Download Role of Asparagine Synthetase and Asparagyl

[CANCER RESEARCH 41, 3104-3106,
0008-5472/81
/0041-OOOOS02.00
August 1981]
Role of Asparagine Synthetase and Asparagyl-transfer
RNA Synthetase in
the Cell-killing Activity of Asparaginase in Chinese Hamster Ovary
Cell Mutants1
Mary M. Y. Waye2 and Clifford P. Stanners3
Department of Medical Biophysics,
University of Toronto, and the Ontario Cancer Institute, Toronto, Ontario M4X 1K9, Canada
ABSTRACT
The cell-killing activity of asparaginase on three classes of
Chinese hamster ovary cell mutants was examined: a mutant
which overproduces asparagine Synthetase (AH5); mutants
defective in asparagine synthetase (N3 and N4); and mutants
conditionally defective in asparagyl-transfer
RNA synthetase
(Asn 3, Asn 7, and Asn 9). The overproducer was more
resistant to the cell-killing activity of asparaginase than wildtype Chinese hamster ovary cells, while mutants defective in
asparagine synthetase were more sensitive. Surprisingly, the
asparagyl-transfer
RNA synthetase mutants were even more
sensitive to asparaginase than the asparagine synthetase mu
tants. In a preliminary survey of four human lymphoid cell lines
(RPMI 8402, RPMI 8392, B46M, and Molt-4F) which showed
dramatically different asparaginase sensitivity, however, sen
sitivity to the cell-killing activity of asparaginase was correlated
with reduced levels of asparagine synthetase and not with
reduced levels of asparagyl-transfer RNA synthetase.
INTRODUCTION
The reason for variability in clinical response to the cancer
chemotherapeutic agent asparaginase is unknown at present
(4, 17). This is perhaps not surprising as the basic mechanism
for cell killing by the drug is also unknown. Evidence has been
presented in support of cell-killing mechanisms involving se
vere starvation for asparagine (3, 8), cleavage of cell surface
protein at asparagine residues (5), and disruption of the cellular
energy-producing reactions (9). Regardless of mechanism, the
efficacy of asparaginase will obviously be affected by the
extracellular and intracellular levels of asparagine. These levels
are determined in part by the intracellular biosynthetic enzyme
asparagine synthetase, and some of the variability in response
to asparaginase has been ascribed to variability in the activity
of this enzyme.
We have recently reported the isolation of mutants of CHO"
cells with reduced or absent asparagine synthetase activity
(18). Evidence was presented in support of the hypothesis that
the mutations were in the structural gene for the enzyme (18).
We have also isolated CHO mutants with conditional defects in
asparagyl-tRNA synthetase (15, 16), an essential enzyme for
cellular protein synthesis. As the latter mutants are both tem
perature sensitive and auxotrophic for asparagine (15, 16), it
' Supported by grants from the National Cancer Institute of Canada and Grant
MT 1877 from the Medical Research Council of Canada.
2 Research student of the National Cancer Institute of Canada.
3 To whom requests for reprints should be addressed.
' The abbreviations used are: CHO, Chinese hamster ovary; WT, wild type.
Received September 30, 1980; accepted May 7, 1981.
3104
was of interest to compare the sensitivity of the 2 types of
mutants having the same genetic background to the cell-killing
activity of asparaginase. Any differences in sensitivity to as
paraginase between mutants with different enzymatic lesions
could provide an explanation for some of the variability in drug
sensitivity of human lymphoid cell lines.
MATERIALS
AND METHODS
Chemicals. Asparaginase, or asparagine amidohydrolase,
or Kidrolase (commercial name), EC 3.5.1.1 type EC2, partially
purified from Escherichia coli, was obtained from Poulenc,
Ltd., Montreal, Quebec, Canada (specific activity, approxi
mately 200 units/mg). This relatively impure enzyme prepara
tion is used clinically and hydrolyzes asparagine to aspartic
acid but also contains a low level of glutaminase activity [about
3% of the asparaginase activity (9)]. It should be pointed out
that our tissue culture medium contains a relatively high con
centration of glutamine (292 mg/liter) which would minimize
any anticellular effects of the contaminating glutaminase. Also,
the cell-killing activity of the preparation was strongly reduced
by the inclusion of asparagine in the treatment medium (Chart
1).
Radioactive isotopes were obtained from Amersham/Searle
Corp., Oakville, Ontario, Canada; amino acids were obtained
from GIBCO, Burlington, Ontario, Canada; liver tRNA was
obtained from A. Hampel, Northern Illinois University.
Cells and Culture Conditions. The wild-type CHO strain,
Gat", isolated by McBurney and Whitmore (10), will be referred
to as the WT strain. This strain was also used by Thompson ef
al. (15, 16) and Waye and Stanners (18) to isolate asparagyltRNA synthetase mutants (Asn 3, Asn 7, and Asn 9) and
asparagine synthetase mutants (N3 and N4), respectively.
AH5, isolated from fsH1 (13), is a CHO cell mutant that over
produces asparagine synthetase (7) and was obtained from S.
Arfin, University of California, Irvine. Human T-cell lines (RPMI
8402 and Molt-4F) and human B-cell lines (RPMI 8392 and
B46M) were obtained from J. Minowada (11) (Roswell Park
Memorial Institute, Buffalo, N. Y.). CHO cells were maintained
in suspension culture at 34°using «-modified minimal essential
medium (12) with asparagine-H2O at 50 /¿g/ml and supple
mented with 10% fetal calf serum. Human lymphoid cells were
maintained in static suspension cultures at 37° in the same
growth medium. Cell concentrations were determined using an
electronic particle counter.
Assay for Asparagine Synthetase Activity. Asparagine syn
thetase activity was measured by determining the ability of
sonicated whole-cell extracts to synthesize [14C]asparagine
from [14C]aspartic acid as a function of time as described by
Arfin ef al. (1). Reactions were carried out taking determina-
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RESEARCH
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Asparagine Synthetase and Asparagyl-tRNA Synthetase in Asparaginase Cell Killing
tions at 0, 20, 40, and 60 min and were linear up to 40 min.
Separation of the asparagine formed from aspartic acid was
carried out by the electrophoretic method of Horowitz et al. (8)
or by a Dowex 1-acetate column (7). The authenticity of the
asparagine spot was tested by its susceptibility to hydrolysis
with asparaginase. Protein determination was performed using
the dye-binding protein assay of Bradford (2). The percentage
of conversion of aspartic acid to asparagine per mg of extract
protein per hr was determined from the slopes of straight lines
relating the percentage of conversion with time.
Assay for Asparagyl-tRNA Synthetase Activity. AsparagyltRNA synthetase activity was measured by determining the
amount of [14C]asparagine accepted by rat liver tRNA at 34°
as a function of time, using the supernatant fractions obtained
from centrifugation of detergent (Nonidet P-40)-lysed cells for
30 min at 30,000 x g. Details of this procedure have been
reported by Thompson ef al. (15, 16).
RESULTS
The measured activities of asparagine synthetase and asparagyl-tRNA synthetase for the cell lines used in this study
are presented in Table 1. Considering the CHO cell mutants,
asparagine synthetase activities ranged from less than 2% of
the activity of WT cells for the N3 mutant to 2 to 3 times WT
activity for the asparagyl-tRNA synthetase mutants to 11 times
WT activity for the superproducer line, AH5. Asparagyl-tRNA
synthetase activities, measured at a permissive temperature
for the fs mutants, showed very low levels relative to WT cells
for the Asn 3 and Asn 7 mutants as reported previously (15,
16). These low levels are presumably due to inactivation of the
more labile defective enzyme in the mutants during preparation
of the cellular extracts. The asparagine synthetase mutant
(N3), on the other hand, showed higher than WT asparagyltRNA synthetase activity.
The cell-killing activity of different doses of asparaginase on
Table 1
Asparagine synthetase and asparagyl-tRNA synthetase activities of various cell
lines
lineAH5WTN3N4Asn
Cell
3Asn
7Asn
9RPMI
8402MOLT-4FRPMI
8392B46MAsparagine
syn
thetase*11
synthetase0ND"1
±21
0.1<0.020.4
±
0.22.50.3<0.010.09ND3.3
±
0.12.0
±
0.12.3
±
0.22.7
±
0.4<0.1<0.11.6
±
sensitivity'_+++
++
+++
++
++
++
+_-
0.63.1
±
0.30.3
±
0.21.3
±
0.030.3
±
±0.4Asparagyl-tRNA ±0.2Asparaginase
The values shown represent the activity relative to WT cells, with the 90%
confidence intervals for repeat determinations. The activity of WT cells was 2.2
nmol asparagine per mg protein per hr.
The values shown represent the activity relative to WT cells, with the 90%
confidence intervals for repeat determinations. The activity of WT cells was 35
pmol [14C]asparagine incorporated per mg protein per min. Asparagyl-tRNA
synthetase activities of all cell lines were first normalized to the overall aminoacyltRNA synthetase activity measured with a mixture of 4 amino acids (phenylalanine, tyrosine, threonine, and valine) to correct for differences in extraction
efficiency from preparation to preparation. The normalization procedure did not
affect the qualitative conclusions drawn.
Semiquantitative estimates of sensitivity for cell survival to asparaginase
obtained from Chart 1a for CHO cell lines and from Ohnuma ef al. (11) for the
human lymphoid cell lines; —,relatively insensitive; +, relatively sensitive.
d ND, not determined.
AUGUST 1981
,Asn9
IO
" O
IO*
Asporogìnose concenlration(IU/ml)
IO'5
io-
KI' io'!
MfJ
Chart 1. a, dose-response curve of cellular colony-forming ability to asparag
inase in asparagine-deficient
medium. Different serial concentrations of cells
were plated in plastic tissue culture dishes containing complete growth medium
for treatment with asparaginase and for outgrowth of surviving colonies. After the
cells were attached, the medium was changed to growth medium lacking aspar
agine and supplemented with 10% dialyzed fetal calf serum. Various concentra
tions of asparaginase were added to the cells, and the cultures were incubated
at either 34°(for AH5 and Asn 3) or 37°(for Asn 7, Asn 9, WT, N3, and N4) for
65 hr. After washing with 2 rinses of phosphate-buffered saline (6), the cells were
incubated in complete «-modified minimal essential medium at 34°(for AH5, Asn
3, Asn 7, and Asn 9) and at 37° (for N3, N4. and WT) for 10 days. After
incubation, the number of visible colonies was scored and expressed as a fraction
of that obtained with asparagine-supplemented
medium without asparaginase. b.
dose-response curve of cellular colony-forming ability to asparaginase in aspar
agine-supplemented medium. Different serial concentrations of cells were plated
in complete growth medium containing asparagine -H2O at 50 /ig/ml. After the
cells were attached, various concentrations of asparaginase were added to the
medium, and the cultures were incubated at 37° for 54 hr. After washing with
one rinse of a-modified minimal essential medium lacking asparagine, the cells
were incubated in a-modified mimimal essential medium containing asparagineH2O at 200 /ig/ml and 10% fetal calf serum at 34°for 10 days. Colonies were
scored as in a. In both a and b, the plating efficiency at any particular survival
level was found to be constant over a range of cell numbers plated of about 103.
these CHO cell lines maintained in asparagine-free medium for
a fixed exposure time of 65 hr, as assessed by colony-forming
ability, is shown in Chart 1a. The viability of the asparagine
synthetase superproducer, AH5, was unaffected by even the
highest dose of asparaginase used, whereas WT cells showed
some loss of viability at very high drug doses. The asparagine
synthetase mutants, N3 and N4, were predictably much more
sensitive to asparaginase than WT cells. The asparagyl-tRNA
synthetase mutants, Asn 3, Asn 7, and Asn 9, when maintained
under semipermissive conditions (37°; asparagine-free me
dium), were found to be even more sensitive to the cell-killing
activity of asparaginase than the asparagine synthetase mu
tants. This difference is most apparent for Asn 3, which suffered
relatively little damage from the incubation at semipermissive
conditions in the absence of asparaginase.
To confirm these results using incubation conditions which
themselves had no toxic effect on any of the asparagyl-tRNA
synthetase mutants, cultures were exposed to asparaginase in
medium containing normal levels of asparagine. The results
shown in Chart 1b again show that the mutants deficient in
asparagyl-tRNA synthetase were far more sensitive to the cellkilling activity of asparaginase than the mutants deficient in
asparagine synthetase.
These results raised the possibility that the variability in
asparaginase sensitivity of human leukemic cells could be due
to defects in both asparagine synthetase and asparagyl-tRNA
3105
M. M. Y. Waye and C. P. Stanners
synthetase. Two human T-cell lines, RPMI8402 and MOLT-4F,
which were much more sensitive to asparaginase than 2 human
B-cell lines, RPMI 8392 and B46M (11), however, showed low
levels of asparagine synthetase only (Table 1).
DISCUSSION
The recent isolation of both asparagine synthetase (18)- and
asparagyl-tRNA synthetase (15, 16)-deficient mutants in the
same cell line, CHO, has enabled us to examine a possible role
of both these enzymes in determining asparaginase sensitivity
of mammalian cells. We find that the conditionally defective fs
asparagyl-tRNA synthetase mutants were far more sensitive to
asparaginase than the defective asparagine synthetase mu
tants, even when maintained under fully permissive conditions.
The cell-killing action of the drug on the former mutants is
probably due to the progressive permanent inactivation of this
essential protein biosynthetic enzyme by extreme asparagine
starvation as reported recently in detail for several aminoacyltRNA synthetase CHO cell mutants under nonpermissive con
ditions (14).
What are the possible clinical ramifications of this observa
tion? It would seem unlikely that human neoplastic cells would
have strong mutations leading to inactive aminoacyl-tRNA synthetases, as these would be lethal. Weak mutations, producing
no defects in these enzymes under normal conditions, however,
are possible and would, as we have shown, lead to greatly
increased sensitivity to drugs such as asparaginase. We found
no evidence for asparagyl-tRNA synthetase mutations in 2
human T-lymphoid leukemic cell lines which were highly sen
sitive to asparaginase but feel that more sensitive tests for
cryptic mutations in these enzymes and results with a much
wider spectrum of human neoplastic cells are required before
any conclusions can be drawn. We are currently developing
such tests using our CHO cell mutants as a guide.
ACKNOWLEDGMENTS
We thank Dr. S. Arfin for providing AH5 and Dr. J. Minowada for providing the
human lymphoid cell lines. We thank W. A. Mehring for technical assistance.
3106
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