Note: Descriptions are shown in the official language in which they were submitted.
Background of the Invention
Studies with the murine mammary tumor model have established
the ~easibility of using plasma-concentrations of viral protein
to assess the presence and status of solid tumor7 The viral
protein utilized for such studies was a 52,000 dalton glycopro-
tein (gp 52) isolated from the murine mammary tumor virus (MMTV)
using affinity chromatography. ~vailability of purified gp 52
by the a~oresaid procedure allowea development of ~ radioimmuno-
assay to this protein sensitive to plasma levels down to 0.1 ng/
100 ~1. See in regard to the above Ritzi et al., Virology, 75,
188 (1976) and Ritzi et al., Proc. Natl. Acad. Sci. USA, 73,
No. 11,4190 (1976)
The relationship between the mammary tumors and the plasma
levels of gp 52 were found to be as follows:
(a) tumor-bearing mice, male or female, showed markedly
elevated (100-1000 ng/ml) levels of gp 52 as a free soluble
protein in the plasma and the mean concentration increased with
average tumor siz~;
-- 1 --
- .,
... . . . .
.
~. ;~ ' '
88
(b) the presence of another malignanc~ (leukernia) did not
result in any change o~ gp 52 le~els in the plasma;
(cl mammary tumor tissue located ~y transplantation
outside the mammary g~ is also detected by highlplasma gp 52
leveIs;
(d) low (2-10 ng/ml) plasma levels of gp 52 are found in
tumor free mice whether they orginate from strains characterized
by high or low frequencies of spontaneous mammary tumors;
(e) tumor-free lactating females exhibit the normally low
level~ of plasma gp 52 despite the fact that their milk contains
an average of 20,000 ng/ml of this protein; and
(f) the circulatory clearance time of gp 52 in tumorous
animals is sufficiently rapid (a half-life of 4-6 hr.) to
suggest a requirement for continued replenishment to maintain
the high levels observed.
The MMTV model provides the basis for establishing the
feasibility of utlizing a viral protein "marker" in plasma
for monitoring the presence and status of human breast cancer.
The u~e of a viral protein marker would be distinguishable from
assays, such as disclosed in U.S. Patent 3,999,944, which rely
on detecting antigen induced leukocyte adherence inhibition caused
by tumor specific cell mediated immunity.
Description of the Invention
; The present invention relates to a method ~or detecting the
presence and status o~ cancer in humans by assaying ~or certain
tumor specific viral related proteins in plasma samples and
novel reagents use~ul therein. Such method is thus useful in
diagnosis as in initial screening programs ~or early detection
of the disease, in therapy as in evaluating the status of the
disease after surgical, radiation and/or chemothe~apeutic
- 2 -
:
L.~ 8~88
treatment ~nd in pxogno5is such as ~n det~cting the poss~bility
of xecurrence or metast~ses,
Viral related proteins which can be employed as markers
for the detect~on of cancer, particularly breast cancer,
include viral enzymes such as RNA-dependent DNA polymerase
(reverse transcriptase) or alternatively a tumor associated
protein which is of viral oriyin.
A first aspect of the present invention therefore is
lQ directed to the detection of human breast cancer by utilizing
RNA-dependent DNA polymerase as the plasma marker.
It has been previously known in the art that human breast
tumor particles possessmany of the features characteristic of
RNA tumor viruses. In addition to the expected size (600S) and
density (1.16g/ml), these features include possession of an
outer membrane and an inner one surrounding a "core" containing
a DNA polymerase and a large molecular weight (70S) RNA possess-
ing detectable homology to the RNA's of the mouse marMnary tumor
virus (MMTV) and of the Mason-Pfizer Monkey Virus (MPMV).
The purification and characterization of the DNA polymerase
from the human breast cancer particles has now been accomplished
and forms a part of the present invention. Key properties of
this enzyme are very similar to those of the reverse
transcriptases found in MMTV and MPMV. Thus, like these viral
enzymes, the purified human breast cancer DNA polymerase exhibits
the following three features that together di~tinguish the
known viral reverse transcriptases from normal cellular DNA
polymerases: a) a strong preference for oligo(dT):poly(rA) over
oligo(dT~:poly(dA) as a template for the synthesis of poly(dT);
b) the acceptance of the highly specific ol~go(dG):poly(rCm)
as a template for the formation o~ poly~dG); c) the abilîty to
-- 3 --
,
use a viral ~NA (AMV~ as a template to ~ash~on a faith~ul DNA
cornplementary cop~. The re~emblance o~ the human enzyme to the
reverse transcriptases of MMTV and MPMV extends further in its
possessing a molecular weight of 70,000 daltons and in its
pre~erence for Mg~ over Mn~. To date, an enzyme with these
properties has not been detected in normal breast tissues or in
benign tumors of the breast.
Isolation of human breast cancer DNA polymerase was
accomplished by homogenizing breast cancer tissue and layering
over discontinuous sucrose gradients~ The density region at 1.16-
l.l9g/cc was pooled, diluted and centrifuged. Suspension of
the pellets were fractionated by polyacrylamide agarose gel
filtration. Fractions found active by DNA polymerase assay were
pooled and chromatographed over a phosphocellulose column.
Elution with a linear gradient of a O.lM to 0.5M phosphate buffer
pH 7.2. The main peak o~ polymerase activity was pooled and the
enzyme concentrated by dialysis.
The purified human breast cancer DNA-polymerase can be
utilized to develop a diagnostic assay for human breast cancer
in a number of ways. It can be used to elicit human breast can-
cer DNA-polmerase specific antibodies by injecting the purified
enæyme preferably in an emulsion of complete Freund's adjuvant
into a suitable host animal such as a rabbit, guinea pig, goat,
horse, etc. over a period of time and then bleeding the host
(usually after booster injections have been given) ko yield the
desired antisera.
Additionally, the purified enzyme can be employed as a
~` substrate for radio-iodination to yield the [125~ enzyme. A
suita~le procedure for radioiodination ~nvolves treating the
enzyme with [125~ -3(4-hydroxphenyl) propionic acid N-
~r
,
,
hydrox~succini~ide ester ~Bolton~Hunter reagentl ~ollowed by
puri~ication over a G-100 column.
The a~oresaid ant~body and labelled enzyme can be utilized
in a rad~oimmunoassay ~or human breast cancer DNA-polymerase
in~human plasma samples. Suitable radioi~nunoassay procedures
are known in the art. Thus, for example, an analogous
procedure which can be employed is described by Ritzi et al,,
Virology 75, 188 (1976). In such a procedure a buffered sample
was treated with the antisera, i.e., rabbit anti-DNA
polymerase and then after incubation for about 45 minutes at 37C
the rr25~ -labelled enzyme was added. The sample was incubated
for a further two hours at 37C and the bound radioactivity was
separated from the free by the addition o normal IgG (rabbit)
and a sufficient amount of second antibody (goat anti-rabbit
IgG) to yield optimal preciptation of the IgG (rabbit).
The concentration of D~A-polymerase in the sample can be
determined by comparing the counts of radioactivity observed in
the bound and/or free fractions to a standard curve obtained by
utilizing different known amount of DNA-polymerase in the same
assay procedure.
In an alte~nate procedure, the enzyme concentration in
plasma samples can be determined by isolating the enzyme and
measuring for enzymatic activity~ Isolation can be readily
accomplished by ammoni.um sulphate treatment o~ the plasma sample
to precipitate the enzyme ~ollowed by affinity chromatography
~he reconstituted precipitate through a column of SEPHAROSE bead
to which a synthetic template for the enzyme had been covalently
bonded. Suitable templates ~or this purpose include
polyriboadenylate (poly(rA) or poly (2'-O-methylcytidylate)
(poly(rCm)~ Elut~on o~ the enzyme from the column is
accomplished using a gradient of 0.01 M KCl to 1.0 M KCl in
* Trademark
.OlM phosphate bu~fered at pH 7.2. The D~A-polymerase activity
of the isolated enzyme can be assayed using the same assay
procedures employed in the following the purification of the
enzyme from breast cancer tissue discussed previously.
A further embodiment of the method of the present invention
relates to the discovery of a human tumor associated protein which
is of viral origin. One such protein can be demonstrated in
substantial concentration in the intercellular spaces of
human breast cancer tissue specimens and also circulating in the
plasma of breast cancer patients. Tumor associated protein appears
to be excess protein produced either by the virus after it has
infected the breast cell or by the cell itself under viral
control. It is therefore a further aspect of this invention to
assay fox the presence of this tumor associated protein in
plasma samples as a diagnostic and prognostic test for
cancer, such as, breast cancer.
The isolation of tumor associated protein from homogenized
cancer tissue can be carried out by a combination of affinity
chromatography and column chromatography. The affinity column
comprises Concavalin A* coupled to Sepharose 4B* and is an article
of commerce (Con A Sepharose). Elution of the protein from the
affinity column is accomplished using buffered O~methyl-D-manno-
side solution. Additional purification of the protein is
accomplished by DEAE cellulose chromatography. The aforesaid
procedures are directly analogous to the procedures employed by
Ri~zi et al., Virology 75, 188 (1976) for purification of gp 52
from MMTV and are descr;bed in greater detail therein.
* Trademark
--6w
The purified tumor associated protein o~ viral vrigin Gan be
utilized in the same manner as described prev~ously for DNA-poly-
merase for the development of a radioimmunoassay useful in the
detection of breast cancer. Thus, the protein can be injected
into host animals in a known manner to elicit antibodies
specific to the tumor associated protein of viral origin.
Moreover, the purified tumor associated protein can be radio-
labelled, preferably radioiodinated with 125I Bol~on-Hunter
reagent to yield the labelled protein, i.e., 125I-tumor associated
protein r used as the marker in such radioimmunoassay. The
radioimmunoassay procedure used in this aspect of the invention
is not narrowly critical and any conventional technique can be
employed. Preferably, the assay procedure employed will be
the blocking double antibody procedure used for the radioimmuno-
assay of DNA~polymerase, described previously above.
In a further aspect of the present invention, it has now
been discovered that Mason-Pfizer Monkey Virus (MPMV) specific
antibodies cross-react at reasonably high levels with both
human breast cancer DNA-polymerase and human breast cancer viral
origin tumor associated protein. It is thus possible to utilize
such antibodies and radiolabelled antigen, preferably 125I-MPMV,
in the human breast cancer radioimmunoassays of this invention.
Since MPMV can be grown in tissue culture and thus is readily
available in substantial amounts, this provides an especially
convenient source of reagents for the wide~scale application
of this invention.
Thus the present invention provides a human breast cancer
specific viral related protein essentially free ~rom other viral
and breast tissue components, said protein selected from the
group consisting of human breast cancer RNA-dependent DNA
polymerase and human breast cancer viral origin tumor associated
prote~n.
- 7 -
In one aspect the in~ention pro~ides human breast cancer
RN~-dependent DNA polymerase essentially ~ree ~r~ o~her viral
and breast tissue components. In another embodiment the inven-
tion provides 125I~human breast cancer RNA dependent DNA poly-
merase~ In another embod~ment the inVention prov;des human
breast cancer viral orig;n tumor associated prote~n essentially
free from other viral and breast tissue components. In another
embodiment the invention provides 125I-human breast cancer vira]
origin tumor associated protein. In another embodiment the
invention provides an antibody specific to human breast cancer
RNA-dependent DNA polymexase. In anotner embodiment the inven- -
tion provides an antibody specific to human breast cancer viral
origin tumor associated protein.
In yet another aspect of the invention a suitable en-
zyme is covalently bound to the desired antibody. The antibody-
enzyme complex is still enzymatically active and when placed in
contact with tissue specimens that contain the proper antigen
the antihody combines with it. A substrate of the en~yme is
then added which results in the release of a colored precipitate
at the site of activity. In a specific embodiment peroxidase
was coupled to antibodies against Mason-Pfizer viral proteins.
The appearance and distribution of the colored product enable not
only the identification of the presence of the antigens, but to
actually localize it in the malignant cells in the tissue
specimens.
Another method employing this principle involves the
quantitative estimate Oæ the same antigens in the body Eluids
(e.g., plasma), The procedure is as Eollows: 1) coat the
antibody on a sol~d sur;Eace ~e.y., polystyrene~; 2) add a known
volume o~ plasma fxom a patient to the tube and allow the antibody
coated on the surface to pick up any of the relevant antigens
present in the sample; 3~ the sample is then removed and the
~! .
3~
tube washed; 4) add the antibody~enz~me complex and incubate
for attachment; any unabsorbed enzyme-l~nked antihody i5 then
washed out; and 5) add the chromogenic su~strate which gives
either ~he color or the fluorescence which can be measured to
estimate the amount of tumor antigen present. Preferred enzymes
which can be employed lnclude alkal~ne-phosphatase and ~-
galactos~dase, both of which have excellent chromogenic substrates
Further details relating to procedures useful in the
practice of this aspect of the invention are available in the
prior art. See for example U.S. Patent Nos. 4,002,532 and
4,016,0~3.
The several assays which form the method aspects of
this invention may be utilized to detect the presence of breast
cancer in humans. To effectuate such use a statistically
significant number of blood plasma samples from clinically
established breast cancer patients, from normal patients, and~
from patients with benign or non-breast cancer tumors are
assayed by either the direct DNA-polymerase activity method,
the DNA-polymerase immunoassay, or the tumor associated
protein immunoassay. The concentration of marker protein
found in these assays is markedly elevated in the case of the
breast cancer plasma samples when compared to the levels found
for the normal and non-breast cancer tumor samples. It is thus
possible to draw an arbitrary control level line between
concentration levels of marker protein in each assay method
which corresponds to the presence of breast aancer and levels
which correspond to normal or non-breast cancer skates. An
unknown plasma sample can then be evaluated for the possibility
of breast cancer in the subject by as~aying the sample in accord-
~ccordance with one of ~ methods of the pres~lt invention and deten~ng
whether the marker protein is present in a concentrat~on in
.
~.
_ g _
excess of the control level.
Alternatively, a patient's own levels o~ marker protein
can serve as an internal control~ Thus a patient with a
confirmed breast cancer can be assayed before and after the
initiat~on of therapy. A marked drop in the level of the marker
protein would be ;ndicative o~ a ~avorable prognosis of the kreat-
ment. A subsequent substantial ~ncrease in the marker protein
concentration levels would be ~ndicative of a possible
recurrence or metastases of the disease and would allow the
attending physician to initiate therapy at an early time.
Moreover, the effectiveness of such therapy could be monitored
by the instant method.
The present invention is furthex illustrated by reference
to the Examples which follow.
Example 1
Subcellular Fractionation of Breast Tumor Tissue
Depending of the amounts of material available, between
9 and 30 g of tumor were thawed, minced, suspended in four volumes
of cold 5% sucrose (w/v)-TNE (O.OlM Tris-HCl, pH 8.0, 0.15 M
NaCl, 3mM EDTA) and blended in a Silverson homogenizer. The
homogenate was centrifuged at 4000 X g and then 10,000 X g
to remove nuclei and mitochondria, respectively. Trypsin was
added to the post-mitoahondrial supernatent to a final
concentration of 0.5 mg/ml 7 AEter incubation at 20 for 10 min,
proteolytic activity was inhibited by the addition o~ two poly-
peptides, lima bean trypsin inhibitor (one-fold excess) and
Trasylol*(100 KIU/ml), The sample was layered over discontinuous
sucrose gradients composed o 6 ml of 50~ sUcrose -TNE and 8 ml
of 25% sucrose-TNE. Following centrifugat~on at 25,000 rpm for
90 min at 4~ in a Spinco S~-27* rotor, material ~t the 25/50
*Trademark - 10 ~
interface was collected, diluted with TNE, and la~ered over
line~r 20-50% sucrose-TNE ~rad~ents~ The samples were centr~
fuged as above for 16 hr and the di~ferent density regions
collected. The density region ~1.16-1.19 g~cc) in which
RNA tumor viruses localtze was pooled, diluted,and centrifuged
as above for 90 min. The result~ng pellets were resuspended in
approximately 0.6 ml of 0Ol M Tris-HCl, pH 8Ø
Six lots of tumors were processed for enzyme in the manner
described. Four of these ~A, B, C, and D) yielded enough
enzyme to characterize. One preparation (A), a metastatic
liver tumor,c~me from a single patient, all the others being
pooled material from a number of different individuals.
Pol~acrylamide Agarose Gel Filtration
_
The resuspended pellet was solubilized and disrupted at
0 for 15 min by the addition of KCl (to 0.4M), DTT ~dithiothreitol,
to 0.0I M), and a non-ionic detergent such as Triton X-100 ~to
0.6%~. The sample, approximately 0.9 ml, was applied to a 0.9 X
50 cm column of polyacrylamide agarose gel (Ultrogel*AcA44)
equilibrated with 0.3 M potassium phosphate, pH 8.0, in buffer A
(2 mM DTT, 1 mM EDTA, 0.02% Triton X-100, and 10% glycerol).
Elution was with 0.3 M phosphate~ufer A at a flow rate of about
2 ml/hr. Fractions (0~5 ml) were assayed for DNA polymerase and
terminal transferase activity as described below.
Phosphocellulose Chromatography
The peak fractions from the Ultrogeloolumn were pooled
(3 ml) and Trasylol was added to a concentration of 100 KIU/ml.
The sample was dialyzed against 0.01 M potassium phosphate,
. pH 7.2, in bu~er A until the phosphate concentration was less
than 0.02 M and then was loaded onto a 0.9 X 10 cm phosphocel-
lulose column ~Whatman* P-ll) e~uilibrated with the same buffer.
.^0
*Tra~rk
q~he colum, was washed with 30 ml o~ the O.Ol M phosphate
buffer and the enzyme activi~y was eluted with a 120 ml linear
gradient of O~Ol M to U.5 M pot~ssium phosphate buf~er A,
pH 7.2, at a flow rate of l4~ml/hr. Fractions (1.2ml)
~ere assayed for both DN~ pol~merase and terminal transferase
activities, The ma~n peak of polymerase activity was pooled
and Trasylol was added to lO0 KIU~ml, This enzyme ~raction
(called PC enzyme) was concentrated by dialysis at
0~ ag~inst an osmotically active, high molecular weight
synthetic polymer such as ~quacide*ll-A,
Glycerol Gradient Centrifugation
For estimation of molecular weight, the concentrated
PC enzyme was diluted three-fold with O.l M potassium phosphate,
pH 8.0, and layered on a linear 10-30% glycerol gradient
containing Ool M potassium phosphate, pH 8.0, 2 mM DTT, and
0.02% Triton X-lO0. Centrifugation was at 48,000 rpm fox 12 hr
at 1 in a Spinco SW-50.l rotor. Fractions were collected from
the bottom and assayed for reverse transcriptase activity with
oligo(dG)~poly(,rC) as template. Bovine serum albumin served
as a sedimentation marker in a parallel gradient.
' DNA PoIymerase Assays
Assay mixtures for polymerase activity with synthetic
polymer templates contained in ~lO0 ~ 5 ~ mol Tr~s-HCl,
p~ 8.0, O.S ~mol MgCl2l O~l ~mol DTT, and the following
combinations of polymer and dNTP~-04. ~g oligo(dG):
poly~rC) or oligo(dG):poly(rCm), 0.02~mol dCTP and l.0 nmol
[ H] dGTP (4000 cpm/pmoll; 0.4~g oligo(dT):po1y(rA) or
oligo(dT~:pol~(,dAl~ 0.02~ mol dATP and l.0 mmol [3H~ dTTP
(,4000 cpm~pmoll. In reactions w~th oligo(dG):poly(rCm),
MnC12 (0'~02 ~moll replaced MgC12.
*Trademark - l2
88
A5sa~s emplo~in~ AMY RNA cont~ined (.in 100 ~ 5 pmol
Tris-HClr pH 8~0~ 0~8 ~ol MgC12, U,l ~mol DTT~ lQ/~y actinomycin
D (Sigma co~p.), 5 ~g distamycin ~ ~C~lb~ochem) 2 ~g AMV 70S'
RN~, 0.1 ~g oligo (dT~ 12 1~' O.li~mol each o~ dATP,
dGTP and dTTP, and 5 nmol [3H] d~TP ~1.5 X 1~4 cpm~pmol).
All reactions were incubated at 36v for 15-30 min and were
terminated by the addition of 0~5 ml cold 0.067 M sodium
pyrophosphate - 1 M sodium phosphate, pH 7.2, followed by 0.5 ml
cold 80~ TCA. Acid-insoluble radio activity was collected on
membrane filters and measured in a scintillation counter.
Terminal deoxynucleotidyl transferase activity was measured
by the polymerization of [3H] dGTP in the absence of a complemen-
tary polymer template. Reactions were carried out as described
above except that polymer dNTP combination was replaced with
0.4 ~g oligo(dG)10 18 plus 0.02 ~mol dC~P and 1.0 nmol [3H] dGTP.
,
All synthetic oligo- and polynucleotides tritiated dGTP,
dTTP, and dCTP were articles of commerce. AMV 70S RNA was
isolated from the purified virus as described by Marcus et al.
Virology 71, 242 (1976).
Hybridization Re_ctions
Procedures for the hybridization reac~ion~ and their analysis
with S nuclease have been reported by Weiss et al. supra.
Conditions for Cs2SO4 equilibrium density centrifugation as
disclosed by Axel et al., Natuxe 235, 32 (1972) were modified
by the addition of 0.02~ sodium N-lauroyl sarcosinate and ~0 ~g
each of E. coli DNA and RNA to the gradients.
esults
The data described below are based on independent enzyme
. ~ .
- 13 -
isol~tions ~Xom ~ouX dif~e~en~ tu,m,or collecti.on~, ~abeled A
through D~.~ The beh.~ior du~ng ~xactionati.on and the properties
of the breast tumor pol~m,e~se di.d not vary sign~ficantly
from one prepaxation to another~
Polymerase preparation A was ~solated ~rom a metastatic
les~on in the liver of a patient with breast cancer. Nine grams
of tumox were homogenized and the particulate material, bending
at a density of 1.16-1.19 g/ml, was collected as
describe~above. The recovered pellet was solubilized by the
addition of Triton X-100 and then fractionated through a
polyacrylamide~agarose gel ~Ultrogen ACA-443 column. Each
column ~raction was assayed for 1) DNA polyermase with oligo
(dG)12 18poly(rC) as a template and 2) for terminal transferase,
using oligo(.dG)12-18 as a primer. Three peaks (two major and
one minor) of DNA polymerase activity were observed and it was
evident that the first major peak also contained terminal
transferase. The two major peaks were found (Table 1) to contain
90~ of the applied polymerase activity and 3~ of the protein
as measured by the fluorescamine procedure of Udenfriend et al.,
Science 178, 871 (1972).
38
P~ ~ ~ X ~C
~ R~ ~ ~D 00
u~ m
~1~ ~1 h
a) ~ 0~ oo w S:~
~ E~ ' _l ~1 a) ~ ~
~ O L ~ I ~ P
_.
o` . Y 2
," U~
~ ~ P ,~0
~ ~ _i , h o O h ~ N
~ I I i N
, ` ~:' '
:`:
X - 15 ~
.` .
To examine the reality of the separation of the pol~m~se activities
observed abwe, the two major pe~ks were pooled, dialy ~ to reduce the phosphate
buffer concentration, and then chromatographed through a phosphocellulose column(PC) with a linear (0.01 M to 0.5 M) phosphate gradient. me polymerase
activities were seen to again resolve into two major peaks one
eluting at 0.08 M phosphate and the other at 0.18 M. It will be
noted that again the second major polymerase peak i5 devoid
of terminal transferase activity. The latter splits into two
peaks, one associated with the first DNA polymer~se activity
at 0.08 M phosphate, and another eluting by itself at 0.23 M
phosphate.
The second (O.l~M phosphate~ peak of polymerase activity
observed on the PC column is not found in normal tissues (3
samples of breast tissue and 3 samples from spleens) or in `'
benign ~ibroadenomas of the breast (3 pools o~ 3-4 fibn~e~
each). and is thus the DNA polymerase unique to breast cancer
tissueO The fractions composing peak ~ of the PC column are
found to contain 65% of the applied DNA polymerase activity
and 8% of the protein. These fractions are pooled and
concentrated as described above to yield the breast tumor
polymerase. Table 1 summarizes the yields of activity and
protein at each of the three steps in a typical purification.
Example 2
Evidence that the Breast Cancer Polymeras~ is a Reverse
: Transcri~tase
There are several useul criteria which distinguish the
reverse transcriptases of the RNA tumor viruses from normal
`. mammalian DNA polymerases. The viral reverse transcriptases
show a pre~erence for oligo~dT):poly~rA) over oligo(d~
` poly(dA) and they al~o accept oligo~dG):poly(rC) and oliyo~dG):
poly(rCm) as excellent templates or the synthesis of poly(dG~.
Another, and more diagno~tic characterlstic, is the ability
- 16
,
..
'il~8~38
o~ a reVerse t~nscriptase to use ~ heteropolymeriC RNA to
direct the synthesis of a faithful co~ple~entary DNA as
demonstrated by proper back-hybridization of the cDNA to the
template used in the synthesis.
The responses of four of the breast cancer polymerases
to the synthetic polyribonucleoti~es are summariæed in Table 2.
The results show a pattern of activities completely consistent
with that obtained with reverse transcriptases isolated from
authentic animal RNA tumor viruses. Thus, ln all cases, oligo
(dT~:poly(rA~ is superior to oliyo(dT~:poly~dA) for the synthesis
of poly(dT). Further, both oligo~dG):poly~rC~ and oligotdG):
poly(rCn) were excellent templates for the formation of
poly~dG). It should be noted that in addition to the four
breast tumor enzyme preparations descri~ed here, many others
: (more than 50) obtained from additional patients in an ongoing
effort have been examined in the same way at different stages of
purity using varlous methods of fractionation and they all
exhibited the response pattern described in Table 2,
,,~
: - 17 ~
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.
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o ~ o o o z; z 8
~ r'1
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,1 ~ ~ ~ o~ . . U
S-J ~1 N O --I E-l E~ a) ~
Q ~ ~. o o o Z Z 0 ~
~ ~8
o ~ ~ ~ al ~
~ ~ ~ ~ o
0 ~ . u~ I d O CO ~ ~
o ~ ~o
~r o ~r er O ~ a~ O
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~ ~ o . o o ;7 o U ~ ~ ~
1~ .rJ o ~ E'
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.` ~ 000~
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~r~ N ~r ~ ~`1 O~ 0 (I)
r er r~) o,~
0 ~1 ~ o u~ u) o td~
~ ' ~ O ' O ~ OO
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~n ~ o~: o
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~1 O O O O ~ O
.~ Q~ a) t~~ ~ ~ ~ ul O ~n
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, ' .
The operational definition of a reverse t~anscriptase
requIres the demonstxation that it can use a heteropolymeric
RNA to make a DN~ transcript. lhe response ~Table 3)
o~ the breast cancer polymerase to the RNA of the avian myelo-
blastosis virus (AMV) is that expected ~rom the synthesis of a
heteropolymer~c DNA. Leaving out any one, or all of the
required three unlabeled deoxyribosidetrisphosphates leads to
the same virtual disappearance of synthetic activity as occurs
on omission of the RNA template.
' ' : . . . ' '
J,J O r-~ N Ll~ D ~: ~
. ~ I rl r-l ~J ~
~1 . p:~ ~1 0 0 ~ O O ~ 151 ~>
. o o c~ o c~ o a~
. ~ .N
~'5~ ~
~ ~ ~
O O ~I N _I O ~ C
0 ~3 ~¢ N O O O O O ~ ~ 5'1
~ Q~ ~ ~ O O O O O ~ a
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a ~
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.;
The most telling test of a putatiVe reVerse transcriptase
reaction comes from an exam~nat~on of the f~delity of the DNA
transcript, This requires ~solation of the I 3H3 DNA product
and challenging it in anneal;ng react~on with the ~NA template
used ~n the synthesis. To thiS end, a 2-ml reaction was run
for 15 min with the DNA polymerase preparation A and AMV-RNA
as the template, leading to the synthesis of approx;matelY
3 ng ~3~ DNA at ~ X 107 cpm~g. The [3~] DNA product was
purified and recovered by the usual procedures and then annealed
with AMV and RLV 70S RNAs. The outcome was examined ~y
separation in Cs2SO4 gradients and by resistance to Sl nuclease.
Both methods yielded less than 5~ annealing to the unrelaked
RLV-RNA and between 80 and 85% hydribidzation to AMV-RNA, the
template used to direct the synthesis. These results suggest
therefore that the [ ~] DNA is a singLe-stranded complement
of the AMV-RNA. A more informative examination of the [3H]
DNA product is provided by a kinetic examination of the annealing
reaction. A comparison was made of the annealing kinetics to
AMV-RNA of two [3H] DNA products, one synthesized under the
2~ direction of AMV-RNA by AMV reverse transcriptase and the other
synthesized by the human breast cancer polymerase instructed
by the same template. It was found that the kinetics of
annealing to AMV-RNA of the two DNA products are indistinguish-
able. Thus, the human enzyme is every bit as efficient in
reverse transcribing AMV-RNA as is the homologous reverse
transcriptase purified rom the avian virus.
O ~39~93~ the Bxeast Cancer Pol~merase
~ariations in t~rature and pH were ~ned for their effects
on ~he acti~ities of several preparations o~ the breast cancer polymerase
using oligo~dG):poly(rCJ and oligo~dT):poly(rA) as templates.
The max~mal rate of polymerization occurred at 37 ;n 5 mM
~gCl2 over a pH range of 7.~ to 8.5.
X
The divalent ion requirements ~ reverse tr~nscriptases are
of some interest since they ser~e,~o ~ de the,v~ruses o~
origin ~nto different groups. Thus~ all six stra~ns of MMTV
from a variety of sources contain a reverse transcriptase show-
ing a strong preference for Mg~+ as compared with Mn . The
same holds true ~or the Mason~P~izer monkey virus, the bromo-
deoxyurid~ne-induced guinea pig v~rus and the bovine leukemia
virus, In contrast, the reverse transcriptases of the murine
leukem~a and sarcoma viruses function much more effectively in
the presence of Mn~. For the human breast cancer enzyme,
Mn at its optimum yields only about one seventh of the
activity attainable with Mg~+. It is clear that as between
the murine mammary tumor and leukemia viruses, the human breast
cancer enzyme shows a divalent ion requirement most closely
resembling the mammary tumor virus reverse transcriptases.
The molecular size of the breast cancer enzyme was
estimated by sedimentation through a linear (10-30%) glycerol
gradient. Ths enzyme was located by assaying fractions
with ~MV-RNA as template. ~l~he enzyme activity sediments
between 5S and 6S, slightly faster than the bovine serum
albumin marker, placing the molecular weight at around 70,000
daltons. This result is also consistent with the relative
elut~on positions of these same proteins on ultrogel. A
number of en~yme preparations from different breast tumor
sources yielded identical sedimentation values.
'~
Materials and Methods
Viruses Mason-Pfizer monkey virus was propagated in a
__
suspension culture of the noxmal human lymphocytic cell line
NC-37 and concentrated as previously descrlbed by Schlom and
Spiegelm~nf Proc. Nat. Acad. Sci. U.S.A. 68, 1613 (1971). The
; virus was further puri~ied by centri~ugation through an 8-ml
column of 20% glyercol in TN~ buf~er (0.01 M Tris-HCl, pH 8.3,
, ~2 -
0.15 M N~Cl, 0~002 EDTA~ onto ,a, pad o~ 100~ ylyercol at 98,000
X ~ for 60 min at 4, The viral pellet was taken up ~n TN~
buffer and spun to equil~brium ~n a continuous 20~50~ sucrose
gradient ~n TNE at 98,000 X g for 16 hr. The particles banding
between densIt~es of 1.14-1.19 g/ml were collected, diluted, and
centr~fuged at 98,000 X g ~or 45 min at 4. The pellet was
used ~mmediately for DNA polymerase purification.
Avian myeloblastos~s virus ~AMV, BAI straln-A), simian
sarcoma virus stra~n-l (SSV-l), Friend leukemia virus (FLV),
feline leukemia virus (FeLV), and Rauscher leukemia virus ~RLV)
were also used in this Example. All viral concentrates were
purified as described a~ove.
Preparation of RNA-lnstructed DNA Polymerase from Human
Mali~nant Breast' Tumor ,
Reverse transcriptase from human malignant breast tumor
was prepared as previously described by Ohno et al., Proc.
Natl. Acad. Sci. U.S.A. 74, 764 (1977) from particles purified
by isopycnic separation. After disruption by incubation in
0.2% Triton X~100 for 15 min at 0, some of the samples were
analyzed for endogenous polymerase activity as well as oligo(dG):
poly(rC) and oligo(dT):poly(rA) directed synthesis of poly(dG)
and poly (rA), respectively. The disrupted virus density regions
chromatographed on a polyacrylamide agarose column (Ultrogel
AcA44, LKB,Co.) and the eluted enzyme peak loaded on a phos-
phocellulose (Whatman pll) column. The enzyme was eluted with
0,01 to 0.5 M potassium phosphate gradient and concentrated as
described by Ohno et al. supra.
' Preparatio'n o~ ~iral Re'gions ~om Human Leukem~a and Hodgkin's
''S'pl'e'en
Spleens from patients with human chronic lymphocytic
leukemia (CLL~ r chron~c myelogenous leukemia (CML), and
Hodgkin~s lymphoma were use~ as the sources o~ viral density
region preparations, and the polymerase act~ties were
analyzed by endogenous k~netics as described by Witkins et al.,
- 23 ~
.
,
:
Proc. Natl. Acad. Sci. U~s.A~ 72, 4133 ~lg75).
Preparation of MPMV Polymerase
The MPMV pellet prepared as described above ~viruses) was
resuspended onto 0.05 M Tris-HCl, pH 9.2, 0.001 M EDTA and Z
M KCl, son~cated, and then centrifuged at Y8,000 X g for l20
min. The pellet was used to prepare purified DN~ polymerase by
column chromatography as described previous1y by Witkins et al~,
supra.
Preparation of Antiserum
Antiserum against MPMV-DNA was induced in New Zealand white
rabbits. Three cycles of immunization were required to achieve
the desired titer of anti-polymerase IgG. In each cycle the
enæyme ~l X 103 pmoles of TMP incorporated per min) was
emulsified with an equal ~ol of Freund's adjuvant and injected
into ~he two hind footpads. This was followed by two additional
similar inoculations given at two-week intervals in the same
sites.
Sera were fractionated by chromatography on a Sephadex*
G-200, O.l M Tris-HCl, pH 8.0~ Rabbit gamma glohulins were
identified serologically by immuno-diffusion with goat anti-
rabblt IgG antiserum. The relevant fractions were concentrated
by ammonium sulfate precipitation (50~ saturation) and dialyzed
against O.l M Tris-HCl, pH 8O0~ The protein concentration of
the ~gG fraction was measured by the Lowry procedure.
* Trademark
.
. .
f~
DN~ Polymerase Assays
Assay mixtures for polymerase act~ity ~7ith synthet~c polymer
templates contained (in 100 ~1~: 5 ~mol Tris-HCl, pH 8.0, 0,5 ~mol
MgC12, 0.1 ~mol DTT, and the ~ollowing com~lnations of polymer
and dNTPs: 0.4 yg oligo~dGl:poly(rC~ or ol~go(dG~: poly(rCm),
0702 ~mol dCTP and 1.0 nmol [3H] dGTP ~4000 cpm/pmol), V.4 ,ug
oligo(dT~:poly(rA] or ol~go(dTI:poly(dA), 0.02 ,umol dATP and 1.0
nmol [ H] dTTP (4000 cpm~pmol), In reaction with oligo(dG):poly
(rCm), MnC12 (0.02 ~mol) replaced MgC12.
Assays using endogenous RNA contained (in 100 jul): 5 ~mol
Tris-HCl, pH 8.0, O.8 ~mol MgCl~, 0.1 ,umol DTT, 10 ,ug actinomycin
D 5 ug distamycin A, 0.1 ~g oligo (dT)12-18, 0.1 ,umol
dATP, dGTP, and dTTP, and 5 nmol [3H] dCTP ~1.5 X 104 cpm/pmol).
All reactions were incubated at 36 for 15-30 min as indi-
cated, and were terminated by the addition of 0.5 ml cold
0.067 M sodium pyrophosphate, 1 M sodium phosphate, pH 7.2,
follow~d by 0.5 m. cold 80~ TCA. Acid-insoluble radioactivity
was collected on membrane filters and measured in a scintillation
counter.
Terminal deoxynucleotide transferase activity was measured
by the polymerization o~ 13H] dGTP in the absence of a complemen-
tary polymer template, the latter being replaced by 0.4 ~g oligo
( G)10-18- ....
The Effect of'Antibody on DNA Polymerase'Activities
Reaction mixtures ~or the neutralization of DNA polymerase
activity (total vol 55 ~1) contained in addition to 25 ~g of
bovine serum albumin (BSA) and DNA polymerase, the indicated
amount 125-150 ,ug) of purified IgG fraction. The buffer used
was 0.01 M Tris-HCl, pH 8.0, 0.15 M of potassium chloride.
After 15 min incubation at 4, a polymerase assay was carried
out uslng oligo(dG):poly(rC) as described above. In certain
instances, the e~fect of antibody on the activlty by the
endogenous RNA was examined.
- 2s
,:: .
Detection of ~nti~ody-enz e ~omple~es; ~n Glycerol Gradients
The concentrated enzyme (reverse transcriptase ~r~m MP~V
or from human malignant breast tumors~ fractions were diluted
~hree-flow with 0~1 M potassium phosphate, pH 8.0~ contain~ng
0.5 mg~ml of bov~ne serum album~n, and ~he indica~ed amounts
of puri~ied Ig~ fractions were added. After incubation for 15
min at 4, the samples were layered over a 1~-30% glyercol gradi-
ent adjusted to 0.1 M potassium phosphate, pH 8.0, .OOZ M
d~thiothreitol and 0.02% Triton X-lO0. ~l~he samples were
sedimented at 48,000 rpm for lZ hr in a Spinco S~50-l rotor at
1. Fractions were collected dropwise from the bottom of the
tubes and the enzyme act~vity assayed as described above.
''Results
A ComParison of the Effects o~ Anti-MPMV DNA Polymerase IgG on a
Var'iety o'f~~Pol'ymer'ases
e effects of the anti-MPMV ~NA polymerase on a nu~r of viral DNA
poi~nerases and on the corresponding enzyme of the human breast
cancer particles were compared. In these experiments, isopycnical-
"
ly banded particles, purified as described in Materials andMethods, were employed as a source of the DNA polymerase and
oligo(dG):poly(rC) was used as the template. lhe antibody
inhi~its the MPMV-DNA polymerase more than ~0% and ach~eves a
26% inhibition of the DNA polymerase associated with the human
breast cancer particles. In contrast, no detectable effect is
observed on the DNA polymeraces of any of the other animal
oncornaviruses, including avian myeloblastosis virus (AMV~,
~auscher and Friend murine leukemia viruses ~RLV and FLV),
feline leukemia virus (FeLV), a simlan sarcoma virus ~SSV-l), or
murine mammary tumor virus ~MTV).
- Z6
':
'Speci~i_it~ o~:the'Tnh~ on by ~the'Anti-MPMV Pclymerase IgG
~ he next issue exam~ned centered on whethex the lnhibition
obser-ved w~th the breast cancer particle enzyme was confined to
this malignancy. As already known in the art, spleens from
pat~ents with mesenchymal cancers constitute a convenient
source of particle enzyme, and these were chosen for immunologic
comparison. The particle fractions were prepared and the
endogenous polymerase activities were assayed as previously
described for human breast tumors and for spleens involved
in mesenchymal neoplasias. At least five instances of each
kind of neoplastic tissue were examined and typical results
are sho~m in Table 1.
,, ~7 _
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a~ ~ ~ ~ ~ ~ cO ~ D aJ ~ 3 ~
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h ~ ~ O
U~
O
U~ ~ ~1 ~ a 1` O ~ u~
Ll H dP --i t~ t'~l O 11~ O I h ::1
a~ ~ ~ ~ o a~ ~ ta o ~
h ;~ . p, ~ ~
~ a~ ~ ~
O ::1 ~ri ~ U~ D r~l 00 .
~ ~ O V t` H N 00 ID O )-I ~ 0
O 0 J~ 3~ 0
g ~~ ~P OD ~1 ~1~ U') , 0 0 D~
~ ~ ~ ~ O O~D In O ~
H Ei--I ~ a~ a~ O O al I¢ ~ a) 3
(1) ~ .~ -I -I ~ 0~ ~_
~o 0 ~ ~ co ~ oo o o Ln o r 0 ~
0 ~i ~ ~4 ~ N r ~ O Lt~
S~:/ ,.F~ O V r~ ~r ~ ,J, ~ N ~J rl ~3 tJ~
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~o 0~ 3 ~ ~ ~
P U~ ~1 F~ F~ 0 0 (U
' ~ W ~ ~) ~ o ~ 00
H O ~1 N 0 ~ O H _ 0 4
I ~rl ~ E u7 c~ ~C
~r~ ~~1 t~ ~;4 a~ ~D ~ O In N a ~ X~rl o
O ~ o.
~ ~5~ o . Ln
4ol~ozO~ . U,Q V 0'~
~, x x V C~ ~ a a
a~ ~ ~ ~ a~
E~ ~ ~ ~
S~ U O C.1 Q~ q V h Q
~1 O U~ O ~ ~S 0
a~a
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_I ~1 ~ E ~
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E~ ~ m ~ aH E~ ~:
-- 28 --
. : : ,
':' . ~ . .,
$ignificant ~nhibitions are not seen with the particle en-
z~mes derived from the leukem~c and the lymphoma spleens.
Howe~er, the breast cancer particulate enzymes were inhibited
from 59~ to over 95%. Note that these endogenous reac~ions are
more severely affected by the anti-MPMV polymerase IgG than
the synthesis directed by synthetic templates. As expected,
all the DNA polymerase activities described in Table 1 are
sensitive to ~Nase and resistant to the presence of actinornycin
D (100 ~g/ml~ and distamycin t50 ~y/ml), features characteristic
of RNA-directed DNA polymerase,
The data shown in Table 1 were obtained with the endogenous -
reactions of detergent-disrupted particles isolated from the
indicated neoplastic tissues. For completeness a similar
comparison was carried out using the corresponding purified
enzymes directed by oligo(dG):poly(rC). ~xperiments using
; this approach provided the responses to anti-MPMV polymerase
IgG of the reverse transcriptases purified from the particles
prepared from breast cancers and ~rom a chronic myelogenous
leukemic spleen. Over the whole concentration range of IgG
examined, the leukemic reverse transcriptase is not significantly
affected. In contrast, the anti-MPMV polymerase IgG does
suppress the activity of the hreast cancer reverse transcriptase
at all concentrations tested, achieving a 37~ inhibition at
150 ~g per reaction mixture.
,~ .
Table 2 examines another aspect of the speci~icity of the
interaction by testing the responses o~ normal cellular DNA
polymerases to the anti-MPM~ polymerase IgG,
~ - 29 -
38
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::~ ~ . . . . ~ ~ a
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O H O er o O O z~0
~1 .
~ ~n O u~ ~
,1 0 -d ~1 0
C) ~ dP O O U
.~ ~ '
~0 ~,î
~4 O ~ U~
;C
_l ~
E3 ~ ~ _~ ~ ~ ~ k
~: ~ ~ ~ ~ Q 0 d o
I ~ ~ ~D ~1 0 00 ~rl 0 0 1`
~) ~ ~a ~ ~1 ~ 0 r~
U~ ~ U) U ~ 0
0 ~ 1` 0 a~ ~
Q ~_, ~Y01Ho~d
~ .
11~ . ~ O
S l ~ tl~ ~ h
0 H,f:: O
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a) H C:1 ~ 00 ~D ul a~ a) ,q 0
U ~ Ql N ~ ~ S l ~ ~3:
~I t~) U D ~1 00 D a) ta -
O ~ ~ --~ N _I _1 3 ~I f:C o
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4~ ~ ~ ~) O ~
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0 0 0 0 ,o ~ h
4 1 ~ ~ h $1 h ~1 ~
¦~¦ EO~ ~ ~ o ~
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,_~ a)~ ~~ al :~ o
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: Q) ~ ~ ~ p,, . E~
a~ v :~ u~
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,
-- 3 0
. . .. . .
.`, . . `
Neither preparation o~ DNA polymerase y, whether isolated
from a breast tumor or from the HeLa cell strain, was
detectably ~nhibited, and the same was true for DNA polymerase
~. At the same level the anti-MPMV polymerase IgG suppressed
the breast cancer reverse transcriptase by 40%. Similar
: experiments were carried out with the normal DNA polymerase a
and again have found no evidence of inhibition by the anti-
MPMV polymerase IgG.
The Demonstration by Sedimentation of Complexes between the
Breast Cancer DNA Polymerase and the Anti-Polymerase IgG
It was desirable to see whether ~urther evidence could be
provided for the existence of physical complexes between the
breast cancer reverse transcriptase and the anti-MPMV
polymerase IgG. Fixst, such evidence would add direct support
to the conclusions derived from simple suppression of enzyme
activity. Second, experiments along these lines could
identi~y the basis underlying the apparent inability of the
anti-MPMV DNA polymerase IgG to achieve total neutralization
of the breast cancer reverse transcriptase activity. Basically,
two mechanisms can be offered to explain the incompleteness
o~ the inhibition. One would suggest that the enzyme prepara-
tion is heterogeneous and that only a sub-population forms
inactive complexes with the added IgG. The other would
assume that the population of enzyme molecules is homogenous
in this respect and that all form complexes, which can,
-; howevex, express a fraction of the original activity. ~l~hese
two possibilities are readily distinguishable by a sedimentation
analysis of the enzyme activity before and after reaction
with the relevant IgG.
To monitor the e~fectiveness o~ this approach, a positive
control experiment was carried out with the homologous system
consisting of MPMV-DNA polymerase and its antibody. A negative
X
- 31 -
control w~s incLuded uslny normal (pre-immunized) IgG from the
same rabbit~ A 250~ reaction containing enz,yme and 150 ,uy
of the indicated IgG were incubated at 4 for 15 min as
described in Materials and Methods. The mixture is then layered
on a 10 to 30 96 gradient and centrifuged at 48,000 rpm for 12 hr.
-at 1. Fractions are then collected from the bottom and
assayed for reverse transcriptase and ~or the presence of IgG
by immunodiffusion. Incubation with normal IgG was found
not to change the position (tube 131 of the peak o~ MPMV reverse
transcriptase activity with respect to the external marker
bovine serum albumin (BSA). ~l~he enzyme still sediments at a
velocity corresponding to a molecular weight of 70,000 daLtons.
In this same gradient, the IgG was located in tubes 10, 11, and
12. Incubation of the polymerase with 150 jug of anti-MPMV
polymerase results in an 80% loss of enzyme activity and a
markedly different sedimentation pattern of the residual
activity~ No enzyme is detectable at the original position
close to BSA, all of it appearing as complexes sedimenting
faster than ~ree enzyme or IgG. The IgG is now detected by
immunodiffusion in fractions 13, 14, and 15 as well as in
fractions 6 through 10, which encompass the peak of
polymexase activity.
A very similar situation is obtained in the experiments
with the reverse transcriptase purified from human breast
cancer particles. Incubation with normal IgG leaves the
human enzyme in its usual position (tube 15) within one tube
of the BSA marker, and the IgG is found by i~unod,iffusion
in tubes 12 to 14. However, reaction wi-th anti-MP~V
polymerase IgG results in a 45% loss of ackivity an~ shifks
the' residue down the tube as ~'ast-moving complexes found in
fractions 6 through 10 r in which IgG can also be detected
by immunodifusion. IgG is also found in its original posi-
tion (fractions 12 through 14).
- 3~ -
I
It is evident ~rom the results described above that neither
the MPMV reverse transcriptase nor the one isolated ~rom human
breast cancer particles conta;ns a significant proportion of
molecules unable to complex with ant~-MPMV polymerase IgG.
The fact that the enzyme IgG complexes can express some activity
is not a new phenomenon. Indeed, in some reporte~ instances,
such complexes are fully active.
Discussion
The experiments described here show that anti-MP~ DNA
IgG, when present in excess, can completely complex with and
partially inhibit the reverse transcriptase isolated from
human breast cancer particles. The specificity of the
inhibition is supported by the inability of this same antibody
to affect the activities of normal cellular DNA polymerases
or of a variety of reverse transcriptases from animal
oncornaviruses (e.g., AMV, RLV, FLV, FeLV, SSV-l, and MM~rV~.
Further, normal IgG obtained from the same rabbit prior to
immunization does not complex with, or inhibit, either the
MPMV or the human breast cancer reverse transcriptase.
Aside from its etiologic interest, the immunologic
cross-reactivity between the reverse transcriptases of
MPMV and of the human breast cancer partlcles has an
implication of more immediate import. It provides the basis
for examining human breast cancer by procedures of clinical
usefulness. MPMV can ~e produced in tissue culture in yields
adequate for purification of its enzyme and other protein
components. These can in turn be used to generate antisera
for use as specific detecting reayents in immunofluorescent
and ~mmunoperoxidase staining of frozen sections as diagnostic
aids for surgical pathologists. Of further interest is the
use of such development in immunoassays for the system;~c
- 33 -
detect~on o~ immunologXcall~ relate~ pxotein in the plasma
and other body flu;ds o~ pat~ents with cancers, okher than
breast cancer such as lung cancer, stomach, rectal and colon
cancers, ovarian cancer, brain cancer, bone cancer, cancer of
the lymph glands, skin cancer (squamous cell and basal cell
carcinomas and melanoma) and the ltke~ Each of these cancers
has its own distinctive particle associated protein.
34 -
