U.S. patent number 3,755,086 [Application Number 05/114,059] was granted by the patent office on 1973-08-28 for diagnostic method utilizing synthetic deoxyrilionucleotide oligomer template.
This patent grant is currently assigned to Hoffmann-LaRoche, Inc.. Invention is credited to Edgar Philip Heimer.
United States Patent |
3,755,086 |
Heimer |
August 28, 1973 |
DIAGNOSTIC METHOD UTILIZING SYNTHETIC DEOXYRILIONUCLEOTIDE OLIGOMER
TEMPLATE
Abstract
A diagnostic method for the detection of virus-related
neoplastic disease states is described. This method involves
employing synthetic nucleotide oligomers hybridized with RNA-type
polymers as a template for assaying RNA-dependent DNA polymerase
activity. RNA-dependent DNA polymerase activity has been found to
be specifically characteristic of several neoplastic disease states
including human leukemia. In a preferred embodiment the instant
method employs synthetic thymidylic acid oligomers (d-pT)
hybridized with polymeric ribonucleotide rA.
Inventors: |
Heimer; Edgar Philip (Cedar
Grove, NJ) |
Assignee: |
Hoffmann-LaRoche, Inc. (Nutley,
NJ)
|
Family
ID: |
22353164 |
Appl.
No.: |
05/114,059 |
Filed: |
February 9, 1971 |
Current U.S.
Class: |
435/6.1;
435/194 |
Current CPC
Class: |
C12Q
1/68 (20130101); C12Q 1/48 (20130101); C12Q
1/68 (20130101); C12Q 2521/107 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12Q 1/48 (20060101); G01n
031/14 () |
Field of
Search: |
;195/28N,13.5R |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
3597318 |
August 1971 |
Sutherland et al. |
|
Other References
Hackh's Chemical Dictionary, 3rd Ed. (1944) p. 672 .
Spiegelman et al., "Nature" 228: 430-432 (Oct. 31, 1970) .
Gallo et al., "Nature" 228: 927-929 (Dec. 5, 1970).
|
Primary Examiner: Tanenholtz; Alvin E.
Assistant Examiner: Hensley; Max D.
Claims
I claim:
1. A method for detecting RNA-dependent DNA polymerase activity in
a purified mammalian or avian cellular extract or plasma sample
which method comprises adding said purified sample to a DNA
polymerase assay reaction mixture, said reaction mixture being
particularly distinguished in containing a template consisting of a
synthetic deoxyribonucleotide oligomer containing 2 to 24
nucleotide units per oligomer molecule hybridized with a RNA-type
polymer selected from the group consisting of rA, rU, rG, rC and
rI, and deoxyribonucleoside triphosphates complementary to said
RNA-type polymer, at least one of which is isotopically labeled;
incubating said mixture whereby polymeric product is formed under
the direction of said template; terminating reaction in said
mixture; and measuring the incorporation of labeled nucleotide into
said polymeric product wherein the amount of said incorporation is
proportional to the presence of RNA-dependent DNA polymerase
activity.
2. The method of claim 1 wherein said synthetic nucleotide oligomer
is a linear thymidine polynucleotide.
3. The method of claim 2 wherein said synthetic nucleotide oligomer
is d-pT.sub.6.
4. The method of claim 2 wherein said synthetic nucleotide oligomer
is d-pT.sub.8.
5. The method of claim 2 wherein said synthetic nucleotide oligomer
is d-pT.sub.9.
6. The method of claim 2 wherein said synthetic nucleotide oligomer
is d-pT.sub.3.
7. The method of claim 1 wherein said RNA-type polymer is rA.
8. The method of claim 1 wherein said sample is obtained from human
leukemia cells.
9. The method of claim 1 wherein said sample is a mammalian
cellular extract obtained from whole blood which has been purified
by running it through a glycerol gradient.
10. The method of claim 9 wherein said reaction mixture comprises
100-200 .mu.g. of purified sample, 1-5 .mu.g. of said template; and
is 4 .times. 10.sup.-.sup.2 M with respect to K.sup.+and 6
.times.10.sup.-.sup.3 M with respect to Mg.sup.+.sup.+; and
contains 800 m.mu.moles/ml. of two deoxyribonucleotides
complementary to said template, at least one of which is
isotopically labeled having a specific activity of about 500
cpm/pmole.
11. The method of claim 1 wherein said sample is a purified plasma
obtained from whole blood purified by treatment with Kieselguhr and
said reaction mixture comprises 25 .mu.g. of said purified sample;
is 0.006 M in Mg.sup.+.sup.+is 0.04 M in K.sup.+, is 0.008 M in a
first complementary nucleoside triphosphate and is 0.00016 M in an
isotopically labeled second complementary nucleoside triphosphate
having a specific activity of about 100 cpm/pmole, and contains
about 1 .mu.g. of said synthetic template.
Description
BACKGROUND OF THE INVENTION
It has recently been demonstrated that an RNA-dependent DNA
polymerase is present in the virions of Rauscher mouse leukemia
virus and Rous sarcoma virus, both viruses being RNA tumer viruses
(Baltimore, Nature, 226, 1209-11 [1970]). The template for the
RNA-dependent DNS polymerase was shown to be the viral RNA.
Activity of this polymerase was determined by employing a standard
DNA polymerase assay utilizing the incorporation of radioativity
from .sup.3 H-TTP (thymidine triphosphate) into an acid-insoluble
(polymeric) product as the mode of measurement. It was postulated
that all RNA tumor viruses tested have such RNA-dependent DNA
polymerase activity.
In a contemporaneous publication, Temin and Mizutani (Nature, 226,
1211-13 [1970]) confirmed Baltimore's finding with respect to Rous
sarcoma virus and extended this discovery to avian myeloblastosis
virus (AMV). Furthermore, they reported that RNA-dependent DNA
polymerase activity was not present in supernatant of normal cells
even if treated with detergent which served to increase polymerase
activity ten-fold in infected cell supernatants.
Most recently, Gallo, Yang and Ting reported that an RNA-dependent
DNA polymerase analogous to that found in RNA tumor viruses above
had been found in lymphoblasts obtained from humans suffering from
acute leukemia whereas lymphoblasts produced from lymphocytes
obtained from normal subjects were shown to be devoid of such
activity (Nature, 228, 927-29 [1970]). Additionally, Gallo et al.
discovered that this RNA-dependent DNA polymerase did not have
specificity for viral RNA. They are able to employ not only
mammalian RNA, but also the synthetic ribopolynucleotide poly
rA:poly rU as template for this activity. The use of synthetic
polymeric DNA-RNA hybrids and RNA-RNA duplexes as templates for
oncogenic DNA polymerase assay was reported by Spiegelman et al.
(Nature, 228, 430-32 [1970 ]).
These findings enhance the possibility of an eventual discovery of
a preventive and/or therapeutic treatment for disease states whose
etiology involves RNA-dependent DNA polymerase activity. However,
of more immediate practicality is the use of these discoveries as a
basis for a diagnostic method to screen populations for the
presence of such diseases, to monitor the effectiveness of present
treaments for these diseases in patients known to be afflicted or
to monitor patients who have obtained remissions of their diseases
so as to alert the treating physicians of the initiation of a
relapse so that treatment can be reinstated prior to the exhibition
of clinical symptoms. The basic problem preventing the utilization
of such an assay on a practical clinical level, however, is the
fact that the template materials used in assaying for the
RNA-dependent DNA polymerase activity, i.e., viral RNA, mammalian
(rat liver) RNA, and synthetic ribonucleotides (poly rA:poly rU)
are obtainable only in small quantities in the laboratory and thus
are prohibitively expensive. It is evident that a low-cost source
of template material is necessary in order to make effective use of
any diagnostic technique directed to the assay of RNA-dependent DNA
polymerase activity.
BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to an improved diagnostic method for
the detection of those virus-related neoplastic disease states in
whose etiology RNA-dependent DNA polymerase activity is likely to
be involved. Examplary of such disease states are Rous sarcoma,
Rauscher mouse Leukemia, avian myeloblastosis and human leukemia.
In the diagnostic method of this invention known procedures for the
assay of RNA-dependent DNA polymerase activity using modified
standard reactive mixtures are utilized wherein the incorporation
of a labeled nucleotide, i.e., .sup.3 H--TTP into an acid insoluble
fraction is monitored. However, the selective template for the
RNA-dependent DNA polymerase activity in the instant method is a
synthetic nucleotide oligomer hybridized with an RNA-type polymer.
Since both components of this template are readily available and
are relatively inexpensive, the diagnostic method of this invention
may be readily employed in he clinic to screen subjects suspected
of having the aforesaid diseases or to monitor treatment in
patients known to be suffering from such diseases.
The synthetic nucleotide oligomers useful in the preparation of a
RNA-dependent DNA polymerase selective template are preferably
linear thymidine polynucleotides containing from 2 to 24 thymidine
nucleotide units per molecule, i.e., d-pT.sub.2 to d-pT.sub.24,
most preferably d-pT.sub.3 to d-pT.sub.9. These linear nucleotide
oligomers are known compounds and methods for their preparation,
separation and purification and described in detail in the papers
by Khorana and Vizsolyi, J. Am. Chem. Soc., 83, 675-85 (1961) and
Narang et al., J. Chem. Soc., 90, 2702 (1968). For the purpose of
the diagnostic method of this invention tri-, hexa-, octa- and
nona-nucleotides d-pT.sub.3, d-pT.sub.6, d-pT.sub.8 and d-pT.sub.9
are preferred. While the higher nucleotide oligomers are preferred
because of their greater activity in the assay this is balanced by
the fact that the lower oligomers are more readily available and
thus less expensive.
The second component of the template useful in the present
invention are the RNA-type polymers, such as, for example, rA, rU,
rG, RC and rI, a most particularly preferred polymer is rA, RNA
type polymers are articles of commerce and thus readily available.
Hybridization of the aforesaid two components to form the desired
template is conveniently carried out by the addition of
approximately equimolar amounts (on a monomer basis) of the
nucleotide oligomer and the RNA-type polymer (100 .mu.g/ml. in 0.01
M Tris-HCl, pH 7.4), making the solution 0.2 M with respect to NaCl
and allowing the mixture to stand for 15 minutes at room
temperature.
The assay method of the present invention can employ purified
extracts of neoplastic cellular material or leukemic plasma for
which the RNA-dependent DNA polymerase activity is to be
determined. For purposes of illustration standard assay procedures
for determining DNA polymerase activity in accordance with the
method of the present invention is given utilizing alternative
sample sources.
A suitable assay procedure for neoplastic cellular material, i.e.,
human leukemic cells derived from whole blood is as follows:
Heparinized blood samples (5-10 ml. whole blood) are kept at
4.degree.C., and after separation the buffy coat is removed and
transferred into a mortar, pre-cooled in dry ice. The material is
thoroughly ground with a pre-cooled pestle and the contents
transferred and weighed. It is then taken up (2ml, per gram) in TM
buffer (0.1 M Tris, pH 8.3, 0.01 M MgCl.sub. 2, 0.002 M
dithiothreitol [DTT]). The homogenized suspension is spun down in
an SS-34 rotor in a Sorvall contrifuge for 30 min. at 305,000 xg.
The supernatant is removed and the pellet is taken up in phosphate
buffer (0.01 M potassium phosphate, pH 8). A convenient volumn is 1
ml. of buffer per gram of original buffy coat.
The suspension obtained in this manner is further purified by
running it through a glycerol gradient. The purification is done by
layering 17 ml. of the buffy coat suspension in a 37 ml.
polyallomer centrifuge tube on top of a 10 to 30 percent glycerol
gradient (12 ml.) containing 0.01 M potassium phosphate and 0.003 M
DTT, and running it for 3 h. at 25,000 xg. in a Spinco centrifuge
with a SW 27 rotor. The glycerol gradient is made over a 100
percent glycerol pad (8 ml.). The material that collects on top of
the pad, after centrifugation, is removed (usually about 2 ml. of
suspension per tube). The suspension is then thoroughly homogenized
with the help of a syringe and canula. If the suspension contains a
large amount of protein and is very viscous, it is diluted with
0.01 M Tris pH 8.3. An aliquot of this material is used for a
"Lowry" protein determination.
If the buffy coat suspension is small in quantity, the glycerol
gradient purification can be done either in a SW 41 or SW 501
rotor. For example, a 0.3 ml. buffy coat suspension can be purified
using 4.4 ml. of a 10 to 30 percent glycerol gradient with 0.3 ml.
100 percent glycerol pad in a 501 rotor at 50,000 xg. for 11/2
hours.
In a standard DNA polymerase enzyme assay, 100-200 .mu.g. of
protein prepared above contained in 80 .mu.l. with 0.01 M Tris (pH
8.3) is employed. The solution is made 1 percent with respect to
Nonidet P-40, a non-ionic detergent, and incubated for 30 minutes
at 0.degree.C. The reaction is then carried out in a total volume
of 125 .mu.l. The incubation mixture contains 1-5 .mu.g. of the
synthetic template and is 5 .times. 10.sup.-.sup.2 M in Tris (pH
8.3), 4 .times. 10.sup.-.sup.2 M with respect to K.sup.+and 6
.times. 10.sup.-.sup.3 M with respect to Mg.sup.+.sup.+. The
mixture also contains the two deoxyribonucleoside triphosphates
complementary to each member of the synthetic template. At least
one of the two nucleoside triphophates is isotopically labeled.
Thus, if the template were poly d-pT:rA, then cold dATP and labeled
dTTP would be added. Cold deoxynucleoside triphosphate
corresponding to the labeled one, i.e., TTP, is added until the
concentration of this species equals that of the complementary
triphosphate, i.e., both species are present at a concentration of
800 m.mu. moles/ml. The labeled triphosphate may be .sup.3 H--TTP,
.alpha.-.sup.32 P-TTP or .sup.14 C-TTP. Preferably .sup.3 H-TTP is
employed and is used at a specific activity of 100-500 cpm/pmole.
For routine assays, a 20 minute reaction is employed.
A second purification procedure utilizing leukemic plasma as a
sample is as follows:
Heparinized whole blood is centrifuged to obtain a clear plasma.
The following steps were then conducted at a temperature of
0.degree.-4.degree.C. A total of 200 ml. of the plasma is mixed
with 2 gm. of Kieselguhr and centrifuged at 1,800 xg. for 10
minutes. The supernatent is filtered through a Buchner funnel
containing a layer of Kieselguhr over filter paper. To remove the
virus from the supernatent, the plasma is centrifuged against a 10
ml. glycerol pad in a Spinco SW-25.2 rotor at 75,000 xg. for 1
hour. The virus is removed from top of the glycerol pad by pipet
and diluted with 40 ml. of 0.15 m NaCl-0.01 M Tris pH 8.8. The
above glycerol purification is repeated three times.
A standard general assay system for the sample prepared by the
second purification method utilizes the following procedure. About
25 .mu.g. of the purified sample is initially incubated in 50
.mu.l. of 1-2 percent Nonidet P-40, 0.003 M DTT and 0.01 M Tris-HCl
(pH 8.3) for 10-30 minutes at 0.degree.C. There is then added the
following components (assuming a poly d-pT:rA template is being
used) in an amount to achieve the respective concentration or
quantities:
Tris-HCl (pH 7.5-9.0, i.e., 8.3) .05M MgCl.sub.2 .006M DTT .002M
dATP .0008M .sup.3 H-TTP .00016M KCl .04M poly d-pT:rA template 1
.mu.g.
The total final volume of the above mixture is 125 .mu.l. While the
above procedure is particularly useful in assay of AMV, it is
within the skill of the art to adapt the second purification method
to utilize other sample materials. For example, Rauscher murine
leukemia virus, Rous sarcoma virus and Mouse mammary tumor virus
can be assayed by modifying the second purification procedure. A
particulate-free fluid sample from a host infected with any of the
foregoing viruses is treated at 0.degree.-4.degree.C. in the
following procedure.
The fluid sample is layered over a 100 percent glycerol pad in a
centrifuge tube and centrifuged at 95,000 xg. for 70 minutes. The
material layered on the glycerol pad is transferred to the top of a
25-50 percent sucrose gradient in a centrifuge tube and centrifuged
for 3 hours at 95,000 xg. The virus band is removed and diluted in
0.01 M Tris-HCl (pH 8.3), 0.1 M NaCl and 0.002 M EDTA (mixture
identified as TNE). This mixture is centrifuged at 95,000 xg. for 2
hours to pellet the virus. The virus pellet is suspended in TNE and
utilized as a sample in the second method assay procedure as
described previously.
In both general methods described above the DNA polymerase activity
in the respective reaction mixtures is determined by measuring
labeled nucleotide uptake into an acid-insoluble polymer in a
manner known per se. For example, the reaction mixture is incubated
for about 20-30 minutes at from 30.degree.-45.degree.C., i.e., at
37.degree.C. and the reaction is then terminated with 0.5 ml. of
cold water and 0.3 ml. of a trichloroacetic acid solution
comprising equal volumes of 100 percent trichloroacetic acid and
saturated solution of sodium orthophosphate-sodium pyrophosphate.
The precipitate is collected by filtration or centrifugation,
washed with water and then counted by appropriate known methods
employing liquid scintillation techniques. It is also possible to
isolate polymeric product by passing the incubation mixture over an
appropriate gel column, recovering the polymeric product in the
exclusion volume and measuring the isotope content by known
techniques.
While several specific general assay methods have been described,
it is within the skill of the art to modify general assay
procedures known in the art to utilize the present synthetic
templates of the instant invention.
The assay method of the present invention is further illustrated by
the following examples.
Example 1
This example demonstrates the template activity of various
synthetic templates in an assay utilizing AMV as the source of
RNA-dependent DNA polymerase. The templates employed were the
following linear thymidylic acid oligomers: d-pT.sub.2, d-pT.sub.3,
d-pT.sub.4, d-pT.sub.5, d-pT.sub.6, d-pT.sub.7, d-pT.sub.8 and
d-pT.sub.9 ; each respectively hybridized with poly rA by the
procedure described previously. Also tested were poly dT:rA, poly
dT, poly rA, d-pT.sub.9 and d-pT.sub.3 for the sake of comparison
of template activity.
The BAI strain A myeloblastosis virus plasma was obtained by tissue
culture propagation and was purified according to the second
purification method in this specification. A total of 26.7 .mu.g.
of protein was employed for each template tested. The labeled
.sup.3 H-TTP used had a specific activity of 100 cpm/pmole. The
incubation time was 20 minutes at 37.degree.C. The polymerization
product was precipitated, filtered through nitrocellulose filters,
the filters washed with water and dried. The filter was counted in
a BBOT scintillation fluid and the results obtained are summarized
in the following table.
TABLE I
Template Cpm poly dT:rA 62,679 d-pT.sub.9 :rA 177,312 d-pT.sub.8
:rA 147,706 d-pT.sub.7 :rA 156,225 d-pT.sub.6 :rA 178,397
d-pT.sub.5 :rA 75,788 d-pT.sub.4 :rA 40,311 d-pT.sub.3 :rA 80,076
d-pT.sub.2 :rA 9,974 poly dT 2,499 poly rA 471 d-pT.sub.9 8,702
d-pT.sub.3 3,383
it is seen from the above that synthetic templates utilizing a
synthetic thymidylic acid oligomer can be employed successfully in
an assay for the measurement of RNA-dependent DNA polymerase
activity. A substantial number of such templates unexpectedly
exhibit superior template activity compared to the polymeric dT:rA.
Control runs with polymeric or synthetic unhybridized oligomeric
materials are also shown and are seen to provide generally
negligible activity levels when used as templates in this
assay.
Example 2
This example demonstrates the template activity of various
synthetic templates in an assay employing RNA-dependent DNA
polymerase obtained from human leukemic cells by means of the first
purification method described in the specification. A total of 100
.mu.g. of protein was employed using a 20 minute incubation period
at 37.degree.C. Labeled .sup.3 H-TTP having a specific activity of
500 cpm/pmole was used. The polymerization product was prepared for
counting by the same procedure as in Example 1. Results of these
experiments are summarized in Table 2 below.
TABLE 2
Template Cpm poly dT:rA 794 p-dT.sub.9 :rA 688 p-dT.sub.8 :rA 543
p-dT.sub.7 :rA 478 p-dT.sub.6 :rA 450 p-dT.sub.5 :rA 730 p-dT.sub.4
:rA 472 p-dT.sub.3 :rA 387 p-dT.sub.9 383
this example demonstrates the utility of the synthetic templates of
the present invention in an assay employing cellular RNA-dependent
DNA polymerase as sample. The templates of the present invention
can thus be used to determine or monitor the presence of
RNA-dependent DNA polymerase in human cells.
* * * * *