(CANCER RESEARCH 42, 4964-4969, December 1982]
0008-5472/82/0042-0000$02.00
Putative Transformation-dependent
Proteins in the Blood Plasma of
Tumor-bearing Rats and Cancer Patients1
Dorothy E. Schumm2 and Thomas E. Webb
Department ot Physiological
Columbus, Ohio 43210
Chemistry, The Ohio State University College of Medicine, and Ohio State University Comprehensive
ABSTRACT
By use of a biochemical assay, we detected the existence in
blood plasma of tumor-bearing rats the presence of factor(s)
low or absent in normal rat plasma. The present report de
scribes parameters which influence the appearance of this
factor during carcinogenesis and development and also dem
onstrates its presence in the plasma of human cancer patients.
The "plasma factor" was detectable within 48 hr of treatment
of rats with a carcinogenic regimen of dimethylnitrosamine or
thioacetamide. The activity in this initial transient phase, which
was maximal at 3 weeks, was followed by a second progressive
increase in activity, which, after 8 to 10 months, attained a
level 5 to 6 times the initial increase. The latter persistent
increase appears to parallel tumorigenesis. The factor showed
a small and very transient increase during the early stages of
liver regeneration after two-thirds partial hepatectomy or CCU
intoxication, with normalization of the plasma activity within 15
days. The appearance of the factor in circulation coincided
with that of a M, 60,000 phosphoprotein. A factor with similar
activity has been detected in the conditioned culture medium
in which human cancer cells were grown and was present in
significant concentration in the plasma of 100 cancer patients
with a wide variety of primary (and metastatic) tumors. On
Sepharose 6B chromatography, it appeared to have a M, of
about 70,000. The factor was absent or low in nonconditioned
medium, in the plasma of normal controls and in patients free
of active cancer (i.e., leukemia patients in remission). The
plasma factor(s) appears to be an embryonic protein, since a
protein(s) with similar activity is present in the plasma of preg
nant rats at 14-day gestation and in the blood of the newborn
rat; the factor rapidly disappears postpartum, reaching control
levels by 4 days.
INTRODUCTION
Transformed cells produce a large number of proteins which
are absent in the normal adult cells from which they originate.
Some of these proteins are released from tumor cells, enter
the circulation, and have been used as markers for the exis
tence of specific types of cancer. a-Fetoprotein, normally syn
thesized by mammalian embryos early in development (6), was
discovered in 1963 in experimental liver tumors (1). Since that
time, it has been used as a diagnostic test to detect primary
liver cancer and teratocarcinoma in humans (2, 5). Carcinoembryonic antigen is another tumor-derived protein which has
' Supported by a grant from the Ohio Division of the American Cancer Society
and by Grant CA 30627 to the authors: also supported by Grant P-30-CA-1605809 to Ohio State University Comprehensive Cancer Center from the NIH, De
partment of Health and Human Services.
* To whom requests for reprints should be addressed.
Received March 25, 1982; accepted August 19. 1982.
4964
Cancer Center,
been used in cancer detection. It was first described by Gold
and Freedman (7) in 1965 and is currently used for the diag
nosis of cancer of the digestive system (22). As important as
these proteins have become in cancer detection, their use is
limited by the narrow spectrum of tumor types of which they
are characteristic. What is needed is a protein or group of
proteins which occur in most, if not all, malignant diseases and
in only a limited number of nonmalignant conditions.
We have developed a cell-free system for the transport of
functional mRNA and rRNA which is modulated by proteins
produced by transformed cells (16-19). Samples of plasma,
drawn from rats and mice bearing a variety of chemically
induced primary and transplantable tumors, caused a marked
increase in the release of messenger-like RNA from rat liver
nuclei into the medium of the cell-free system (19). The plasma
factor was shown to be nondialyzable and heat labile with a
short functional half-life in vivo. Since it appeared to be neither
tumor nor species specific, this plasma factor may represent a
protein useful in the development of a general tumor detection
test. Therefore, further experiments have been carried out to
confirm and extend these initial studies. The release of these
factors to the circulation has been analyzed during tumorigen
esis in an animal model. Here, we provide extensive data which
show that similar factors are indeed released from tumors to
the circulation in cancer patients.
MATERIALS
AND METHODS
Tissue and Plasma Sources. Normal liver used for the preparation
of the cell-free system was obtained from fasted male Sprague-Dawley
rats weighing 250 g. Nuclei were prepared by homogenizing the nuclei
in 2.3 M sucrose:3.3
mM calcium acetate after a 30-min in vivo
prelabeling of the nRNA with 50 ¿idiof [6-'"C]orotic
acid (specific
activity, 323 ftCi/mg; New England Nuclear, Boston, Mass.) as de
scribed previously (17). Cytosol was prepared from a supernatant
(100,000 x gìof a 1:2 liver homogenate and was dialyzed overnight
against a buffer containing 50 HIM Tris (pH 7.5), 25 mM KCI, and 2.5
mM MgCI2(17).
Plasma from experimental animals was prepared from heparinized
blood drawn by cardiac puncture. The carcinogenic regimens involved
a course of 9 daily i.p. injections of 50 mg thioacetamide per kg body
weight (Apache Chemicals, Seward, III.) (8) or a single i.p. injection of
dimethylnitrosamine
(redistilled;
Eastman Kodak Co., Rochester,
N. Y.) at 5.0 mg/kg body weight 24 hr after a two-thirds partial
hepatectomy (4). CCU intoxication of the rats was achieved by admin
istering a 1.25-ml/kg dose of CCU:sesame oil (1:1) by stomach tube
(14). Human plasma samples and patient histories were obtained
through the Tumor Procurement Laboratory of the Comprehensive
Cancer Center of The Ohio State University.
Cell-free System. The cell-free system consisted of 5 x 106 prelabeled nuclei per ml of medium containing 5 mg dialyzed cytosol protein
per ml, 50 mM Tris-HCI (pH 7.5), 25 mw KCI, 2.5 mM MgCI?, 0.5 mw
CaCI2, 0.3 mM MnCI2, 5.0 mM NaCI, 2.5 mM Na2HPO4, 5.0 mw
CANCER
RESEARCH
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VOL. 42
Transformed Cell-dependent
spermidine, 2.0 mM dithiothreitol, 2.0 mM ATP, 2.5 mM phosphoenolpyruvate, 35 units pyruvate kinase per ml, and 300 jig low-molecularweight yeast RNA per ml. This mixture, with or without the addition of
up to 100 p\ of plasma, was incubated at 30° for 30 min. The nuclei
were removed by centrifugation,
and the resulting supernatant was
precipitated with 5% trichloroacetic acid. The precipitate was washed
with ethanol, dissolved in solubilizer, and counted in liquid scintillant.
Results are reported as percentage of increase in RNA release per mg
protein added. When results are given as units of activity, 1 unit is
defined as a 1% increase in RNA release per mg protein added.
Alternately, the nuclei-free supernatant was extracted with phenol, and
fe
È
1 io£ 8-1
the RNA was separated on a sucrose density gradient as described
previously (2).
Phosphoprotein Analysis. A female rat carrying a 4-month DMBA3induced mammary tumor was given an injection of 1 mCi of 32P¡.
Three
hr later, blood was drawn, and plasma was prepared, diluted 1:2, and
subjected to polyacrylamide gel electrophoresis (2).
Molecular Weight Determination. Twenty-eight ml of plasma from
human patients which contained a high concentration of the factor
were fractionated by ammonium sulfate. The material precipitating
between 30 and 60% saturation was dissolved in 3 ml of buffer
containing 50 mM Tris (pH 7.5), 25 mM KCI, and 2.5 mM MgCI2 and
dialyzed overnight against the same buffer. The material was then
fractionated on a column of Sepharose 6B using the same buffer. Onemi samples were collected, and 100 /¿I
of this were assayed in the cellfree system. Reference standards were ferritin (M, 440,000), bovine
serum albumin (M, 68,000), and lysozyme (M, 14,300).
RESULTS
In an earlier study, we identified regulatory macromolecules
in the peripheral blood plasma of animals bearing a variety of
chemically induced primary and transplantable tumors in rats
and mice (19). These regulatory molecules were assayed by
their ability to modulate the release of prelabeled RNA from
isolated nuclei in a cell-free system. These plasma factors were
protein in nature, could be precipitated with streptomycin sul
fate, and fractionated on DEAE columns (18). Although the
plasma factor appeared early after the inoculation of tumor
cells and appeared to increase in concentration with increasing
tumor load, the relationship between tumor induction by car
cinogens and the plasma factor was not explored.
Previous studies indicated that the effect of carcinogen treat
ment could be detected by protein changes in the cytosol of
the liver as early as 24 hr after treatment and persisted for at
least 4 months (16). In order to determine whether the plasma
factor showed a similar response, a time course study of the
appearance of this factor was conducted. Blood samples were
drawn starting 24 hr after completion of treatment with either
thioacetamide, a weak hepatocarcinogen, or dimethylnitrosamine, a strong carcinogen. Plasma, freshly prepared from these
blood samples, was dialyzed and then assayed in the cell-free
system for its ability to modulate mRNA release. Chart 1 shows
the results of that experiment. Even though they differ in their
carcinogenic potency, the early time course was similar, but
the amount of factor at all time points was greater for the
stronger carcinogen. For both carcinogens, the plasma factor
was detectable within 48 hr after treatment. During the initial
transient phase, the activity rose to a maximum at about 18 to
21 days and then decreased, reaching a minimum at about 6
weeks before the persistent increase of the second phase was
observed. The amount of factor in the plasma during the second
! The abbreviation
DECEMBER
1982
used is: DMBA. 7.12-dimethylbenz(a)anthracene.
Protein
4-
je
y
20
40
60
369
12
DAYS
MONTHS
Chart 1. Time course of the appearance of the plasma factor after carcinogen
treatment. Blood was drawn by cardiac puncture at various times after treatment.
Plasma was prepared and assayed in the cell-free system for its ability to
stimulate RNA release. •¿,
plasma from animals treated with a carcinogenic
regimen of dimethylnitrosamine; G, plasma from animals treated with thioacetamide; O, plasma from animals treated with dimethylnitrosamine
without partial
hepatectomy, a noncarcinogenic regimen. Results are the average of 3 to 5 rats.
phase increased at a rate considerably less than that seen in
the initial rise. However, 8 to 10 months posttreatment, the
plasma activity in this second phase was 5- to 6-fold higher
than the maximum observed in the initial phase. If animals were
treated with the same dose of dimethylnitrosamine without the
accompanying partial hepatectomy, i.e., a noncarcinogenic
regimen, the factor in the plasma did not rise above the back
ground of the normal controls.
Both carcinogenic regimens used in these studies produce
considerable liver damage. It was possible that the results
observed in the early phase reflected liver damage and con
sequent regeneration rather than carcinogenesis. In order to
test this possibility, the appearance of the plasma factor was
followed after a two-thirds partial hepatectomy, sham opera
tion, or CCI4 intoxication. As can be seen in Chart 2, both
partial hepatectomy and CCI4 intoxication caused a small tran
sient increase in the factor, but in neither case did the amount
equal that observed in the carcinogenesis regimen. The maxi
mum concentration of factor occurred at 4 days after partial
hepatectomy and returned to normal by Day 7. For CCI4 intox
ication, the maximum was reached by Days 7 and 8 and
returned to near control values by Day 15. Sham operation did
not cause a detectable increase in the levels of the factor.
Furthermore, the prolonged increase in plasma factor (second
phase) observed in carcinogen-treated
rats was absent in
partially hepatectomized and CCI4-treated animals. No increase
was observed at any time in the sham-operated animals.
Tumors, in contrast to normal tissues, often produce fetal
proteins, and such proteins serve as diagnostic tumor markers;
a-fetoprotein in primary liver cancer and carcinoembryonic
antigen in cancer of the digestive tract are examples. There
fore, an attempt was made to identify the factor in pregnant,
lactating, and newborn rats. As anticipated (Table 1), plasma
prepared from blood drawn from female rats in midpregnancy
(14 days) showed a very significant (16-fold) increase of 4.1%
in RNA transport per mg of plasma protein added to the cellfree system. Plasma prepared from female rats 4 days post4965
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D E. Schumm and T. E. Webb
Table 1
Bioassay of tissue culture media and tissue from normal, perinatal, pregnant,
and tumor-bearing rats
of activ
ity
RNArelease
(% of
/mgplasma
proteinadded)0.25
sourceControl
Tissue
ratPlasmaLiverPregnant
0.51.64.10.43.65.1
±
rat14-day
gestation4-day
postpartumNewborn
0.5
old)PlasmaLiverModificationNoneNoneNoneNoneNoneNoneUnits
rat (4 days
o:
o
z
a«
5
IO
I5
20
Culture medium
Conditioned by growth of
BoWo cells
Nonconditioned medium
TIME (days)
Chart 2. Time course of the appearance of the plasma factor after partial
hepatectomy, sham operation, and CCI4 intoxication. Blood was drawn by cardiac
puncture at various times after treatment. Plasma was prepared and assayed in
the cell-free system. •¿.
plasma from rats after a two-thirds partial hepatectomy;
D, plasma from rats after CCU intoxication; O, plasma from rats after a sham
partial hepatectomy. Results are the average of 3 to 5 rats.
Plasma from rat with s.c. hep
atoma 5123D
Before removal of s.c. tu
mor
48 hr after surgical removal
3 wk later with local meta
static tumor
partum had values of the plasma factor in the range of the
normal controls (0.4). However, the plasma from 4-day-old rats
contained sufficient factor to cause a 3.6% increase in trans
port per mg of added protein; the liver contained 5.1% increase
per mg. In contrast, adult liver showed an increase of only
1.6% per mg. The factor was also found in the 30 to 60%
ammonium sulfate fraction of culture medium in which BoWo
human carcinoma cells had been grown. It was nearly absent
from a similar fraction of nonconditioned medium. This confirms
that the factor is produced by the tumor cells rather than being
induced in normal host cells by the presence of a tumor.
The data in Table 1 also indicate that the factor(s) is rapidly
cleared after surgical resection of a transplantable rat hepatoma (5123D) inoculated previously at a single s.c. site. How
ever, in this particular experiment, the concentration of the
plasma factor dropped 4- to 5-fold to near basal level within 48
hr and then increased again, attaining a value 8-fold above the
basal level within 3 weeks. This latter increase coincided with
the appearance of a second (metastatic) tumor in close prox
imity to the site of the tumor which was surgically removed. It
should be noted that, although this hepatoma ¡sa transplanta
ble tumor, it has a moderately slow growth rate (12).
The data in Table 1 also provide some information on the
appearance of the factor in the plasma of rats during DMBAinduced mammary tumorigenesis. By 10 months, the plasma
concentration of the factor is elevated 4- to 6-fold over the
basal level. According to the protocol used, mammary tumors
begin appearing within 6 weeks. It is significant that the release
of this excess nRNA in response to the plasma factor is, like
the transport of mRNA and rRNA, essentially totally dependent
on the presence of an energy source. Deletion of the latter
from the assay results in a marked inhibition of RNA release.
Also, as shown in Table 1, the plasma factor from DMBAtreated rats is heat labile and, like the material from the culture
medium, is precipitated at between 30 and 60% saturation with
Plasma from rats bearing
DMBA-induced mammary
tumors
4 mos. after DMBA
6 mos. after DMBA
10 mos. after DMBA
10 mos. after DMBA
4966
Plasma from rat bearing 4-mo.
DMBA-induced mammary
tumor
Plasma heated
Plasma fractionated
Precipitation by 30-60%
(NH4)2S04
Precipitation by 30-60%
(NH4)2S04
0.67
0.21
None
3.6
None
None
0.8
2.1
None
None
None
Assay minus ATP
4.6
6.8
11.6
0.1
45°for 5 min
60°for 5 min
Precipitation by 0-30%
(NH4)2S04
Precipitation by 30-60%
(NH4)2SO4
3.6
0.9
0.5
2.7
ammonium sulfate. It has no detectable ribonuclease
activity.4
Other data on its physical properties have been presented
previously (18, 19).
In 1979, Senger et al. (21) reported the existence of a M,
58,000 phosphoprotein which was secreted into the growth
medium by a variety of transformed mammalian cells. These
proteins were species specific and capable of being phosphorylated in vitro. To determine whether the factor we were
studying exhibited similar properties, we prepared plasma from
a DMBA-induced mammary tumor-bearing rat which had been
given an injection of 32P¡
3 hr earlier. Chart 3 shows the result
of an acrylamide gel electrophoresis of a 1:2 dilution of that
plasma. The plasma from a tumor-bearing animal gave a radio
active peak corresponding to a phosphorylated protein of M,
60,000. Antibody prepared to a partially purified fraction of
this protein, according to the procedure of Senger ef al. (21),
eliminated the peak from the gel and removed most, but not
all, of the plasma factor activity as measured in the cell-free
system. Plasma from a normal control rat showed only a small
4 T. Palayoor, D. Schumm, and T. Webb, unpublished
CANCER
observations.
RESEARCH
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VOL. 42
Transformed Cell-dependent
1000
~
N.pòX^
n800
'
NV °N.
\i
Ourwith system has been investigated.
theprevious
nuclei in
indicatedlabeled
tumor-bearing animals (7, 9)
studiesthat
containingthe
mean size ofcell-free RNA released to medium
inits tumor factor was considerably larger than that released
shiftoccurred
absence. In order to determine whether a similar size
in the presence of plasma from human cancer pa
tients,
cell-freesystem
released labeled RNA was extracted from the
after incubation in the
orTable
presence of buffer (control)
•¿
240
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20
NUMBERChart
GEL SLICE
u
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2
patientsPrimary Bioassay of plasma from cancer
0
Tumor-bearing
3. Polyacrylamide gel electrophoresis of MP-labeled plasma.
3hr
and control rats were given injections of [32P]P¡,
and blood was drawn
electrophoresis.G.
later. Plasma was prepared, diluted 1:2, and subjected to gel
froma
plasma from a rat bearing a DMBA-induced mammary tumor; •¿,
plasma
developa
control animal; A, plasma from a rat treated with DMBA which failed to
sulfate.radioactive
tumor. SOS, sodium dodecyl
siteOvaryIn tumor
mgNo.
plasma4
Sex
1.42
system.Preliminary
similarbiological evidence indicated that a factor with
inthe
activity in the cell-free system could be detected
greatervolume
plasma of human cancer patients (20). However, a
toobtain of plasma from the human patients was necessary
plasma.We
a response equivalent to that observed for rat
TumorProcurement
analyzed 100 plasma samples, obtained from the
Center,which Laboratory of the Comprehensive Cancer
yearsoldincluded 41 males ranging in age from 20 to 81
canbe
and 59 females ranging in age from 19 to 76 years. As
(greaterthan
seen from the data in Table 2, positive responses
patientswith
3% release per mg protein) were obtained from
primarytumors.
confirmed active disease due to a variety of
andwomen
There was no difference in the values for men
ofbreast with the same tumor type and, with the exception
metastasis.In
tumors, little difference with the existence of
48hr
general, patients who had received chemotherapy within
patientswhose
of the sample being drawn had values lower than did
.4withoutlast therapy had been at least 1 week before (4.8 ±1
ovariantumors;
recent prior treatment versus 1.8 and 2.5 for
coloncancer).
3.9 ± 0.4 versus 2.7 ± 0.5 for women with
theconcentration
This decrease could be due to a true reduction of
thebioassay
of the plasma factor or an interference in
consistedofby the chemotherapeutic agent(s). Controls
oldermale
10 normal healthy young adult males (1 .83 ±0.09), 2
andone
patients with benign prostate tumors (2.2 and 2.3),
withthrombophlebitis
normal pregnancy patient (2.9). Plasma from a patient
value.All
also gave a negative (basal)
slightlyhigher
patients with cancers in remission had values only
±0.1 than controls (2.1 for Hodgkin's lymphoma and 2.2
valuesrose
5 for acute myelogenous leukemia). In relapse, these
3.5.Although
to 3.7 and
inresponse the identity of the excess labeled RNA released
itseffect
to the plasma factor has not been established,
on the size distribution of the labeled RNA released fromthe
F
ofMetasrelease/Age
hr) prior
tasis
treat(yr)
present
ment
26-67
+
1.8.2.51F
57, 66
+
3.41 F
64
2.84 F
43
remissionCervixBreastEndometriumVulvaLungColonHodgkin
1.02 F
34-54
+
3.62
4.93
.72
3.82
trianglesrepresent
peak in the M, 60,000 range. The closed
hadbeen the results of testing the plasma of a rat which
failedtogiven a carcinogenic regimen of DMBA but which
sameanimal
develop a tumor after 5 months. Plasma from this
cell-free showed a small but still positive response in the
DECEMBER 1982
Protein
F
F
F
F
F
F
F
36, 48
56, 65
40-70
43, 72
61,66
71-76
42
F
F
F
M
M
73
57
26-68
52-71
'
40
1F
+2.21
6.13 M
0.46 F
M
52
58
56-62
46-81
+
+
+
60.51
53-56
+
41.01
6.11
2.83
1
0.74
1.01
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+2.31 M
66
M
31
M
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+
+
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%
4.8
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4.1
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+
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± 1.0
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2.7
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2.1
+2.9
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lymphomaRemission
s
RelapseAcute
myelogenousleukemia
3.74
1
0.151 M
RemissionRelapseNon-Hodgkin's
lym
32-55
3.54 F
0.081 F
53
24-59
+
M
60
+
56
50
59
49
55
+
+
50
66
+
phomaKidney
+2.01
1F
+2.81 M
3.12 F
M
36,
1
3.21
M
EsophagusTestesLarynxTongueMaxillary
+2.61 M
2.42 M
**.o1
M
RNi
+
+
+5.52 M
27
+
3.51 M
60
+
+4.31 M
64
+
4.81 F
45
3.71 M
63
3.41 F
37
sinusOral
M
76
+
cavity
1
F
64
PalatePharynxFaceParotid 3.61F
+3.21
34
+
4.91 M
69
M
50
+
gland
1F
1.91
38
antrumGlottisBladderLeiomyosarcomaThyroidPyreforme
Maxillary
2.91 M
50
+
M
65
+
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3.81
19
+
3.81F M
45
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3.61
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70
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sinus
12.91 F
67
SkinMelanomaUnknownRecent(<48
3.81F M
51
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-
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4967
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1982 American Association for Cancer Research.
D E. Schumm and T. E. Webb
tumor plasma. The results of fractionating this RNA on a
sucrose density gradient are shown in Chart 4. As can be seen,
the pattern for the control plasma is similar to that for added
buffer. The RNA transported in the presence of human tumor
plasma is skewed to heavier sizes.
Chart 5 shows the results of a Sepharose 6B separation of
a 30 to 60% ammonium sulfate fraction of pooled human
plasma. The plasma selected included those samples which
had been shown previously to contain a high concentration of
the factor. When assayed in the cell-free system, the majority
of the activity was found in a peak having a M, of 60,000 to
70,000. Since there are several smaller peaks and the main
one is quite skewed, this may represent the existence of
multiple protein species, all exhibiting a similar biological activ
ity.
DISCUSSION
Carcinogen treatment causes a biphasic appearance of a
plasma factor in the peripheral blood. The first phase occurs
immediately after carcinogen treatment and may be due to
damage and regeneration of the liver. This damage could
release the normal cytoplasmic factors which we have shown
modulate RNA transport in liver. Another possibility is some
form of reversible transformation which may involve immune
surveillance. In fact, cells prepared from target organs within
2 days of exposure to carcinogens have been shown to have
malignant potential, i.e., higher fibrinolytic activity and pro
longed survival in agar (3). The second phase, which begins 6
weeks after carcinogen exposure, appears to be due to the
release of a factor by a steadily increasing number of tumor
cells. Although the biological activity of the factors in the 2
phases is similar, they may not be identical proteins.
The factor appears to be produced by the transformed cells
themselves rather than by a host-tumor or host-carcinogen
interaction, since it appeared in the culture fluid in which
transformed cells were grown. Considering its occurrence in
normal pregnancy and in newborn blood and liver, the factor
may be a fetal protein but is definitely not identical to a-
10
N
Q 8
X
FRACTION
20
NUMBER
30
Chart 4. Sucrose density gradient of RNA transported in the presence and
absence of human tumor plasma. •¿,
plasma from a patient with metastatic colon
cancer; O. plasma from a normal control; D, sample to which only buffer had
been added.
4968
40
•¿20
cÃ-1.2 i
in
I
.a
.6 •¿
.4
.2 •¿
tc
u
IO
20
30
40
50
60
TO
FRACTION NUMBER
Chart 5. Sepharose 6B chromatography of human plasma. Plasma containing
the factor was pooled, and the material precipitating between 30 and 60%
ammonium sulfate saturation was applied to a column of Sepharose 6B. •¿,
assay
of 100-/il fractions in the cell-free system. Molecular weight curve (O) was based
on ferritin (44 x 10"), bovine serum albumin (6.8 x 10"), and lysozyme (1.4 x
10").
fetoprotein, carcinoembryonic antigen, or the /S-protein (11). It
may be related to the M, 60,000 phosphoprotein identified in
medium conditioned by tumor cells (15), since the appearance
of the factor coincides with that of a M, 60,000 phosphoprotein.
An antibody made to the partially purified phosphoprotein
significantly diminished the activity of the factor in the cell-free
system.
Ratei ef al. (13) have shown that, after feeding of 3'-methyl4-dimethylaminoazobenzene,
a small RNA, normally restricted
to the nucleus, is released to the cytoplasm both in vivo and in
vitro. This RNA, which is a small molecule in the nucleus, is
released as part of a much larger molecule during carcinogenesis. A similar release of large, perhaps incompletely proc
essed, RNA was observed when plasma from tumor-bearing
animals (8) or human cancer patients (this study) was added to
the cell-free system. This material may not be true mRNA,
since little change in the polyadenylate-containing
RNA has
been observed immediately after carcinogen treatment (9).
As indicated in our initial publication (19), we found it essen
tial to test for the tumor-specific factor in plasma rather than
serum, since the preparation of serum destroyed the factor to
varying degrees. While the present survey of human cancer
patients was in progress, Le Maire eÃ-al. (10) reported a
stimulation of RNA transport activity when serum from patients
with a variety of tumor types was added to our cell-free system.
All cancers gave positive results except for some mammary
tumors which produced lower than expected values. In our
hands, we found no such discrepancy with plasma from pa
tients with mammary tumors as compared with tumors at other
sites. In order to ensure consistency, it is highly recommended
that these tests be run on plasma rather than serum.
A general tumor or transformed cell detection system should
be rapid and sensitive and respond to most cancers and few
nonmalignant conditions. The results presented here indicate
that this test responds positively to a wide range of cancers
and gives nearly basal values for plasma from patients with
benign conditions and cancers in remission. Normal preg
nancy, a nonmalignant condition in which fetal proteins are
found in the circulation, also gives positive results. Thus, the
test is highly sensitive with a good positive predictive value in
CANCER
RESEARCH
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VOL. 42
Transformed Cell-dependent
detecting patients with active malignant disease. Its specificity
has not yet been thoroughly tested with a large number of nontumor-bearing patients. From the results found so far, care
must be taken in interpreting the results, especially when other
degenerative or regenerative conditions may be present. The
purification of the protein or proteins involved and the prepa
ration of a radioimmunoassay will greatly add to the ease and
reproducibility of the assay and may reduce the number of
nonmalignant positive responses.
It will be of utmost importance to establish the relationship of
these plasma factors to the many growth factors recently
identified. As pointed out by Potter (15), many of these proteins
appear to be normal embryonic products the production of
which by transformed cells is related to their partially blocked
differentiation, i.e., partially blocked ontogeny.
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Putative Transformation-dependent Proteins in the Blood
Plasma of Tumor-bearing Rats and Cancer Patients
Dorothy E. Schumm and Thomas E. Webb
Cancer Res 1982;42:4964-4969.
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