Vol.
167,
No.
February
28,
BIOCHEMICAL
1, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
258-264
AMPLIFICATION OF THE GENES FOR BOTH COMPONENTS OF RIBONUCLEOTIDE REDUCTASE IN HYDROXYUREA RESISTANT NANNALIAN CELJS Robert
A.R.
Hurta
and Jim A. Wright1
Departments of Biochemistry and Microbiology, Department of Internal Medicine and Manitoba Institute of Cell Biology, University of Manitoba, 100 Olivia Street Winnipeg, Manitoba, Canada R3E OV9 Received
January
18,
1990
Ribonucleotide reductase catalyzes the formation of deoxyribonucleotides from ribonucleoside diphosphate precursors, and is a rate-limiting step in the synthesis of DNA. The enzyme consists of two dissimilar subunits usually called Ml and M2. The antitumor agent, hydroxyurea, is a specific inhibitor of DNA synthesis and acts by destroying the tyrosyl free radical of the M2 subunit of ribonucleotide reductase. Two highly drug resistant cell lines designated HR-15 and HR-30 were isolated by exposing a population of mouse L cells to increasing concentrations of hydroxyurea. HR-15 and HR-30 cells contained elevated levels of ribonucleotide reductase activity, and were 68 and 103 times, respectively, more resistant than wild type to the cytotoxic effects of hydroxyurea. Northern and Southern blot analysis indicated that the two drug resistant lines contained elevated levels of M2 mRNA and M2 gene copy numbers. Similar studies with Ml specific cDNA demonstrated that HR-15 and HR-30 cell lines also contained increased Ml message levels, and showed Ml gene amplification. Mutant cell lines altered in expression and copy numbers for both the Ml and M2 genes are useful for obtaining information relevant to the regulation of ribonucleotide reductase, and its role in DNA synthesis and cell proliferation. 0 1990 Academic Press, Inc.
The antitumor synthesis useful cells
agent,
and arrests tool
in
cell
tiator
(5,6). (7),
process
for
reductase,
range
studies
it
hydroxyurea which
is
whom correspondence
is
has
0006-291X/90 $1.50 Copyright 0 1990 b.v Academic Press, Inc. All rights of reproduction in any form reserved.
(1,2),
inhibitor making
Hydroxyurea as well
shown promise
agent
in treating
of psoriasis
the highly
should
(3). tumors,
proliferation
responsible
a specific
and has been used
of solid
as a myelosuppressive the
is
at S phase
(4),
Furthermore,
and in controlling action
growth
synchrony
of a wide
leukemia
'To
cell
by a diffusion
treatment
hydroxyurea,
for
regulated the
de novo
be addressed.
258
of DNA the
enters
clinically as acute
drug
mammalian in the
and chronic
as a radiation polycythemia (9).
enzyme
The primary ribonucleotide
conversion
a
potenvera
(8), site
of ribonucleo-
of
Vol.
167,
No.
tides
1, 1990
BIOCHEMICAL
to deoxyribonucleotides
This
reaction
plays
is
called
cells,
(1,2),
rate-limiting
an important
mammalian
role this
for
in the enzyme
Ml and M2 (1,2,10).
of 170,000 Protein
a dimer
stoichiometric required
amounts for
activity
regulation
contains
Ml is
of non-heme
iron
the
isolation
ribonucleotide problem lead
has been reductase
in
the
to the
properties
treatment
development are
novel
molecular
cells
directly
selected
agent,
interest
MATEXIALS
AND METHODS
for
as a selective
(15),
sites
is
In this
involving
ribonucleotide
resistance
to high
(11).
tyrosyl of
free the
has been
in cell
a major
free culture
in
clinical processes
drug report reductase
concentrations
radical
tyrosyl
modifications
molecular
exhibiting
(15-17).
weight
and contains
agent
with
and the
populations
In
of hydroxyurea
destruction
enzyme
often
a molecular
binding
lines
the
(1,2).
of 88,000
Drug resistance
of cancer of cell
alterations
with
and a unique
cell
(1,2).
of obvious
antitumo:c
useful
resistant
of DNA.
components
The mode of action
very
of drug
synthesis
division
a dimer
weight
COMMUNICATIONS
and therefore
and effector
to involve --in vivo and in vitro within the M2 protein (13,14).
Hydroxyurea for
the
of cell
shown both radical
for
two dissimilar
a molecular
(12,13).
RESEARCH
DNA synthesis,
substrate with
BIOPHYSICAL
required
Protein
and possesses
M.2 is
AND
that
resistant we describe in mammalian of the
hydroxyurea.
Cell lines and culture conditions. Cells were routinely cultured at 37°C on plastic tissue culture plates (Lux Scientific, Ltd.) in a-minimal essential medium (Flow Laboratories, Ltd.) supplemented with 10% fetal calf serum (Gibco, Ltd.) and antibiotics (18). The procedure used to isolate mouse cell lines with increasing drug resistance characteristics have been described (19). Starting with a wild type population of mouse L cells, the following hydroxyurea concentrations were used in the selections: 0.35 mM; 1.3 mM; 1.5 ml"l; 2.0 mM; 3.0 mM; 4.0 mM; 5.0 mM; 15.0 r&l (HR-15); 30 mM (HR30). Cells were frozen in the presence of growth medium containing 5% dimethyl sulfoxide at each selection step, and those selected between 0.35 mM and 5.0 mM drug have been characterized in detail (20). The drug resistance properties of the HR-15 and HR-30 cell lines presented in this report are stable and remained essentially unchanged during approximately 12 months of continuous culture in the absence of drug selection. The relative colony forming efficiency was defined as the ability to form colonies in the presence of hydroxyurea divided by that ability in the The D10 value is the drug concentration which absence of drug (21). reduces the relative colony forming efficiency to 10% (21).
Genomic DNA was prepared from logarithmically Nucleic acid analysis. growing cells by phenol-chloroform extraction (22), and total cellular RNA was isol.ated from logarithmically growing cells by the guanidium-cesium chloride method (23). Southern and Northern hybridizations were performed Ncol as previously described (e.g. 13,18,20), using a 32P-labelled generated fragment containing the cDNA of clone 65 (Ml) or the Pst 1 fragment of clone 10 (M2) (24). Loading of RNA was determined by probing and densitometric analysis was performed using a with p-actin cDNA (20), Beckman D/J-8 gel scanning spectrophotometer. 259
Vol.
167,
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
and assay of ribonucleotide reductase. Enzyme preparations containing 1 to 4 mg of protein/ml were used to assay for ribonucleotide reductase by a modified method of Steeper and Stuart (25) using [14C] CDP as substrate and snake venom phosphodiesterase to hydrolyze the nucleotides as we have previously described (19,20,26). Preparation
RESULTS AND DISCUSSION Sensitivity
to
cell
HR-15
lines,
mouse L cell gradually
and HR-30 were
population
variant
lines
assaying
to the
colony
tions. selected
lines
cytotoxic
were
of
effects
ability these
very
in
the HR-15
and HR-30 populations
15.5
mM drug.
The DlO value
103-fold
less
reductase
for
to hydroxyurea
levels
HR-15
reductase
/
5.00
(1,2).
8.0 HYDROXYUREA
1.
effects
cell
the wild shown
drugs
that
11.0
14.0
drug
concentra-
Both
drug
of
10.2
were
mM and 0.15
68-
and
line.
cell
frequently
In agreement
by
was only
lines type
two
of hydroxyurea,
DlO values
and HR-30 than
and related
in Fig.
and the compared
mouse L cells
We have
activity.
of ribonucleotide
: 2:o
the
in
type were
type
of
as outlined
cytotoxic
exhibiting
wild
the presence
of various
shown
to the
to hydroxyurea
Ribonucleotide
resistance
are
resistant
sensitive
of the wild
of the parental
that
sensitive
in
the presence
resistant
indicating
a drug
of hydroxyurea
studies
with
hydroxyurea,
hydroxyurea
of hydroxyurea,
The sensitivities
forming
The results
from
selections
concentrations
and Methods".
stable
isolated
by step-wise
increasing
"Materials
Phenotypically
hydroqurea.
lines
selected
possess with
elevated
previous
17.0
I 20.0
(mbl)
Fie. 1. Relative colony forming abilities in the absence and presence of various concentrations of hydroxyurea of wild type (A), HR-15 (w) and Hi30
(0)
cell
lines.
260
mM
Vol.
167,
No.
1, 1990
BIOCHEMICAL
Table
Cell
1:
Lines
AND
Ribonucleotide
type
Table
1 shows
activity,
HR-15 whereas
elevation
cell
showed
that
were
cell
30 cell
respectively.
'Lines,
blot
cell
lines
both
HR-15
situation.
and drug (Fig.
levels
2A)
of M2 message estimated
levels
analysis,
M2
in HR-15 and HR-
using
Ml specific and showed
of Ml message
in the
drug
resistant
Densitometric
that lines
measurements in HR-l5
concentration
message
A Southern
genes.
cell
in Fig.
4A and B.
obvious
that
the
with
M2 cDNA is
showed
and HR-30 scans lines.
analysis
Hind
III
in Fig.
banding
of bands
amplified
A Southern to the that
and
mouse
wild
in both
type
clear
to the wild drug
type
cells in
Ml cDNA is line,
copy number
and
type
resistant
and HR-30
HR-
amplifica-
amplification
with
HR-15
Ml gene
type, endo-
The wild with
in
probed
the
3.
M2 gene
blot
of wild
restriction
pattern,
12 to 14-fold
When compared indicated
blot
DNA, when compared
Ml gene was amplified
analysis
the
shown
similar
of approximately
and HR-30
in HR-15
type
in the type
the Ml
2B),
in Ml
Densitometric
estimates
Therefore,
a
the
(Fig.
increase
with
and probed
Densitometric
encoding to analyze
measurements
blot
situation.
nuclease,
HR-15
used
interesting
type
DNA, digested
gave
in
about
of M2 mRNA levels
above wild
was also
15 and HIR-30 genomic
with
increase
contained
in wild
differences
elevations
reductase
tion
wild
respectively.
Ribonucleotide
mutant
line
were
analysis
Northern
to the wild
a 7- and lo-fold
the
to parental
The cDNA clones
Densitometric
lines.
probe
significant
cells,
lines
a 4-fold
HR-30
reductase
and 150-fold
cDNA as hybridization
HR-30
approximately
resistant
considerable
three of 125-
showed
cell
when compared
levels.
Northern
mRNA increases
were
showed
resistant
of the Ml and M2 transcripts
there
in the
there
cells
message
lines.
when compared
hydroxyurea
of ribonucleotide
amounts
present
3.87 23.1
activity,
the more
reductase
and M2 components resistant
Fold Increase
in activity.
Ribonucleotide
relative
both
CDP reductase
mouse L cells.
enzyme
that
COMMUNICATIONS
Activity
22.6
increased
23-fold
Reductase
0.98 3.79
HR-30
exhibited
RESEARCH
Enzyme Activity (nmole CDP reducedfir/mg)
Wild type HR-15
observations
BIOPHYSICAL
it
the
shown
was cells.
was increased
and HR-30 cells by approximately 3- and 4-fold, respectively. resistance to very high concentrations of hydroxyurea in mouse L
261
Vol.
167,
No.
BIOCHEMICAL
1, 1990
AND
BIOPHYSICAL
-18 8
COMMUNICATIONS
l8S
11 a
02
RESEARCH
-2.3
b
c
-2.0
03
B
A
(kb) a
C
b
Fie. 2. Northern blots of M2 and Ml mRNA in the wild type and drug resistant cell lines. For Northern blots, 20 pg of total cellular RNA isolated from wild type and hydroxyurea resistant variants were run on 1% Loading agarose formaldehyde gels as indicated in Materials and Methods. of RNA was determined by reprobing with ,9-actin cDNA as described. Northern blot of M2 mRNA: (a) wild type, (b) HR-15 and (c) HR-30. E Northern blot of Ml &A: (a) wild type, (b) HR-15 and (c) HR-30. The Autoradiograms were exposed positions of 28s and 18s rRNA are indicated. for 24 hours at -70°C with intensifying screens. Fip. 3. Southern blot and drug resistant cell digested to completion labelled Pst 1 fragment Methods. Autoradiograms ing screens. The lanes
cells
was
accompanied
cleotide
reductase
it
is
of
step
much in
of
activity
cells
resistance,
cell
of
for
Ml
(1,2).
gene The
through by
and
both
genes
selection
key drugs
M2 mRNA to
expression
Ml
its
study gene
of
ribonu-
the
ribonucleotide activity
clearly
profound
role
in
with
potential
the
and
M2
antitumor
can
presence
262
of
that
upon
synthesis
of
gene
DNA
makes
amplification hydroxyurea, in
but
drug-resistant
modifications achieved
step-wise
the
chemotherapeutic
agent,
be
a rate-
Alterations
effects
rarely
shows
is
(1,2).
relatively
amplification in
enzyme
have
levels the
occur
mammalian
proliferation can
designing
resistance
this
cell
activity
present
of
because and
Elevated
accompany in
regulation
synthesis
(1,26),
target (27-29).
frequently
the
reductase the
a logical
expression
amplifications
importance
DNA
ribonucleotide
changes
obvious
Understanding
limiting in
by
genes in genomic DNA of the wild type fig of high molecular weight DNA were Blots were hybridized with a 32Pas described in Materials and for 48 hours at -70°C with intensifytype; (b) HR-15 and (c) HR-30.
reductase.
Conclusions.
biology
analysis of M2 lines. Twenty with Hind III. of the M2 cDNA were exposed are: (a) wild
at increases
in very
high in
Ml
gene drug
Vol.
167,
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
23.1 6.4 6.7 4.3
2.3 2.6
:kb) -
a
a
b
c
Fig. 4:. Southern blot analysis of Ml genes in genomic DNA of the wild type and drug-resistant cell lines. Twenty pg of high molecular weight DNA were digested to completion with EcoRl a or Hind III m. Blots were hybridized with a 32 P-labelled Ncol fragment of the Ml cDNA as described in Materials and Methods. Autoradiograms were exposed for 72 hours at -70°C with intensifying screens. The lanes are: (a) wild type, (b) HR-15 and (c) HR-30 and m: (a) wild type, (b) HP:-15 and (c) HR-30.
hydroxyur-ea resistance selection
concentrations. involving without
This
Ml gene further
indicates
amplification
genetic
for
the
first
can occur
manipulation
time
by direct
(30,31).
Mutants
that
drug
drug of this
in the expression of both the Ml and M2 genes provide a type p altered valuable approach for investigations of the complex regulation of mammalian ribonucleotide
reductase
and its
key role
in
the
synthesis
of DNA (1,2,32).
ACKNOWLELXXENTS Financial support for this work was provided by the National Cancer Institute of Canada, and the Natural Sciences and Engineering Research We thank the Sellers Foundation, Department Council of Canada (to J.A.W.). of Internal Medicine, University of Manitoba for a postdoctoral fellowship for R.A.R.H.. Bela Tiwari's assistance with colony-forming assays was greatly appreciated, and we thank Bob Choy and Arthur Chan for helpful discussion. J.A.W. is a Senior Research Scientist of the National Cancer Institute of Canada. REFERENCES 1. 2.
3.
Wright, J.A. (1989). Int. Encyclopedia of Pharmacol. and Therapeut. 128, 89-111. Wright, J.A., McClarty, G.A., Lewis, W.H. and Srinivason, P.R. (1989). In Drug Resistance in Mammalian Cells. Vol. 1. Antimetabolite and cytotoxic analogues. Edited by R. Gupta CRC Press Inc., Boca Raton, Fl. pp 15-27. Ashihara, J. and Baserga, R. (1979). Methods Enzymol. 58, 259-261. 263
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4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(1987). Biochem. Cell Biol. Tagger, A.Y., Boux, J. and Wright, J.A. 65, 925-929. Engstrom, P.F., MacIntyre, J.M., Mittelman, A. and Klaassen, D.J. Am. J. Clin. Oncol. 7, 313-318. (1984). Bolin, R.W., Robinson, W.A., Sutherland, J. and Hamman, R.F. (1982). Cancer 50, 1683-1686. (1983). Piver, M.S., Barlow, J.J., Vongtania, V. and Blumenson, L. Am. J. Obstet. Gynecol. 147, 803-808. Donovan, P.B., Kaplan, M.E., Goldberg, J.D., Tatarsky, I., Nejean, Y., Stilberstein, E.B., Knospe, W.H., Lazlo, J., Mack, K., Berk, P.D. and Wasserman, L.R. (1984). Am. J. Hematol. 17, 329-334. McDonald, C.J. (1981). Pharmacol. Ther. 14, l-24. Hopper, S. (1972). J. Biol. Chem. 247, 3336-3340. Thelander, L., Eriksson, S. and Akerman, M. (1980). J. Biol. Chem. 255, 7426-7432. Thelander, M., Graslund, A. and Thelander, L. (1985). J. Biol. Chem. 260, 2737-2741. McClarty, G,, Chan, A.K., Engstrom, Y., Wright, J.A. and Thelander, L. (1987). Biochemistry 26, 8004-8011. Graslund, A., Ehrenberg, A. and Thelander, L. (1982). J. Biol. Chem. 257, 5711-5715. Stark, G.R. (1986). Cancer Surv. 5, l-23. Stark, G.R., Debatisse, M., Giulotto, E. and Wahl, G.M. (1989). Cell 57, 901-908. (1988). Schimke, R.T. J. Biol. Chem. 263, 5989-5992. Wright, J.A., Alam, T.G., McClarty, G.A., Tagger, A.Y. and Thelander, Somat. Cell Mol. Genet. 13, 155-165. L. (1987). McClarty, G.A., Chan, A. and Wright, J.A. (1986). Somat. Cell Mol. Genet. 12, 121-131. Choy, B.K., McClarty, G.A., Chan, A.K., Thelander, L. and Wright, J.A. (1988). Cancer Res. 48, 2029-2035. Hards, R.G. and Wright, J.A. (1981). J. Cell. Physiol. 106, 309-319. Bin, N. and Stafford, D.W. (1976). Nucleic Acids Res. 3, 2303-2308. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979). Biochemistry 18, 5294-5299. Thelander, L. and Berg, P. (1986). Mol. Cell. Biol. 6, 3433-3442. Steeper, J.P. and Stuart, C.D. (1970). Anal. Biochem. 34, 123-130. Choy, B.K., McClarty, G.A. and Wright, J.A. (1989). Biochem. Biophys. Res. Commun. 162, 1417-1424. Elford, H.L. and van't Riet, B. (1989). Int. Encyclopedia of Pharmacol. and Therapeut. 128, 217-233. Veale, D., Carmichael, J., Cantwell, B.M.J., Elford, H.L., Blackie, R ., Kerr, D.J., Kaye, S.B. and Harris, A.L. (1988). Br. J. Cancer 58, 70-72. Moore, E.C. and Hurlbert, R.B. (1989). Int. Encyclopedia of Pharmacol. and Therapeut. 128, 165-201. Lewis, W.H. and Srinivason, P.R. (1983). Mol. Cell. Biol. 3, 10531061. Cocking, J.M., Tonin, P.N., Stokoe, N.M., Wensing, E.J., Lewis, W.H. and Srinivason, P.R. (1987). Somat. Cell. Mol. Genet. 13, 221-233. Jackson, R.C. (1989). Int. Encyclopedia of Pharmacol. and Therapeut. 128, 89-111.
264
167,
No.
February
28,
BIOCHEMICAL
1, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
258-264
AMPLIFICATION OF THE GENES FOR BOTH COMPONENTS OF RIBONUCLEOTIDE REDUCTASE IN HYDROXYUREA RESISTANT NANNALIAN CELJS Robert
A.R.
Hurta
and Jim A. Wright1
Departments of Biochemistry and Microbiology, Department of Internal Medicine and Manitoba Institute of Cell Biology, University of Manitoba, 100 Olivia Street Winnipeg, Manitoba, Canada R3E OV9 Received
January
18,
1990
Ribonucleotide reductase catalyzes the formation of deoxyribonucleotides from ribonucleoside diphosphate precursors, and is a rate-limiting step in the synthesis of DNA. The enzyme consists of two dissimilar subunits usually called Ml and M2. The antitumor agent, hydroxyurea, is a specific inhibitor of DNA synthesis and acts by destroying the tyrosyl free radical of the M2 subunit of ribonucleotide reductase. Two highly drug resistant cell lines designated HR-15 and HR-30 were isolated by exposing a population of mouse L cells to increasing concentrations of hydroxyurea. HR-15 and HR-30 cells contained elevated levels of ribonucleotide reductase activity, and were 68 and 103 times, respectively, more resistant than wild type to the cytotoxic effects of hydroxyurea. Northern and Southern blot analysis indicated that the two drug resistant lines contained elevated levels of M2 mRNA and M2 gene copy numbers. Similar studies with Ml specific cDNA demonstrated that HR-15 and HR-30 cell lines also contained increased Ml message levels, and showed Ml gene amplification. Mutant cell lines altered in expression and copy numbers for both the Ml and M2 genes are useful for obtaining information relevant to the regulation of ribonucleotide reductase, and its role in DNA synthesis and cell proliferation. 0 1990 Academic Press, Inc.
The antitumor synthesis useful cells
agent,
and arrests tool
in
cell
tiator
(5,6). (7),
process
for
reductase,
range
studies
it
hydroxyurea which
is
whom correspondence
is
has
0006-291X/90 $1.50 Copyright 0 1990 b.v Academic Press, Inc. All rights of reproduction in any form reserved.
(1,2),
inhibitor making
Hydroxyurea as well
shown promise
agent
in treating
of psoriasis
the highly
should
(3). tumors,
proliferation
responsible
a specific
and has been used
of solid
as a myelosuppressive the
is
at S phase
(4),
Furthermore,
and in controlling action
growth
synchrony
of a wide
leukemia
'To
cell
by a diffusion
treatment
hydroxyurea,
for
regulated the
de novo
be addressed.
258
of DNA the
enters
clinically as acute
drug
mammalian in the
and chronic
as a radiation polycythemia (9).
enzyme
The primary ribonucleotide
conversion
a
potenvera
(8), site
of ribonucleo-
of
Vol.
167,
No.
tides
1, 1990
BIOCHEMICAL
to deoxyribonucleotides
This
reaction
plays
is
called
cells,
(1,2),
rate-limiting
an important
mammalian
role this
for
in the enzyme
Ml and M2 (1,2,10).
of 170,000 Protein
a dimer
stoichiometric required
amounts for
activity
regulation
contains
Ml is
of non-heme
iron
the
isolation
ribonucleotide problem lead
has been reductase
in
the
to the
properties
treatment
development are
novel
molecular
cells
directly
selected
agent,
interest
MATEXIALS
AND METHODS
for
as a selective
(15),
sites
is
In this
involving
ribonucleotide
resistance
to high
(11).
tyrosyl of
free the
has been
in cell
a major
free culture
in
clinical processes
drug report reductase
concentrations
radical
tyrosyl
modifications
molecular
exhibiting
(15-17).
weight
and contains
agent
with
and the
populations
In
of hydroxyurea
destruction
enzyme
often
a molecular
binding
lines
the
(1,2).
of 88,000
Drug resistance
of cancer of cell
alterations
with
and a unique
cell
(1,2).
of obvious
antitumo:c
useful
resistant
of DNA.
components
The mode of action
very
of drug
synthesis
division
a dimer
weight
COMMUNICATIONS
and therefore
and effector
to involve --in vivo and in vitro within the M2 protein (13,14).
Hydroxyurea for
the
of cell
shown both radical
for
two dissimilar
a molecular
(12,13).
RESEARCH
DNA synthesis,
substrate with
BIOPHYSICAL
required
Protein
and possesses
M.2 is
AND
that
resistant we describe in mammalian of the
hydroxyurea.
Cell lines and culture conditions. Cells were routinely cultured at 37°C on plastic tissue culture plates (Lux Scientific, Ltd.) in a-minimal essential medium (Flow Laboratories, Ltd.) supplemented with 10% fetal calf serum (Gibco, Ltd.) and antibiotics (18). The procedure used to isolate mouse cell lines with increasing drug resistance characteristics have been described (19). Starting with a wild type population of mouse L cells, the following hydroxyurea concentrations were used in the selections: 0.35 mM; 1.3 mM; 1.5 ml"l; 2.0 mM; 3.0 mM; 4.0 mM; 5.0 mM; 15.0 r&l (HR-15); 30 mM (HR30). Cells were frozen in the presence of growth medium containing 5% dimethyl sulfoxide at each selection step, and those selected between 0.35 mM and 5.0 mM drug have been characterized in detail (20). The drug resistance properties of the HR-15 and HR-30 cell lines presented in this report are stable and remained essentially unchanged during approximately 12 months of continuous culture in the absence of drug selection. The relative colony forming efficiency was defined as the ability to form colonies in the presence of hydroxyurea divided by that ability in the The D10 value is the drug concentration which absence of drug (21). reduces the relative colony forming efficiency to 10% (21).
Genomic DNA was prepared from logarithmically Nucleic acid analysis. growing cells by phenol-chloroform extraction (22), and total cellular RNA was isol.ated from logarithmically growing cells by the guanidium-cesium chloride method (23). Southern and Northern hybridizations were performed Ncol as previously described (e.g. 13,18,20), using a 32P-labelled generated fragment containing the cDNA of clone 65 (Ml) or the Pst 1 fragment of clone 10 (M2) (24). Loading of RNA was determined by probing and densitometric analysis was performed using a with p-actin cDNA (20), Beckman D/J-8 gel scanning spectrophotometer. 259
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and assay of ribonucleotide reductase. Enzyme preparations containing 1 to 4 mg of protein/ml were used to assay for ribonucleotide reductase by a modified method of Steeper and Stuart (25) using [14C] CDP as substrate and snake venom phosphodiesterase to hydrolyze the nucleotides as we have previously described (19,20,26). Preparation
RESULTS AND DISCUSSION Sensitivity
to
cell
HR-15
lines,
mouse L cell gradually
and HR-30 were
population
variant
lines
assaying
to the
colony
tions. selected
lines
cytotoxic
were
of
effects
ability these
very
in
the HR-15
and HR-30 populations
15.5
mM drug.
The DlO value
103-fold
less
reductase
for
to hydroxyurea
levels
HR-15
reductase
/
5.00
(1,2).
8.0 HYDROXYUREA
1.
effects
cell
the wild shown
drugs
that
11.0
14.0
drug
concentra-
Both
drug
of
10.2
were
mM and 0.15
68-
and
line.
cell
frequently
In agreement
by
was only
lines type
two
of hydroxyurea,
DlO values
and HR-30 than
and related
in Fig.
and the compared
mouse L cells
We have
activity.
of ribonucleotide
: 2:o
the
in
type were
type
of
as outlined
cytotoxic
exhibiting
wild
the presence
of various
shown
to the
to hydroxyurea
Ribonucleotide
resistance
are
resistant
sensitive
of the wild
of the parental
that
sensitive
in
the presence
resistant
indicating
a drug
of hydroxyurea
studies
with
hydroxyurea,
hydroxyurea
of hydroxyurea,
The sensitivities
forming
The results
from
selections
concentrations
and Methods".
stable
isolated
by step-wise
increasing
"Materials
Phenotypically
hydroqurea.
lines
selected
possess with
elevated
previous
17.0
I 20.0
(mbl)
Fie. 1. Relative colony forming abilities in the absence and presence of various concentrations of hydroxyurea of wild type (A), HR-15 (w) and Hi30
(0)
cell
lines.
260
mM
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1, 1990
BIOCHEMICAL
Table
Cell
1:
Lines
AND
Ribonucleotide
type
Table
1 shows
activity,
HR-15 whereas
elevation
cell
showed
that
were
cell
30 cell
respectively.
'Lines,
blot
cell
lines
both
HR-15
situation.
and drug (Fig.
levels
2A)
of M2 message estimated
levels
analysis,
M2
in HR-15 and HR-
using
Ml specific and showed
of Ml message
in the
drug
resistant
Densitometric
that lines
measurements in HR-l5
concentration
message
A Southern
genes.
cell
in Fig.
4A and B.
obvious
that
the
with
M2 cDNA is
showed
and HR-30 scans lines.
analysis
Hind
III
in Fig.
banding
of bands
amplified
A Southern to the that
and
mouse
wild
in both
type
clear
to the wild drug
type
cells in
Ml cDNA is line,
copy number
and
type
resistant
and HR-30
HR-
amplifica-
amplification
with
HR-15
Ml gene
type, endo-
The wild with
in
probed
the
3.
M2 gene
blot
of wild
restriction
pattern,
12 to 14-fold
When compared indicated
blot
DNA, when compared
Ml gene was amplified
analysis
the
shown
similar
of approximately
and HR-30
in HR-15
type
in the type
the Ml
2B),
in Ml
Densitometric
estimates
Therefore,
a
the
(Fig.
increase
with
and probed
Densitometric
encoding to analyze
measurements
blot
situation.
nuclease,
HR-15
used
interesting
type
DNA, digested
gave
in
about
of M2 mRNA levels
above wild
was also
15 and HIR-30 genomic
with
increase
contained
in wild
differences
elevations
reductase
tion
wild
respectively.
Ribonucleotide
mutant
line
were
analysis
Northern
to the wild
a 7- and lo-fold
the
to parental
The cDNA clones
Densitometric
lines.
probe
significant
cells,
lines
a 4-fold
HR-30
reductase
and 150-fold
cDNA as hybridization
HR-30
approximately
resistant
considerable
three of 125-
showed
cell
when compared
levels.
Northern
mRNA increases
were
showed
resistant
of the Ml and M2 transcripts
there
in the
there
cells
message
lines.
when compared
hydroxyurea
of ribonucleotide
amounts
present
3.87 23.1
activity,
the more
reductase
and M2 components resistant
Fold Increase
in activity.
Ribonucleotide
relative
both
CDP reductase
mouse L cells.
enzyme
that
COMMUNICATIONS
Activity
22.6
increased
23-fold
Reductase
0.98 3.79
HR-30
exhibited
RESEARCH
Enzyme Activity (nmole CDP reducedfir/mg)
Wild type HR-15
observations
BIOPHYSICAL
it
the
shown
was cells.
was increased
and HR-30 cells by approximately 3- and 4-fold, respectively. resistance to very high concentrations of hydroxyurea in mouse L
261
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No.
BIOCHEMICAL
1, 1990
AND
BIOPHYSICAL
-18 8
COMMUNICATIONS
l8S
11 a
02
RESEARCH
-2.3
b
c
-2.0
03
B
A
(kb) a
C
b
Fie. 2. Northern blots of M2 and Ml mRNA in the wild type and drug resistant cell lines. For Northern blots, 20 pg of total cellular RNA isolated from wild type and hydroxyurea resistant variants were run on 1% Loading agarose formaldehyde gels as indicated in Materials and Methods. of RNA was determined by reprobing with ,9-actin cDNA as described. Northern blot of M2 mRNA: (a) wild type, (b) HR-15 and (c) HR-30. E Northern blot of Ml &A: (a) wild type, (b) HR-15 and (c) HR-30. The Autoradiograms were exposed positions of 28s and 18s rRNA are indicated. for 24 hours at -70°C with intensifying screens. Fip. 3. Southern blot and drug resistant cell digested to completion labelled Pst 1 fragment Methods. Autoradiograms ing screens. The lanes
cells
was
accompanied
cleotide
reductase
it
is
of
step
much in
of
activity
cells
resistance,
cell
of
for
Ml
(1,2).
gene The
through by
and
both
genes
selection
key drugs
M2 mRNA to
expression
Ml
its
study gene
of
ribonu-
the
ribonucleotide activity
clearly
profound
role
in
with
potential
the
and
M2
antitumor
can
presence
262
of
that
upon
synthesis
of
gene
DNA
makes
amplification hydroxyurea, in
but
drug-resistant
modifications achieved
step-wise
the
chemotherapeutic
agent,
be
a rate-
Alterations
effects
rarely
shows
is
(1,2).
relatively
amplification in
enzyme
have
levels the
occur
mammalian
proliferation can
designing
resistance
this
cell
activity
present
of
because and
Elevated
accompany in
regulation
synthesis
(1,26),
target (27-29).
frequently
the
reductase the
a logical
expression
amplifications
importance
DNA
ribonucleotide
changes
obvious
Understanding
limiting in
by
genes in genomic DNA of the wild type fig of high molecular weight DNA were Blots were hybridized with a 32Pas described in Materials and for 48 hours at -70°C with intensifytype; (b) HR-15 and (c) HR-30.
reductase.
Conclusions.
biology
analysis of M2 lines. Twenty with Hind III. of the M2 cDNA were exposed are: (a) wild
at increases
in very
high in
Ml
gene drug
Vol.
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No.
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BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
23.1 6.4 6.7 4.3
2.3 2.6
:kb) -
a
a
b
c
Fig. 4:. Southern blot analysis of Ml genes in genomic DNA of the wild type and drug-resistant cell lines. Twenty pg of high molecular weight DNA were digested to completion with EcoRl a or Hind III m. Blots were hybridized with a 32 P-labelled Ncol fragment of the Ml cDNA as described in Materials and Methods. Autoradiograms were exposed for 72 hours at -70°C with intensifying screens. The lanes are: (a) wild type, (b) HR-15 and (c) HR-30 and m: (a) wild type, (b) HP:-15 and (c) HR-30.
hydroxyur-ea resistance selection
concentrations. involving without
This
Ml gene further
indicates
amplification
genetic
for
the
first
can occur
manipulation
time
by direct
(30,31).
Mutants
that
drug
drug of this
in the expression of both the Ml and M2 genes provide a type p altered valuable approach for investigations of the complex regulation of mammalian ribonucleotide
reductase
and its
key role
in
the
synthesis
of DNA (1,2,32).
ACKNOWLELXXENTS Financial support for this work was provided by the National Cancer Institute of Canada, and the Natural Sciences and Engineering Research We thank the Sellers Foundation, Department Council of Canada (to J.A.W.). of Internal Medicine, University of Manitoba for a postdoctoral fellowship for R.A.R.H.. Bela Tiwari's assistance with colony-forming assays was greatly appreciated, and we thank Bob Choy and Arthur Chan for helpful discussion. J.A.W. is a Senior Research Scientist of the National Cancer Institute of Canada. REFERENCES 1. 2.
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Wright, J.A. (1989). Int. Encyclopedia of Pharmacol. and Therapeut. 128, 89-111. Wright, J.A., McClarty, G.A., Lewis, W.H. and Srinivason, P.R. (1989). In Drug Resistance in Mammalian Cells. Vol. 1. Antimetabolite and cytotoxic analogues. Edited by R. Gupta CRC Press Inc., Boca Raton, Fl. pp 15-27. Ashihara, J. and Baserga, R. (1979). Methods Enzymol. 58, 259-261. 263
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