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Use of t cell cloning to detect in vivo mutations induced by cyclophosphamide.

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757
USE OF T CELL CLONING TO DETECT IN VIVO
MUTATIONS INDUCED BY CYCLOPHOSPHAMIDE
ROBERT G. PALMER, CAROLINE A. SMITH-BURCHNELL, BRYAN K. PELTON,
WINSOME HYLTON, and A. MICHAEL DENMAN
By cloning T cells, mutations at the hypoxanthine-guanine phosphoribosyltransferase locus were
quantified in peripheral blood lymphocytes of 12 patients with connective tissue diseases receiving long-term
cyclophosphamide. Frequency of mutation was higher
than in control subjects and was related to the duration
of therapy; therefore, some cells with mutations are
long-lived, and these cells accumulate in the peripheral
circulation. Mutation frequency was also independently
related to age. The results indicate that even low doses of
cyclophosphamide are mutagenic and may explain, in
part, why these patients are at risk of drug-induced
malignancy.
Cyclophosphamide is a useful immunosuppressive agent in patients with connective tissue diseases. It
is administered in low doses to minimize the risk of
drug-induced malignancy; nevertheless, cancer may
occur (1,2). The chromosome damage can be measured
as increased chromosome aberrations in some patients
(3) and as high sister chromatid exchange frequencies in
all patients receiving this drug (4). These methods only
detect the effects of chemical mutagens, however, and
do not detect specific mutations.
One mutation that may be readily detected in
From the Connective Tissue Diseases Research Group,
Division of Rheumatology, Clinical Research Centre and Northwick
Park Hospital, Harrow, Middlesex, United Kingdom.
Robert G. Palmer, DM; Caroline A. Smith-Burchnell, BSc;
Bryan K. Pelton, PhD; Winsome Hylton, MSc; A. Michael Denman, FRCP.
Address reprint requests to Robert G. Palmer, DM, Consultant Rheumatologist, Dudley Road Hospital, Dudley Road, Birmingham, B18 7QH, UK.
Submitted for publication February 19, 1987; accepted in
revised form November 23, 1987.
Arthritis and Rheumatism, Vol. 31, No. 6 (June 1988)
vitro is at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus. Mutated cells with absent
or nonfunctional enzyme (HGPRT - cells) can be
selected by growth in medium containing thioguanine.
These cells survive, and normal cells die in the presence of thioguanine. Results of short-term cultures
with bulk lymphocytes, using incorporation of tritiated
thymidine as the endpoint, have suggested that cyclophosphamide leads to increased mutations in patients
with rheumatic diseases (3,and cancer patients
treated with chemotherapy may have high frequencies
of mutation, as demonstrated by autoradiography (6).
But variations in cell dynamics mean it is sometimes
difficult to interpret and quantify the results of shortterm cultures in selective media.
In contrast, the T cell cloning technique used to
detect HGPRT- mutants has a clear-cut endpoint:
Cell clones originating from single cells grown with or
without selective media can easily be identified and
quantified (7-9). Spontaneous mutations are present in
1 in 105-106 peripheral blood lymphocytes of normal
individuals (10). The T cell cloning technique has
already been used to show that technicians exposed to
ionizing radiations have elevated mutant frequencies
(11). We have used it to show that, when administered
as immunosuppressive therapy to patients with connective tissue diseases, cyclophosphamide induces
mutations in large numbers of blood lymphocytes. The
HGPRT activity of cloned cells was assayed and the
cells of origin determined by immunofluorescence.
PATIENTS AND METHODS
Patients and controls. Mutant frequencies were determined in peripheral blood lymphocytes from 12 patients with
PALMER E T AL
758
connective tissue diseases who had received continuous
daily cyclophosphamide for periods of 1-84 months. Their
diagnoses were systemic lupus erythematosus, rheumatoid
arthritis, or vasculitis. Controls were 13 patients with the
same diagnoses and 7 healthy individuals. Diagnoses, ages,
and details of therapy are shown in Table 1. None of the
patients or controls were receiving any other treatment
known to damage chromosomes.
T cell cloning and detection of mutants. Techniques
similar to those described by Morley et a1 (7,8) were followed. Briefly, separated blood lymphocytes were incubated
overnight in fresh medium. For each experiment, 2 microtiter plates with 96 wells each were required. The technique of
limiting dilution was used to add 3 cells in RPMI and human
serum to each well of one of the plates, to determine the
cloning efficiency without thioguanine. Then, 2 x lo4 cells
Table 1. Clinical data and laboratory observations on cyclophosphamide-treated connective tissue disease patients and controls*
Group and
patient no.
Months Cloning
of
efficiency
Diagnosis Age therapy
(%)
Mutation
frequency
Cyclosphosphamide
1
2
3
4
5
6
7
8
9
10
11
12
CTD controls
1
2
3
4
5
6
7
8
9
10
11
12
13
Healthy
controls
1
2
3
4
5
6
7
RA
SV
sv
RA
SLE
sv
sv
sv
sv
SLE
sv
sv
SLE
RA
RA
RA
RA
sv
SLE
RA
sv
SLE
sv
sv
SLE
-
46
63
56
48
46
67
49
14
5
1
39
58
39
32
38
53
56
71
59
75
55
30
53
59
30
43
37
24
35
38
25
32
52
32
27
5
11
13
66
33
84
52
14
3
-
-
-
-
-
-
-
-
26
8
19
26
9
9
25
6
7
31
38
4
1.4 X
1.2 x
1.4 x
2.5 X
1.4 x 10-4
5.5 x 10-4
5.7 x 10-5
1.0 x ]OW5
5.4 x 1 0 - 4
1.8 x 10-4
6.6 X
3.2 x 10-5
25
13
7
13
30
14
16
4
10
1.7
5.7
4.2
7.6
1.6
1.7
x
x 10-5
x 10-~
x lo-’
X
9
x
1.5 x
1.6 x
3.7 x
3.2 x
5.6 x
4.5 x
6.4 x
35
19
23
30
4
15
9
5.1 x
3.0 x
7.2 x
1.0 x
1.7 x
1.7 x
4.6 x
4
10
5
lo-’
10-~
10-5
lo-’
10-5
and thioguanine (at a final dilution of 5 x 10-6M) were
added to the wells of the second plate. Interleukin-2 (2%
lymphocult T-HP; Biotest, Frankfurt, FRG), 0.5% phytohemagglutinin (PHA), and 1 x lo4 irradiated feeder cells
(HGPRT-) were also present in each well. After 14 days of
incubation, the plates were read by visual assessment with
an inverted microscope. Cells that had cloned formed a
continuous layer in the bottom of the well. The single reader
of the plates was “blinded” to the diagnostic and drug
categories of the samples. The cloning efficiency with and
without thioguanine was calculated by the formula:
1
(Total number of wells)
- x log,
N
(Number of empty wells)
where N = number of cells addediwell. The mutant frequency was calculated as the ratio of the 2 cloning efficiencies.
Uptake of tritiated thymidine to validate visual assessment of clones. At the end of the 14-day incubation period, 1
pCi of tritiated thymidine was added to each well. Cells were
harvested after 4 hours, and uptake was determined by liquid
scintillation counting. Results were expressed as counts per
minute per well.
HGPRT assay. The conversion of 14C-hypoxanthine
to 14C-inosine monophosphate (I4C-IMP) by HGPRT was
measured by the method of Craft et a1 (12). Thymidine
triphosphate was present to inhibit 5’-nucleotidase activity.
The assay was performed on sonicated cells from 18-day
cultures that had been subcultured after 14 days. Hypoxanthine and IMP were separated by high-voltage paper electrophoresis, and IMP activity was determined by liquid
scintillation counting. Results were expressed as nmoles of
IMP/hour/mg of protein. The assay was linear for time and
protein.
Hypoxanthine-aminopterin-thymidine(HAT) sensitivity as a measure of HGPRT activity. Cells from 18-day
cultures (previously subcultured at day 14) were incubated
with HAT media supplement (Sigma, St. Louis, MO) in
RPMI medium together with 15% human serum, 0.5% PHA,
5 x lo4 irradiated feeder cells (HGPRT-), and interleukin-2
(1% lymphocult T-HP; Biotest). Control wells contained no
HAT medium. Thioguanine (final concentration 5 x 10-6M)
was added to the thioguanine-resistant clones. After 3 days,
tritiated thymidine (1 pCi) was added to 100 p1 of cell
suspension (in duplicate) for 6 hours, and uptake was determined by liquid scintillation counting.
Immunofluorescence studies on clones. A standard
technique (13) was followed to analyze cells with the fluorescence-activated cell sorter. Four monoclonal antibodies were
used: CD4 (T helper cells), CD8 (T suppressor cells), CD3
(pan-T cell marker), and CD19 (pan-B cell marker).
10-5
RESULTS
10-5
Findings in the patients and controls are shown
in Table 1 and Figure 1. Analysis of variance was
performed to determine the independent effects of
diagnosis and treatment on the frequency of mutation
indicated that there was no significant effect of diagnosis (P = 0.2) and no significant interaction between
lo-’
lo-’
lo-’
* RA = rheumatoid arthritis; SV = systemic vasculitis; SLE =
systemic lupus erythematosus; CTD = connective tissue disease;
- = not applicable.
T CELL CLONING
759
i
l
.I
'i
0
0
r
0
0
I
10
I
20
I
30
1
40
I
I
50
60
I
70
I
80
1
90
Duration of cyclophosphamide therapy (mos. )
a
0
I
0
a
c
Normal
controls
o
'i
0
Figure 2. Increase in cyclophosphamide-induced mutations with
duration of therapy (P < 0.05).
I
1
CTD
controls
Cydophosphamide
Figure 1. Frequency of hypoxanthine-guanine phosphoribosyltransferase mutations in connective tissue disease (CTD) patients treated
with cyclophosphamide and in controls. Each point represents a
single observation. Vertical bars indicate geometric means f 2
SEM.
diagnosis and cyclophosphamide therapy (P = 0.5).
However, cyclophosphamide led to a significant increase in mutant frequency (P < 0.OOOl). The frequency
of mutation in the disease controls did not differ significantly from that in the healthy controls (P = 0.2).
For the patients who received cyclophosphamide, multiple regression analysis was used to demonstrate that mutation frequency was related to the
duration of therapy (P < 0.05) and age (P < 0.02), but
not significantly related to cloning efficiency ( P =
0.08). The relationship between mutation frequency
and duration of therapy is shown in Figure 2. For the
connective tissue disease controls, the mutation frequency was again related to age (P < 0.02), but not to
cloning efficiency (P = 0.06). For the healthy controls,
the mutant frequency was not related to age or cloning
efficiency.
The visual assessment of clones by use of the
inverted microscope was validated by tritiated thymidine uptake. The high uptake of wells containing clones
was clearly differentiated from the low uptake of wells
that contained cell debris only (ranges 6 x lo2 to 5 x
lo4 cpm and 5 x 10' to 5 x lo2 cpm, respectively).
HGPRT activity and HAT sensitivity were determined in some clones. Thioguanine-resistant clones
had no significant enzyme activity and were sensitive
to HAT medium; thioguanine-sensitive clones had
normal enzyme activity and were resistant to HAT
medium (results not shown).
T cell phenotype was determined by immunofluorescence for 11 clones. Nine were CD4+ and 2
were CD8+; 3 clones that were CD3+ were also
CD19-. No clones had mixtures of markers, and there
were no differences in the distribution of markers
between thioguanine-sensitive and thioguanine-resistant clones.
DISCUSSION
Low-dose cyclophosphamide induces HGPRT
mutations in patients with connective tissue diseases.
Since the damaging effect of cyclophosphamide on
PALMER ET AL
DNA is nonspecific, it is likely that many other mutations also occur. This may account, in part, for its
oncogenic potential. This effect is dependent on the
duration of therapy. The longer it is administered, the
more cells with mutations are found in the peripheral
circulation. Drug-induced mutations are, therefore,
cumulative. We have already shown that chlorambucil, another alkylating agent, induces chromosome
damage-measured as increased sister chromatid exchange frequencies-in a duration-dependent manner
(14). Our suspicion that these drugs induce long-term
genetic damage associated with cell mutation has been
confirmed. Others have shown that HCPRT mutations
increased with dose in nuclear medicine technicians,
but not in radiotherapy technicians, who had occupational exposure to ionizing radiation (11). The 2 patients who had been treated for 3 months or less, and
possibly some individuals who had received longer
treatment, had mutant frequencies that were within
the normal range. Although sister chromatid exchange
frequencies in these patients would be high (4), it is
possible that short courses of cyclophosphamide lead
to little or no long-term chromosome damage. The
frequency of spontaneous mutations in our healthy
controls was approximately 1 in lo5. This is consistent
with the observations of others (10).
The mean frequency of mutations in patients
receiving cyclophosphamide was greater than 1 in lo4.
This may be an underestimate of the true frequency in
these patients, because it is likely that only the healthiest
of the cells damaged by cyclophosphamide will be able
to grow in vitro and clone over a 14-day period. It is
anticipated that many mutant cells with extensive genetic damage might be present in vivo, but that these
cells would not divide and would even die in vitro. The
mutant frequency in the disease controls was slightly,
but not significantly, higher than that in the healthy
controls. Perhaps greater differences would have been
demonstrated if larger groups had been studied; this
possibility is being explored.
Mutant frequencies also increase with the age
of the treated and untreated patients. This effect was
not demonstrable for the healthy controls, in whom
the age span was narrower. The effect of age has also
been observed by others, using the less sensitive
autoradiographic technique (15).
We used the technique of limiting dilution in our
study. Three cells were added to each well. The
maximum cloning efficiency was 35%; therefore, it is
likely (but not proven) that the cell layers observed at
the end of 14 days in culture were clonal in origin and
were not mixed populations. Immunofluorescence
demonstrated that all wells tested contained T cells
only. The cells of most wells were CD4 + ; the remainder were CD8+. No wells contained cells of both
phenotypes. Others have observed a preponderance of
T helper-positive clones (lo), although some workers
have found approximately equal numbers of each
phenotype (16). We have been able to demonstrate
that our thioguanine-resistant clones have little or no
functioning HGPRT enzyme and that thioguaninesensitive clones have normal enzyme function.
The results suggest that there was an inverse
relationship between mutant frequency and cloning
efficiency without thioguanine. Mutant frequency is
calculated as the ratio of cloning efficiency with thioguanine to cloning efficiency without thioguanine. This
may imply that under adverse conditions (low cloning
efficiency), thioguanine-resistant cells selected by the
medium from large numbers of cells (most of which are
thioguanine sensitive) are more able to survive than
small numbers of cells plated without thioguanine.
This does not affect the interpretation of the results,
however, because the ranges of cloning efficiencies
within the 3 groups studied were similar.
It is interesting that perhaps those patients with
the highest number of mutations are also those most at
risk of drug-induced neoplasia. Followup studies may
help clarify the relationship between the mutagenic
and oncogenic potentials of cytotoxic drugs.
ACKNOWLEDGMENTS
We thank the following staff of the Clinical Research
Centre: Jennifer Allsop for performing the enzyme assays, Dr.
Andrew Edwards for assistance with the immunofluorescence
studies, and Stephen Burman for help with data analysis.
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375-382, 1974
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