Reprinted from

Proc. Nat. Acad. Sci. USA
Vol. 69, No. 2, pp. 485-439, February 1972

RNA in Human Leukemic Cells Related to the RNA of a Mouse Leukemia Virus

(leukocytes/RNA-DNA hybridization/Rauscher virus/polysomal RNA)

R. HEHLMANN, D. KUFE, AND 8. SPIEGELMAN

Institute of Cancer Research, College of Physicians and Surgeons, Columbia University, New York City, N.Y. 10032

Contributed by S. Spiegelman, December 7, 1971

ABSTRACT Molecular hybridization with radioactively
labeled DNA complementary to the RNA of the Rauscher
leukemia virus was used to probe for homologous RNA in
the polysome fraction of human leukemic cells. The leu-
kocytes of 24 out of 27 patients examined contained RNA
possessing homology to that of the mouse leukemia agent,
but not to that of the unrelated viruses causing mammary
tumors in mice or myeloblastosis in chickens. Further, no
control human leukocytes or other adult and fetal tissues
showed significant levels of the leukemia-specific RNA.
It would appear that human leukemic cells contain RNA
sequences homologous to those found in a viral agent
known to cause leukemia in an experimental animal. The
fact that human sarcomas have also been shown to con-
tain this type of RNA points to a remarkable parallelism
in the leukemias and sarcomas of mice and men.

 

The search for evidence implicating oncogenic RNA viruses
in human neoplasia constitutes a major endeavor in present-
day cancer research. One approach recently explored stemmed
from an attempt to determine via RNA-DNA hybridiza-
tion (1) whether human breast carcinomas contain RNA mole-
cules homologous to an analogous virus of proven oncogenic
potential. Human breast cancer was chosen for the initial
effort because of the recent observations (2) in human milk of
particles similar to the mouse mammary tumor virus and the
demonstration (ref. 3 and manuscript in preparation) that the
human particles also contain the enzyme and high molecular
weight (70S) RNA characteristic of the animal RNA tumor
viruses.

Asa necessary prelude to attempts with human material,
we examined the technical feasibility of using the molecular
hybridization technique with mouse mammary tumors and the
corresponding viral agent as a suitable experimental model.
Hybridizations with radioactively labeled DNA comple-
mentary to the RNA of the mouse mammary tumor virus
revealed (4) that it was in fact possible to identify in tumor
extracts viral-specific RNA in the fraction of ribosomes
actively engaged in protein synthesis.

The experiments with the mouse model produced the de-
tailed technology required to handle human tissue specimens.
The mouse mammary tumor virus was chosen to produce the
radioactively labeled DNA needed to search for homologous
RNA in human tumors. The results obtained (5) were satis-
fyingly clear-cut. Of 29 malignant breast tumors examined,
67% showed unmistakable evidence of RNA that can
hybridize with mouse mammary tumor virus DNA. None of
the 30 preparations from normal or nonmalignant breast tis-
sues contained detectable quantities of this type of RNA.

 

Abbreviation: RLV, Rauscher leukemia virus.

435

Further, the malignant RNA samples that hybridized with
mouse mammary tumor virus DNA showed no ability to com-
plex with DNA complementary to the RNA of two leukemo-
genic agents, the avian myeloblastosis virus and the murine
Rauscher leukemia virus.

These last observations in particular gave added credence to
the belief that the positive responses observed between RNA
from malignant human breast tumors and the mouse—viral
DNA were specific and meaningful. In any event, as soon as
it became apparent that the experiments with breast tumor
material were yielding interesting results, the present investi-
gation into human leukemia, and a parallel one in human
sarcomas (manuscript in preparation), were instituted.

A particle of established leukemogenic potential, the
murine Rauscher leukemia virus (RLV), was chosen to pro-
duce the radioactively labeled DNA required. We report here
that 89% of leukemic patients in the active phase of the
disease contained, in their leukocytes, RNA _ possessing
homology to that of the Rauscher leukemia virus but not to
the RNAs of the unrelated mouse mammary tumor virus or the
avian myeloblastosis virus. Further, no control leukocytes or

other tissues examined showed significant levels of this specific
RNA.

METHODS

Leukocytes were obtained by fractionation of citrated blood
samples in inverted syringes. Leukemic cells prepared in other
laboratories were sent to us frozen in dry ice. The cells were
collected by centrifugation, washed in saline, and then sus-
pended in 0.01 M Tris-HCl (pH 7.4)-0.15 M NaCl-2 mM
MeCl, containing 5% sucrose. They were ruptured with 5-12
strokes of a Dounce homogenizer. Cell membranes and nuclei
were removed and the cytoplasmic pellets were collected and
used for the isolation of polysomal RNA (4). All annealings
were at 37°C in 50% formamide, to minimize fragmentation
of the RNA during the hybridization. Hybrids were detected
by isopyenic separation in Cs,SO, gradients (6).

RESULTS

For comparison, and to see what might be optimally expected
with human material, it was of interest to examine the out-
comes of analogous hybridizations in instances of two animal
leukemias known to be associated with a viral agent. Fig. 14
shows the result of hybridization of RLV-[}H]DNA to poly-
somal plus monosomal RNA, hereafter designated polysomal
RNA, prepared from the spleen of an RLV-infected mouse.
A peak of HJDNA, corresponding to about 3% of the input
DNA, is found in the RNA region of the density gradient.
The density position of the hybrid indicates that the RNA
436 Microbiology: Hehlmann et al.

A ova

e

.

§ ab a \7
a

2 oO
z 16

RNA

 

~] 5 iO 15 20, 25
Fraction Number
bya

5rB
°
°
“x 4 170
§3 160 9
‘a $
2 2 150 2
” <
\ 40.
130

 

   

5 10 I 20 25
Fraction Number

Fre. 1. (A) CseS0, density profile of Rauscher leukemia virus
(RLV) ([H]DNA hybridized to polysomal RNA obtained from
the spleen of an RLV-infected mouse. RLV-[*H] DNA was syn-
thesized as follows: A 1-ml reaction mixture, containing 100 pg
of viral protein purified (6) from plasma, 50 ymol of Tris-
HCl (pH 8.3)-40 umol KCl-6 pmol MgCl-2.5 pmol dithio-
threitol-0.00125% NP-40 detergent (Shell Chemical Co.)-
100 umol (each) of dGTP, dATP, and dCTP-5 xX 104 pmol of
([H]TTP (8000 cpm/pmol), was incubated at 37°C for 180 min.
After addition of 0.5% sodium dodecyl sulfate and extraction
with an equal volume of phenol-cresol, the [*H]DNA product
was purified by Sephadex G-50 chromatography and treated with
0.5 M NaOH at 43°C for 24 hr to hydrolyze any viral RNA pres-
ent.

In studies such as these, it must be shown before use that the
radioactive DNA product bands solely in the DNA density
region of a Cs,SQ, gradient and that it hybridizes with homol-
ogous RNA and not to normal cellular RNA.

The RNA (designated as pRNA) used in the hybridizations is
derived from a cytoplasmic pellet consisting of a mixture of
monosomes and polysomes (4). Suitable care must be exercised
to insure the complete removal of protein contaminants. For the
preparation of polysomal RNA, the spleen was disrupted by a
Silverson homogenizer at 4°C in two volumes of 5% sucrose in
TNM buffer [0.01 M Tris-HCl (pH 7.4)-0.15 M NaClL-2 mM
MgCl]. The suspension was centrifuged at 20,000 g for 15 min
at 0°C. The supernatant fluid was then layered on 20 ml of 25%
sucrose in TNM buffer containing 200 ug of polyvinyl sulfate
per ml and spun for 180 min at 180,000 x g in a 60 Ti rotor
(Spinco). The pellet was resuspended in TNM buffer with 0.5%
sodium dodecyl sulfate and the RNA was extracted twice with
an equal volume of cresol-phenol (pH 8.0). Nucleic acid was
precipitated from the aqueous phase by addition of two volumes
of ethanol and 0.1 volume of 4 M NaCl. The polysomal RNA was
redissolved in 50% formamide-50% 5mM EDTA. The [SH] DNA
was denatured by incubation at 80°C for 15 min in 70% form-
amide and subsequent quick chilling. 480 ug of polysomal RNA
were hybridized to 2000 cpm of RLV-[*H]DNA in 60 ul of 0.4 M
NaCl containing 50% formamide. The reaction was incubated for
18 hr at 37°C, mixed with 10 ml of 50%-saturated Cs:SO,
(starting density 1.52) and centrifuged at 44,000 epm for 60 hr
at 20°C in a 50 Ti rotor (Spinco). 0.4-ml (8-drop) Fractions were
collected through an 18-gauge needle from the bottom of the
tube and assayed for Cl,CCOOH-precipitable radioactivity.

Proc. Nat. Acad. Sci. USA 69 (1972)

 

 

DNA Tr \
A ; c
3 e 6 {
° Ne? LS 5b
2 _~ ‘Te Qa < 41.7 &
e° * eg € 41.6 &
§ 152 2 3t lise
Sj, RNA 442 x 2 —
x ‘ * 41.4
° 1.3 t+ RNA
, he 4 L qs
1 5 10 15 20 2 15 10 mi) 20 25
Fraction Number Fraction Number
DNA
DNA eto :
7 5
2
< 4
@ §3
28
Ze
274
5 10 1 2 25 1 5 10 15 20 25

 

 

  

Fraction Number Fraction Number

Fie. 2. (A-D) Cs.SO, density profiles of RLV-[7H]DNA
hybridized to polysomal RNAs of four leukemic samples. Poly-
somal RNA was isolated from buffy coats of patients showing
clinical manifestation of acute lymphocytic leukemia (A, C, and
D) and of acute myelogenous leukemia (B). The cells were dis-
rupted with a Dounce homogenizer, and cytoplasmic pellets were
prepared as described under Fig. 1B. 300 ug of polysomal RNA
were hybridized to RLV-[?H]DNA in 60-ul volumes, and the
reactions were analyzed by Cs.SO, density centrifugation.

molecule in the complex is much larger than the 4 8-6 S aver-
age size of the product DNA. Fig. 1B shows the similar out-
come with a rat ascitic monocytic leukemia, originally in-
duced (7) with 20-methylcholanthrene, that produces low
levels of a C-type particle (8) of unknown specificity. Again,
about 3-4% of the input [7HJDNA is found in the RNA region
at the concentration of polysomal RNA used in the annealing
reaction. Similar results have been obtained in hybridizations
of RLV-[}H]DNA with polysomal RNA from cat lymphocytes
infected with feline leukemia virus and hamster cells (AT2)
transformed with the Moloney sarcoma virus.

Fig. 2A—D shows representative profiles in Cs,SO, gradients
of annealing reactions between RLV-[3H|DNA and poly-
somal RNA preparations derived from human-leukemic
leukocytes (buffy coats). As may be seen, the results are com-
parable to those obtained with the animal systems (Fig. 1A
and B). In Fig. 2A, 15% of the input RLV-DNA is hybridized;
the other three samples gave 3-5% formation of the hybrid
complex. Again, as in the hybridizations with polysomal RNA
from the animal leukemic material (Fig. 14 and B), the loca-
tions of the complexes in the gradients of Fig. 2 indicate that
the RNA is much larger than the DNA, and determines
the density of the DNA-RNA hybrid structure.

The positive responses observed (Fig. 2A—D) with the
leukemic polysomal RNAs are in contrast to the negative

 

Data are expressed in this and subsequent figures as cp10m,
counts per 10 min.

(B) Cs,SO, equilibrium gradient centrifugation of RLV-
(SH]DNA annealed to 600 ug of polysomal RNA obtained from
ascites cells of a rat leukemia. The cells were collected from the
rat ascitic fluid, washed in phosphate-buffered saline (pH 7.4)
by low-speed centrifugation (2500 rpm, Sorvall), and resuspended
in two volumes of TNM buffer containing 5% sucrose. The cells
were disrupted by 5-12 strokes of a Dounce homogenizer, and
treated asin part A.
Proc. Nat. Acad. Sct. USA 69 (1972)

reactions with polysomal RNAs from normal leukocytes
(Fig. 3A), from phytohemagglutinin-stimulated lymphocytes
(Fig. 3B), from leukocytes of a leukemic patient in clinical
remission (Fig. 3C), and from human fetal lung (Fig. 3D).
In none of these samples does one find significant amounts
of [7H]DNA in the RNA region of the density gradient.

An informative way to compare positive and negative re-
actions is via a concentration curve, as shown in Fig. 4. Here,
the same amount of RLV-[*H]DNA is annealed to increasing
concentrations of polysomal RNA and the percent of [*H]-
DNA complexed to the RNA is determined by isopycnic sep-
aration in Cs,SQ, gradients. It is clear that no detectable re-
sponse with polysomal RNA from normal leukocytes is ob-
served, even at 14 mg of RNA per ml. On the other hand, the

percent of RLV-DNA complexed to polysomal RNA from the

buffy coat of a patient with acute myelogenous leukemia shows

3H cplOm x10?

3H cplOm x10?

 

no evidence of saturation within the concentration range
tested. Several leukemic buffy coats were obtained in amounts
sufficient to permit the determination of such concentration
curves, and comparable results were obtained. It should be
further noted that similar concentration curves were also run
with polysomal RNA preparations from phytchemagglutinin-
stimulated lymphocytes with results not significantly different
from those seen with normal leukocytes (Fig. 4).

Since it is impractical to present the Cs:SO, gradient profile
of every sample examined, a more convenient recording of
our findings was used. After correction for background counts,
the sum of the tritium counts in the RNA density region (be-
tween densities 1.62 and 1.68 g/ml) was used to measure the
amount of DNA complexed to RNA. To achieve the accuracy
desired, 10-min counts were taken on each sample. An opera-
tional mean background and its standard deviation (S) were
empirically determined for individual machines by the total
counts/10 min of three tubes in the negative regions (e.g.,
tubes 2, 3, and 4) of each of 60 gradients. The convention was
adopted that all specimens with less than three standard
deviations of the background mean counts/10 min would be
considered negative.

   

   

; 4
5 Si 8 |
°
4 70S 4 17
16 9
3 609 25 6g
508 & > 1s 2
2 3 Se an
140s 3 s
| 130702! 13
5 10 6 20 2 1 5 ©) 6 20 2

Fraction Number Fractiyn Number

wu
+
ann

3Heplom x IO?
N
{7} Aysuag

NN 14a

5 lo 15 20 25 I 5 10 iS 20 25
Fraction Number Fraction Number

Fic. 3. (A-D) Cs.8C, density centrifugation of RLV-[*H]-
DNA after annealing to polysomal RNA isolated from (A) normal
white buffy coat, (B) phytohemagglutinin-stimulated lympho-
cytes (C), buffy coat of a leukemic patient in clinical remission,
and (D) fetal lung. The polysomal RNA input was 300 yg, ex-
cept in (C) where 1000 zg were used per 60-! hybridization re-
action. Subsequent analysis on Cs:SO, was described in Fig. 1.

i RNA

w

 

Viral RNA in Human Leukemia Cells 437

Table 1 lists the leukemic buffy coats tested for RNA com-
plementary to RLV-DNA, with results recorded as the total
counts/10 min (corrected for background) in the RNA density
region, and as multiples of the mean background standard
deviation. The samples came from patients in the active
phase of the disease, as determined by differential counts of
their peripheral leukocytes, and include acute myelogenous
leukemia, acute lymphocytic leukemia, chronic lymphocytic

 

 

Taste l. Test for viral-specific RN A in human leukemic cells
RNA-region Counts per
Tissue Counts/10min 10 min/S Reaction
Acute lymphatic

leukemias
(Y) 402 4.7 +
(Hl) 515 6.0 +
(E) 387 4.5 +
(V) 233 2.7 _
(Gr) 1413 - 16.4 +
(8) 308 3.6 +
(W) 832 9.7 +
(O) 393 4.6 +
(Bx) 1271 14.8 +

Chronic lymphatic

leukemias
(LSes) 478 5.6 +
(LSn) 328 3.8 +

Acute myelogenous

leukemias
(B) 631 7.3 +
(D) 813 9.5 +
(Ar) 564 6.6 +
(GM) 912 10.6 +
(WSs) 118 1.4 _
(Hes) 682 7.9 +
(Hi) 450 5.2 +
(L) 684 8.0 +
(R) 361 4.2 +
(C) 322 3.7 +
(M) 665 7.7 +
(G) 220 2.6 —
(F) 1315 15.3 +

Monocytic

leukemias
(WR) 528 6.1 +
(RG) 590 6.9 +
(Sy) 550 6.4 +

 

Results of hybridization reactions between RLV-[3H]DNA
and polysomal RNA isolated from human leukemic cells. In two
cases (H and LS) cells were taken in 1- and 3-year intervals. 200—
1000 zg of polysomal RNA of each sample were hybridized to
2000 epm of RLV-[%H]DNA and the reactions were analyzed by
Cs.80, equilibrium centrifugation. The amount of DNA banding
in the RNA region of the gradient (between densities 1.62 and
1.68) was then determined. The results are expressed as counts/
10 min (corrected for background) banding in the RNA region
for each RNA sample tested, and as multiples of S, the operational
standard deviation (see text and legend to Fig. 5). The annealing
reaction is considered positive only if the counts/10 min per RNA
region is greater than 3S, thus providing 99.9% confidence sta-
tistically. Of the 27 samples tested, 89% were positive.
438 Microbiology: Hehlmann ei al.

 

 

5
2
J
4
3 .
s Acute Myelogenous Leukemia~,
53
z=
3
G2
3 °
x
as |
Normal Leukocytes 7
oO 2 O°
7 T T T + T T i 7
100 200 300 400 500 600 700 800 900

RNA (ug /6Ou!)

Fie. 4. Comparison of hybridization reactions between
RLV-[*H]DNA, leukemic polysomal RNA, and normal buffy-
coat polysomal RNA at various RNA concentrations. The in-
dividual annealing reactions were analyzed by Cs;SOQ, density
centrifugation, and the percent DNA hybridized was determined
by the counts/10 min of [7H]DNA (corrected for background)
banding in the RNA region (between densities 1.62 and 1.68)
of the gradients.

leukemia, and monocytic leukemia. Fig. 5 presents a conve-
nient pictorial summary of the reactions observed with leuke-
mic (Table 1) and various control tissues.

Of the 27 leukemic samples examined, only three fell in the
negative range. The remaining 24 showed positive reactions,
with some exceeding those observed with the animal. leukemic
material (Fig. 1A and B). In contrast, none of the polysomal
RNA preparations from 33 normal tissues gave a reaction that
could be unambiguously designated as positive. These prep-
arations include RNAs from normal leukocytes, phytohemag-
glutinin-stimulated lymphocytes, and various normal human
adult and fetal tissues.

The fact that 89% of the leukemic polysomal RNAs yielded
unmistakable positive hybridizations with RLV-DNA,
whereas none of the 33 control tissues exhibited this type of
response, argues for the specificity of the annealing reactions.

Further support for this conclusion can be obtained by the
use of a [7H]DNA complementary to the RNA of the mouse
mammary tumor virus or to that of the avian myeloblastosis
virus. We have shown (manuscript in preparation) that
RLV-DNA and the homologous viral RNA do not crosshy-
bridize significantly with the corresponding nucleic acids of
either the mammary tumor virus or the avian agent. If the
annealing reaction is specific, one would not expect a leukemic
polysomal RNA, positive for a reaction with RLV-DNA to
show the ability to hybridize either with mouse mammary
tumor viral-DNA or avian myeloblastosis viral-DNA. Fig. 6
shows that these expectations are realized. None of three
other leukemic polysomal RNAs giving a positive (550-910
counts/10 min above background) reaction with RLV-DNA
responded to either of the other two viral DNAs.

Stimulated by the known viral etiology of animal leukemias
(9, 10), others have sought for similar evidence in the human
disease. Particles resembling the C-type viruses of the avian
and murine leukemias have been occasionally detected by
electron microscopy (11). Of perhaps more immediate rele-
vance is the report (12) of components in human leukemic cells
and sera that can interact with fluorescent-labeled antibodies
against the Rauscher leukemia virus.

The present investigation is not directly aimed at resolving

Proc. Nat. Acad. Sct. USA 69 (1972)

the issue of a viral etiology of human cancer. It is concerned
rather with determining whether viral-related information, in
terms of sequence similarities, can be detected in human
neoplastic cells.

It is worth explicitly noting the questions that remain un-
answered by the data described here. They provide no measure
of the degree of homology between the RLV-DNA and the
RNA detected in human leukemic cells. To settle this issue
will require the synthesis of RLV-DNA in amounts adequate
to permit comparative saturation curves of labeled RLV-RNA
and the relevent RNA strands from human leukemic cells.
The fact that hybrids are found in the RNA region of the
density gradient implies that the RNA is much larger than the
DNA product. However, experiments must now be designed
and performed that will determine how large this RNA is,
how much viral sequence it contains, and whether the viral
information is covalently linked to normal cellular message.
The latter point is particularly relevant to whether the viral
information is integrated into the genome of the cancer cell.
Finally, more highly purified mRNA fractions will be re-
quired to make the quantitative aspects more certain. In par-
ticular, it is apparent that a negative outcome of a hybridiza-

Human Leukemias Normal Human Tissues

 

 

 

 

 

 

 

 

 

 

 

 

7
e
I6
e
57 «
Hl °
10
7 °.
9
8 id
e
$7 e
SO Eie 8
& ar)
x5
a » .
4 ele
e
ary ° a, 8
2
. we] 2
{ Fiala) o
ALL CLL AML ML »o & &
2 > 8
ag 2 ££ 8 3
ee 68 6 =5 ~3
55 € € 33 23
25 3 3 @ er

Fie. 5. Results of hybridization reactions with RLV-[*H]-
DNA and polysomal RNA from leukemic and normal human
cells. The RNA was derived from leukemic and normal leuko-
cytes, phytohemagglutinin-stimulated lymphocytes (A), a human
lymphocyte cell line, NC37 (VY), lymph nodes, other adult tissues:
liver (A), spleen (X), intestine (©), striated muscle (1), and
fetal tissues: liver (A), lung (V), limbs (©), placenta (CG). The
reactions were then subjected to Cs:SO, equilibrium density
centrifugation as described under Fig. 1. The amount of [7H] DNA,
expressed as cpl0m corrected for background banding in the
density region of RNA (between densities 1.62 and 1.68), was
determined for each reaction. An operational mean and standard
deviation (S) were determined for each counter by the total
cp10m of three tubes [e.g., 2, 3, 4] of each of 60 gradients. The
ep10m [?H]DNA corrected for background banding in the RNA
region of the gradient was then divided by the appropriate oper-
ational standard deviation. Any reaction with less than 3S
cp10m in the RNA region is considered negative, thus providing
99.9% confidence that those reactions retained as positive
(greater than 3S) are statistically significant. ALL, acute lym-
phatic leukemia; CLL, chronic lymphatic leukemia; AML,
acute myelogenous leukemia; ML, monocytic leukemia.
Proc. Nat. Acad. Sci. USA 69 (1972)

5b N ONA AL?
A NN
° 4b e Ne? 169
x a
§3t . y=
3 s
-

T
2
=

/
L
o

il

 

hs L L
| 5 10 15 20 25
Fraction Number
B ve

wh

nm
T

T

SHeplom x tO?
Ui

 

4l4¢

RNA we I 3 ~

re a leeee L i 1

| 5 10 15 20. 25

Fraction Number

Fie. 6. Cs,80.1 equilibrium gradient centrifugation of (A)

mouse mammary tumor virus [*H]DNA and (B) avian myelo-

blastosis virus [*H]DNA hydridized (each) to 400 wg of human

leukemic polysomal RNA. Hybridization conditions and Cs2804
gradient analysis as in Fig. 1.

 

 

tion test cannot be accepted as evidence for absence of the
relevant RNA.

From the data presented, one can conclude at best that the
probability of finding RNA homologous to Rauscher leukemia
viral RNA is much greater in human leukemic cells than in
normal tissues. Indeed, if the provirus (13) or oncogene
hypotheses (14) are valid, some part of this information might
be found with more sensitive tests in presumably normal cells
derived from fetal tissues or phytohemagelutinin-stimulated
lymphocytes, both of which have been reported (14, 15) to
possess the group-specific antigen of the mammalian leukemo-
genic viruses.

While the experiments described here do not constitute
definitive proof of a viral etiology of human leukemia, they
provide rather compelling evidence for the presence in human
leukemic cells of RNA posessing sequences homologous to
those found in a viral agent known to cause leukemia in an
experimental animal. From what we know of animal systems,

Viral RNA in Human Leukemia Cells 439

one would predict that a similar situation would be found in
human sarcomas, a prediction that has been confirmed and
will be detailed in another publication.

We thank the following for supplying cell material: Dr. M.
Stoker (Imperial Cancer Research Fund, London), Dr. R. Powles
(Royal Marsden Hospital, London), Dr. R. Gallo (National In-
stitutes of Health, Bethesda, Md.), Dr. J. F. Holland (Roswell
Park Memorial Institute, Buffalo, N.Y.), Drs. J. H. Burchenal
and H. F. Oettgen (Sloan-Kettering Institute, New York), and
Drs. E. F. Osserman and M. Farhangi (Institute of Cancer Re-
search, Columbia University, New York). We greatly appreciate
the excellent technical assistance of Jeanne W. Meyers, Lee E.
Hindin, and Mildred Rothenberg. This research was supported
by the National Institutes of Health, National Cancer Institute,
Special Virus Cancer Program Contract 70-2049 and Research
Grant CA-02332.

1. Hall, B. D. & Spiegelman, 8. (1961) Proc. Nat, Acad. Sct,
USA 47, 137-146.

2. Schlom, J., Spiegelman, S. & Moore, D. H. (1971) Nature
231, 97-100.

3. Schlom, J. & Spiegelman, 8. (1971) Science 174, 840-843.

4. Axel, R., Schlom, J. & Spiegelman, S. (1972) Proc. Nat. Acad.
Sci. USA 69, in press.

5. Axel, R., Schlom, J. & Spiegelman, S., Nature, in press.

6. Spiegelman, 8., Burny, A., Das, M. R., Keydar, J., Schlom,
J., Travnicek, M. & Watson, K. (1970) Nature 227, 563-
567.

7. Shay, H., Gruenstein, M., Marx, H. E. & Glazer, L. (1951)
Cancer Res. 11, 29-34.

8. Weinstein, R. 8. & Moloney, W. C. (1965) Proc. Soc. Exp.
Biol. Med. 118, 459-461.

9. Ellermann, V. & Bang, O. (1908) Centralb. f. Bakt., Abty. I
46, 595-610.

10. Gross, L. (1970) in Oncogenic Viruses (Pergamon Press,
Oxford, England).

11. Dmochowski, L., Yumoto, T., Grey, C. E., Hales, R. L.,
Langford, P. L., Taylor, H. G., Freireich, E. J., Schullen-
berger, C. C., Shively, J. A. & Howe, C. D. (1967) Cancer
20, 760-777.

12. Fink, M. A., Karon, M., Rauscher, F. J., Malmgren, R. A.
& Orr, H. C. (1965) Cancer, 18, 1317-1321.

13. Temin, H. M. (1971) J. Nat. Cancer Inst. 46, III-VII.

14. Huebner, R. J., Kelloff, G. J., Sarma, P. S., Lane, W. T.,
Turner, H. C., Gilden, R. V., Oroszlan, S., Meier, H., Myers,
D. D. & Peters, R. L. (1970) Proc. Nat. Acad. Sci. USA 67,
366-376.

15. Hellman, A. & Fowler, A. K. (1971) Nature New Biol.
233, 142-144,