integrity for the RNA
function.

That RNA was taken up as macromolecules was further supported by analysis
of C¹⁴-L-RNA and C¹⁴-T-RNA (ratio of activities about 10:1). Chromatograms
of both C¹⁴-L-RNA and C¹⁴-T-RNA hydrolysates were made by the techniques
used for yeast RNA [16]. C¹⁴-orotic acid is supposed to be incorporated into the
C¹⁴-uridylic and C¹⁴-cytidylic acids and C¹⁴-adenosine into the C¹⁴-adenine of the
C¹⁴-L-RNA. If the macromolecules of C¹⁴-L-RNA enter the cells without preliminary
degradation, the ratios of radioactivity of C¹⁴-adenine and C¹⁴-cytidylic and
C¹⁴-uridylic acids of the C¹⁴-T-RNA should be equal or close to those found in the
C¹⁴-L-RNA. Table 3 shows the distribution and ratios of radioactivity in the
RNA components. It can be seen that the ratios of C¹⁴-L-RNA and C¹⁴-T-RNA
are similar within experimental error.

So far as RNA function is concerned, untreated L-RNA lowered the tumor-producing
potentiality of the ascites cells to 20 per cent in Swiss Webster mice
and to 10 per cent in the Rockefeller Swiss mice. This is in contrast to the 50 to
62 per cent reduction observed in the series in which cells were incubated with
RNAse (column 5, Table 2). It should be emphasized that the experiment with
RNAse was repeated many times, and the inhibitory effect of RNAse never approached
that of the untreated L-RNA.

Fate of the injected cells: Viable cells in the liver RNA-treated population varied
from 40 to 80 per cent. When a half million of these cells were inoculated intramuscularly
into the thigh, the frequency of tumors was only 10 to 20 per cent.
The immediate question is what happens to the majority of the injected cells?
One possibility is that the liver RNA contributed something to the cells which invoked
an immunological response leading to their elimination. If this were the
case, there should be a gradual decrease in the number of cells, particularly no
organized structure should appear at the site of injection. Figure 2 shows a low-power
view of the site of injection of untreated ascites cells. The luxuriant growth
of the ascites tumor cells is conspicuous, and invasion into the muscle bundle has
begun. Numerous mitotic figures can easily be seen under higher magnification
(Fig. 3a) but are seldom found in the RNA-treated cells. In contrast to Figure 3a,
there appears to be some sort of cell grouping (Fig. 3b). Four days later, the
number of the identifiable injected cells is greatly reduced, but those remaining
have oriented themselves into an organized pattern (Fig. 4). It seems then that
some of the RNA-treated cells went through stages of progressive specialization
instead of being expelled. A detailed analysis of this differentiation will be reported
elsewhere.

Possible mechanism of the tumor inhibition: The findings presented above have
demonstrated that the L-RNA induced cellular change. Microscopic examinations
do not reveal the nature of the change prior to the injection. There was, however,

Under our experimental conditions, the synthesis of serum albumin-like protein was directly related to RNA concentration. If protein were contaminating the RNA used, higher concentrations would carry more protein. Accordingly, the observed increment in C¹⁴-albumin might be explained either by a C¹⁴-leucine exchange in the contaminating protein [12] or by the influence of incorporated RNA on protein synthesis. The former , however, is unlikely because cells treated with either serum albumin alone or T RNA plus albumin did not show this increase.

Discussion:

It is known that protein synthesis requires the participation of transfer
or carrier RNA, of "messenger" RNA, and of microsomal or structural RNA [23].
However, the chemical identification and the relationships among these RNAs
are poorly understood at the present time. With this in, mind, our experiments on
the biological potentiality of RNA have utilized RNA isolated from the whole

FIGt. 5.-Effect of mouse liver RNA on the incorporation of DL-leucine -C¹⁴ into protein
in the L-RNA-treated ascites cells during 2 1/2 hr.

In vivo, the untreated ascites cells resumed active mitotic division shortly after
inoculation. On the third or fourth day, they began to invade the neighboring
muscle bundles and reached the size of a chestnut by the tenth day. The host
died within three to four weeks. In contrast, 80 to 90 per cent of the injections
with L-RNA-treated cells did not give rise to tumors. The failure was correlated
with the loss of two biological markers for cancer cells, namely, a high rate of
mitotic division and invasiveness. Since cell growth is inversely proportional to
cell differentiation, the loss of these properties suggests that differentiation might
have occurred. This appears true of cells (Fig. 4) which still remain in the muscle
tissue eight days after injection. Apparently most of them are gone. The disappearance
may possibly be due to one or a combination of three factors: (1)
non-viable injected cells may have been removed by phagocytes, (2) the viable
cells that have acquired the capacity to synthesize serum albumin may have been
relocated, and (3) some cells may even have been "assimilated" by the neighboring
muscle tissue. Points (2) and (3) will constitute the subject of another publication.

The concept of "stopping tumor growth through differentiation" is an old one.
The present work is actually the beginning of a new approach. This approach has
recently been extended to rat hepatoma in experiments by DeCarvalho and Rand [10],
in which the L-RNA-treated hepatoma cells gave rise to slightly haemorrhagic
nodules scattered in the mesentery in 85 per cent of the injected rats and in 15
per cent formed a solid tumor. Histologically, the nodules showed a more orderly
cordonal organization with the tendency to radiate around a vein. The cells
contained glycogen in a polarized distribution resembling normal livers.

The manner by which RNA acts upon the cells is not clearly known. In view
of the induced biosynthesis of specifie protein, however, it may be assumed that it
enters the cells without loss of biological potentiality. Evidence supporting this
hypothesis comes from our enzymatic studies showing the requirement of macro-molecular
integrity for RNA function, the correspondence in radioactivity ratios
of the hydrolytic components between C¹⁴-L-RNA and C¹⁴-T-RNA isolated from
the C¹⁴-L-RNA-treated tumor cells, and the autoradiographic demonstration of
radioactivity within the treated cells. Once in the cells, the exogenous RNA
would perhaps be functioning in a fashion similar to infectious RNA. The synthesis
of new protein is correlated with the appearance of new morphological entities.

Summary:

It has been shown that the treatment of the Nelson mouse ascites
cells with ribonucleic acid in suitable concentrations reduced tumor formation
from 97 per cent to 10-20 per cent. This reduction was correlated with induced
cellular change rather than regression caused by cell death and/or immunological
response. The change was found to be accompanied by an induced biosynthesis
of specific protein. Apparently hepatoma cells under proper influence of liver
RNA gave rise to an organoid structure resembling normal liver [10].

That the effect of L-RNA was specific was demonstrated by its sensitivity to
RNAse and indifference to DNAse and by the inactivity of T-RNA. The entrance
of macromolecular RNA into the treated cells was indicated by the presence
of C¹⁴ in cells treated with C¹⁴-L-RNA and by the correspondence of the ratios of
labelled bases in the administered C¹⁴-L-RNA and in the recovered C¹⁴-T-RNA.

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