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L-Asparaginase effects on intact murine leukemia cells and on isolated cell plasma membranes
Vol. 48, No. 1, 1972
BIOCHEMICAL
L-ASPARAGINASE
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EFFECTS ON INTACT MURINE LEUKEMIA CELLS
AND ON ISOIATFD David
Kessel
CELL PLASMA MEMBRANES and H. Bruce
Bosmann
Department of Pharmacology and Division of Oncology, University of Rochester Medical Center Rochester, New York 14642 Received
May
19,
1972
Summary
The enzyme L-asparaginase catalyzes solubilization of a minor portion of the total glycoprotein of isolated plasma membranes from the L517gY murine leukemia. Such a solubilization was not found using intact L5178Y cells, or using cells or plasma membranes of the asparaginaseresistant L5178Y/ASP cells. The mechanism of asparaginase action therefore involves inhibition of glycoprotein and protein synthesis required for maintenance of membrane structure, rather than a direct lytic action on preformed membranes of the intact cell. The enzyme L-asparaginase activity,
as summarized
of exogenous synthesis that
supplies
in cells
has marked
in recent
L-asparaginase
has a selective
fetuin tial
hydrolysis (4)
solubilization
present
of cell
examination
membranes;
the
cell
were
lines
Methods
of effects
for
described, Plasma
membranes
cated,
cells
3 H-L-fucose
were
along
were
prepared
labeled
12 hours,
with
and again
Cop.vright 0 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
for
as described
35
substan-
has prompted cells
L5178Y/ASP
and L5178YJASP these
have been
cells (7).
injection collection
the
and on isolated
by Dods et al.
before
to
derivatives,
(7) describing
isolating
by intraperitoneal 2 hours
on synthesis
and the resistant
L5178Y
methods
in vivo
report
enzyme on intact
maintaining
evidence
the enzyme was found asparagine
L5178Y
of protein
effect
by L-asparaginase
asparaginase-sensitive
The procedures
inhibition
inhibitory
A recent
of the
depletion
We have reported
Rxcthemore,
membranes
here.
to a rapid
and early
(6).
employed
Enzyme-catalyzed
molecular-weight
and asparaginyl-tRNA
and immunosuppressive
synthetase.
(4,5). of two high
(l-3).
leads
asparagine
of asparaginyl-glycoproteins catalyze
reviews
of L-asparagine lacking
anti-tumor
(5). Where
indi-
of 250 uCi of
of cells
from
Vol. 48, No. 1, 1972
BIOCHEMICAL
tumor-bearing animals. hydrate,
Cur methods for measuring anthrone-reactive
fucose, and sialic
cell surface glycoprotein
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
carbo-
acid, together with the procedure for isolating bound by papain-sensitive
linkages,
have been
reported (8). Plasma membranepreparations containing glycoprotein labeled with 3 H-L-fucose were prepared from cells exposed to this radioactive precursor in vivo as described above. buffer
These membraneswere incubated in 0.1 M Tria
at pH 7.2 for 30 min with the specified
ase (EC-2, derived from Escherichia &, zation of radioactivity,
concentration
and supplied by Merck).
and of anthrone-positive
measured. Cells were centrifuged
of L-asparagin-
material
Solubili-
and protein was
at 105,000 x g and the supernatant was
analyzed for released 3R-fucose radioactivity,
anthrone positive
material,
and protein. Intact
cells,
in Earle's salts
previously
(5 x lo7 cells/l0
enzyme for 30 min.
of the cell pellets
by centrifugation,
Results
The effluent
were suspended
and acid-
was measured. The supernatant
was concentrated in vacua and fractionated
BioRad P-100 gel.
in viva,
ml) and treated with 10 or 100 U/ml of
The cells were collected
insoluble radioactivity fluid
labeled with %-L-fucose
on a 1 x 45 cm column of
was monitored for radioactivity.
When L5178Y or L5178Y/ASP cells containing labeled glycoproteins were treated with asparaginase in vitro,
of radioactive
material
into the supernatant fluid
Analysis of the supernatant fluids weight radioactivity
no significant
was found (Table 1).
showed that the released high molecular-
had an average molecular weight of 100,000.
or treated tubes, more radioactivity
In control
was released from L5178Y cells,
which may be related to the higher level of cell-surface cells as comparedwith L5178Y/ASP (Table 2). cell surface glycopeptides in the resistant cells indicates
release
This finding
glycoprotein
a finding in these
of lower levels of
cells compared to the sensitive
that L-asparaginase resistance is a consequence of lower
36
Vol. 48, No. 1, 1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1. Results of treatment of 3H-fucose labeled intact with asparaginase.
Cell line
Enzyme level (U/d>
L517BY
Cell pellet
High mol. wt. radioactivity in the supernatant fluid
(counts/min)
(counts/min)
0
35,000
600
10
36,500 34,575
575 610
21,600 20,100
310 295
20,700
305
100
L5178Y/AsP
cells
0 10
100
Cells (50 mg, wet weight) were incubated with specified levels of enzyme for 30 min; the cells were then collected and acid-insoluble radioactivity associated with cell pellets was measured. The supernatant fluid was desalted on PlOO gel columnsand the high molecular weight radioactivity (average mol. wt. 10,000) was measured. Low molecular weight material (mol. wt. under 500) represented approximately 3000 counts/min from each supernatant, and was chromatographically identified as fucose.
Table 2.
Cell-surface
glycoprotein
Anthrone c-e/P)
Cell Line
in papain-sensitive
linkages
Fucose (UP/d
Sialic acid c.dl3)
L5178Y
480
175
206
L5178Y/ASP
130
56
80
Cells (300 mg wet weight) were treated with papain (g), and release of glycoprotein into the mediumwas measured. Units are in terms of galactose equivalents (anthrone test) and micrograms of fucose or sialic acid released per gram of cells, wet weight. levels of plasma membraneglycoproteins, for actinomycin D resistant glycoproteins
(g,9).
cells,
the exact opposite of the finding
which have higher levels of plasma membrane
The lower levels of surface glycoproteins
37
in the
Vol. 48, No. 1, 1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 3. Effect of L-asparaginase on murine leukemia cell plasma membranes. Data from 2 experiments. Enzyme Wml>
Cell Line L517BY/ASP
Anthrone (M/P)
isolated
Protein (Lowry) b.dg)
0 1 10 100
a 2 3
0 0 0 0
L5178Y
0 1 10 100
2 12 26 23
0 11 20 15
L5178Y/ASP
0 1 10 100
0 0 0 0
1 1 2 1
0 1
6 22 46 34
1 32 46 33
L5178Y
10
100
Isolated plasma membraneswere incubated with specified levels of enzyme for 30 min, and release of anthrone-reactive materFa1 (galactose units) and protein was measured. Units are in terms of micrograms of galactose and micrograms of protein released per gram of wet cells from which the membraneswere derived.
L-asparaginase resistant
cell line may be the result
of adaptation or selec-
tion for plasma membraneswhich do not require asparaginyl-glycoproteins. That is, sialyl
the lower amounts of anthrone-positive glycopeptide
reflect
material
released by papain from the L5178Y/ASP cells
the fact that the L-asparaginase resistant
essentially
devoid of asparaginyl-glycoproteins
these glycoproteins
and fucosyl and
which L-asparaginase inhibits
(Table 2) may
cell plasma membranesare
since it is the synthesis of (4,5).
Treatment of isolated plasma membranesfrom L5178Y cells with asparaginase resulted in the release of small amounts of anthrone-positive and protein
into an acid-soluble
fraction
(Table 3).
This result was not
found using isolated plasma membranesfrom L5178Y/ASP cells.
38
material
Furthermore,
BIOCHEMICAL
Vol. 48, No. 1, 1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on 3 H-fucose
Table 4. Effect of L-asparaginase labeled cell plasma membranes L5178Y (counts/min)
Enzyme Level (D/ml)
L5178YjASP (counts/min)
0
290
284
1
430
285
10
630
273
100
810
285
Isolated plasma membranes from cells labeled in vivo with 3H-L-fucose were incubated with asparaginase for 30 min; release of radioactivity into the supernatant fluid was measured. Each experiment represents approximately 1 mg of plasma membranes per tube; total radioactivity was 23,000 counts/min/mg of plasma membrane protein.
treatment
with
asparaginase
L5178Y solubilized (Table
4).
using
some radioactivity,
We could
not
phase-contrast
or isolated
detect
membranes
The data
L-asparaginase. could
such a release
resulting therefore,
from
treatment
in a preferential glycoproteins
(4,5).
and have now reported
using
membrane postulated
Jasin that
treatment
minor
not
intact
so affected
L5178Y
in vivo
with cell
lysis
cells
30 minutes.
of glycoprotein
by the action
of
by the
enzyme,
nor
The rapid
cell
death
asparaginase
(10)
resulting
cannot,
from
an action
structures. that
treatment
inhibition
of synthesis
and Prager
(11)
treatment
cells.
membranes
intact
for
amounts
from
L5178YjASP
of either
of L-asparaginase
were
isolated
using
L5178Y membranes
to an immediate
and early
not found
show that
isolated
of L5178Y
of the enzyme on preformed previously
here
membranes
in the L5178Y plasma
after
100 U/ml
membranes
be found
be attributed
We have
with
from
L5178YfASP
a result
(1000X)
presented
are released
plasma
any alterations
microscopy
plasma
Discussion
of fucose-labeled
with
39
with
asparaginase
of asparaginyl-
have confirmed asparaginase
results
this
impairs
observation synthesis
of
Vol. 48, No. 1, 1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
immunoglobulin IgG, an asparaginyl-glycoprotein, synthesis of other asparaginyl-glycoproteins rapidly
by spleen.
plastic
would be expected to result
in
Thus a mechanismof action of L-asparaginase may be inhibi-
tion of synthesis of asparaginyl-glycoproteins synthesis in rapidly
of
which play an important role in
turning over plasma membraneintegrity
early cell lysis.
Inhibition
which in turn inhibits
membrane
turning over cell plasma membranes. The fact that neo-
cells are somewhatmore responsive to L-asparaginase may result
their more rapid division,
the lesser likelihood
of neoplastic
cells
from
to be in
the GOphase of growth, and the more rapid membraneturnover of neoplastic cells. This work was supported in part by NIH grants CA 12085, CA 11242, Ca 11198, CA 13320, and GM 15190. HBB is a recipient of a Research Career Development Award from NI@lS. We thank Regina Bruns, Delena DeHond, Kenneth R. Case, and Richard Ball for excellent technical assistance. References 1. 2.
Broome, J. D., TranaNY Acad. Sci. 30 (l968) 690. Cooney, D. A. and Handschumacher,R. E., Ann. Rev. Pharmacol. 10 (1970) 421.
3. 4. 5. 6. 7. 8. 9. 10. 11.
Prager, M. D., in Oncology 1970, Vol. II, p. 850 (ed. R. L. Clark et al.), Chicago: Year Book Medical Publishers, 1971. Bosmann, Ii. B., and Kessel, D., Nature 226 (1970) 850. Kessel, D. and Bosmann, H. B., PEBSLett. 10 (1970) 85. Kessel, D., Biochim. Biophys. Acta 240 (1971) 554. Dods, R. P., Essner, E., and Barclay, M., Biochem. Biophys. Res. Commun. 46 (1972) 1074. Kessel, D. and Bosmann,Il. B., Cancer Res. 30 (1970) 2695. Bosmann,H. B., Nature 233 (1971) 566. Hofer, K. G., DeBenedetto, J., and Hughes, W.L., 2. Krebsforsch. 74 (1970) 34. Jasin, H. E. and Prager, M. D., Clin. Exp. Immunol. 10 (1972) 515.
40
BIOCHEMICAL
L-ASPARAGINASE
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EFFECTS ON INTACT MURINE LEUKEMIA CELLS
AND ON ISOIATFD David
Kessel
CELL PLASMA MEMBRANES and H. Bruce
Bosmann
Department of Pharmacology and Division of Oncology, University of Rochester Medical Center Rochester, New York 14642 Received
May
19,
1972
Summary
The enzyme L-asparaginase catalyzes solubilization of a minor portion of the total glycoprotein of isolated plasma membranes from the L517gY murine leukemia. Such a solubilization was not found using intact L5178Y cells, or using cells or plasma membranes of the asparaginaseresistant L5178Y/ASP cells. The mechanism of asparaginase action therefore involves inhibition of glycoprotein and protein synthesis required for maintenance of membrane structure, rather than a direct lytic action on preformed membranes of the intact cell. The enzyme L-asparaginase activity,
as summarized
of exogenous synthesis that
supplies
in cells
has marked
in recent
L-asparaginase
has a selective
fetuin tial
hydrolysis (4)
solubilization
present
of cell
examination
membranes;
the
cell
were
lines
Methods
of effects
for
described, Plasma
membranes
cated,
cells
3 H-L-fucose
were
along
were
prepared
labeled
12 hours,
with
and again
Cop.vright 0 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
for
as described
35
substan-
has prompted cells
L5178Y/ASP
and L5178YJASP these
have been
cells (7).
injection collection
the
and on isolated
by Dods et al.
before
to
derivatives,
(7) describing
isolating
by intraperitoneal 2 hours
on synthesis
and the resistant
L5178Y
methods
in vivo
report
enzyme on intact
maintaining
evidence
the enzyme was found asparagine
L5178Y
of protein
effect
by L-asparaginase
asparaginase-sensitive
The procedures
inhibition
inhibitory
A recent
of the
depletion
We have reported
Rxcthemore,
membranes
here.
to a rapid
and early
(6).
employed
Enzyme-catalyzed
molecular-weight
and asparaginyl-tRNA
and immunosuppressive
synthetase.
(4,5). of two high
(l-3).
leads
asparagine
of asparaginyl-glycoproteins catalyze
reviews
of L-asparagine lacking
anti-tumor
(5). Where
indi-
of 250 uCi of
of cells
from
Vol. 48, No. 1, 1972
BIOCHEMICAL
tumor-bearing animals. hydrate,
Cur methods for measuring anthrone-reactive
fucose, and sialic
cell surface glycoprotein
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
carbo-
acid, together with the procedure for isolating bound by papain-sensitive
linkages,
have been
reported (8). Plasma membranepreparations containing glycoprotein labeled with 3 H-L-fucose were prepared from cells exposed to this radioactive precursor in vivo as described above. buffer
These membraneswere incubated in 0.1 M Tria
at pH 7.2 for 30 min with the specified
ase (EC-2, derived from Escherichia &, zation of radioactivity,
concentration
and supplied by Merck).
and of anthrone-positive
measured. Cells were centrifuged
of L-asparagin-
material
Solubili-
and protein was
at 105,000 x g and the supernatant was
analyzed for released 3R-fucose radioactivity,
anthrone positive
material,
and protein. Intact
cells,
in Earle's salts
previously
(5 x lo7 cells/l0
enzyme for 30 min.
of the cell pellets
by centrifugation,
Results
The effluent
were suspended
and acid-
was measured. The supernatant
was concentrated in vacua and fractionated
BioRad P-100 gel.
in viva,
ml) and treated with 10 or 100 U/ml of
The cells were collected
insoluble radioactivity fluid
labeled with %-L-fucose
on a 1 x 45 cm column of
was monitored for radioactivity.
When L5178Y or L5178Y/ASP cells containing labeled glycoproteins were treated with asparaginase in vitro,
of radioactive
material
into the supernatant fluid
Analysis of the supernatant fluids weight radioactivity
no significant
was found (Table 1).
showed that the released high molecular-
had an average molecular weight of 100,000.
or treated tubes, more radioactivity
In control
was released from L5178Y cells,
which may be related to the higher level of cell-surface cells as comparedwith L5178Y/ASP (Table 2). cell surface glycopeptides in the resistant cells indicates
release
This finding
glycoprotein
a finding in these
of lower levels of
cells compared to the sensitive
that L-asparaginase resistance is a consequence of lower
36
Vol. 48, No. 1, 1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1. Results of treatment of 3H-fucose labeled intact with asparaginase.
Cell line
Enzyme level (U/d>
L517BY
Cell pellet
High mol. wt. radioactivity in the supernatant fluid
(counts/min)
(counts/min)
0
35,000
600
10
36,500 34,575
575 610
21,600 20,100
310 295
20,700
305
100
L5178Y/AsP
cells
0 10
100
Cells (50 mg, wet weight) were incubated with specified levels of enzyme for 30 min; the cells were then collected and acid-insoluble radioactivity associated with cell pellets was measured. The supernatant fluid was desalted on PlOO gel columnsand the high molecular weight radioactivity (average mol. wt. 10,000) was measured. Low molecular weight material (mol. wt. under 500) represented approximately 3000 counts/min from each supernatant, and was chromatographically identified as fucose.
Table 2.
Cell-surface
glycoprotein
Anthrone c-e/P)
Cell Line
in papain-sensitive
linkages
Fucose (UP/d
Sialic acid c.dl3)
L5178Y
480
175
206
L5178Y/ASP
130
56
80
Cells (300 mg wet weight) were treated with papain (g), and release of glycoprotein into the mediumwas measured. Units are in terms of galactose equivalents (anthrone test) and micrograms of fucose or sialic acid released per gram of cells, wet weight. levels of plasma membraneglycoproteins, for actinomycin D resistant glycoproteins
(g,9).
cells,
the exact opposite of the finding
which have higher levels of plasma membrane
The lower levels of surface glycoproteins
37
in the
Vol. 48, No. 1, 1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 3. Effect of L-asparaginase on murine leukemia cell plasma membranes. Data from 2 experiments. Enzyme Wml>
Cell Line L517BY/ASP
Anthrone (M/P)
isolated
Protein (Lowry) b.dg)
0 1 10 100
a 2 3
0 0 0 0
L5178Y
0 1 10 100
2 12 26 23
0 11 20 15
L5178Y/ASP
0 1 10 100
0 0 0 0
1 1 2 1
0 1
6 22 46 34
1 32 46 33
L5178Y
10
100
Isolated plasma membraneswere incubated with specified levels of enzyme for 30 min, and release of anthrone-reactive materFa1 (galactose units) and protein was measured. Units are in terms of micrograms of galactose and micrograms of protein released per gram of wet cells from which the membraneswere derived.
L-asparaginase resistant
cell line may be the result
of adaptation or selec-
tion for plasma membraneswhich do not require asparaginyl-glycoproteins. That is, sialyl
the lower amounts of anthrone-positive glycopeptide
reflect
material
released by papain from the L5178Y/ASP cells
the fact that the L-asparaginase resistant
essentially
devoid of asparaginyl-glycoproteins
these glycoproteins
and fucosyl and
which L-asparaginase inhibits
(Table 2) may
cell plasma membranesare
since it is the synthesis of (4,5).
Treatment of isolated plasma membranesfrom L5178Y cells with asparaginase resulted in the release of small amounts of anthrone-positive and protein
into an acid-soluble
fraction
(Table 3).
This result was not
found using isolated plasma membranesfrom L5178Y/ASP cells.
38
material
Furthermore,
BIOCHEMICAL
Vol. 48, No. 1, 1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on 3 H-fucose
Table 4. Effect of L-asparaginase labeled cell plasma membranes L5178Y (counts/min)
Enzyme Level (D/ml)
L5178YjASP (counts/min)
0
290
284
1
430
285
10
630
273
100
810
285
Isolated plasma membranes from cells labeled in vivo with 3H-L-fucose were incubated with asparaginase for 30 min; release of radioactivity into the supernatant fluid was measured. Each experiment represents approximately 1 mg of plasma membranes per tube; total radioactivity was 23,000 counts/min/mg of plasma membrane protein.
treatment
with
asparaginase
L5178Y solubilized (Table
4).
using
some radioactivity,
We could
not
phase-contrast
or isolated
detect
membranes
The data
L-asparaginase. could
such a release
resulting therefore,
from
treatment
in a preferential glycoproteins
(4,5).
and have now reported
using
membrane postulated
Jasin that
treatment
minor
not
intact
so affected
L5178Y
in vivo
with cell
lysis
cells
30 minutes.
of glycoprotein
by the action
of
by the
enzyme,
nor
The rapid
cell
death
asparaginase
(10)
resulting
cannot,
from
an action
structures. that
treatment
inhibition
of synthesis
and Prager
(11)
treatment
cells.
membranes
intact
for
amounts
from
L5178YjASP
of either
of L-asparaginase
were
isolated
using
L5178Y membranes
to an immediate
and early
not found
show that
isolated
of L5178Y
of the enzyme on preformed previously
here
membranes
in the L5178Y plasma
after
100 U/ml
membranes
be found
be attributed
We have
with
from
L5178YfASP
a result
(1000X)
presented
are released
plasma
any alterations
microscopy
plasma
Discussion
of fucose-labeled
with
39
with
asparaginase
of asparaginyl-
have confirmed asparaginase
results
this
impairs
observation synthesis
of
Vol. 48, No. 1, 1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
immunoglobulin IgG, an asparaginyl-glycoprotein, synthesis of other asparaginyl-glycoproteins rapidly
by spleen.
plastic
would be expected to result
in
Thus a mechanismof action of L-asparaginase may be inhibi-
tion of synthesis of asparaginyl-glycoproteins synthesis in rapidly
of
which play an important role in
turning over plasma membraneintegrity
early cell lysis.
Inhibition
which in turn inhibits
membrane
turning over cell plasma membranes. The fact that neo-
cells are somewhatmore responsive to L-asparaginase may result
their more rapid division,
the lesser likelihood
of neoplastic
cells
from
to be in
the GOphase of growth, and the more rapid membraneturnover of neoplastic cells. This work was supported in part by NIH grants CA 12085, CA 11242, Ca 11198, CA 13320, and GM 15190. HBB is a recipient of a Research Career Development Award from NI@lS. We thank Regina Bruns, Delena DeHond, Kenneth R. Case, and Richard Ball for excellent technical assistance. References 1. 2.
Broome, J. D., TranaNY Acad. Sci. 30 (l968) 690. Cooney, D. A. and Handschumacher,R. E., Ann. Rev. Pharmacol. 10 (1970) 421.
3. 4. 5. 6. 7. 8. 9. 10. 11.
Prager, M. D., in Oncology 1970, Vol. II, p. 850 (ed. R. L. Clark et al.), Chicago: Year Book Medical Publishers, 1971. Bosmann, Ii. B., and Kessel, D., Nature 226 (1970) 850. Kessel, D. and Bosmann, H. B., PEBSLett. 10 (1970) 85. Kessel, D., Biochim. Biophys. Acta 240 (1971) 554. Dods, R. P., Essner, E., and Barclay, M., Biochem. Biophys. Res. Commun. 46 (1972) 1074. Kessel, D. and Bosmann,Il. B., Cancer Res. 30 (1970) 2695. Bosmann,H. B., Nature 233 (1971) 566. Hofer, K. G., DeBenedetto, J., and Hughes, W.L., 2. Krebsforsch. 74 (1970) 34. Jasin, H. E. and Prager, M. D., Clin. Exp. Immunol. 10 (1972) 515.
40