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Selective resistance of L1210 cell lines to inhibitors directed at the subunits of ribonucleotide reductase
SELECTIVE RESISTANCE OF L1210 CELL LINES TO INHIBITORS DIRECTED AT THE SUBUNITS OF RIBONUCLEOTIDE REDUCTASE GAY L. CARTER and JOSEPH G. CORY 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
INTRODUCTION
Ribonucleotide reductase (EC 1.17.4.1) is a key enzyme in the replication of DNA. 2'-Deoxyribonucleoside 5'-triphosphates, which are the substrates for D N A polymerase (EC 2.7.7.7), are generated via a de n o v o pathway in which ribonucleoside 5'-diphosphates are reduced to the corresponding 2'-deoxyribonucleoside 5'-diphosphates followed by phosphorylation to the dNTPs. This pathway is shown in Figure 1. The reaction catalyzed by ribonucleotide reductase is the rate-limiting step in D N A synthesis and it is to be expected that there would be critical properties of this
enzyme which would serve to make this an extremely delicate metabolic step. These properties include: (a) the enzyme activity is expressed as a function of the cell cycle (1); (b) the enzyme consists of two non-identical protein subunits encoded by different genes (2--6); (c) the genes, in terms of protein products, are not coordinately expressed as the cells pass through the cell cycle (7-9); (d) the enzyme is allosterically regulated by dNTPs acting as either positive or negative effectors (10-13); (e) the two subunits making up the active enzyme species do not have the same half-lives (14); and (f) the two subunits making up the active enzyme species can be specifically and independently inhibited/inactivated by specific classes of ribonucleotide reductase inhibitors (15-19). There have been many reports of the development of resistance of various mammalian cell types to ribonucleotide reductase inhibitors (20--31) and to agents such as aphidicolin (32, 33) which cause alterations at the ribonucleotide reductase site. In this report we compare the drug sensitivity/cross-resistance of L1210 cell lines resistant to specific classes of ribonucleotide reductase inhibitors and the properties of ribonucleotide reductase in the cell-free extracts from these cells to those of wild-type L1210 cells. 123
G. L. CARTER and J. G. CORY
124 ADP
•
dADP
=
GDP
-
dGDP
2
CDP
-
dCDP
~
UDP
-
dUDP
--dTMP
~ dATP. d G T P ~ - dCTP
- DNA
~--dTTP
FIG. 1. De novo pathway for synthesis of 2'-dcoxyribonucleoside 5'-triphosphates. Ribonucleoside 5'-diphosphates arc reduced via ribonucleotide reductase (1) to dNDPs: the dNDPs are phosphated to dNTPs by nuclcosidcdiphosphate kinase (2); dCMP deaminase activity (3) provides dUMP, the substrate for thymidylatesynthase (4); DNA [x~lymerase(5) catalyzes the polymerizationof dNTPs in the presence of template and primer.
METHODS AND M A T E R I A L S
Growth of LI210 cells and selection of resistant clones. Wild type L1210 cells were grown in RPMI 1640 culture medium which was supplemented with 10% horse serum, gentamicin sulfate (50 rag/l) and sodium bicarbonate (2 g/I). The cells were grown in a humidified incubator maintained at 37°C and 95% air/5% CO 2. The original culture was purchased from the American Type Culture Collection. The ED2 and HU-7 cell lines were selected as previously described (29, 31). The Y-8 cell line was selected by growing the wild-type L1210 cells in deoxyadenosine, 100 ~M/EHNA, 5 ~M and then plating out on soft agar. The colonies which grew out were regrown in liquid culture in deoxyadenosine, 100 p,M/EHNA, 5 t~M. The cells were then grown in deoxyadenosine, 300/zM/EHNA, 5 txM. Preparation of cell-free extracts from wild-type and resistant LI210 cell lines. The wild-type and resistant L1210 cells were grown to mid-log phase and collected by centrifugation. The cells were washed in phosphatebuffered saline, resuspended in Tris-HCI buffer, 0.02 M, pH 7 containing dithioerythritol, 1 raM. The cells were homogenized and the homogenates centrifuged at 21,000 x g for 1 hr at 4°C. The supernatant fractions were passed over Dowex-l-acetate columns to remove endogenous nucleotides and the eluants were precipitated with ammonium sulfate (80% saturation) and dialyzed. Assay of ribonucleotide reductase activities. CDP reductase was assaye d using the Dowex-l-borate method of Steeper and Steuart (34). The assay mixture was as described by Carter and Cory (31). A D P reductase was assayed using the method of Cory et al. (35) with the assay mixture as described by Carter and Cory (31).
DRUG-RESISTANTLI210CELLS
125
[14C]Cytidine metabolism in L1210 cells. Wild-type, HU-7, Y-8 and ED2 L1210 cells werc grown in culture to mid-log phase. The cells were collected by centrifugation and resuspended in fresh medium to a concentration of 1.6 × IIY' ceils/ml. The cells were incubated in the presence and absence of ribonucleotide reductase inhibitors for 1 hr at 37°C in 95% air/5% CO 2. [14C]Cytidine (0.5 p.Ci, 350 mCi/nmol) was added and the cells werc incubated for an additional hour. The cells were collected and treated as previously described to separate the acid-soluble, RNA and DNA fractions (29). Deoxycytidinc nucleotides were isolated from the acid-soluble nucleotide pools (29). Materials. The tissue culture medium (RPMI 1640), horse serum and NaHCO 3 were purchased from GIBCO. Hydroxyurea and the nucleosides were purchased from Sigma Chemical Company. 4-Methyl-5-amino1-formylisoquinoline thiosemicarbazone (MAIQ) and 2,3-dihydro-lHimidazo(1,2-b)pyrazole (IMPY) were obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute, through the assistance of Dr. Nancita R. Lomax. Desferal (Df) was a gift from Ciba-Geigy Corporation. 1lsoquinolylmethylene-N-hydroxy-N'-aminoguanidine tosylate (HAG-IQ) and 2-fluorodeoxyadenosine were gifts from Drs. Eric J. Lien and John A. Montgomery, respectively. Erythro-9-(2-hydroxy-3-nonyi)adenine (EHNA) and 8-aminoguanosine (8-AGuo) were purchased from Burroughs-Wellcome Company and Calbiochem-Behring, respectively. The radiochemicals, [14C]CDP and [3H]ADP. were purchased from New England Nuclear.
RESULTS AND DISCUSSION Generation of LI210 Cell Lines Resistant to Ribonucleotide Reductase lnhibitors Earlier studies had shown that combinations of drugs simultaneously directed at the non-heine iron and effector-binding subunits of ribonucleotide reductase resulted in strong synergistic inhibition of L1210 cell growth in culture. For example, as shown in Figure 2, a combination of hydroxyurea and deoxyadenosine (16) or IMPY and deoxyguanosine (17) caused synergistic inhibition of L1210 cell growth and synergistic cytotoxicity. In carrying out colony forming assays with these drug combinations it was observed that several colonies of L1210 cells grew out on soft agar in the combination of dAdo/EHNA/IMPY/Desferal which essentially completely inhibited L1210 cell growth (Fig. 3). These colonies were "picked" and grown out in liquid culture and further subjected to the selective pressure of higher drug concentrations of deoxyadenosine and
126
G. L. CARTER and J. G. CORY
CDP
'~ d C D P
HU IMPY MAIQ HAG - IQ
dATP dGTP F-dATP
FIG. 2. Subunit nature of mammalian ribonucleotide reductase. Ribonucleotide reductase consists of two non-identical protein subunits, coded for by different genes. The non-heine iron (NHi) and effector-binding (EB) subunits can be specifically and independently inhibited by the drugs as indicated.
o o A •
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O
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it) w
tO
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~i~~_/dolOlp[H~;lDP/~JjYiNiOD/l ~
,
DAYS IN CULTURE
FIG. 3. Growth of LI210 cells in culture. LI210 cells were grown in culture in the presence and ab~nce of drugs as indicated. The four-drug combination (Q) contained dAdo, 15 V.M/EHNA, 5 V.M/IMPY, 190 p,M/Desferal, 20 V.M.
LI210 Ceils
LI210 Cells
LI210 CeUs
"resistant" LI210 clones
"resistant" LI210 clones
HU. 0 SrnM HU-5
HU.O75~ HU-7
X ~ . I OmM HU-IO
~IMPY.IOOIOf,SO J ~A,~o.3OO/EHNA. s ~l.do.IBO0/ EH N A / Y-8
ED-I
~l~tl~lt~y. 4.4~tE/Mt~ 0N~~ ED-2
FIG. 4. Selection of LI210 cells resistant to inhibitors of ribonucleotide reductase.
DRUG-RESISTANT LI210 CELLS
127
IMPY at fixed concentrations of EHNA and Desferal. These resistant cell lines were referred to as ED1 and ED2 cell lines. The diagram in Figure 4 shows the selection procedures for the L1210 cell lines resistant to deoxyadenosine/IMPY (ED2), hydroxyurea (HU-7) and deoxyadenosine (Y-8). These drug-resistant L1210 cell lines had the same doubling times as the wild-type L1210 cells. This was an especially important feature of these L1210 cell lines since ribonucleotide reductase is a cell-cycle specific enzyme.
Effects of Ribonucleotide Reductase Inhibition on LI210 Cell Lines The effects of hydroxyurea, IMPY, deoxyadenosine and deoxyguanosine on wild-type L1210 cells and HU-7, Y-8 and ED2 resistant cell lines are shown in Figures 5--7. As can be seen in Figure 5, the HU-7 cell line was very resistant to hydroxyurea and IMPY (approximately 10-fold increase in IC5o) with essentially no increase in resistance to either deoxyadenosine or deoxyguanosine. The Y-8 cell line (Fig. 6) showed marked resistance to deoxyadenosine (approximately 100-fold), a 3.5-fold increase in resistance to deoxyguanosine and a similar increase in resistance to hydroxyurea (2-fold) and IMPY (4-fold). The Y-8 cell line showed a high degree of cross-resistance to 2-fluorodeoxyadenosine (data not shown). The ED2 cells were highly resistant to deoxyadenosine with a corresponding cross-resistance to deoxyguanosine (Fig. 7). In addition, these cells showed a 5- to 6-fold increase in resistance to hydroxyurea and IMPY. These data show: (a) that the cells (HU-7) selected for resistance to hydroxyurea were likewise highly cross-resistant to IMPY, but were still sensitive to deoxyadenosine and deoxyguanosine; (b) that the cells (Y-8) selected for resistance to deoxyadenosine did not display the same degree of cross-resistance to deoxyguanosine and were still relatively sensitive to hydroxyurea and IMPY although there were increases in their IC.~o'srelative to the wild-type cells; and (c) that the L1210 cells which had been selected in the presence of both deoxyadenosine and IMPY (ED2) showed marked cross-resistance to deoxyguanosine and 2-fluorodeoxyadenosine with lesser cross-resistances to IMPY (5.5-fold) and hydroxyurea (6.1-fold). The effects of MAIO and HAG-IQ, potent inhibitors of ribonucleotide reductase, on these cell lines were also studied. As seen in Figure 8, there was little or no change in the sensitivities of the HU-7, Y-8 and ED2 cell lines, relative to the wild-type L1210 cells lines, to these ribonucleotide reductase inhibitors. All of these data are summarized in Table 1. Drug-resistant cell lines were generated in response to the selective pressure of specific inhibitors directed at the individual subunits of ribonucleotide reductasc. The cross-resistance patterns of these cells depended on the agent to which the resistance had been developed. It is particularly intriguing that these cell lines generated
G. L. CARTER and J. G. CORY
128
O01
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-7
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oor •
. ~
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-
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o ~o
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FIG. 5. Growth of wild-type and HU-7 LI210 cell lines in the presence of hydroxyurea, IMPY, deoxyadenosine or deoxyguanosine. The wild-type and HU-7 L1210 cells were seeded at approximately 150,000 cells/well (24-well plate; 2 ml) in the presence of drug as indicated. EHNA was added to inhibit adenosine deaminase; 8-aminoguanosine was added to inhibit purine nucleoside phosphorylase. After 72 hr, aliquots were removed and cell counts made.
in r e s p o n s e to inhibitors o f ribonucleotide reductase and which s h o w m a r k e d resistance to either h y d r o x y u r e a or d e o x y a d e n o s i n e w e r e not resistant to the inhibitors, M A I Q and H A G - I Q . Previous studies have indicated that M A I Q (36) and H A G - I Q (37) may have different m e c h a n i s m s of inhibition of r i b o n u c l e o t i d e reductase c o m p a r e d with hydroxyurea and I M P Y e v e n though they are both directed at the n o n - h e m e site. For e x a m p l e , iron-chelating agents p o t e n t i a t e d the effects of hydroxyurea and I M P Y but c o m p l e t e l y reversed the inhibition caused by M A I Q (36). It has been reported that hydroxyurea and M A I Q inhibit r i b o n u c l e o t i d e reductase
DRUG-RESISTANT LI210 CELLS I001
129
IOC 8-AGuo,25~M
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FIG. 6. Growth of wild-type and Y-8 LI210 cell lines in the presence of deoxyadenosine, deoxyguanosine, IMPY or hydroxyurea. The wild-type and Y-8 cells were seeded at approximately 150,(X)0cclls/well (24-well plate; 2 ml) in the presence of drugs as indicated. activity by consuming the frec radical on the non-heme iron subunit (22, 38) which would indicate the same site of inhibition on the same subunit. Using the approach of Elford (39), we compared the rates of consumption of the free radical on diphenylpicrylhydrazyl by hydroxyurea and M A I Q . These date are shown in Figure 9. Although M A I Q is at least 100 times more active than hydroxyurea as an inhibitor of ribonucleotide reductase activity and tumor cell growth, hydroxyurea was 5 times more active than M A I Q
G. L. CARTER and J. G. CORY
130
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DEOXYGUANOSINE,mM
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0'4
HYDROXYUREA.mM
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FIG. 7. Growth of wild-type and ED2 LI21() cell lines in the presence of deoxyadenosine, deoxyguanosine, IMPY or hydroxyurea. The wild-type and ED2 LI210 cells were seeded at approximately 15(I,000 cells/well (24-well plate; 2 ml) in the presence of drug as indicated. in i n t e r a c t i n g with the free radical on d i p h e n y l p i c r y l h y d r a z y l . W h e t h e r this r e p r e s e n t s a basis for the d i f f e r e n c e seen in d r u g sensitivities in the r e s i s t a n t cells is u n c l e a r at this t i m e .
Properties of Ribonucleotide Reductase in Cell-Free Extracts From WildType and Resistant L1210 Cell Lines T h e b i o c h e m i c a l b a s e s for the resistance of the H U - 7 , Y-8 a n d E D 2 cells to specific i n h i b i t o r s o f r i b o n u c l e o t i d e r e d u c t a s e i n h i b i t o r s h a v e b e e n s t u d -
DRUG-RESISTANT LI210 CELLS
131
\
,\ \
ae °~o
=
\WT°~\ eHU'7
\\.
~°
20
40
~.WT ~ O Z
~oWT~o'¢- 8 20 40 60 MAIO,uM
I0
2.0
3.0
FIG. 8. Effect of MAIO on wild-type, HU-7, Y-8 and ED2 LI210 cell lines. The LI210 cells were seeded at approximately 150,000 cells/well (24-well plate; 2 ml) in the presence of MAIQ as indicated. After 72 hr, aliquots were removed and cell counts made.
030
e-o 0 0.05 I
0.1
0.3 DRUG,mM
0.5
FIG. 9. Effects of hydroxyurea and MAIQ on the free radical on diphenylpicrylhydrazyl. The loss of the free radical, as measured by the decrease in absorbance at 518 nm, was determined at several concentrations of hydroxyurea or MAIQ. as indicated. These rates were followed at 25°C. TABLE I. ICso VALUES FOR INHIBITION OF WILD-TYPE LI210 CELLS AND REDUCTASE INHIBITORS CELL LINES ICso, #M Drug Deoxyadenosine/EHNA Deoxyguanosine/8-AGuo F-Deoxyadenosine Hydroxyurea IMPY/Df MAIQ HAG-IQ
Wild type
HU-7
Y-8
ED2
33 136 1.9 77 198 2. i 2.5
52 116 p 2000 1470 2.4 3.4
>3000 480 >16 1.55 790 3.5 3.5
1000 >!000 37 475 1090 3.0 2.0
132
G. L. CARTER and J. G. CORY
led. R i b o n u c l e o t i d e reductase activity in cell-free extracts from the H U - 7 treated cells was markedly elevated. C D P and A D P reductase activities were both elevated as secn in Figure 10. H o w e v e r , the two activities were not coordinately increased. A D P reductase activity was increased 13-fold while the C D P reductase was increased only 5-fold. In a c o m p a r i s o n of the properties of the ribonucleotide reductase in cell-free extracts it was f o u n d that there were no differences in the inhibitory effects of h y d r o x y u r e a , I M P Y , d A T P , d T F P or d G T P between the H U - 7 ceils and the wild-type L1210 cells. T h a t is, the e n z y m e from the H U - 7 cells was still sensitive to the effects of h y d r o x y u r e a and I M P Y , even though intact cells showed a high degrce of resistance to the growth-inhibitory effects of h y d r o x y u r e a
ADP Reductose
1.50
HU-7
c_ E o ~O.75 o E t-
/
/ WT
o15 0.50
/
o
c
Ii0
'
CDP Reductose
.c_ E ,~ 0.25
'
/
e/HU-7
o/" o15
PROTEIN. mg
,~o
FIG. I0. CDP and ADP reductase activities in cell-free extracts from wild-type and HU-7 LI210 cells. Cell-free extracts were prepared, passed over Dowex-l-acetate and assayed for CDP and ADP reductase activities.
D R U G - R E S I S T A N T LI210 C E L L S
133
and IMPY. It was not expected that there would be any changes in the effects of d A T P , d'lq'P or d G T P as negative modulators of ribonucleotide reductase from these hydroxyurea-resistant cells. Other hydroxyurea-resistant L1210 cell lines were also studied. It was shown that HU-5 and HU-10 cell lines had increases in reductase activity, but at levels lower than the HU-7 cells. The degree of resistance of the LI210 cells to growth inhibition by hydroxyurea correlated with the level of increase in reductase activity (31). Ribonucleotide reductase activity in extracts from the Y-8 cells was only slightly increased (less than 2-fold) relative to the extracts from the wild-type cells. This was seen for both CDP and A D P reductase activities. However, in contrast to the reductasc activity in the extracts from HU-7 cells, the ribonucleotide reductase from the Y-8 cells was essentially insensitive to d A T P as a negative effector. These data are shown in Figure 11. On the other hand, the CDP rcductase activity from the Y-8 cells was inhibited by d G T P to the same extent as the enzyme from wild-type cells. In the ED2 cells, CDP reductase activity was elevated as much as 9-fold over that of the wild-type cells. However, there was essentially no increase in A D P reductase activity as measured in the same cell-free extracts. As seen in Figure 12, the ribonucleotide reductase from the ED2 cells was essentially unaffected by d A T P at concentrations as high as 150/.tM whereas the CDP reductase activity in the wild-type cells was completely inhibited by d A T P at this concentration. On the other hand, the CDP reductase activity from the ED2 cells was sensitive to dGTP. CDP reductase activity in the extracts from wild-type and ED2 cells was inhibited to the same extent by hydroxyurea and IMPY. The data obtained from the studies with the cell-free extracts from the .m
~
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120
i
i
2 0 400 dGTP,JJM
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FIG. 11. Effects of d A T P and d G T P on C D P reductase activities in LI210 cell lines. Crude extracts were prepared from the wild-type and Y-8 LI210 cell lines. C D P reductase activity was determined in the presence of d A T P or d G T P as indicated.
134
G. L. CARTER and J. G. CORY I00
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FIG. 12. Effects of dATP and dGTP on CDP reductasc activitics in LI210 cell lines. Crude extracts were prepared from wild-type and ED2 LI210 cell lines. CDP reductase activity was determined in the presence of dATP or dGTP as indicated. The control activities for wild-type and ED2 cell lines were: in the left panel, 0.08 and 0.29 nmol/30 min/mg protein respectively: and in the right panel, 0.06 and 0.46 nmol/31)min/mg protein, respectively.
H U - 7 a n d E D 2 cell lines showed that these cells had e l e v a t e d levels of r i b o n u c l e o t i d e reductase, possibly due to increases in the n o n - h e m e iron a n d e f f e c t o r - b i n d i n g s u b u n i t s , respectively. Cell-free extracts from the H U - 7 a n d E D 2 cells were mixed a n d assayed for C D P a n d A D P reductase activities. As seen in T a b l e 2, the m i x t u r e of extracts from H U - 7 a n d E D 2 cells gave C D P a n d A D P reductase activities which were much in excess of the activity expected o n the basis of simple a d d i t i o n of activities. This w o u l d s u p p o r t the n o t i o n that the H U - 7 cells had excess n o n - h e m e iron s u b u n i t while the E D 2 cells had excess e f f e c t o r - b i n d i n g s u b u n i t . W h e n the extracts from H U - 7 a n d Y-8 cells were mixed there was no increase in activity b e y o n d the simple a d d i t i o n of the activities from the two extracts consistent with the lack of increase of reductase activity in the Y-8 cells. TABLE 2. EFFECT OF MIXING CELL-FREE EXTRACTS FROM HU-7 AND ED2 LI210 CELL LINES Crude extract HU-7, 20 t.tl ED2, 40 ~1 HU-7, 20 #1 + ED2, 40 gl
Reductase activity CDP
(nmol/30 rain) ADP
0.038 0.059 0.161 (0.097)*
0.078 0.089 0.358 (0.167)*
*Simple summation of activities for each extract.
135
DRUG-RESISTANT L1210 CELLS
Incorporation of [t4C]Cytidine into DNA for Wild-Type, HU-7, 1"-8 and ED2 L1210 Cell Lines The incorporation of [14C]cytidine into DNA depends entirely on the level of flux of cytidine via CDP through the intracellular activity of ribonucleotide reductase to dCTP. The effects of ribonucleotide reductase inhibitors on the formation of [~4C]deoxycytidine nucleotides and incorporation into DNA from [14C]cytidine were studied in wild-type, HU-7, Y-8 and ED2 cells. As seen in Table 3, hydroxyurea, IMPY and deoxyadenosine markedly inhibited the formation of dcoxycytidine nucleotides and incorporation into DNA of the wild-type L1210 cells (experiments I and II). In the HU-7 cell line, there was only 40% inhibition of the formation of deoxycytidine nucleotides by either hydroxyurea or deoxyadenosine. This lack of inhibition, in view of the sensitivities of the enzyme in cell-free extracts to hydroxyurea or dATP, is probably related to the marked increase in reductase activities which was observed (Fig. 10). [14C}Cytidine reduction to deoxycytidine nucleotides in the Y-8 cell line was inhibited by hydroxyurea but relatively insensitive to deoxyadenosine/EHNA. These data are consistent with the fact that there was no increase in reductase activity in the Y-8 cells, no change in inhibition of reductase activity in cell-free extracts by hydroxyurea and the loss of sensitivity to feedback inhibition by dATP in these extracts. The metabolism of cytidine in thc ED2 cell line, which had TABLE 3. METABOLISM OF [z4C]CYTID1NE IN WILD-TYPE, HU-7, Y-8 AND ED2 L1210 CELL LINES IN THE PRESENCE OF RIBONUCLEOTIDE REDUCTASE INHIBITORS dCyd + DNA cpm/106 cells
% control
WT + HU, 200/gM + dAdo, 200/aM/EHNA, 5/.tM
3768 300 343
100 8 9
HU-7 + HU, 200//M + dAdo, 200/~M/EHNA, 5/~M
3761 2310 2200
100 61 59
Y-8 + HU, 200/~M + dAdo, 200/~M/EHNA, 5/./M
2212 287 1759
100 13 80
WT + IMPY, 360 ~aM/Df, 40/~M + dAdo, 600/~M/EHNA, 5 pM
1640 183 0
100 11 0
ED2 + IMPY, 360 #M/Dr, 40/t/M + dAdo, 600/~M/EHNA, 5/./M
3730 1004 1190
100 61 73
Experiment I
136
G.L. CARTER and J. G. CORY
been generated in response to the pressure of elevated levels of both IMPY and deoxyadenosine, was inhibited modestly compared with the wild-type cells (experiment II). These data are consistent with the fact that there was an increase in reductase activity and the loss of sensitivity to dATP. Recent studies have shown that hydroxyurea-resistant cell lines have elevated levels of the non-heme iron subunit, the mRNA for the non-berne iron subunit and in some cases amplification of the gene for the non-heine iron subunit (6, 40). Our data with the HU-7 LI210 cells are consistent with these studies. Caras and Martin (41) have shown the mutant $49 iymphoma cell line which has ribonucleotide reductase activity insensitive to feedback inhibition by dATP has an altered sequence in the cDNA for the effector-binding subunit (a guanine to adenine transition). Our data from the Y-8 and ED2 cell lines are compatible with this observation. From our data it is clear that the specificity of resistance to ribonucleotide reductase inhibitors depends on the drug to which the resistance has been developed and the subunit to which the drug is directed. The resistance has been shown to be due to elevated levels of ribonucleotide reductasc activity (HU-7 and ED2) or altered mechanisms of feedback control (Y-8 and ED2). The molecular bases for these observations are currently under study.
SUMMARY LI210 cell lines were generated which were resistant to specific ribonucleotide reductase inhibitors. Hydroxyurea-resistant L1210 cells (HU-7) were cross-resistant to IMPY but sensitive to deoxyadenosine and deoxyguanosine. Deoxyadenosine-resistant L1210 cells (Y-8) were cross-resistant to 2-fluorodeoxyadenosine and showed only a small increase in resistance to hydroxyurea or IMPY. L1210 cells which were generated in the presence of deoxyadenosinelEHNAIIMPYlDesferal were markedly resistant to deoxyadenosine, deoxyguanosine and 2-fluorodeoxyadenosine with moderate increases in resistance to IMPY. The HU-7, Y-8 and ED2 cell lines were sensitive to the inhibitory effects of MA[Q and HAG-IQ. The HU-7 LI210 cell line had elevated levels of ribonucleotide reductase activity and this activity showed normal inhibition by hydroxyurea, IMPY, dATP, dGTP and dTTP. The Y-8 LI210 cell line did not have elevated levels of ribonucleotide reductase activity, but had altered allosteric properties relative to dATP. The ED2 LI210 cell line had elevated levels of ribonucleotide reductase activity and had altered allosteric properties relative to dATP. These data show that resistance to ribonucleotide reductase inhibitors is specifically generated in response to the particular drug. The biochemical
DRUG-RESISTANT LI210 CELLS
137
basis can be related to either increased levels of ribonucleotide reductase activity or loss of feedback control by d A T P or both. ACKNOWLEDGEMENTS
This work was supported by grants from the USPHS, National Cancer Institute CA-27398 and CA-42070 and the Phi Beta Psi Sorority. REFERENCES I. 2. 3. 4. 5.
6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17.
M. K. TURNER, R. ABRAMS and i. LIEBERMAN, Levels of ribonucleotide reductase activity during the division cycle of the L cell. J. BioL Chem. 243, 3725-3728 (19681. E. C. MOORE. Components and control of ribonucleotide reductase systems of the rat, Advan. Enzyme Regul. 15, 101-114 11977). J. G. CORY, A. E. FLEISCHER and J. B. MUNRO, iil, Reconstitution of ribonucleotide reductasc from Ehrlich tumor cells, J. Biol. Chem. 253, 2898--2901 11978). C.-H. CHANG and Y.-C. CHENG, Substrate specificity of human ribonucleotide reductase from Molt-4F cells, Cancer Res. 39, 5081-5086 119791. 1. W. CARAS, B. B. LEVINSON, M. FABRY, S. R. WILLIAMS and D. W. MARTIN, JR., Cloned mouse ribonucleotide reductase subunit MI cDNA reveals amino acid sequence homology with Escherichia coli and herpes virus ribonucleotide reductases, J. Biol. Chem. 260, 7015-7022 (19851. L. THELANDER and P. BERG, Isolation and characterization of expressible eDNA clones e n c ~ i n g the MI and M2 subunits of mouse ribonucleotide reductase, Mol. Cell. Biol. 6, 3,133-3442 (19861. J. G. CORY and A. E. FLEISCHER, Noncoordinate changes in the components of ribonucleotide reductase in mammalian cells, J. Biol. Chem. 257. 1263-1266 (1982). S. ERIKSSON and D. W. MARTIN, JR., Ribonucleotide reductase in cultured mouse lymphoma cells. Cell cycle-dependent variation in the activity of subunit protein M2. J. Biol. Chem. 256, 9436--9440 (1981). T. YOUDALE, L. FRAPP1ER, J. F. WHITFIELD and R. A. RIXON, Changes in the cytoplasmic and nuclear activities of the ribonucleotide reductase holoenzyme and its subunits in regenerating liver cells in normal and thyroparathyroidectomized rats, Can. J. Biochem. Cell Biol. 62, 914---919 119841. E. C. MOORE and R. B. HURLBERT, Regulation of mammalian deoxyribonucleotide biosynthesis by nucleotides as activators and inhibitors. J. Biol. Chem. 241, 4802-4809. C.-H. CHANG and Y.-C. CHENG, Effects of nucleoside triphosphates on human ribonucleotide reductase from Molt-4F cells, Cancer Res. 39, 5087-5092 (19791. S. ERIKSSON. L. THELANDER and M. AKERMAN, Allosteric regulation of calf thymus ribonucleoside diphosphate reductase, Biochemi~try 18, 2948--2952 11979). J. G. CORY, D. A. REY, G. L. CARTER and P. E. BACON, Nucleoside 5'-diphosphates as cffcctors of mammalian ribonucleotidc reductase. J. Biol. Chem. 260, 12001-12007 (19851. E. H. RUBIN and J. G. CORY, Differential turnover of the subunits of ribonucleotide reductase in synchronized LI210 cells. Cancer Res. 46, 6165-6168 119861. J. G. CORY and A. E. FLEISCHER, Specific inhibitors directed at the individual components of ribonucleotide reductase as an approach to combination chemotherapy. Cancer Res. 39, 4600--4604 (19791. A. SATO, G. L. CARTER, P. E. BACON and J. G. CORY, Effects of combinations of drugs having different modes of action at the ribonucleotide reductase site on the growth of LI210 cells in culture, Cancer Res. 42, 4353--4357 (19821. A. SATO, P. E. BACON, S. W. SCHNELLER and J. G. CORY, Effect of combinations of deoxyguanosine and 8-aminoguanosine with 2,3-dihydro-lH-imidazo[1,2-bJpyrazole on LI210 cell growth in culture, Biochem. Pharmacol. 33, 689--691 (19841.
138
G. L. CARTER and J. G. CORY
18. A. SATO, J. MONTGOMERY and J. G. CORY, Synergistic inhibition of leukemia LI210 cell growth in vitro by combinations of 2-fluoroadenine nucleosides and hydroxyurea or 2,3-dihydro-lH-pyrazolo[2,3-a]imidazole, Cancer Res. 44, 3286-3290 (19841. 19. G. WECKBECKER, A. WECKBECKER, E. J. LIEN and J. G. CORY, Effects of N-hydroxy-N'-aminoguanidine isoquinoline in combination with other inhibitors of ribonucleotide reductase on L1210 cells, J. Natl. Cancer Inst. 8(}, 491-496 (1988). 20. W. H. LEWIS and J. A. WRIGHT, Altered ribonucleotide reductase activity in mammalian tissue culture cells resistant to hydroxyurea. Biochem. Biophys. Res. Commun. 60,926-933 (19741. 21. W. H. LEWIS and J. A. WRIGHT, Isolation of hydroxyurea-resistant CHO cells with altered levels of ribonucleotide reductase, Somat. Cell Genet. 5, 83--96 (19791. 22. L. AKERBLOM, A. EHRENBERG, A. GRASLUND, H. LANKINEN, P. REICHARD and L. THELANDER, Overproduction of the free radical of ribonucleotide reductase in hydroxyurea-resistant mouse fibroblast 3T6 cells, Proc. Natl. Acad. Sci. U.S.A. 78, 2159-2163 (1981). 23. J. A. WRIGHT and J. G. CORY, Alterations in the components of ribonucleotide reductase in hydroxyurea-resistant hamster cells, Biosci. Rep. 3,741-748 (1983). 24. G . A . McCLARTY, A. K. M. CHAN and J. A. WRIGHT, Characterization of a mouse cell line selected for hydroxyurea resistance by a stepwise procedure: Drug-dependent overproduction of ribonucleotide reductase activity, Somat. Cell Mol. Genet. 12,121-131 (1986). 25. R . G . HARDS and J. A. WRIGHT, N-carbamoyloxyurea-resistant Chinese hamster ovary cells with elevated levels of ribonucleotide reductase activity, J. Cell Physiol. 106, 3(N--319 (1981). 26. J. A. WRIGHT and W. H. LEWIS, Evidence of a common site of action for the antitumor drugs, hydroxyurea and guanazole, J. Cell Physiol. 83, 437--440 (1974). 27. B. ULLMAN, L. J. GUDAS, S. M. CLIFT and D. W. MARTIN, JR., Deoxyadenosine metabolism and cytoxicity in cultured mouse T-lymphoma cells: A model for immunodeficiency disease, Cell 14, 365-375 (1978). 28. B. ULLMAN, S. M. CLIFT, L. J. GUDAS, B. L. LEVINSON, M. A. WORMSTED and D. W. MARTIN, JR., Alterations in deoxyribonucleotide metabolism in cultured cells with ribonucleotide reductase activities refractory to feedback inhibition by 2'-deoxyadenosine triphosphate. J. Biol. Chem. 255, 83118-8314 (1980). 29. J . G . CORY and G. L. CARTER, Leukemia LI210 cell lines resistant to ribonucleotide reductase inhibitors, Cancer Res. 48,839-843 (1988). 30. B. ULLMAN, L. J. GUDAS, I. W. CARAS, S. ERIKSSON, G. L. WEINBERG, M. A. WORMSTED and D. W. MARTIN, JR., Demonstration of normal and mutant protein M 1 subunits of deoxyGTP-resistant ribonucleotide reductase from mutant mouse lymphoma cells, J. Biol. Chem. 256, 10189-10192 (1981). 31. G. L. CARTER and J. G. CORY, Cross-resistance patterns in hydroxyurea-resistant leukemia L1210 cells, Cancer Res. 48, 5796-5799 (19881. 32. C. L. K. SABOURIN, P. F. BAYES, L. GLATZNER, C. C. CHANG, J. E. TROSKO and J. A. BOEZI, Selection of aphidicolin-resistant CHO cells with altered levels of ribonucleotide reductase, Cell. Genet. 7,255-268 (1981). 33. D. AYUSAWA, K. IWATA and T. SENO, Alteration of ribonucleotide reductase in aphidicolin-resistant mutants of mouse FM3A cells with associated resistance to arabinosyladenine and arabinosylcytosine, Somat. Cell Genet. 7, 27-42 (1981). .34. J. R. STEEPER and C. D. STEUART. A rapid assay for CDP reductase activity in mammalian cell extracts, Anal. Biochem. 34, 123-130 (1970). 35. J. G. CORY, F. A. RUSSELL and M. M. MANSELL, A convenient assay for ADP reductase using Dowex-l-borate columns, Anal. Biochem. 55,449-456 (19731. 36. J. G. CORY, L. LASATER and A. SATO, Effect of iron-chelating agents on inhibitors of ribonucleotide reductase, Biochem. Pharmacol. 30,979-984 (19811. 37. J . G . CORY, G. L. CARTER. P. E. BACON, A. TANG and E. J. LIEN, Inhibition of ribonucleotide reducta~ and L121(1 cell growth by N-hydroxy-N'-aminoguanidine derivatives, Biochem. Pharmacol. 34, 2645-2650 (1985).
DRUG-RESISTANT LI2111 CELLS 38.
139
L. THELANDER and A. GRASLUND, Mechanism of inhibition of mammalian ribonucleotide reductase by the iron chelate of l-formylisoquinoline thiosemicarbazone. Destruction of the tyrosine free-radical of an enzyme in an oxygen-requiring reaction, J. Biol. Chem. 258, 4063--41K~6(1983). 39. H.L. ELFORD, B. VAN'T RIET, G. L. WAMPLER, A. L. LIN and R. M. ELFORD, Regulation of ribonucleotide reductase in mammalian cells by chemotherapeutic agents, Advan. Enzyme Regul. 19, 151-168 11981). 40. J. A. WRIGHT, T. G. ALAM, G. A. McCLARTY, A. Y. TAGGER and L. THELANDER, Altered expression of ribonucleotide reductase and role of M2 gene amplification in hydroxyurea-rcsistant hamster, mouse, rat and human cell lines, Somat. Cell Mol. Genet. 13, 155-165 (1987). 41. I. W. CARAS and D. W. MARTIN, JR., Molccular cloning of thc cDNA for a mutant mouse ribonuclcotide rcductase M1 that produces a dominant mutator phenotypc in mammalian ceils. Mol. Cell Biol. 8, 2698-2704 (1988).
INTRODUCTION
Ribonucleotide reductase (EC 1.17.4.1) is a key enzyme in the replication of DNA. 2'-Deoxyribonucleoside 5'-triphosphates, which are the substrates for D N A polymerase (EC 2.7.7.7), are generated via a de n o v o pathway in which ribonucleoside 5'-diphosphates are reduced to the corresponding 2'-deoxyribonucleoside 5'-diphosphates followed by phosphorylation to the dNTPs. This pathway is shown in Figure 1. The reaction catalyzed by ribonucleotide reductase is the rate-limiting step in D N A synthesis and it is to be expected that there would be critical properties of this
enzyme which would serve to make this an extremely delicate metabolic step. These properties include: (a) the enzyme activity is expressed as a function of the cell cycle (1); (b) the enzyme consists of two non-identical protein subunits encoded by different genes (2--6); (c) the genes, in terms of protein products, are not coordinately expressed as the cells pass through the cell cycle (7-9); (d) the enzyme is allosterically regulated by dNTPs acting as either positive or negative effectors (10-13); (e) the two subunits making up the active enzyme species do not have the same half-lives (14); and (f) the two subunits making up the active enzyme species can be specifically and independently inhibited/inactivated by specific classes of ribonucleotide reductase inhibitors (15-19). There have been many reports of the development of resistance of various mammalian cell types to ribonucleotide reductase inhibitors (20--31) and to agents such as aphidicolin (32, 33) which cause alterations at the ribonucleotide reductase site. In this report we compare the drug sensitivity/cross-resistance of L1210 cell lines resistant to specific classes of ribonucleotide reductase inhibitors and the properties of ribonucleotide reductase in the cell-free extracts from these cells to those of wild-type L1210 cells. 123
G. L. CARTER and J. G. CORY
124 ADP
•
dADP
=
GDP
-
dGDP
2
CDP
-
dCDP
~
UDP
-
dUDP
--dTMP
~ dATP. d G T P ~ - dCTP
- DNA
~--dTTP
FIG. 1. De novo pathway for synthesis of 2'-dcoxyribonucleoside 5'-triphosphates. Ribonucleoside 5'-diphosphates arc reduced via ribonucleotide reductase (1) to dNDPs: the dNDPs are phosphated to dNTPs by nuclcosidcdiphosphate kinase (2); dCMP deaminase activity (3) provides dUMP, the substrate for thymidylatesynthase (4); DNA [x~lymerase(5) catalyzes the polymerizationof dNTPs in the presence of template and primer.
METHODS AND M A T E R I A L S
Growth of LI210 cells and selection of resistant clones. Wild type L1210 cells were grown in RPMI 1640 culture medium which was supplemented with 10% horse serum, gentamicin sulfate (50 rag/l) and sodium bicarbonate (2 g/I). The cells were grown in a humidified incubator maintained at 37°C and 95% air/5% CO 2. The original culture was purchased from the American Type Culture Collection. The ED2 and HU-7 cell lines were selected as previously described (29, 31). The Y-8 cell line was selected by growing the wild-type L1210 cells in deoxyadenosine, 100 ~M/EHNA, 5 ~M and then plating out on soft agar. The colonies which grew out were regrown in liquid culture in deoxyadenosine, 100 p,M/EHNA, 5 t~M. The cells were then grown in deoxyadenosine, 300/zM/EHNA, 5 txM. Preparation of cell-free extracts from wild-type and resistant LI210 cell lines. The wild-type and resistant L1210 cells were grown to mid-log phase and collected by centrifugation. The cells were washed in phosphatebuffered saline, resuspended in Tris-HCI buffer, 0.02 M, pH 7 containing dithioerythritol, 1 raM. The cells were homogenized and the homogenates centrifuged at 21,000 x g for 1 hr at 4°C. The supernatant fractions were passed over Dowex-l-acetate columns to remove endogenous nucleotides and the eluants were precipitated with ammonium sulfate (80% saturation) and dialyzed. Assay of ribonucleotide reductase activities. CDP reductase was assaye d using the Dowex-l-borate method of Steeper and Steuart (34). The assay mixture was as described by Carter and Cory (31). A D P reductase was assayed using the method of Cory et al. (35) with the assay mixture as described by Carter and Cory (31).
DRUG-RESISTANTLI210CELLS
125
[14C]Cytidine metabolism in L1210 cells. Wild-type, HU-7, Y-8 and ED2 L1210 cells werc grown in culture to mid-log phase. The cells were collected by centrifugation and resuspended in fresh medium to a concentration of 1.6 × IIY' ceils/ml. The cells were incubated in the presence and absence of ribonucleotide reductase inhibitors for 1 hr at 37°C in 95% air/5% CO 2. [14C]Cytidine (0.5 p.Ci, 350 mCi/nmol) was added and the cells werc incubated for an additional hour. The cells were collected and treated as previously described to separate the acid-soluble, RNA and DNA fractions (29). Deoxycytidinc nucleotides were isolated from the acid-soluble nucleotide pools (29). Materials. The tissue culture medium (RPMI 1640), horse serum and NaHCO 3 were purchased from GIBCO. Hydroxyurea and the nucleosides were purchased from Sigma Chemical Company. 4-Methyl-5-amino1-formylisoquinoline thiosemicarbazone (MAIQ) and 2,3-dihydro-lHimidazo(1,2-b)pyrazole (IMPY) were obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute, through the assistance of Dr. Nancita R. Lomax. Desferal (Df) was a gift from Ciba-Geigy Corporation. 1lsoquinolylmethylene-N-hydroxy-N'-aminoguanidine tosylate (HAG-IQ) and 2-fluorodeoxyadenosine were gifts from Drs. Eric J. Lien and John A. Montgomery, respectively. Erythro-9-(2-hydroxy-3-nonyi)adenine (EHNA) and 8-aminoguanosine (8-AGuo) were purchased from Burroughs-Wellcome Company and Calbiochem-Behring, respectively. The radiochemicals, [14C]CDP and [3H]ADP. were purchased from New England Nuclear.
RESULTS AND DISCUSSION Generation of LI210 Cell Lines Resistant to Ribonucleotide Reductase lnhibitors Earlier studies had shown that combinations of drugs simultaneously directed at the non-heine iron and effector-binding subunits of ribonucleotide reductase resulted in strong synergistic inhibition of L1210 cell growth in culture. For example, as shown in Figure 2, a combination of hydroxyurea and deoxyadenosine (16) or IMPY and deoxyguanosine (17) caused synergistic inhibition of L1210 cell growth and synergistic cytotoxicity. In carrying out colony forming assays with these drug combinations it was observed that several colonies of L1210 cells grew out on soft agar in the combination of dAdo/EHNA/IMPY/Desferal which essentially completely inhibited L1210 cell growth (Fig. 3). These colonies were "picked" and grown out in liquid culture and further subjected to the selective pressure of higher drug concentrations of deoxyadenosine and
126
G. L. CARTER and J. G. CORY
CDP
'~ d C D P
HU IMPY MAIQ HAG - IQ
dATP dGTP F-dATP
FIG. 2. Subunit nature of mammalian ribonucleotide reductase. Ribonucleotide reductase consists of two non-identical protein subunits, coded for by different genes. The non-heine iron (NHi) and effector-binding (EB) subunits can be specifically and independently inhibited by the drugs as indicated.
o o A •
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O
I,(2
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it) w
tO
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~i~~_/dolOlp[H~;lDP/~JjYiNiOD/l ~
,
DAYS IN CULTURE
FIG. 3. Growth of LI210 cells in culture. LI210 cells were grown in culture in the presence and ab~nce of drugs as indicated. The four-drug combination (Q) contained dAdo, 15 V.M/EHNA, 5 V.M/IMPY, 190 p,M/Desferal, 20 V.M.
LI210 Ceils
LI210 Cells
LI210 CeUs
"resistant" LI210 clones
"resistant" LI210 clones
HU. 0 SrnM HU-5
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X ~ . I OmM HU-IO
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~l~tl~lt~y. 4.4~tE/Mt~ 0N~~ ED-2
FIG. 4. Selection of LI210 cells resistant to inhibitors of ribonucleotide reductase.
DRUG-RESISTANT LI210 CELLS
127
IMPY at fixed concentrations of EHNA and Desferal. These resistant cell lines were referred to as ED1 and ED2 cell lines. The diagram in Figure 4 shows the selection procedures for the L1210 cell lines resistant to deoxyadenosine/IMPY (ED2), hydroxyurea (HU-7) and deoxyadenosine (Y-8). These drug-resistant L1210 cell lines had the same doubling times as the wild-type L1210 cells. This was an especially important feature of these L1210 cell lines since ribonucleotide reductase is a cell-cycle specific enzyme.
Effects of Ribonucleotide Reductase Inhibition on LI210 Cell Lines The effects of hydroxyurea, IMPY, deoxyadenosine and deoxyguanosine on wild-type L1210 cells and HU-7, Y-8 and ED2 resistant cell lines are shown in Figures 5--7. As can be seen in Figure 5, the HU-7 cell line was very resistant to hydroxyurea and IMPY (approximately 10-fold increase in IC5o) with essentially no increase in resistance to either deoxyadenosine or deoxyguanosine. The Y-8 cell line (Fig. 6) showed marked resistance to deoxyadenosine (approximately 100-fold), a 3.5-fold increase in resistance to deoxyguanosine and a similar increase in resistance to hydroxyurea (2-fold) and IMPY (4-fold). The Y-8 cell line showed a high degree of cross-resistance to 2-fluorodeoxyadenosine (data not shown). The ED2 cells were highly resistant to deoxyadenosine with a corresponding cross-resistance to deoxyguanosine (Fig. 7). In addition, these cells showed a 5- to 6-fold increase in resistance to hydroxyurea and IMPY. These data show: (a) that the cells (HU-7) selected for resistance to hydroxyurea were likewise highly cross-resistant to IMPY, but were still sensitive to deoxyadenosine and deoxyguanosine; (b) that the cells (Y-8) selected for resistance to deoxyadenosine did not display the same degree of cross-resistance to deoxyguanosine and were still relatively sensitive to hydroxyurea and IMPY although there were increases in their IC.~o'srelative to the wild-type cells; and (c) that the L1210 cells which had been selected in the presence of both deoxyadenosine and IMPY (ED2) showed marked cross-resistance to deoxyguanosine and 2-fluorodeoxyadenosine with lesser cross-resistances to IMPY (5.5-fold) and hydroxyurea (6.1-fold). The effects of MAIO and HAG-IQ, potent inhibitors of ribonucleotide reductase, on these cell lines were also studied. As seen in Figure 8, there was little or no change in the sensitivities of the HU-7, Y-8 and ED2 cell lines, relative to the wild-type L1210 cells lines, to these ribonucleotide reductase inhibitors. All of these data are summarized in Table 1. Drug-resistant cell lines were generated in response to the selective pressure of specific inhibitors directed at the individual subunits of ribonucleotide reductasc. The cross-resistance patterns of these cells depended on the agent to which the resistance had been developed. It is particularly intriguing that these cell lines generated
G. L. CARTER and J. G. CORY
128
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-7
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FIG. 5. Growth of wild-type and HU-7 LI210 cell lines in the presence of hydroxyurea, IMPY, deoxyadenosine or deoxyguanosine. The wild-type and HU-7 L1210 cells were seeded at approximately 150,000 cells/well (24-well plate; 2 ml) in the presence of drug as indicated. EHNA was added to inhibit adenosine deaminase; 8-aminoguanosine was added to inhibit purine nucleoside phosphorylase. After 72 hr, aliquots were removed and cell counts made.
in r e s p o n s e to inhibitors o f ribonucleotide reductase and which s h o w m a r k e d resistance to either h y d r o x y u r e a or d e o x y a d e n o s i n e w e r e not resistant to the inhibitors, M A I Q and H A G - I Q . Previous studies have indicated that M A I Q (36) and H A G - I Q (37) may have different m e c h a n i s m s of inhibition of r i b o n u c l e o t i d e reductase c o m p a r e d with hydroxyurea and I M P Y e v e n though they are both directed at the n o n - h e m e site. For e x a m p l e , iron-chelating agents p o t e n t i a t e d the effects of hydroxyurea and I M P Y but c o m p l e t e l y reversed the inhibition caused by M A I Q (36). It has been reported that hydroxyurea and M A I Q inhibit r i b o n u c l e o t i d e reductase
DRUG-RESISTANT LI210 CELLS I001
129
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FIG. 6. Growth of wild-type and Y-8 LI210 cell lines in the presence of deoxyadenosine, deoxyguanosine, IMPY or hydroxyurea. The wild-type and Y-8 cells were seeded at approximately 150,(X)0cclls/well (24-well plate; 2 ml) in the presence of drugs as indicated. activity by consuming the frec radical on the non-heme iron subunit (22, 38) which would indicate the same site of inhibition on the same subunit. Using the approach of Elford (39), we compared the rates of consumption of the free radical on diphenylpicrylhydrazyl by hydroxyurea and M A I Q . These date are shown in Figure 9. Although M A I Q is at least 100 times more active than hydroxyurea as an inhibitor of ribonucleotide reductase activity and tumor cell growth, hydroxyurea was 5 times more active than M A I Q
G. L. CARTER and J. G. CORY
130
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FIG. 7. Growth of wild-type and ED2 LI21() cell lines in the presence of deoxyadenosine, deoxyguanosine, IMPY or hydroxyurea. The wild-type and ED2 LI210 cells were seeded at approximately 15(I,000 cells/well (24-well plate; 2 ml) in the presence of drug as indicated. in i n t e r a c t i n g with the free radical on d i p h e n y l p i c r y l h y d r a z y l . W h e t h e r this r e p r e s e n t s a basis for the d i f f e r e n c e seen in d r u g sensitivities in the r e s i s t a n t cells is u n c l e a r at this t i m e .
Properties of Ribonucleotide Reductase in Cell-Free Extracts From WildType and Resistant L1210 Cell Lines T h e b i o c h e m i c a l b a s e s for the resistance of the H U - 7 , Y-8 a n d E D 2 cells to specific i n h i b i t o r s o f r i b o n u c l e o t i d e r e d u c t a s e i n h i b i t o r s h a v e b e e n s t u d -
DRUG-RESISTANT LI210 CELLS
131
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,\ \
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40
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FIG. 8. Effect of MAIO on wild-type, HU-7, Y-8 and ED2 LI210 cell lines. The LI210 cells were seeded at approximately 150,000 cells/well (24-well plate; 2 ml) in the presence of MAIQ as indicated. After 72 hr, aliquots were removed and cell counts made.
030
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0.1
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0.5
FIG. 9. Effects of hydroxyurea and MAIQ on the free radical on diphenylpicrylhydrazyl. The loss of the free radical, as measured by the decrease in absorbance at 518 nm, was determined at several concentrations of hydroxyurea or MAIQ. as indicated. These rates were followed at 25°C. TABLE I. ICso VALUES FOR INHIBITION OF WILD-TYPE LI210 CELLS AND REDUCTASE INHIBITORS CELL LINES ICso, #M Drug Deoxyadenosine/EHNA Deoxyguanosine/8-AGuo F-Deoxyadenosine Hydroxyurea IMPY/Df MAIQ HAG-IQ
Wild type
HU-7
Y-8
ED2
33 136 1.9 77 198 2. i 2.5
52 116 p 2000 1470 2.4 3.4
>3000 480 >16 1.55 790 3.5 3.5
1000 >!000 37 475 1090 3.0 2.0
132
G. L. CARTER and J. G. CORY
led. R i b o n u c l e o t i d e reductase activity in cell-free extracts from the H U - 7 treated cells was markedly elevated. C D P and A D P reductase activities were both elevated as secn in Figure 10. H o w e v e r , the two activities were not coordinately increased. A D P reductase activity was increased 13-fold while the C D P reductase was increased only 5-fold. In a c o m p a r i s o n of the properties of the ribonucleotide reductase in cell-free extracts it was f o u n d that there were no differences in the inhibitory effects of h y d r o x y u r e a , I M P Y , d A T P , d T F P or d G T P between the H U - 7 ceils and the wild-type L1210 cells. T h a t is, the e n z y m e from the H U - 7 cells was still sensitive to the effects of h y d r o x y u r e a and I M P Y , even though intact cells showed a high degrce of resistance to the growth-inhibitory effects of h y d r o x y u r e a
ADP Reductose
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PROTEIN. mg
,~o
FIG. I0. CDP and ADP reductase activities in cell-free extracts from wild-type and HU-7 LI210 cells. Cell-free extracts were prepared, passed over Dowex-l-acetate and assayed for CDP and ADP reductase activities.
D R U G - R E S I S T A N T LI210 C E L L S
133
and IMPY. It was not expected that there would be any changes in the effects of d A T P , d'lq'P or d G T P as negative modulators of ribonucleotide reductase from these hydroxyurea-resistant cells. Other hydroxyurea-resistant L1210 cell lines were also studied. It was shown that HU-5 and HU-10 cell lines had increases in reductase activity, but at levels lower than the HU-7 cells. The degree of resistance of the LI210 cells to growth inhibition by hydroxyurea correlated with the level of increase in reductase activity (31). Ribonucleotide reductase activity in extracts from the Y-8 cells was only slightly increased (less than 2-fold) relative to the extracts from the wild-type cells. This was seen for both CDP and A D P reductase activities. However, in contrast to the reductasc activity in the extracts from HU-7 cells, the ribonucleotide reductase from the Y-8 cells was essentially insensitive to d A T P as a negative effector. These data are shown in Figure 11. On the other hand, the CDP rcductase activity from the Y-8 cells was inhibited by d G T P to the same extent as the enzyme from wild-type cells. In the ED2 cells, CDP reductase activity was elevated as much as 9-fold over that of the wild-type cells. However, there was essentially no increase in A D P reductase activity as measured in the same cell-free extracts. As seen in Figure 12, the ribonucleotide reductase from the ED2 cells was essentially unaffected by d A T P at concentrations as high as 150/.tM whereas the CDP reductase activity in the wild-type cells was completely inhibited by d A T P at this concentration. On the other hand, the CDP reductase activity from the ED2 cells was sensitive to dGTP. CDP reductase activity in the extracts from wild-type and ED2 cells was inhibited to the same extent by hydroxyurea and IMPY. The data obtained from the studies with the cell-free extracts from the .m
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o
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o ~
o
WT
11. D
o
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6~0 ' dATP,.tlM
"
120
i
i
2 0 400 dGTP,JJM
i
FIG. 11. Effects of d A T P and d G T P on C D P reductase activities in LI210 cell lines. Crude extracts were prepared from the wild-type and Y-8 LI210 cell lines. C D P reductase activity was determined in the presence of d A T P or d G T P as indicated.
134
G. L. CARTER and J. G. CORY I00
IOC
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I I 150 dGTP, ~M
O
3
60
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I 300
I
FIG. 12. Effects of dATP and dGTP on CDP reductasc activitics in LI210 cell lines. Crude extracts were prepared from wild-type and ED2 LI210 cell lines. CDP reductase activity was determined in the presence of dATP or dGTP as indicated. The control activities for wild-type and ED2 cell lines were: in the left panel, 0.08 and 0.29 nmol/30 min/mg protein respectively: and in the right panel, 0.06 and 0.46 nmol/31)min/mg protein, respectively.
H U - 7 a n d E D 2 cell lines showed that these cells had e l e v a t e d levels of r i b o n u c l e o t i d e reductase, possibly due to increases in the n o n - h e m e iron a n d e f f e c t o r - b i n d i n g s u b u n i t s , respectively. Cell-free extracts from the H U - 7 a n d E D 2 cells were mixed a n d assayed for C D P a n d A D P reductase activities. As seen in T a b l e 2, the m i x t u r e of extracts from H U - 7 a n d E D 2 cells gave C D P a n d A D P reductase activities which were much in excess of the activity expected o n the basis of simple a d d i t i o n of activities. This w o u l d s u p p o r t the n o t i o n that the H U - 7 cells had excess n o n - h e m e iron s u b u n i t while the E D 2 cells had excess e f f e c t o r - b i n d i n g s u b u n i t . W h e n the extracts from H U - 7 a n d Y-8 cells were mixed there was no increase in activity b e y o n d the simple a d d i t i o n of the activities from the two extracts consistent with the lack of increase of reductase activity in the Y-8 cells. TABLE 2. EFFECT OF MIXING CELL-FREE EXTRACTS FROM HU-7 AND ED2 LI210 CELL LINES Crude extract HU-7, 20 t.tl ED2, 40 ~1 HU-7, 20 #1 + ED2, 40 gl
Reductase activity CDP
(nmol/30 rain) ADP
0.038 0.059 0.161 (0.097)*
0.078 0.089 0.358 (0.167)*
*Simple summation of activities for each extract.
135
DRUG-RESISTANT L1210 CELLS
Incorporation of [t4C]Cytidine into DNA for Wild-Type, HU-7, 1"-8 and ED2 L1210 Cell Lines The incorporation of [14C]cytidine into DNA depends entirely on the level of flux of cytidine via CDP through the intracellular activity of ribonucleotide reductase to dCTP. The effects of ribonucleotide reductase inhibitors on the formation of [~4C]deoxycytidine nucleotides and incorporation into DNA from [14C]cytidine were studied in wild-type, HU-7, Y-8 and ED2 cells. As seen in Table 3, hydroxyurea, IMPY and deoxyadenosine markedly inhibited the formation of dcoxycytidine nucleotides and incorporation into DNA of the wild-type L1210 cells (experiments I and II). In the HU-7 cell line, there was only 40% inhibition of the formation of deoxycytidine nucleotides by either hydroxyurea or deoxyadenosine. This lack of inhibition, in view of the sensitivities of the enzyme in cell-free extracts to hydroxyurea or dATP, is probably related to the marked increase in reductase activities which was observed (Fig. 10). [14C}Cytidine reduction to deoxycytidine nucleotides in the Y-8 cell line was inhibited by hydroxyurea but relatively insensitive to deoxyadenosine/EHNA. These data are consistent with the fact that there was no increase in reductase activity in the Y-8 cells, no change in inhibition of reductase activity in cell-free extracts by hydroxyurea and the loss of sensitivity to feedback inhibition by dATP in these extracts. The metabolism of cytidine in thc ED2 cell line, which had TABLE 3. METABOLISM OF [z4C]CYTID1NE IN WILD-TYPE, HU-7, Y-8 AND ED2 L1210 CELL LINES IN THE PRESENCE OF RIBONUCLEOTIDE REDUCTASE INHIBITORS dCyd + DNA cpm/106 cells
% control
WT + HU, 200/gM + dAdo, 200/aM/EHNA, 5/.tM
3768 300 343
100 8 9
HU-7 + HU, 200//M + dAdo, 200/~M/EHNA, 5/~M
3761 2310 2200
100 61 59
Y-8 + HU, 200/~M + dAdo, 200/~M/EHNA, 5/./M
2212 287 1759
100 13 80
WT + IMPY, 360 ~aM/Df, 40/~M + dAdo, 600/~M/EHNA, 5 pM
1640 183 0
100 11 0
ED2 + IMPY, 360 #M/Dr, 40/t/M + dAdo, 600/~M/EHNA, 5/./M
3730 1004 1190
100 61 73
Experiment I
136
G.L. CARTER and J. G. CORY
been generated in response to the pressure of elevated levels of both IMPY and deoxyadenosine, was inhibited modestly compared with the wild-type cells (experiment II). These data are consistent with the fact that there was an increase in reductase activity and the loss of sensitivity to dATP. Recent studies have shown that hydroxyurea-resistant cell lines have elevated levels of the non-heme iron subunit, the mRNA for the non-berne iron subunit and in some cases amplification of the gene for the non-heine iron subunit (6, 40). Our data with the HU-7 LI210 cells are consistent with these studies. Caras and Martin (41) have shown the mutant $49 iymphoma cell line which has ribonucleotide reductase activity insensitive to feedback inhibition by dATP has an altered sequence in the cDNA for the effector-binding subunit (a guanine to adenine transition). Our data from the Y-8 and ED2 cell lines are compatible with this observation. From our data it is clear that the specificity of resistance to ribonucleotide reductase inhibitors depends on the drug to which the resistance has been developed and the subunit to which the drug is directed. The resistance has been shown to be due to elevated levels of ribonucleotide reductasc activity (HU-7 and ED2) or altered mechanisms of feedback control (Y-8 and ED2). The molecular bases for these observations are currently under study.
SUMMARY LI210 cell lines were generated which were resistant to specific ribonucleotide reductase inhibitors. Hydroxyurea-resistant L1210 cells (HU-7) were cross-resistant to IMPY but sensitive to deoxyadenosine and deoxyguanosine. Deoxyadenosine-resistant L1210 cells (Y-8) were cross-resistant to 2-fluorodeoxyadenosine and showed only a small increase in resistance to hydroxyurea or IMPY. L1210 cells which were generated in the presence of deoxyadenosinelEHNAIIMPYlDesferal were markedly resistant to deoxyadenosine, deoxyguanosine and 2-fluorodeoxyadenosine with moderate increases in resistance to IMPY. The HU-7, Y-8 and ED2 cell lines were sensitive to the inhibitory effects of MA[Q and HAG-IQ. The HU-7 LI210 cell line had elevated levels of ribonucleotide reductase activity and this activity showed normal inhibition by hydroxyurea, IMPY, dATP, dGTP and dTTP. The Y-8 LI210 cell line did not have elevated levels of ribonucleotide reductase activity, but had altered allosteric properties relative to dATP. The ED2 LI210 cell line had elevated levels of ribonucleotide reductase activity and had altered allosteric properties relative to dATP. These data show that resistance to ribonucleotide reductase inhibitors is specifically generated in response to the particular drug. The biochemical
DRUG-RESISTANT LI210 CELLS
137
basis can be related to either increased levels of ribonucleotide reductase activity or loss of feedback control by d A T P or both. ACKNOWLEDGEMENTS
This work was supported by grants from the USPHS, National Cancer Institute CA-27398 and CA-42070 and the Phi Beta Psi Sorority. REFERENCES I. 2. 3. 4. 5.
6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17.
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DRUG-RESISTANT LI2111 CELLS 38.
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L. THELANDER and A. GRASLUND, Mechanism of inhibition of mammalian ribonucleotide reductase by the iron chelate of l-formylisoquinoline thiosemicarbazone. Destruction of the tyrosine free-radical of an enzyme in an oxygen-requiring reaction, J. Biol. Chem. 258, 4063--41K~6(1983). 39. H.L. ELFORD, B. VAN'T RIET, G. L. WAMPLER, A. L. LIN and R. M. ELFORD, Regulation of ribonucleotide reductase in mammalian cells by chemotherapeutic agents, Advan. Enzyme Regul. 19, 151-168 11981). 40. J. A. WRIGHT, T. G. ALAM, G. A. McCLARTY, A. Y. TAGGER and L. THELANDER, Altered expression of ribonucleotide reductase and role of M2 gene amplification in hydroxyurea-rcsistant hamster, mouse, rat and human cell lines, Somat. Cell Mol. Genet. 13, 155-165 (1987). 41. I. W. CARAS and D. W. MARTIN, JR., Molccular cloning of thc cDNA for a mutant mouse ribonuclcotide rcductase M1 that produces a dominant mutator phenotypc in mammalian ceils. Mol. Cell Biol. 8, 2698-2704 (1988).