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Uridine Incorporation into the Media and RNA of Cultured Rheumatoid Synovial Cells.

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Uridine Incorporation into the Media and RNA of Cultured
Rheumatoid Synovial Cells
Arthur I. Grayrel
The incorporation of 3H-uridine into the media and RNA of rheumatoid
synovial cell cultures, with and without pretreatment with Sbromo-deoxyuridine (BUDR) and Actinomycin D, has been examined by sucrose density
gradient centrifugation without providing evidence for the presence of an
infectiousagent within these cells.
There has been considerable speculation concerning the possible role of an infectious agent
in the etiology of rheumatoid arthritis. Indirect
evidence for such an agent in rheumatoid synovial cells propagated in cell culture has been
sought by means of technics previously used in
the investigation of tumor viruses (I). The
method involves the use of sucrose density gradient centrifugation to isolate radioactively labeled particles from the culture medium of cells
incubated with W-uridine, a method -used to
demonstrate both viruses (2) and mycoplasma (3) in human cell strains. The incorporation of uridine into the RNA of these
cells has also been examined.
MATERIALS AND METHODS
Synovial membranes obtained from patients with rheumatoid arthritis undergoing reconstructive surgery were
From the Department of Medicine, Montefiore Hospital
and the Albert Einstein College of Medicine, Bronx, NY.
Supported by a grant from the John A. Hartford Foundation, Inc.
ARTHUR I. CRAYZEL, MD: Head, Division of Rheumatology, Montefiore Hospital and Medical Center, Associate
Professor of Medicine, Albert Einstein College of Medicine,
Bronx, NY.
Reprint requests should be addressed to: Dr. Arthur I.
Grayzel, Montefiore Hospital and Medical Center, 111
East 210th Street, Bronx, NY 10467.
Submitted for publication Aug 14, 1972; accepted Jan
18, 1973.
grown in explant culture; the cells that grew out were then
subcultured several times. The cultures were maintained in
Dulbecco-Vogt modified Eagle's medium containing 10%
untreated calf serum under 5% C 0 2 in room air. For specific experiments 2 to 3 x 106cellsgrown o n 100-mm Falcon petri dishes containing 5 ml of medium without calf serum were incubated with 50 fiCi of 'H-uridine with a specific activity of 27 Ci/mmole (Schwarz BioResearch). At
the end of 24 hours the medium from 2 such plates was
pooled and chilled to 00 C. An equal volume of cold saturated ammonium sulfate was added drop-wise with constant
mixing, taking care not to allow the temperature to exceed
3'C. The mixture was allowed to stand for 30 minutes in
the cold and then centrifuged at 30,OOOg for 10 minutes.
The precipitate was dissolved in 1 ml of buffer (0.01 M
Tris-HCI, 0.001 M EDTA at pH 7.1) and layered on top of
a linear density gradient of 15 to 60% (w/v) sucrose in buffer. The gradient was centrifuged at 25,000 rpm for 20 to 24
hours at 4" C in a SW 25.1 rotor (Spinco Division, Beckman Instruments). One-milliliter fractions were collected
and radioactivity was determined by adding 1 mg of carrier
bovine serum albumin and 1 ml of cold 10% trichloracetic
acid (TCA). The samples were allowed to stand at 4 O C for
30 minutes and the precipitate collected_on0.t5-p cellulose
membrane filters (Schleicher and Schuell). The filters were
washed with large volumes of 5% TCA followed by water,
dissolved in 10 ml of Bray's solution (4) and counted. Small,
0.1 ml, aliquots of the original fractions were removed for
the determination of sucrose concentration by refraction. In
other experiments cells were exposed to 5-bromodeoxyuridine (BUDR, from Schwan Mann) 20 r g / d for
7 days and 2% dimethyl sulfoxide (DMSO) for the last 4
days before incubation with 'H-uridine. In a few experiments the cells were incubated with 10 &i/5 ml of 'Hthymidine (16 Ci/mmole, Schwarz BioResearch) in place of
3H-uridine. Studies of the incorporation of uridine into
RNA were performed as described by Girard (5).
Arthritis and Rheumatism, Vol. 16, No. 3 (May-June 1973)
419
GRAYZEL
800 -1.28
- 1.24
-1.20
m
400;F\
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-1.08
- 1.04
u
t
'
I ' '
-
2=
2
-
5
200-
o
ported to activate RNA tumor viruses in animal
cell systems (6, 7) and treatment with 5-iodo2'deoxyuridine (IUDR) plus DMSO has been
used to activate candidate viruses from human
tumor cells (8). The presumed viruses isolated
from human tumor cells by these methods (1,2)
as well as animal RNA tumor viruses such as
Rous sarcoma and Rous associated virus have a
density of 1.16 to 1.18 g/cu mm (9), while the
common strains of mycoplasma have a density
of 1.23 g/cu mm (3).
' '
$
I
Fig 1. Sucrose density gradient analysis of the
radioactivity incorporated into the medium of a
rheumatoid synovial cell strain incubated with
3H-uridine after prior treatment with 5-bromo-deoxyuridine (BUDR) and dimethyl sulfoxide (DMSO).
RESULTS
A total of 6 different strains of rheumatoid
synovial cells were incubated with 'H-uridine,
an additional 3 strains with 3H-thymidine and a
third group of 6 strains was incubated with 3Huridine after treatment with BUDR and
DMSO. Treatment with BUDR has been re-
- 1.28
-1.24
gions of the gradient in which known viruses
band. The single exception revealed a very
modest peak of862 counts/min at a density if
1.23 g/cu mm suggesting a mycop~asma.hi^
strain was cultured for mycoplasma on Hayflick's medium but none was found. The strain
did not survive beyond the fourth subculture
which was examined in the gradient so no further studies could be performed, whereas the
mean number of subcultures for rheumatoid cell
strains in this laboratory is 15. A representative
sucrose density gradient profile of the medium
from a rheumatoid synovial cell strain incubated with 3H-uridine after prior treatment
E
E
10
20
30
FRACTION NUMBER
Fig 2 (left). Sucrose density gradient profile of the medium from the same cell strain shown in
Figure 1 infected with M hyorhinis, titer unknown, and (right) Newcastle disease virus (NDV).
1@TCD/106 cells. A heterogeneous population of NDV has been observed after ammonium sulfate precipitation and density gradient centrifugation (10).
420
Arthritis and Rheumatism, Vol. 16, No. 3 (May-June 1973)
URlDlNE INCORPORATION
with BUDR and DMSO is shown in Figure 1.
For comparison, a similar analysis of the same
cell strain incubated with a known mycoplasma, M hyorhinis, is shown in Figure 2 (left)
and with Newcastle disease virus in Figure 2
(right).
T h e pattern of uridine incorporation into the
RNA extracted from human rheumatoid and
nonrheumatoid cell strains was also examined.
There were no differences noted between the
two types of synovial cells. Many RNA viruses
can replicate in the presence of Actinomycin D
which inhibits host cell DNA-dependent RNA
synthesis. No significant synthesis of RNA was
detected in rheumatoid synovial cells incubated
with 'H-uridine in the presence of 5 pg/ml of
Actinomycin D.
With the exeeption of a single rheumatoid
cell strain showing a small peak of )H-uridine
incorporation in the region of a sucrose density
gradient corresponding to the density of mycoplasma, these experiments do not provide evidence for the productive infection of rheumatoid
synovial cells. They do not exclude the possibility that these cells have been altered by an infectious agent in a manner analogous to transformation by oncogenic viruses.
A study with similar observations has recently been reported by Person et al (1 1).
REFERENCES
1. Todaro GJ, Zeve V, Aaronson SA: Cell culture
technique in the search for cancer viruses of
man. In Vitro 6:355-361, 1971
2. Todaro GJ, Zeve V, AaronsonLSA: Virus in cell
culture derived from human tumour patients.
Nature (London) 226: 1047-1049,1970
3. Todaro GJ, Aaronson SA, Rands E: Rapid detection of mycoplasma-infected cell cultures. Exp
Cell Res 65:256-257, 1971
4. Bray G: A simple efficient liquid scintillator for
counting aqueous solutions in a liquid xintillation counter. Anal Biochem 1:279-285, 1960
5. Girard M: Isolation of ribonucleic acids from
mammalian cells and animal viruses, Methods
in Enzymology. Vol 12, Part A. Edited by L
Grossman, K Moldave. New York, Academic
Press, 1967, pp 581-586
6. Lowy DR, Rowe WP, Teich N, et al: Murine
leukemia virus: high-frequency activation in
vitro by 5-iodqdeoxyuridine and 5-bromodeoxyuridine. Science 174:155-156,1971
7. Aaronson SA, Todaro GJ, Scolnick EM: Induction of murine C-type viruses from clonal lines of
virus-free BALB/3T3 cells. Science 174:157159,1971
8. Stewart SE, Krasnic G Jr, Draycott C, et al: Activation of viruses in human tumors by 5iododeoxyuridine and dimethylsulfoxide. Science
175:198-1 99, 1972
9. Robinson HL: Isolation of noninfectious particles containing Rous sarcoma virus RNA from
the medium of Rous sarcoma virus transformed
nonproducer cells. Proc Natl Acad Sci USA
57: 1655-1662, 1967
10. Minocha HC, Consigli RA, Eisenstark A: Concentration and purification of Newcastle disease
virus. Am J Vet Res 29:877-882, 1968
11. Person DA, Sharp JT, Rawls WE: Attempts to
identify viruses and mycoplasmas in connective
tissue diseases. Arthritis Rheum 16: 125-126,
1973 (abstr)
Arthritis and Rheumatism, Vol. 16, No. 3 (May-June 1973)
421
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rna, uridine, media, culture, incorporation, synovial, rheumatoid, cells
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