Download Leukemia LI 210 Cell Lines Resistant to

[CANCER RESEARCH 48, 839-843, February 15, 1988]
Leukemia LI 210 Cell Lines Resistant to Ribonucleotide ReducÃ-aseInhibitors1
Joseph G. Cory2 and Gay L. Carter
Division of Medical Oncology, Department of Internal Medicine, University of South Florida College of Medicine, H. Lee Moffitt Cancer Center and Research Institute,
Tampa, Florida 33612
determination of the |1( '|,,,s, ' at least five drug concenlrations were
ABSTRACT
used. Cell aliquols were laken after 3 days for cell counls. Cell counts
were determined in a ( 'miller Counter, Model ZBI.
Leukemia LI 210 cell lines, EDI and ED2, were generated which were
resistant to the cytotoxic effects of deoxyadenosine/erylhro-9-{2hydroxyl-3-nonyl)adenine
and deoxyadenosine/erythro-9-(2-hydroxyl-3non)Hack-nine plus 2,3-dihydro-l//-pyrazole|2,3al¡midazole/Desferal,
re
spectively. The EDI and ED2 were characterized to show that these cell
lines had increased levels of ribonucleotide reducÃ-ase as measured by
CDP reduction. The reducÃ-ase activity in crude cell-free extracts from
the EDI and ED2 cells was not inhibited by dATP. For CDP reducÃ-ase,
the activation by adenylylimido diphosphate and inhibition by dGTP and
d'lTP in these extracts from the EDI and ED2 cells were the same as
Generalion of EDI and ED2 Cell Lines. LI210 cells were incubaled
in Ihe presence of deoxyadenosine, 30 MM/EHNA, 5 MMplus IMPY,
100 MM/Desferal, 30 MMfor 24 h. The cells (75 cells/dish) were Ihen
plaled on sofl agar (Sea-Kern agar, 0.275% with 20% horse serum).
Colonies which grew out were picked after 21 days and cultured in
liquid medium in increasing concentrations of dAdo/EHNA plus
IMPY/Df. After inilial studies utilizing [l4C]cylidine conversion to
| "C'jdeovyeylidinc and incorporalion inlo DNA indicated that mosl of
Ihe resistance was to dAdo/EHNA, Ihe cells were divided inlo iwo
groups. The EDI cells were generated by growing the cells in liquid
medium in which Ihe concentrâtion of dAdo was increased al a conslanl
concentration of EHNA (5 MM).The ED2 cells were generated by
growing Ihe cells in liquid medium in which Ihe concentrane us of dAdo
and IMPY were bolh increased al conslanl concentrations of EHNA
(5 MM)and Df (48 MM).The cells were periodically replated on soft agar
and the surviving colonies plucked and recultured in liquid medium.
The mosl resislani cell lines were mainiained as Ihe slock EDI and
ED2 cell lines.
Preparai inn of Crude Cell-free Exlracls. Equal numbers of wild-lype
EDI, and ED2 cells in midlog phase were laken for Ihe preparation of
Ihe crude exlracts. The cells were pelleted by centrifugation at 500 x g
for 10 min. The cells were then washed two times with cold phosphatebuffered saline and recenlrifuged. The cell pellets were resuspended in
Tris-HCl buffer, 0.02 M, pH 7.0, conlaining dilhioerylhrilol, 1 min al
a concentration of 5 x 10" cells/ml. After swelling for 5 min on ice, Ihe
cell suspension was homogenized wilh a motor-driven Teflon pesile (20
slrokes). The homogenale was centrifuged for 1 h at 21,000 x g al 4"( '.
for the wild-type L1210 cells. The EDI and ED2 cells were highly crossresistant, as measured by growth inhibition, to deoxyguanosine/8-aminoguanosine, 2-fluorodeoxyadenosine,
and 2-fluoroadenine arabinoside.
While the ED2 cells showed resistance to 2,3-dihydro-lff-pyrazole[2,3a)-imidazole/Desferal
(6-fold), the EDI and ED2 cell lines showed
less resistance to hydroxyurea, 4-methyl-S-amino-l-formylisoquinoline
thiosemicarbazone, and the dialdehyde of inosine. These data indicate
that the mechanisms of resistance to the ribonucleotide reducÃ-ase inhib
itors are related to the increased level of ribonucleotide reducÃ-aseactivity
and to the decreased sensitivity of the effector-binding subunit to dATP.
INTRODUCTION
In earlier studies, it was shown that combinations of drugs
directed at the non-heme iron and effector-binding subunits of
ribonucleotide reducÃ-asegave synergistic inhibilion of L1210
cell growth in culture (1-3). Further studies using colony form
ing assays were carried out to show that these combinations of
drugs directed at Ihe subunils of ribonucleolide reducÃ-asealso
resulted in synergistic cytotoxicity (4). In the course of these
studies several colonies grew out in the presence of drug con
centrations which killed greater than 99% of the cells. These
clones were picked and cultured in liquid medium in the pres
ence of the same drugs. Selection of the specific clones was
based on the resislance of the cell lines to growth inhibition
and to resistance to inhibition of [14C]cytidine conversion to
The supernalanl fluid was applied lo a Dowex-1 -acelale column which
had been prewashed wilh Tris-HCl buffer conlaining dilhioerylhrilol,
1 HIM.The enzyme exlract was washed from the column with 2 volumes
of Tris-HCl/dithioerylhrilol. The eluanl was Ihen irealed with ammo
nium sulfate to 80% saturation. The precipitate was collected by cen
trifugation, dissolved in Tris-HCl/dithioerylhritol, and dialyzed againsl
Ihe same buffer. The purpose of Ihe Dowex-1-acelale chromalography
was lo remove endogenous nucleolides which mighl interfere wilh Ihe
assay. The ammonium sulfate step al 80% saturation was done lo
concenlrate the protein after Ihe Dowex step. The recovery of aclivily
was in excess of 100% due lo Ihe removal of low molecular u eight
substances. Aliquots of Ihe dialyzed extracts were quick frozen in an
acelone-dry ice balh and slored al -90°C. No loss of CDP reducÃ-ase
deoxycytidine and incorporalion inlo DNA in inlacl cells in Ihe
presence of the ribonucleotide reductase inhibitors.
In this paper, we describe iwo of ihese cell lines in lerms of
their sensitivily to a variety of ribonucleotide reductase inhibi
tors.
MATERIALS AND METHODS
Growth of LI210 Cells in Culture. LI 210 cells were grown in suspen
sion cuiiure in RPMI 1640 culture medium supplemented with 10%
horse serum, gentamicin sulfate (50 mg/liter), and sodium bicarbonate
(2 g/liler). The cells were seeded al 1.75 x IO5cells/ml in Ihe presence
and absence of drugs. The cells were grown at 37°Cin a humidified
incubator gassed with 90% air/10% CO2. All cell cultures were set up
in triplicale. Aliquols (1.0 ml) were taken daily for growth curves. For
Received 8/3/87; revised 10/16/87; accepted 11/13/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This research was supported by grants CA 27398 and CA 42070 from the
USPHS, National Cancer Institute, and the Grand Chapter of the Eastern Star.
2To whom requests for reprints should be addressed, at Department of Internal
Medicine, MDC Box 19, USF College of Medicine, 12901 Bruce B. Downs Blvd.,
Tampa, FL 33612.
aclivily was observed in the frozen samples.
Assay of Ribonucleotide ReducÃ-aseAclivily. CDP reducÃ-aseaclivily
was assayed by Ihe method of Sleeper and Steuart (5) using Dowex-1borale columns lo separate deoxycytidine from cylidine. The assay
mixlure for CDP reducÃ-aseconlained in a final volume of 0.15 ml
[14C]-CDP, 0.03 MCi, 1.87 nmol; AMP-PNP, 1500 nmol; magnesium
acelale, 600 nmol; sodium phosphate buffer, pH 7.0, 1000 nmol; and
enzyme extract. After a 30-min reaction period, Ihe reducÃ-aseassay
was slopped by healing for 4 min in a boiling waler bath. The samples
were cooled and treated with snake venom (6) as previously described.
All reductase assays were carried out in triplicate. The specific act ivities
were compared on a per mg protein basis ralher lhan on a per cell basis.
The assay was linear wilh time for at least 40 min. The activily measured
3The abbreviations used are: [ICjjo, concentration of drug required to inhibit
cell growth or reductase activity by 50%; AMP-PNP, adenylylimido diphosphate;
AGuo, 8-aminoguanosine; Df, Desterai: EHNA, erythro-9-(2-hydroxyl-3nonyl)adenine; F-araA, 2-fluoroadenine arabinoside; F-dAdo, 2-fluoro-2'-deoxyadenosine; IMPY, 2,3-dihydro-l//-pyrazole[2,3ajimidazole;
Inox, dialdehyde de
rivative of inosine; MAIQ, 4-methyl-S-amino-l-formylisoquinoline
thiosemicar
bazone; dAdo, deoxyadenosine; dGuo, deoxyguanosine; F-dATP, 2-fluoro-dATP.
839
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DRUG-RESISTANT
L1210 CELL LINES
was linear with respect to the protein added when the protein concen
tration was in excess of 600 Mg/rnl (90 (¿g/tube)in the reaction mixture.
This was observed with extracts from the wild-type, EDI, and ED2
cells. The differences in enzyme activities in the extracts from the wild
type, EDI, and ED2 cells were observed using either equal volumes of
extracts or equal amounts of protein in the assays.
("•qCytidineMetabolism in L1210 Cells. L1210 cells were collected
by centrifugation and resuspended in fresh culture medium (10 ml) at
a concentration of 1.6 x IO6cells/ml. The cells were incubated in the
presence and absence of drugs for 30 min at 37°Cin air/CO2 (95/5%).
[14C]Cytidine (0.5 MCi, 350 mCi/mmol) was added to each flask and
the cells were incubated for an additional 60 min. The cells were
collected and subjected to the Schmidt-Thannhauser procedure (7) to
separate the acid-soluble, RNA, and DNA fractions. The acid-soluble
fraction was neutralized, lyophilized, and treated with snake venom (8).
The [14C)deoxycytidine was separated from the [14C]cytidine by chromatography on Dowex-1-borate columns. All flasks were set up in
triplicate.
Adenosine Deaminase and Kinase Activities. Adenosine deaminase
activity was determined using a continuous spectrophotometeric assay
at 25°C.The conversion of adenosine to inosine was measured at 265
nm. The assay mixture consisted of adenosine, 0.063 mM in 0.02 M
Tris-HCl, pH 7.O. The reaction was started by the addition of the crude
cell-free extract which had been passed over a Dowex-1-acetate column
and dialyzed against 0.02 M Tris-HCl, pH 7.O. The assays were run
with equal amounts of extract protein (0.3 mg/tube). Kinase activity
was determined using [8-'4C]deoxyadenosine (45 mCi/mmol) as the
substrate. The crude cell-free extracts after passage over Dowex-1acetate columns and dialysis against 0.02 M Tris-HCl, pH 7.0 were
incubated with deoxycoformycin (57 fig/ml) for 1 h on ice. The reaction
mixture contained in a final volume of 0.095 ml [14C]deoxyadenosine
(0.2 ^Ci); ATP (285 nmol); magnesium acetate (190 nmol); sodium
phosphate buffer, 950 nmol, pH 7.0; and deoxycoformycin-treated
enzyme extract, 0.3 mg protein/tube). Aliquots (5 fil) were taken at 10,
20, and 30 min of incubation at 25°Cand spotted directly on thin-layer
cellulose plates. These reaction aliquots were spotted on the thin-layer
cellulose plates over carrier dAMP and deoxyadenosine standards. The
thin-layer cellulose plates were developed with a butanol:water (86:14)
solvent. In this system dAMP and other nucleotides remain at the
origin while deoxyadenosine moves with an Rr of 0.5. The dAMP
(origin) and deoxyadenosine areas were scraped directly into scintilla
tion vials and counted for the radioactivity. The reactions were linear
over the 30-min period measured. All assays were carried out in
triplicate.
Materials. The LI210 cell line was purchased from the American
Type Culture Collection (Rockville, MD). The RPMI 1640 culture
medium, horse serum, and sodium bicarbonate were purchased from
Grand Island Biological Co. (Grand Island, NY). IMPY, Inox, and
MAIQ were obtained from the Drug Synthesis and Chemistry Branch,
Division of Cancer Treatment, National Cancer Institute through the
assistance of Dr. Nancita R. Lomax. EHNA was purchased from
Burroughs-Wellcome, Research Triangle Park, NC. Desierai was a gift
from Ciba-Geigy Corp., Summit, NJ. Deoxycoformycin was a gift from
Warner-Lambert Co., Ann Arbor, MI. Hydroxyurea, AMP-PNP, deox
yadenosine, and gentamicin sulfate were purchased from Sigma Chem
ical Co., St. Louis, MO. 8-Aminoguanosine was purchased from Calbiochem-Behring, La Jolla, CA. 2-F-Deoxyadenosine and 2-fluoroadenine arabinoside were gifts from Dr. John A. Montgomery, Southern
Research Institute, Birmingham, AL. 1-Isoquinolylmethylene-A'-hydroxy-/V"-aminoguanidine-tosylate was obtained from Dr. Eric J. Lien,
University of Southern California, Los Angeles, CA. [14C]Cytidine (350
mCi/mmol) and ['4C]CDP (513 mCi/mmol) were purchased from
determine if combinations of inhibitors directed at the nonheme iron and effector-binding subunits of ribonucleotide reductase resulted in synergistic cell kill, it was observed that
several different colonies grew out in the presence of cytotoxic
doses of dAdo/EHNA or dAdo/EHNA plus IMPY/Df combi
nations. The LI210 cell line which was selected for resistance
to dAdo/EHNA was then grown in increasing concentrations
of dAdo/EHNA. This cell line was called EDI. Another cell
line was selected for further study because of its resistance to
the combination dAdo/EHNA plus IMPY/Df. This cell line
was also subjected to increasing concentrations of dAdo and
IMPY with the EHNA and Df concentrations being held con
stant. This cell line was designated as ED2.
As seen in Fig. 1, the combination of dAdo/EHNA which
caused essentially complete inhibition of wild-type LI210 cell
growth had no effect on the growth of EDI cells. Likewise, the
ED2 cell line was refractory to the growth-inhibitory effects of
the combination dAdo/EHNA plus IMPY/Df. The doubling
times of the EDI and ED2 cell lines were the same as for the
wild-type LI210 cells (10 to 12 h). The resistance traits in the
EDI and ED2 cell lines were stable. Even after passage for over
a month in drug-free medium, the resistance phenotypes in the
EDI cells to dADo/EHNA and in the ED2 cells to dAdo/
EHNA plus IMPY/Df were as strong as the cells maintained
in the presence of these drug combinations.
Ribonucleotide ReducÃ-aseActivity in LI 210 Cell Lines. CDP
reducÃ-aseactivity was determined in the wild-type L1210 cells
and in the resistant cell lines, EDI and ED2. Cell-free extracts,
after passage over Dowex-1-acetate, precipitation with ammo
nium sulfate (80%), and dialysis were used as the source of
enzyme. Increases in specific activities as large as 9-fold were
seen in the EDI and ED2 cells compared with the wild-type
cells. As seen in Fig. 2, the CDP reducÃ-ase activily in ihe
exlracls from Ihe EDI and ED2 cells was much less sensilive
lo feedback inhibilion by dATP. The [IC]50s for dATP for Ihe
reducÃ-aseaclivity in the exlracls from Ihe EDI and ED2 cells
were grealer lhan 150 UMcompared wilh ihe [IC]50of less than
20 fiM for dATP for reducÃ-aseaclivity from wild-type LI210
cells. On the other hand, the [IC]50s for dGTP were essentially
the same for the reducÃ-aseaclivities from these three LI210
cell lines although some differences were seen in the percentage
of conlrol versus Ihe [dGTP] curves. The Kas for Ihe aclivalion
of CDP reducÃ-aseby AMP-PNP were Ihe same (0.4 to 0.5 mM)
EDI. dAdo/EHNA
DAYS IN CULTURE
Research Products Int. Co., Mount Prospect, IL and New England
Nuclear Corp., Boston, MA, respectively. [8-14C]-Deoxyadenosine (45
mCi/mmol) was purchased from Moravek Biochemicals, City of Indus
try, CA.
RESULTS
Resistance of LI 210 Cell Lines to Ribonucleotide ReducÃ-ase
Inhibitors. During the soft-agar studies being carried out to
E02 .dAdo/EHNA/IMPY/Df
DAYS IN CULTURE
Fig. 1. Growth of L1210 cells in culture. LI210 cell lines were grown in
culture in the presence and absence of drugs. Each control or drug-treated cell
condition was set up in triplicate. Aliquots were taken at daily intervals for cell
counts. Left, wild-type LI 210 cells in the absence of drugs (O) and in the presence
of dAdo, 400 pM/EHNA, 5 »M(•);EDI cells in the absence of drugs (D) and in
the presence of dAdo. 800 ^M/EHNA, 5 >iM(•);right, wild-type L1210 cells in
the absence of drugs (O) and in the presence of dAdo, 400 ^M/EHNA, 5 MM/
IMPY. 450 fiM/Df, 48 JÕM
(•);ED2 cells in the absence of drugs (D) and in the
presence of dAdo/EHNA/IMPY/Df
at the same concentrations as for wild-type
cells (•).
840
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DRUG-RESISTANT
LI210 CELL LINES
IK'
ED2. HU
. MAIO
2.0
ID
o l.O
•
WT
o EOI
•
ED2
X
ËO.S
E05
o 50
u
a«
\
2
4
DAYS IN CULTURE
DAYS IN CULTURE
dATP.uM
UGTP.jjM
Fig. 2. Effect of dATP and dGTP on CDP reducÃ-aseactivities in L1210 cell
lines. Crude extracts were prepared from wild-type (WT), EDI, and ED2 L1210
cell lines. CDP reducÃ-aseactivily was delermined as described under "Malcriáis
and Methods" excepl lhat dATP or dGTP was added at the final concentralions
as indicaled. The control activilies for Ihe wild-lype. EDI, and ED2 cell lines
were (left), 0.08, 0.43, and 0.29 and (right), 0.06, 0.32, and 0.46 nmol/30 min/
mg prolein, respectively.
Fig. 3. Growth of LI210 cells in cullure. LI210 cell lines were grown in
cullure in the presence and absence of drugs. Each conlrol or drug-treated
condition was set up in triplicate. Aliquots were taken at daily intervals for cell
counts. Left, wild-type cells in ihe absence of drugs (O) and in the presence of
hydroxyurea (HU), 400 ßM
(•)and ED2 cells in the absence of drugs (D) and in
the presence of hydroxyurea, 400 ^M (B); righi, wild-type cells in the absence of
drugs (O) and in the presence of MAIQ, 3 /<M(•)and ED2 cells in the absence
of drug (D) and in the presence of MAIQ, 3 /iM (•).
Table 1 Concentralions of drugs required to inhibit LI 210 cell growth in culture
„MEDI>1000*
for the reducÃ-aseactivities in the extracts from these three cell
lines (data not shown).
In cell-free extracts prepared from the EDI and ED2 cell
lines, ribonucleotide reducÃ-aseactivily showed Ihe same degree
of sensilivily to hydroxyurea and IMPY as did the reducÃ-ase
activily in Ihe wild-lype LI210 cells (dala noi shown).
Cross-Resistance of EDI and ED2 Cell Lines to Ribonucleo
tide ReducÃ-aseInhibitors. The EDI and ED2 cell lines, selecled
for resislance lo dAdo/EHNA and dAdo/EHNA plus IMPY/
Df, respeclively, were sludied for iheir sensitivities lo a wide
range of ribonucleolide reducÃ-aseinhibilors. A combinalion of
dGuo and AGuo would be expecled lo generate intracellular
levels of dGTP lo inhibil reducÃ-aseaclivily (9); F-dAdo would
lead to the formalion of F-dATP (10). The ED2 cell line was
highly resislanl lo reducÃ-aseinhibilors direcled al Ihe effeclorbinding subunil (F-dAdo or dGuo/AGuo) and Ihe cells grew as
well as Ihe conlrol cells. Sludies wilh Ihe EDI cell line gave
very similar dala in lerms of resislance lo these agenls (dGuo/
AGuo or F-dAdo). The EDI and ED2 cell lines also showed
resislance lo F-araA (dala noi shown). On Ihe olher hand, the
EDI and ED2 cell lines were less resistanl lo ribonucleolide
reducÃ-aseinhibilors direcled al Ihe non-heme iron subunil. Fig.
3 shows ihe dala for Ihe effecls of hydroxyurea or MAIQ on
Ihe growlh curves for ihe ED2 cell line. Essentially the same
data were oblained for ihe EDI cell line. The [IC]50s were
delermined for Ihese and olher reducÃ-aseinhibilors and Ihese
dala are summarized in Table 1. The EDI and ED2 cell lines
showed Ihe grealesl degrees of resislance lo inhibilors direcled
at the effector-binding subunit (13- to >25-fold). Resistance to
Inox was minimal. The increases in [IC]50s for the inhibilors
directed at the non-heme iron subunit were much less, ranging
from a 1.7-fold increase for MAIQ in ED2 cells lo a 6.8-fold
increase for IMPY/Df in ihe ED2 cells.
Adenosine Deaminase and Deoxyadenosine Kinase Activities
in 1,1210 Cell Lines. The levels of adenosine deaminase aclivily
were essentially the same in the wild-type, EDI, and ED2 cell
lines (46, 47, and 53 nmol/min/mg protein, respeclively). Like
wise Ihere was no change in Ihe kinase aclivily using deoxyadenosine as Ihe substrate. The kinase studies were carried out
with exlracls preincubaled wilh deoxycoformycin to inaclivale
Ihe deaminase aclivily. These values were 18.4, 20.0, and 22.0
DrugdAdo/EHNA"
175*
>1000*
dGuo/AGuo"
F-dAdo"
2.3
30
Inox"
125
340
Hydroxvurea'iMPY/br1-isoquinolylmethylene-yV-hydroxy420
98
160*
640*
>1000*
37
160
475
1090*
2.51.2[IC|,„,6.83.1ED21000*
6.02.0
W-aminoguanidine-losylate'
MAIQrWildtype40*
" Inhibitors direcled al the effector-binding subunit.
* These [IC]50s are with respect to dAdo, dGuo, or IMPY; in each case the
concentralion of EHNA, AGuo, or Df was held conslant at 5, 25. and 40 /JM,
respectively.
f Inhibitors direcled al Ihe non-heme subunit.
Table 2 Metabolism off'CJcytidine
in LI210 cell lines
lypeEDICytidine
Drug (UM)Wild
conversion)Conlrol
IMPY/Df (360/40)
dAdo/EHNA (600/5)
to deoxycytidine (%
1.0
2.3(230)°
0.5(50)
0(0)
(290)
1.1 (110)
0.7(70)
3.1(310)ED22.9 3.8 (380)
Cytidine to deoxycytidine to DNA (cpm/IO6 cells)
Conlrol
1640
3010(184)
3730(227)
IMPY/Df (360/40)
183(11)
572(35)
1004(61)
dAdo/EHNA (600/5)
0(0)
1114(68)
1190(73)
°Numbers in parentheses, percentage of control of wild-lype LI 210 cells.
pmol/min/mg prolein for ihe wild-type, EDI, and ED2 cells,
respeclively.
[l4C]Cytidine Metabolism in LI 210 Cell Lines. The conversion
of [uC]cylidine lo deoxycylidine and incorporalion inlo DNA
was delermined in the wild-type, EDI, and ED2 cell lines. In
addilion, Ihe effecls of Ihe drug combinalions IMPY/Df and
dAdo/EHNA on [14C]cylidine metabolism in these cells were
sludied. As seen in Table 2, Ihe EDI and ED2 cells showed
increases (2- lo 3-fold) in CDP reducÃ-aseaclivily in ihe inlacl
cells as measured by Ihe increased formation of deoxycytidine.
Wilh Ihe drug combinalion dAdo/EHNA, Ihe formalion of
deoxycylidine was complelely blocked in Ihe wild-lype cells,
while Ihe ED 1 and ED2 cells were noi inhibiled, and if anylhing,
Ihe formalion of deoxycylidine was increased in the EDI and
ED2 cells relalive lo Iheir conlrols. The incorporalion of cylidine inlo DNA via deoxycylidine was also increased in Ihe EDI
and ED2 cells. The incorporalion of label inlo DNA in ihe
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DRUG-RESISTANT L1210 CELL LINES
niKNA for Ihe non-heme iron subunit with gene amplification
(27, 28).
As can be seen from Ihe dala in Table 2, ribonucleotide
reducÃ-aseaclivity as measured in intacl cells was elevated 2- lo
3-fold in Ihe EDI and ED2 cells compared lo Ihe wild-lype
1.1210 cells. On Ihe other hand, the incorporation of [I4C]-
EDI and ED2 cells was less sensitive to the inhibition by the
IMPY/Df or dAdo/EHNA combination. There was no change
in the incorporation of cytidine into the RNA of these cells.
DISCUSSION
One of the major problems which arises in the chemotherapy
of human tumors is the development of drug-resistant clones.
There are multiple biochemical bases for the development of
drug resistance to a single agent (11-16). In the studies pre
sented here, clones of LI210 cells grew out in the presence of
inhibitors directed at ribonucleotide reducÃ-ase.The biochemical
bases for the resistance to dAdo/EHNA in the EDI cell line
and the resistance to dAdo/EHNA plus IMPY/Df in the ED2
cell line appear to be due to the increased levels of ribonucleo
tide reducÃ-aseactivity in the EDI and ED2 cell lines and to the
altered effector-binding subunit which renders the ribonucleo
tide reducÃ-aseinsensitive to the negative effector, dATP (Fig.
2). While Ihe enzyme in Ihe extracts from the EDI and ED2
cell lines was not inhibited by dATP, the apparent A', for the
activalor, AMP-PNP was Ihe same as for Ihe wild-lype cells.
This would appear lo be an anomalous finding as il has been
reported that ATP and dATP both bind to the activity and
specificity siles (17). Consequently, il would be expected that
an alteration in the dATP binding site or siles would likewise
alter the ATP-binding sites(s). However, this kind of alteration
(altered dATP binding with no corresponding effect on ATP
binding) has been reported previously (18,19). In some of these
mutant cell lines the sensitivity lo dATP was decreased while
the sensitivity to dGTP and dTTP remained unchanged (18,
19) or conversely in which the sensilivily lo dATP was unaltered
but the sensitivity to dGTP and dTTP was decreased (19). The
reducÃ-asefrom another mutant cell showed decreased sensitiv
ity to dATP but increased binding of the positive effeclor, ATP
(20). As seen from the data in Table 1, Ihe grealer drug
resistances were seen for Ihose agents directed at the effeclorbinding subunit. The exception to this was in the resistance to
Inox. However, previous studies have shown lhat as an inhibitor
of ribonucleotide reducÃ-ase,Inox specifically inhibils the effec
tor-binding subunit (21) bul il also has oilier siles of action (22,
23). The ED2 cell line showed a relatively high degree of
resistance to IMPY/Df and ihis was expecled as ihese cells had
been grown in increasing concenlralions of IMPY. The EDI
and ED2 cell lines showed cross-resislance to F-dAdo and FAraA, consistent wilh altered dATP-binding sile(s). Ribonucle
otide reducÃ-aseas measured by CDP reducÃ-aseaclivity in the
crude extracts from the EDI and ED2 cell lines showed the
same degree of sensitivity to dGTP as did Ihe enzyme from Ihe
wild-type cells (Fig. 2); similar data were obtained with dTTP
(data not shown). The observed cross-resislance lo dGuo/AGuo
in the intact cells was therefore unexpected, based on the
sensitivity of reducÃ-aseactivily in crude exlracls lo dGTP (Fig.
2). Il is possible that the increased | l('|M,s determined for the
cylidine inlo DNA via deoxycylidine nucleolides in Ihe dAdo/
EHNA-irealed EDI and ED2 cells was reduced lo only 30 lo
40% of Ihe incorporation in Ihe untreated EDI and ED2 cells.
However, compared lo Ihe conlrol value (1640 cpm/106 cells)
for Ihe wild-lype LI210 cells, Ihe level of incorporation in the
drug-treated EDI and ED2 cells was 68 to 73% of control value
in the wild-type cells. This level of DNA synlhesis may be
sufficient to support cell division under these condilions ren
dering Ihese cell lines resislanl lo growth inhibilion by Ihese
drugs.
Since Ihe levels of deoxyadenosine kinase and adenosine
(learninase activities were Ihe same in Ihe wild-lype L1210 cells
and Ihe EDI and ED2 cells lines, Ihe potential mechanisms of
lack of activation or increased inactivalion could be ruled oui.
These sludies show lhat mechanisms of resistance to drugs
directed at Ihe subunils of ribonucleolide reducÃ-ase can be
multiple and can be due lo increased levels of reductase activity
and to alterations in the sensitivily lo negative effectors. Studies
are being direcled al determining Ihe levels of Ihe individual
subunils and Ihe nature of the insensilivily to dATP in the
resistanl EDI and ED2 cell lines.
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a combination of the increased levels of reducÃ-ase aclivity
(dGuo/8AGuo;
hydroxyurea; IMPY/Df; 1-isoquinolylmelhylene-TV-hydroxy-yV-aminoguanidine-tosylate; MAIQ) and the
altered effector-binding subunil (dAdo/EHNA; F-dAdo; FaraA). Il has been shown lhal the resistance to hydroxyurea is
related to increased levels of ribonucleotide reducÃ-aseactivity
in the cells rather than lo marked alterations in the sensitivily
of the enzyme to hydroxyurea (24-26). It has been reported
that Ihe hydroxyurea-resislanl
cells have elevaled levels of
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Leukemia L1210 Cell Lines Resistant to Ribonucleotide
Reductase Inhibitors
Joseph G. Cory and Gay L. Carter
Cancer Res 1988;48:839-843.
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