d - cancer research
TRANSCRIPT
Comparison of the Toxicity and Metabolism of
9-/?-D-Arabinofuranosyl-2-
fluoroadenine and 9-ß-D-Arabinofuranosyladenine in Human Lymphoblastoid Cells1
William Plunkett,2 Sherri Chubb, Lillie Alexander, and John A. Montgomery
Department of Developmental Therapeutics. The University of Texas System Cancer Center, M. D Anderson Hospital and Tumor Institute, Houston. Texas 77030 ¡W.P., S. C., L. A ¡,and Organic Chemistry Department, Southern Research Institute, Birmingham. Alabama 35205 ¡J.A. M.]
ABSTRACT
The toxicity and metabolism of 9-/S-D-arabinofuranosyl-2- fluoroadenine (F-ara-A), an adenosine deaminase-resistant nu- cleoside analog, have been compared to those of 9-/8-o-arabi- nofuranosyladenine (ara-A) in the presence of the adenosine deaminase inhibitor, deoxycoformycin. Equal concentrations of F-ara-A and ara-A plus deoxycoformycin produced similar inhibition of growth of CCRF-CEM human lymphoblastoid cells. 9-/S-D-Arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F- ara-ATP) and 9-/?-D-arabinofuranosyladenine 5'-triphosphate
(ara-ATP), were concentratively accumulated intracellularly and exerted their major inhibitory effect on DNA synthesis. Two approaches were used to compare the effect of these nucleo- tide analogs on the DNA-synthetic capacity of whole cells, (a) The concentrations of F-ara-ATP and ara-ATP in cells incu
bated with the respective nucleosides were determined directly at the same time that the DNA-synthetic capacity of the cells in each culture was measured by incorporation of [3H]thymidine
into DNA. Neither compound significantly affected the specific activity of cellular [3H]deoxythymidine triphosphate. (b) The rates of disappearance of F-ara-ATP and ara-ATP from cells
washed free of the nucleosides were determined. These values were used to calculate the cellular concentration of each nu- cleotide analog in cells in which DNA-synthetic capacity was monitored after incubation with F-ara-A or ara-A plus deoxy
coformycin and washed into fresh media. These determinations indicated that the rates of accumulation of F-ara-ATP and ara-
ATP differed and were related to the concentration of the exogenous nucleoside. F-ara-ATP disappeared from cells in drug-free media at several times the rate of the disappearance of ara-ATP. However, at equal cellular concentrations, F-ara-
ATP was slightly more inhibitory to the processes measured by thymidine incorporation than was ara-ATP. We conclude that F-ara-ATP and ara-ATP share a similar mechanism of action and potency of inhibition of DNA synthesis. The fact that F-ara-
A retains its cytotoxic efficacy in the absence of inhibitors of adenosine deaminase provides a rationale for investigating its antitumor properties further.
INTRODUCTION
The therapeutic effectiveness of ara-A3 is limited by its deam-
Received November 15, 1979; accepted April 3, 1980. ' Supported by NIH Grants CA 14528, CA 11520, and RR 05511; Contract
N01 CM 87185; and American Cancer Society Grant CH-130. 2 To whom requests for reprints should be addressed. 1The abbreviations used are: ara-A, 9-/J-D-arabinofuranosyladenine; ara-ATP.
9-/j-D-arabinofuranosyladenine 5'-triphosphate; F-ara-A. 9-/3-D-arabinofuranosyl- 2-fluoroadenine; dCF. 2'-deoxycoformycin or (R)-3-(2-deoxy-/J-o-erythropento- furanosyl)-3,6.7.8-tetrahydroimidazo[4,5-rf][1.3]diazepin-8-ol; RPMI-1640, Ros-
ination by adenosine deaminase to the relatively inactive Q-ß- D-arabinofuranosylhypoxanthine (4). It is now well established
that this inactivation may be minimized and that a higher therapeutic efficacy may be achieved by coadministration of ara-A with any one of several effective inhibitors of adenosine
deaminase on an appropriate treatment schedule (13, 14, 19, 24, 26). However, rational use of this combination drug treat ment may be complicated by the diverse duration of activity (1, 3, 20) and tissue specificity (29, 30) of the deaminase inhibi tors, as well as the pharmacokinetic properties of ara-A and the tissue kinetics of the active metabolite, ara-ATP (20). In
addition, it is clear that the adenosine deaminase inhibitors evoke multiple effects on cellular metabolism (2, 12), that they are immunosuppressive (7, 15), and that they may possess antitumor activity in humans (28).
Thus, the synthesis of F-ara-A (17) and the subsequent
demonstration (6) that this analog is resistant to deamination by adenosine deaminase, yet retains the antitumor activity of ara-A in the presence of a deaminase inhibitor, have evoked a considerable amount of interest. The purpose of the present study was to compare the toxicity of F-ara-A with that of ara-A
in the presence of a deaminase inhibitor and to correlate the biochemical elements that characterize this toxicity, such as the accumulation and flux of the respective active triphosphates and their effects on DNA synthesis. A preliminary report of the results of these investigations has been published (21).
MATERIALS AND METHODS
Materials. ara-A and dCF were provided by the Drug Devel
opment Branch, Division of Cancer Treatment, National Cancer Institute. F-ara-A was synthesized in the laboratory of J. A. Montgomery (16). ara-ATP, used as a standard for high-pres sure liquid chromatography, was purchased from P-L Biochem- icals, Inc. (Milwaukee, Wis.). [/r)effty/-3H]Thymidine (6.0 Ci/ mmol), [5-3H]uridine (25.4 Ci/mmol), and [2-3H]ara-A (18.7 Ci/
mmol) were obtained from ICN Pharmaceuticals, Inc. (Irvine, Calif.). Radioactive ara-A was routinely recrystallized from H?O
before use and shown to be of greater than 99% purity by analysis of the radioactivity eluted by high-pressure liquid chromatography on C,8-/iBondapak (Waters Associates, Inc.,
Milford, Mass.). Schwarz/Mann (Orangeburg, N. Y.) supplied [4,5-3H]leucine (61 Ci/mmol).
Cell Culture Methods. A T-cell line of human lymphoblastoid cells, CCRF-CEM (9), was maintained in exponential growth at 37°in sealed agitated suspension cultures in RPMI-1640 sup-
well Park Memorial Institute Tissue Culture Medium 1640; F-ara-ATP. 9-/Õ-D- arabinofuranosyl-2-fluoroadenine 5'-triphosphate; PBS. phosphate-buffered sa
line (8.1 g of NaCI. 0.22 g of KCI, 1.14 g of Na2HPO4, and 0.27 g of KH?PO,, per liter of H20. pH 7.4).
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W. Plunkett et al.
plemented with 10% fetal calf serum (Grand Island Biological Co., Grand Island, N. Y.). Cell number and volume were deter mined by a Model ZBI electronic particle counter (Coulter Electronics, Inc., Hialeah, Fla.), equipped with a cell-sizing instrument (Model C-1000) which was calibrated with latex
beads 10.0 ¿imin diameter (Coulter Electronics). The effect of arabinosyl nucleosides on cell growth was determined as fol lows. CCRF-CEM cells were adapted for several days of growth in RPMI-1640 supplemented with 10% horse serum (Grand
Island Biological Co.) that had been dialyzed for 3 days against multiple changes of PBS in our laboratory. This medium, in which adenosine deaminase activity contributed by the horse serum is below the limit of detection, supported cell growth at rates comparable to those of RPMI-1640 plus 10% fetal calf serum (population-doubling time, 22 hr). Drugs were prepared and diluted in RPMI-1640 and added in a final volume of 0.5- ml to 3-ml wells of microtiter plates (Flow Laboratories Inc.,
Hamden, Conn.). Just before incubation, cells were washed into RPMI-1 640 supplemented with 20% dialyzed horse serum, and a 0.5-ml aliquot of 5 x 10" cells was added to each well
containing drugs or medium only (control). Following 72 hr of incubation at 37° in a high-humidity 5% CO?:95% air atmo
sphere, each well was diluted with 1 ml PBS, and the total cell number from duplicate cultures was determined. After subtrac tion of the number of cells plated from the final number of cells in control wells, the growth of drug-treated cells was expressed
as a percentage of the control cell growth. Values obtained in replicate experiments generally differed by less than 10%.
Determination of ara-ATP and F-ara-ATP. Cells incubated with arabinosyl nucleosides were diluted with 2 volumes of ice-
cold PBS, harvested by centrifugation, and washed twice in cold PBS. Cells were suspended in 0.5 ml of cold H?O, and 0.5 ml of 0.8 N HCIO4 was added during vigorous mixing. Following a 5-min incubation in an ice bath, the acid-soluble material was
recovered by centrifugation. The pellet was washed with 0.5 ml of 0.4 N HCIO4, and after centrifugation the acid-soluble supernatants were combined and neutralized with KOH. Fol lowing chilling in ice, the KCIO* was removed by centrifugation, and the supernatant was analyzed for nucleotides by the fol lowing high-pressure liquid Chromatographie procedure. A Wa ters Associates ALC-204 high-pressure liquid Chromatograph
equipped with 2 Model 6000A pumps, a Model 660 solvent programmer, and a column of Partisil-10 SAX anion-exchange
resin (25 cm x 4.6 mm; Whatman, Inc., Clifton, N. J.) was used. Cell extracts (0.05 to 1.0 ml) containing 1 to 5 x 106 cell equivalents of acid-soluble material were injected onto the column with the U6K-LC injection system. Nucleoside mono-
and diphosphates were eluted with 60% 0.005 M NH4H2PO4 (pH 2.8) and 40% 0.750 M NH4H?PO4 (pH 3.7) at a flow rate of 2 ml/min for 10 min. A linear gradient from 40 to 100% 0.750 M NH4H?PO4 was then run for 24 min to separate ribonucleo- side triphosphates from ara-ATP and from F-ara-ATP. Eluted
nucleotides were detected by their absorbance at 254 nm by the Model 440 detector and were quantitated with a CDS-111 electronic integrator (VarÃanAssociates, Palo Alto, Calif.). Peak areas were converted to absolute quantities using predeter mined calibration curves that were linear with nucleotide con centration to a lower sensitivity of 25 pmol. Greater sensitivity was achieved if, after incubation of the cells with [3H]ara-A of
known specific activity, the ara-ATP was quantitated by col lecting the column eluate that coeluted with ara-ATP at 0.5-min
intervals into scintillation vials containing 0.5 ml of H?O and 11 ml of Aquasol (New England Nuclear, Boston, Mass.), and by determining radioactivity by scintillation counting (27). Al though we had successfully used published procedures (25) for the quantitation of ara-ATP by absorbance in the past (20),
we were surprised to find that a substantial portion of radio activity derived from [3H]ara-A coeluted with ATP and trailed into the ara-ATP peak. Using high-purity [3H]ara-A in the pres
ence of either dCF or erythro-9-(2-hydroxy-3-nonyl)adenine,
we have observed this phenomenon to varying degrees not only in CCRF-CEM cells but also in Chinese hamster ovary cells, phytohemagglutinin-stimulated human lymphocytes, hu
man bone marrow cells, and several murine ascitic tumors, and normal tissues. We assume that the labeled ATP arises from the labeled purine ring of [3H]ara-A either after deamination of 9-/8-D-arabinofuranosyladenine-5'-monophosphate (23) or by
deamination of the nucleoside by a species of adenosine deaminase, which is incompletely inhibited by these deaminase inhibitors (11). Following deamination, liberation of [3H]hypo-
xanthine is likely and may be reutilized by purine salvage pathways to form ATP. The Chromatographie separation pro cedure described herein minimizes contamination of ara-ATP
with radioactivity associated with ATP. Determining of [3H]dTTP Specific Activity. F-ara-A (100
nmol/ml) or ara-A (100 nmol/ml) plus dCF (10 nmol/ml) was added to cultures of CCRF-CEM cells in exponential growth 10 min before addition of [3H]thymidine (10 nmol/ml; 2.0 x 105
dpm/nmol). After 4 hr of incubation with the drugs, cells were diluted and washed with PBS, and acid-soluble material was
extracted as described above. Ribonucleotides were removed by oxidation with NalO4 and the remaining deoxyribonucleo- tides were fractionated by a high-pressure liquid Chromato graphie assay as described previously (19). The specific activ ity of cellular [3H]dTTP was calculated by dividing the radio
activity associated with the dTTP peak by the nmol of dTTP detected by UV absorbance. The effect of arabinosylnucleo- sides on the incorporation of [3H]dTTP into DNA was deter
mined as described below. Similar procedures were used to calculate the specific activity of [3H]UTP in acid-soluble ex tracts from cells incubated with [3H]uridine.
Incorporation of [3H]thymidine, [3H]Uridine, and [3H]Leu-
cine into Acid-insoluble Material. Cells were incubated with [3H]thymidine, [3H]uridine, or [3H]leucine for varying times to
estimate the amount of DNA, RNA, and protein synthesis in cells treated with arabinosyl nucleosides. Cells were diluted, washed with PBS, and extracted twice with 0.4 N HCIO4 as described above. The acid-insoluble pellet was washed with
5.0 ml of 0.4 N HCICv»before being resuspended in H2O and brought into solution with 2 drops of 1.0 N KOH. A portion of this fraction was added to a scintillation vial containing 11 ml of Aquasol, and the radioactivity was determined by a Beckman Model 2650 liquid scintillation spectrometer. The dpm, deter mined at an average counting efficiency of 35 to 37%, were computed by the instrument with the aid of a preprogrammed external standard quench curve.
RESULTS
Growth Inhibition by Arabinosyl Nucleosides. The effect of various concentrations of F-ara-A and ara-A, alone and in the presence of dCF, on the growth of CCRF-CEM cells is shown
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Comparison of F-ara-A and ara-A
in Chart 1. Cell growth was inhibited 50% by 3 fiM ara-A alone.
However, in the presence of 10 ¿IMdCF, which by itself is not growth inhibitory, the concentration of ara-A required to give comparable inhibition of growth was reduced to 0.2 ¡J.M.The synergistic action of these 2 compounds may be attributed to the inhibition of the appreciable capacity of these cells to deaminate ara-A (0.37 nmol deaminated per 1 x 105 cells per
hr) which effectively results in a longer incubation with higher concentrations. Subsequently, increased cellular ara-ATP lev els accumulate leading to greater growth inhibition (data not shown). F-ara-A alone was also effective at inhibiting cell
growth by 50% at 0.2 JUM,but this activity was not augmented by the addition of dCF. Subsequent experiments will address the metabolism of ara-A in the presence of dCF, since the metabolism of ara-A in the absence of a deaminase inhibitor has been described in other systems (23). In each experiment, cells were exposed to dCF alone for 30 min before addition of ara-A.
Metabolism of Arabinosyl Nucleosides. As in the case with ara-A, the active form of F-ara-A is thought to be the 5'-
triphosphate (6). F-ara-ATP accumulates in cells incubated with F-ara-A and may be detected and quantitated by high-
pressure liquid Chromatographie fractionation of cell extracts (Chart 2). The identical procedure results in a similar separation of ara-ATP from major cellular ribonucleoside triphosphates in extracts of cells incubated with ara-A plus dCF. The cellular
concentration of ribonucleoside triphosphates was not affected in cells incubated with either nucleoside analog. This elution scheme was designed to maximize the resolution of triphos phates at the expense of that of mono- and diphosphates,
which typically comprise less than 10% of the analog metabo lites in these cells.
Both F-ara-A and ara-A are concentratively phosphorylated in CCRF-CEM cells (Chart 3). Assuming a uniform intracellular
100
0.1 0.2 0.5 1.0 2.0 5.0 10 20 50
Arabinosyl nucleoside concentration, ^M Chart 1. Effect of F-ara-A and ara-A alone or in the presence of dCF (10
nmol/ml) on the growth of CCRF-CEM cells. Control cells grew from 0.52 to 3 02 x 10Vml in 72 hr. A. F-ara-A; A, F-ara-A plus dCF: •.ara-A; O, ara-A plus dCF; •10/nMdCF.
.004 -
.002 -
§.002
Chart 2. High-pressure liquid Chromatographie analysis of acid-soluble ex tracts from 1 x 106 CCRF-CEM cells incubated for 4 hr with ara-A (100 nmol/
ml) plus dCF (10 nmol/ml) or with F-ara-A ( 100 nmol/ml).
j5 600 0} <J
200
100
012345
Hours
Chart 3. Accumulation of F-ara-ATP or ara-ATP by cells incubated with F-ara- A (100 nmol/ml) (O) or ara-A (100 nmol/ml) plus dCF (10 nmol/ml) (•), respectively.
distribution and using the observed mean cell volume of 1084 cu urn, F-ara-ATP accumulated to cellular concentrations of greater than 500 /UMby 5 hr, whereas accumulation of ara-ATP
reached a plateau value of about 200 UM by 3 hr. Analysis of extracts from cells subjected to more prolonged incubations with lower concentrations of these analogs (1 to 5 /ÕM)indicated that the triphosphates accumulated to at least 100 times the concentration of the exogenous nucleosides.
The effects of such an incubation on the macromolecular synthesis of these cells are shown in Chart 4. The inhibition caused by ara-A in the presence of dCF was specific for DMA synthesis, but F-ara-A decreased the incorporation of both
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W. Plunkett et al.
uridine and leucine into RNA and protein, respectively, in addition to severely inhibiting DNA synthesis. Analysis of the specific activity of [3H]UTP in the acid-soluble fractions from cells labeled with [3H]uridine indicated a lower activity in each
of the samples from F-ara-A-treated cells than in the corre sponding samples from the controls. After normalization of the incorporation data based on the cellular [3H]UTP specific activ
ity determined at each time, no significant differences were observed between control and F-ara-A-treated cells. Investi gations into the effect of F-ara-A on the specific activity of [3H]UTP or on the incorporation of leucine have not been
extended. uptake and phosphorylation of [3H]thymidine or on the endog
enous pool size of dTTP that produced an alteration in the specific activity of the cellular [3H]dTTP pool could invalidate
this experimental approach for monitoring DMA synthesis. To control for this possibility, cells were incubated with 100 ¡UMF- ara-A or ara-A plus dCF for 4 hr. The effect of this incubation on the specific activity of cellular [3H]dTTP and the ability to
incorporate this nucleotide into DMA were determined (Table 1). The cellular [3H]dTTP specific activity was altered no more
than 10%, even when incorporation into DNA was inhibited by 96%. These results suggest that the quantitation of thymidine incorporation into acid-insoluble material is a reasonably ac curate method for monitoring the effects of arabinosyl nucleo-
sides on DNA synthesis. Correlation of Cellular Nucleotide Analog Levels with In
hibition of DNA Synthesis. The foregoing experiments and those from other studies (6) indicate that, as is the case with its congener ara-A, the active metabolite of F-ara-A is the nucleo- side triphosphate, F-ara-ATP, and that its major metabolic
target is cellular DNA synthesis. Therefore, to evaluate the relative inhibitory potency of ara-ATP and F-ara-ATP in vivo, it
w 0.8 "öj O o 0.6
E 0.4 C
Hours 02
Chart 4 Effect of F-ara-A (100 nmol/ml) or ara-A (100 nmol/ml) plus dCF (10 nmol/ml) on the incorporation of [3H]thymidine, [3H]uridine. or (3H]leucine
into acid-insoluble material of CCRF-CEM cells. A, control; O. F-ara-A; •.ara-A plus dCF.
Table 1 Effect of F-ara-A or ara-A plus dCF on the specific activity of cellular /3H/o7TP
and its incorporation into the DNA of CCRF-CEM cells
CCRF-CEM cells were incubated in the presence of pHJthymidine and either 100 fiM F-ara-A or 100 /IM ara-A. plus 1 fiM dCF for 4 hr. The effect of these compounds on the specific activity of cellular [3H]dTTP and its incorporation into DNA was determined as described in "Materials and Methods."
TreatmentControl
specific activity (104dpm/nmol)4.83(100)a
incorpora tion into DNA(nmol/107
Numbers in parentheses, percentage of control
Chart 5. Accumulation of F-ara-ATP and ara-ATP by CCRF-CEM cells incu bated with various concentrations of F-ara-A or ara-A plus dCF (10 nmol/ml) for 1 hr. Cellular nucleotide levels were determined as described in "Materials and Methods." O, F-ara-ATP; •.ara-ATP. The mean cell volume was 1084 cu /im or 9.24 x 10" cells/ml. The concentration of nucleotides in the cells, in nmol/ml.
may be calculated by multiplying the ordinate values by 0 924.
was necessary to determine and compare the effect of known cellular concentrations of each nucleotide on the cellular DNA- synthetic capacity. Cells were incubated with various concen trations of F-ara-A or ara-A plus dCF (10 nmol/ml) for 50 min before addition of [3H]thymidine for the remaining 10 min of
the incubation. The acid-soluble material was analyzed for nucleotide analogs and the incorporation of [3H]thymidine into
acid-insoluble material was used as an indicator of DNA syn thesis. Chart 5 illustrates that each nucleotide accumulates to similar levels in proportion to the exogenous nucleoside con centration. Calculations indicated that the cellular concentra tion of each nucleotide analog exceeded that of the exoge-
nously added nucleoside at each point, although this was more pronounced at lower concentrations of F-ara-A. This relatively
brief incubation did not detectably alter the mean cell volume of either culture.
In this same experiment, after cells were allowed to accu mulate F-ara-ATP and ara-ATP for 50 min, [3H]thymidine was
added for 10 min to determine the effects of the nucleotide analogs on DNA synthesis. The results of these determinations (Chart 6) indicate that F-ara-A was more inhibitory to thymidine
incorporation than were equal concentrations of exogenously added ara-A in the presence of dCF. This was particularly evident at lower nucleoside concentrations where F-ara-ATP
was found to be at higher concentrations in the cell (Chart 5). The findings from 2 different analyses of the same experi
ment, presented in Charts 5 and 6, are combined in Chart 7 to facilitate comparison of the effect of different cellular concen trations of F-ara-ATP and ara-ATP on DNA synthesis. It appears that, at equal cellular concentrations of nucleotide, F-ara-ATP
was slightly more inhibitory to thymidine incorporation than was ara-ATP. The sensitivity of our assay for F-ara-ATP did not permit determination of the cellular nucleotide concentration when thymidine incorporation was inhibited by 50%. However, the calculated cellular ara-ATP concentration was 3 nmol/1 x 106 cells when thymidine incorporation was inhibited by
50%. Cellular Flux of F-ara-ATP and ara-ATP and the Effect on
DNA Synthesis. The previous experiments were designed to provide a static view of the DNA-synthetic capacity of cells
during a short time interval when the change in cellular nucleo tide analog levels was minimal. The following experiments give
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100
50
u'S
100
Chart 6. Effect of various concentrations of F-ara-A or ara-A plus dCF (10 nmol/ml) on the incorporation of [3H)thymidine (10 nmol/ml; 2.86 x 10s dpm/
nmol) by CCRF-CEM cells. Cells were incubated with the drugs for 50 min before addition of [JH]thymidine for 10 min. Acid-insoluble material was then extracted from the cells as described in 'Materials and Methods. The control value was 352 pmol (3H]thymidine incorporated per IO6 cells per 10 min. O. F-ara-A; •
ara-A plus dCF.
pmol ara-ATP or F-ara-ATP / IxlO6 Cells
Chart 7. Effect of cellular levels of F-ara-ATP and ara-ATP on DMA synthesis by CCRF-CEM cells. The data presented in Charts 5 and 6 have been replotted to facilitate comparison O. F-ara-ATP; •.ara-ATP
a dynamic view of these ongoing processes. Cells were incu bated for 2 hr with F-ara-A or ara-A plus dCF and allowed to accumulate the respective triphosphates before being washed into drug-free media. The cellular nucleotide analog levels were determined at the indicated times (Chart 8). It is evident that F- ara-ATP disappears from the cells at several times the rate at which ara-ATP decays. In 4 experiments, the half-life of F-ara- ATP ranged from 2.5 to 4.7 hr, whereas that of ara-ATP ranged
from 12.9 to 15.3 hr. Knowing that F-ara-ATP may be more inhibitory to DMA
synthesis than is ara-ATP (Chart 7) but disappears more rapidly
from the cell in the absence of exogenous nucleoside (Chart 8), it was of interest to compare the rates at which cells treated with a pulse incubation of F-ara-A and ara-A recover their ability to synthesize DNA. Cells were incubated with F-ara-A or ara-A plus dCF for 1 hr and washed into fresh medium, and
their ability to incorporate [3H]thymidine during a 15 min pulse
was determined at the indicated times (Chart 9). Both cultures display similar kinetics of recovery of DNA-synthetic capacity,
being maximally inhibited for 8 hr and recovering to approxi mately 50% of control values after 14 hr. Cellular nucleotide analog concentrations, determined in the cells taken from each culture immediately after washing into drug-free medium, were 16.7 pmol of F-ara-ATP per 106 cells and 9.6 pmol of ara-ATP per 106 cells. Using the rates of disappearance of each nu
cleotide analog from Chart 8, the cellular concentrations of F- ara-ATP and ara-ATP 14 hr after washing were calculated to be 3.2 and 5.1 pmol/106 cells, respectively. The mean cell
volume of cells treated with each nucleoside analog increased 35% from 882 to 1194 cu ¿imduring the 14-hr incubation in drug-free medium, thus further diluting the concentration of the
intracellular nucleotide analogs. The mean volume of control cells increased slightly to 955 cu /im.
20\
\i>
8Shr16Hours24
Chart 8. Retention of F-ara-ATP and ara-ATP by CCRF-CEM cells. Cells were incubated for 2 hr with F-ara-A (100 nmol/ml) or ara-A (100 nmol/ml) plus dCF (10 nmol/ml) and washed into prewarmed (37°)drug-free media, and the levels
of cellular nucleotide analogs were determined at the indicated times as de scribed in 'Materials and Methods." O. F-ara-ATP; •.ara-ATP
70r
Hours
Chart 9. Recovery of the DNA-synthetic capacity of CCRF-CEM cells after a pulse with F-ara-A (100 nmol/ml) of ara-A (100 nmol/ml) plus dCF (2 nmol/ml). Following incubation for 1 hr, cells were washed into prewarmed (37°)drug-free
media, and the DNA-synthetic capacity was determined as the ability of a portion of the culture to incorporate (3H]thymidine (10 nmol/ml; 2.91 x 105 dpm/nmol)
into acid-insoluble material during a 15-min pulse. This was expressed as a percentage of the control value at each point. The mean control value of the 7 time points was 518 ±34 pmol [3H]thymidine incorporated per 106 cells per 15
min. O. F-ara-A; •.ara-A plus dCF.
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DISCUSSION
The objective of this study was to compare the growth- inhibitory activity and the cellular metabolism of F-ara-A with those of ara-A. Continuous incubation of human lymphoblastoid
cells with these compounds indicated that they inhibited cell growth equally when deaminative detoxification of ara-A was
eliminated by addition of dCF to the cultures (Chart 1). How ever, F-ara-A, which is not a substrate for adenosine deaminase
and the toxicity of which is not augmented by addition of dCF, was equally toxic at one-tenth the concentration of ara-A in the
absence of dCF. Although these compounds are equally potent in cell culture in the presence of dCF, it has been reported that inhibitors of adenosine deaminase do not completely inhibit host tissue enzyme activity in vivo (29, 30) and that substantial deamination of ara-A occurs even after high doses of deami nase inhibitors (20). Since host tissue enzyme comprises the bulk of the deaminase activity in vivo, this residual activity may in part account for the observed differences in therapeutic activity between F-ara-A and ara-A plus dCF in vivo (6). The fact that F-ara-A retains its cytotoxicity in the absence of
inhibitors of adenosine deaminase provides a basis for further investigations of its antitumor properties.
Both F-ara-A and ara-A were concentratively phosphorylated to the respective 5'-triphosphates, the presumed active metab
olite of each compound. F-ara-ATP accumulated to higher cellular concentrations than did ara-ATP after 1 hr of incubation
with low exogenous levels of nucleoside (Chart 5). Cells incu bated with the analogs at higher concentrations accumulated F-ara-ATP at a nearly linear rate for 5 hr, whereas ara-ATP accumulation reached a plateau value of about one-third the 5- hr F-ara-ATP concentration by 3 hr (Chart 3). We have ob
served similar ratios for the accumulation of each nucleotide in phytohemagglutinin-stimulated human lymphocytes (22), but the value for F-ara-ATP:ara-ATP accumulated by normal hu man bone marrow cells approaches 9.4 Differences in the
amount of each nucleotide accumulated may reflect the pos sibility that the 2 nucleosides are initially phosphorylated by separate enzymes (5), as well as the observed differences in the rates of degradation (Chart 8).
The major cnetabolic effect of each nucleoside analog that we have observed is directed at DNA synthesis (Chart 4), upon which F-ara-A is demonstrably more inhibitory than are equal exogenous concentrations of ara-A plus dCF (Chart 6). While the action of ara-A appeared relatively specific, 100 ¡IMF-ara-
A did inhibit the incorporation of leucine into protein (Chart 4), an effect seen at lower drug concentrations in L1210 cells (6). The nature of this effect is not known, although the similar apparent inhibition of RNA synthesis was due to a decrease in the specific activity of [3H]UTP in cells labeled with [3H]uridine.
However, we have not detected a significant effect on the specific activity of [3H]dTTP in these (Table 1) or other cells
(20) that have been treated with arabinosyl nucleosides and subsequently labeled with [3H]thymidine.
The potency of inhibition of DNA synthesis by F-ara-ATP and ara-ATP in whole cells has been determined by 2 different methods. In the first, the F-ara-ATP and ara-ATP levels were determined in cells incubated with the respective arabinosyl nucleosides and compared to the DNA-synthetic capacity of
4 W. Plunkett. S. Chubb, and G. Spitzer, manuscript in preparation.
the same cells (Chart 7). The results indicated that, at intracel-
lular concentrations of 25 to 100 pmol of arabinosyl nucleotide per 1 x 106 cells, F-ara-ATP was slightly more inhibitory to
DNA synthesis than was ara-ATP. The cellular ara-ATP con centration was 3 nmol/1 x 106 cells in cultures inhibited by
50% in DNA-synthetic capacity, but our procedures were not sensitive enough to make a similar calculation for F-ara-ATP. In the second method, cells were allowed to accumulate F-ara- ATP and ara-ATP, the levels of which were determined imme diately after the cells were washed into drug-free media. The
cellular concentration of each nucleotide analog was calcu lated, using previously determined values for intracellular nu cleotide disappearance (Chart 8), as cells in each culture recovered their DNA-synthetic capacity. The calculations indi cate that the cellular concentrations of F-ara-ATP and ara-ATP were 3.2 and 5.1 nmol/106 cells, respectively, at 14 hr, a time
when the cultures had recovered about one-half of their DNA-
synthetic capacity. The values determined by these different methods for the
concentration of ara-ATP present in cells in which DNA synthe
sis was inhibited by 50% are in good agreement. Furthermore, although not directly comparable, these values are similar to the K,'s of isolated mammalian DNA polymerases for ara-ATP,
assuming a mean cell volume of 1000 cu jum (8, 10, 18). The results in Chart 7 and those calculated from Chart 9 suggest that F-ara-ATP may inhibit the processes measured by thymi- dine incorporation slightly more than ara-ATP. It will be of interest to compare the value's that we have obtained for the
inhibition of DNA synthesis by F-ara-ATP and ara-ATP in whole
cells to similar measurements in both permeabilized cells and to the effects of these nucleotides on various isolated DNA polymerases. Although arabinosyl nucleotides derived from ara-A are incorporated into the DNA of whole cells (23), it is not known if this is also true of F-ara-A nucleotides or if this is
part of the mechanism of toxicity of either compound. Finally, the experiments presented here illustrate how deter
minants of the action of nucleotide analogs, such as the rates of nucleotide accumulation, degradation, and inhibitory effi cacy, can interact in a complex manner so that similar inhibition of DNA synthesis and cell growth is evoked by nucleosides that vary widely in these individual determinants. The interactions of these cellular determinants, which are known to differ in other cell types as mediated by pharmacological parameters, will ultimately determine the cytotoxicity and therapeutic effi cacy of these nucleoside analogs.
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6. Brockman, R. W., Schabel, F. M., Jr., and Montgomery, J. A. Biologic activity of 9-/<-D-arabinofuranosyl-2-fluoroadenine, a metabolically stable
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11. Grover. W. R., Jr.. Carr. M. C., and Ivés.D. H Deoxyguanosine kinase Distinct molecular forms in mitochondria and cytosol J. Biol. Chem., 254 2180-2183. 1979.
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13. Lee, S. H., Carón, N., and Kimball, A. P. Therapeutic effects of 9-/i-o-ara- binofuranosyladenine and 2'-deoxycoformycin combination on intracerebral leukemia. Cancer Res., 37: 1953-1955, 1977.
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17 Montgomery, J. A., and Hewson. K Nucleosides of 2-fluoroadenine. J. Med Chem , ;2. 498-504. 1969
18. Okura, A., and Yoshida. S. Differential inhibition of DNA polymerases of calf thymus by 9-/i-o-arabinofuranosyladenine-5'-triphosphate J Biochem (To
kyo), 84: 727-732, 1978. 19 Plunkett, W.. Alexander, L , Chubb, S.. and Loo. T L Biochemical basis of
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20. Plunkett, W , Alexander, L.. Chubb, S., and Loo. T. L Comparison of the activity of 2'-deoxycoformycin and eryf/Jro-9-(2-hydroxy-3-nonyl)adenine in
vivo. Biochem. Pharmacol., 28. 201-206. 1979. 21. Plunkett. W.. Chubb, S., and Alexander, L. Comparison of the action of 9-
/¡-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) with 9-/i-D-arabmofurano- syladenine (ara-A) in CCRF-CEM cells Proc Am Assoc. Cancer Res , 20 137. 1979.
22. Plunkett. W., Chubb. S.. Shewach, D. S.. and Loo. T. L. Comparison of the metabolism of arabinosyladenine and arabinosyl-2-fluoroadenine by human leukocytes. Tex. J. Sci.. 30(suppl. 5): 111-128, 1978.
23. Plunkett, W.. and Cohen. S. S. Metabolism of 9-/J-D-arabinofuranosyladenine by mouse fibroblasts. Cancer Res., 35 415-422. 1975.
24. Plunkett, W., and Cohen, S S. Two approaches that increase the activity of analogs of adenine nucleosides in animal cells. Cancer Res.. 35. 1547- 1554. 1975.
25. Rose. L. M., and Brockman, R W Analysis by high pressure liquid chro- matography of 9-/<-o-arabinofuranosyladenine 5'-triphosphate levels in mu rine leukemic cells J Chromatogr., 733. 335-343. 1977.
26. Schabel, F. M., Jr., Trader, M W., and Laster. W. R . Jr. Increased thera peutic activity of 9-/<-D-arabinofuranosyladenme (ara-A) against leukemia P388 and L1210 by an adenosine deaminase inhibitor Proc. Am Assoc. Cancer Res.. 17: 46. 1976.
27. Shewach, D. S.. and Plunkett. W. Effect of 2'-deoxycoformycin on the biologic half-life of 9-/i-D-arabinofuranosyladenine 5'-triphosphate in CHO
cells. Biochem Pharmacol.. 28 2401-2404. 1979. 28 Smyth, J. F.. Chassin, M. M . Harrap. K. R , Adamson. R H , and Johns. D
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Am. Assoc. Cancer Res.. 20. 47. 1979. 29. Trotta, P. P., Ahland, M. P.. Brown, G. F.. and Balis. M. E. Studies on the
effects of enzyme inhibitors on mouse adenosine deaminase. Mol Pharma col.. 14: 199-209. 1978
30. Trotta, P. P., Tedde, A., and Balis, M. E. In vivo effects of deoxycoformycin on mouse adenosine deaminase and nucleoside phosphorylase. Proc. Am. Assoc. Cancer Res.. 20 284. 1979
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fluoroadenine and 9-ß-D-Arabinofuranosyladenine in Human Lymphoblastoid Cells1
William Plunkett,2 Sherri Chubb, Lillie Alexander, and John A. Montgomery
Department of Developmental Therapeutics. The University of Texas System Cancer Center, M. D Anderson Hospital and Tumor Institute, Houston. Texas 77030 ¡W.P., S. C., L. A ¡,and Organic Chemistry Department, Southern Research Institute, Birmingham. Alabama 35205 ¡J.A. M.]
ABSTRACT
The toxicity and metabolism of 9-/S-D-arabinofuranosyl-2- fluoroadenine (F-ara-A), an adenosine deaminase-resistant nu- cleoside analog, have been compared to those of 9-/8-o-arabi- nofuranosyladenine (ara-A) in the presence of the adenosine deaminase inhibitor, deoxycoformycin. Equal concentrations of F-ara-A and ara-A plus deoxycoformycin produced similar inhibition of growth of CCRF-CEM human lymphoblastoid cells. 9-/S-D-Arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F- ara-ATP) and 9-/?-D-arabinofuranosyladenine 5'-triphosphate
(ara-ATP), were concentratively accumulated intracellularly and exerted their major inhibitory effect on DNA synthesis. Two approaches were used to compare the effect of these nucleo- tide analogs on the DNA-synthetic capacity of whole cells, (a) The concentrations of F-ara-ATP and ara-ATP in cells incu
bated with the respective nucleosides were determined directly at the same time that the DNA-synthetic capacity of the cells in each culture was measured by incorporation of [3H]thymidine
into DNA. Neither compound significantly affected the specific activity of cellular [3H]deoxythymidine triphosphate. (b) The rates of disappearance of F-ara-ATP and ara-ATP from cells
washed free of the nucleosides were determined. These values were used to calculate the cellular concentration of each nu- cleotide analog in cells in which DNA-synthetic capacity was monitored after incubation with F-ara-A or ara-A plus deoxy
coformycin and washed into fresh media. These determinations indicated that the rates of accumulation of F-ara-ATP and ara-
ATP differed and were related to the concentration of the exogenous nucleoside. F-ara-ATP disappeared from cells in drug-free media at several times the rate of the disappearance of ara-ATP. However, at equal cellular concentrations, F-ara-
ATP was slightly more inhibitory to the processes measured by thymidine incorporation than was ara-ATP. We conclude that F-ara-ATP and ara-ATP share a similar mechanism of action and potency of inhibition of DNA synthesis. The fact that F-ara-
A retains its cytotoxic efficacy in the absence of inhibitors of adenosine deaminase provides a rationale for investigating its antitumor properties further.
INTRODUCTION
The therapeutic effectiveness of ara-A3 is limited by its deam-
Received November 15, 1979; accepted April 3, 1980. ' Supported by NIH Grants CA 14528, CA 11520, and RR 05511; Contract
N01 CM 87185; and American Cancer Society Grant CH-130. 2 To whom requests for reprints should be addressed. 1The abbreviations used are: ara-A, 9-/J-D-arabinofuranosyladenine; ara-ATP.
9-/j-D-arabinofuranosyladenine 5'-triphosphate; F-ara-A. 9-/3-D-arabinofuranosyl- 2-fluoroadenine; dCF. 2'-deoxycoformycin or (R)-3-(2-deoxy-/J-o-erythropento- furanosyl)-3,6.7.8-tetrahydroimidazo[4,5-rf][1.3]diazepin-8-ol; RPMI-1640, Ros-
ination by adenosine deaminase to the relatively inactive Q-ß- D-arabinofuranosylhypoxanthine (4). It is now well established
that this inactivation may be minimized and that a higher therapeutic efficacy may be achieved by coadministration of ara-A with any one of several effective inhibitors of adenosine
deaminase on an appropriate treatment schedule (13, 14, 19, 24, 26). However, rational use of this combination drug treat ment may be complicated by the diverse duration of activity (1, 3, 20) and tissue specificity (29, 30) of the deaminase inhibi tors, as well as the pharmacokinetic properties of ara-A and the tissue kinetics of the active metabolite, ara-ATP (20). In
addition, it is clear that the adenosine deaminase inhibitors evoke multiple effects on cellular metabolism (2, 12), that they are immunosuppressive (7, 15), and that they may possess antitumor activity in humans (28).
Thus, the synthesis of F-ara-A (17) and the subsequent
demonstration (6) that this analog is resistant to deamination by adenosine deaminase, yet retains the antitumor activity of ara-A in the presence of a deaminase inhibitor, have evoked a considerable amount of interest. The purpose of the present study was to compare the toxicity of F-ara-A with that of ara-A
in the presence of a deaminase inhibitor and to correlate the biochemical elements that characterize this toxicity, such as the accumulation and flux of the respective active triphosphates and their effects on DNA synthesis. A preliminary report of the results of these investigations has been published (21).
MATERIALS AND METHODS
Materials. ara-A and dCF were provided by the Drug Devel
opment Branch, Division of Cancer Treatment, National Cancer Institute. F-ara-A was synthesized in the laboratory of J. A. Montgomery (16). ara-ATP, used as a standard for high-pres sure liquid chromatography, was purchased from P-L Biochem- icals, Inc. (Milwaukee, Wis.). [/r)effty/-3H]Thymidine (6.0 Ci/ mmol), [5-3H]uridine (25.4 Ci/mmol), and [2-3H]ara-A (18.7 Ci/
mmol) were obtained from ICN Pharmaceuticals, Inc. (Irvine, Calif.). Radioactive ara-A was routinely recrystallized from H?O
before use and shown to be of greater than 99% purity by analysis of the radioactivity eluted by high-pressure liquid chromatography on C,8-/iBondapak (Waters Associates, Inc.,
Milford, Mass.). Schwarz/Mann (Orangeburg, N. Y.) supplied [4,5-3H]leucine (61 Ci/mmol).
Cell Culture Methods. A T-cell line of human lymphoblastoid cells, CCRF-CEM (9), was maintained in exponential growth at 37°in sealed agitated suspension cultures in RPMI-1640 sup-
well Park Memorial Institute Tissue Culture Medium 1640; F-ara-ATP. 9-/Õ-D- arabinofuranosyl-2-fluoroadenine 5'-triphosphate; PBS. phosphate-buffered sa
line (8.1 g of NaCI. 0.22 g of KCI, 1.14 g of Na2HPO4, and 0.27 g of KH?PO,, per liter of H20. pH 7.4).
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W. Plunkett et al.
plemented with 10% fetal calf serum (Grand Island Biological Co., Grand Island, N. Y.). Cell number and volume were deter mined by a Model ZBI electronic particle counter (Coulter Electronics, Inc., Hialeah, Fla.), equipped with a cell-sizing instrument (Model C-1000) which was calibrated with latex
beads 10.0 ¿imin diameter (Coulter Electronics). The effect of arabinosyl nucleosides on cell growth was determined as fol lows. CCRF-CEM cells were adapted for several days of growth in RPMI-1640 supplemented with 10% horse serum (Grand
Island Biological Co.) that had been dialyzed for 3 days against multiple changes of PBS in our laboratory. This medium, in which adenosine deaminase activity contributed by the horse serum is below the limit of detection, supported cell growth at rates comparable to those of RPMI-1640 plus 10% fetal calf serum (population-doubling time, 22 hr). Drugs were prepared and diluted in RPMI-1640 and added in a final volume of 0.5- ml to 3-ml wells of microtiter plates (Flow Laboratories Inc.,
Hamden, Conn.). Just before incubation, cells were washed into RPMI-1 640 supplemented with 20% dialyzed horse serum, and a 0.5-ml aliquot of 5 x 10" cells was added to each well
containing drugs or medium only (control). Following 72 hr of incubation at 37° in a high-humidity 5% CO?:95% air atmo
sphere, each well was diluted with 1 ml PBS, and the total cell number from duplicate cultures was determined. After subtrac tion of the number of cells plated from the final number of cells in control wells, the growth of drug-treated cells was expressed
as a percentage of the control cell growth. Values obtained in replicate experiments generally differed by less than 10%.
Determination of ara-ATP and F-ara-ATP. Cells incubated with arabinosyl nucleosides were diluted with 2 volumes of ice-
cold PBS, harvested by centrifugation, and washed twice in cold PBS. Cells were suspended in 0.5 ml of cold H?O, and 0.5 ml of 0.8 N HCIO4 was added during vigorous mixing. Following a 5-min incubation in an ice bath, the acid-soluble material was
recovered by centrifugation. The pellet was washed with 0.5 ml of 0.4 N HCIO4, and after centrifugation the acid-soluble supernatants were combined and neutralized with KOH. Fol lowing chilling in ice, the KCIO* was removed by centrifugation, and the supernatant was analyzed for nucleotides by the fol lowing high-pressure liquid Chromatographie procedure. A Wa ters Associates ALC-204 high-pressure liquid Chromatograph
equipped with 2 Model 6000A pumps, a Model 660 solvent programmer, and a column of Partisil-10 SAX anion-exchange
resin (25 cm x 4.6 mm; Whatman, Inc., Clifton, N. J.) was used. Cell extracts (0.05 to 1.0 ml) containing 1 to 5 x 106 cell equivalents of acid-soluble material were injected onto the column with the U6K-LC injection system. Nucleoside mono-
and diphosphates were eluted with 60% 0.005 M NH4H2PO4 (pH 2.8) and 40% 0.750 M NH4H?PO4 (pH 3.7) at a flow rate of 2 ml/min for 10 min. A linear gradient from 40 to 100% 0.750 M NH4H?PO4 was then run for 24 min to separate ribonucleo- side triphosphates from ara-ATP and from F-ara-ATP. Eluted
nucleotides were detected by their absorbance at 254 nm by the Model 440 detector and were quantitated with a CDS-111 electronic integrator (VarÃanAssociates, Palo Alto, Calif.). Peak areas were converted to absolute quantities using predeter mined calibration curves that were linear with nucleotide con centration to a lower sensitivity of 25 pmol. Greater sensitivity was achieved if, after incubation of the cells with [3H]ara-A of
known specific activity, the ara-ATP was quantitated by col lecting the column eluate that coeluted with ara-ATP at 0.5-min
intervals into scintillation vials containing 0.5 ml of H?O and 11 ml of Aquasol (New England Nuclear, Boston, Mass.), and by determining radioactivity by scintillation counting (27). Al though we had successfully used published procedures (25) for the quantitation of ara-ATP by absorbance in the past (20),
we were surprised to find that a substantial portion of radio activity derived from [3H]ara-A coeluted with ATP and trailed into the ara-ATP peak. Using high-purity [3H]ara-A in the pres
ence of either dCF or erythro-9-(2-hydroxy-3-nonyl)adenine,
we have observed this phenomenon to varying degrees not only in CCRF-CEM cells but also in Chinese hamster ovary cells, phytohemagglutinin-stimulated human lymphocytes, hu
man bone marrow cells, and several murine ascitic tumors, and normal tissues. We assume that the labeled ATP arises from the labeled purine ring of [3H]ara-A either after deamination of 9-/8-D-arabinofuranosyladenine-5'-monophosphate (23) or by
deamination of the nucleoside by a species of adenosine deaminase, which is incompletely inhibited by these deaminase inhibitors (11). Following deamination, liberation of [3H]hypo-
xanthine is likely and may be reutilized by purine salvage pathways to form ATP. The Chromatographie separation pro cedure described herein minimizes contamination of ara-ATP
with radioactivity associated with ATP. Determining of [3H]dTTP Specific Activity. F-ara-A (100
nmol/ml) or ara-A (100 nmol/ml) plus dCF (10 nmol/ml) was added to cultures of CCRF-CEM cells in exponential growth 10 min before addition of [3H]thymidine (10 nmol/ml; 2.0 x 105
dpm/nmol). After 4 hr of incubation with the drugs, cells were diluted and washed with PBS, and acid-soluble material was
extracted as described above. Ribonucleotides were removed by oxidation with NalO4 and the remaining deoxyribonucleo- tides were fractionated by a high-pressure liquid Chromato graphie assay as described previously (19). The specific activ ity of cellular [3H]dTTP was calculated by dividing the radio
activity associated with the dTTP peak by the nmol of dTTP detected by UV absorbance. The effect of arabinosylnucleo- sides on the incorporation of [3H]dTTP into DNA was deter
mined as described below. Similar procedures were used to calculate the specific activity of [3H]UTP in acid-soluble ex tracts from cells incubated with [3H]uridine.
Incorporation of [3H]thymidine, [3H]Uridine, and [3H]Leu-
cine into Acid-insoluble Material. Cells were incubated with [3H]thymidine, [3H]uridine, or [3H]leucine for varying times to
estimate the amount of DNA, RNA, and protein synthesis in cells treated with arabinosyl nucleosides. Cells were diluted, washed with PBS, and extracted twice with 0.4 N HCIO4 as described above. The acid-insoluble pellet was washed with
5.0 ml of 0.4 N HCICv»before being resuspended in H2O and brought into solution with 2 drops of 1.0 N KOH. A portion of this fraction was added to a scintillation vial containing 11 ml of Aquasol, and the radioactivity was determined by a Beckman Model 2650 liquid scintillation spectrometer. The dpm, deter mined at an average counting efficiency of 35 to 37%, were computed by the instrument with the aid of a preprogrammed external standard quench curve.
RESULTS
Growth Inhibition by Arabinosyl Nucleosides. The effect of various concentrations of F-ara-A and ara-A, alone and in the presence of dCF, on the growth of CCRF-CEM cells is shown
2350 CANCER RESEARCH VOL. 40
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Comparison of F-ara-A and ara-A
in Chart 1. Cell growth was inhibited 50% by 3 fiM ara-A alone.
However, in the presence of 10 ¿IMdCF, which by itself is not growth inhibitory, the concentration of ara-A required to give comparable inhibition of growth was reduced to 0.2 ¡J.M.The synergistic action of these 2 compounds may be attributed to the inhibition of the appreciable capacity of these cells to deaminate ara-A (0.37 nmol deaminated per 1 x 105 cells per
hr) which effectively results in a longer incubation with higher concentrations. Subsequently, increased cellular ara-ATP lev els accumulate leading to greater growth inhibition (data not shown). F-ara-A alone was also effective at inhibiting cell
growth by 50% at 0.2 JUM,but this activity was not augmented by the addition of dCF. Subsequent experiments will address the metabolism of ara-A in the presence of dCF, since the metabolism of ara-A in the absence of a deaminase inhibitor has been described in other systems (23). In each experiment, cells were exposed to dCF alone for 30 min before addition of ara-A.
Metabolism of Arabinosyl Nucleosides. As in the case with ara-A, the active form of F-ara-A is thought to be the 5'-
triphosphate (6). F-ara-ATP accumulates in cells incubated with F-ara-A and may be detected and quantitated by high-
pressure liquid Chromatographie fractionation of cell extracts (Chart 2). The identical procedure results in a similar separation of ara-ATP from major cellular ribonucleoside triphosphates in extracts of cells incubated with ara-A plus dCF. The cellular
concentration of ribonucleoside triphosphates was not affected in cells incubated with either nucleoside analog. This elution scheme was designed to maximize the resolution of triphos phates at the expense of that of mono- and diphosphates,
which typically comprise less than 10% of the analog metabo lites in these cells.
Both F-ara-A and ara-A are concentratively phosphorylated in CCRF-CEM cells (Chart 3). Assuming a uniform intracellular
100
0.1 0.2 0.5 1.0 2.0 5.0 10 20 50
Arabinosyl nucleoside concentration, ^M Chart 1. Effect of F-ara-A and ara-A alone or in the presence of dCF (10
nmol/ml) on the growth of CCRF-CEM cells. Control cells grew from 0.52 to 3 02 x 10Vml in 72 hr. A. F-ara-A; A, F-ara-A plus dCF: •.ara-A; O, ara-A plus dCF; •10/nMdCF.
.004 -
.002 -
§.002
Chart 2. High-pressure liquid Chromatographie analysis of acid-soluble ex tracts from 1 x 106 CCRF-CEM cells incubated for 4 hr with ara-A (100 nmol/
ml) plus dCF (10 nmol/ml) or with F-ara-A ( 100 nmol/ml).
j5 600 0} <J
200
100
012345
Hours
Chart 3. Accumulation of F-ara-ATP or ara-ATP by cells incubated with F-ara- A (100 nmol/ml) (O) or ara-A (100 nmol/ml) plus dCF (10 nmol/ml) (•), respectively.
distribution and using the observed mean cell volume of 1084 cu urn, F-ara-ATP accumulated to cellular concentrations of greater than 500 /UMby 5 hr, whereas accumulation of ara-ATP
reached a plateau value of about 200 UM by 3 hr. Analysis of extracts from cells subjected to more prolonged incubations with lower concentrations of these analogs (1 to 5 /ÕM)indicated that the triphosphates accumulated to at least 100 times the concentration of the exogenous nucleosides.
The effects of such an incubation on the macromolecular synthesis of these cells are shown in Chart 4. The inhibition caused by ara-A in the presence of dCF was specific for DMA synthesis, but F-ara-A decreased the incorporation of both
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W. Plunkett et al.
uridine and leucine into RNA and protein, respectively, in addition to severely inhibiting DNA synthesis. Analysis of the specific activity of [3H]UTP in the acid-soluble fractions from cells labeled with [3H]uridine indicated a lower activity in each
of the samples from F-ara-A-treated cells than in the corre sponding samples from the controls. After normalization of the incorporation data based on the cellular [3H]UTP specific activ
ity determined at each time, no significant differences were observed between control and F-ara-A-treated cells. Investi gations into the effect of F-ara-A on the specific activity of [3H]UTP or on the incorporation of leucine have not been
extended. uptake and phosphorylation of [3H]thymidine or on the endog
enous pool size of dTTP that produced an alteration in the specific activity of the cellular [3H]dTTP pool could invalidate
this experimental approach for monitoring DMA synthesis. To control for this possibility, cells were incubated with 100 ¡UMF- ara-A or ara-A plus dCF for 4 hr. The effect of this incubation on the specific activity of cellular [3H]dTTP and the ability to
incorporate this nucleotide into DMA were determined (Table 1). The cellular [3H]dTTP specific activity was altered no more
than 10%, even when incorporation into DNA was inhibited by 96%. These results suggest that the quantitation of thymidine incorporation into acid-insoluble material is a reasonably ac curate method for monitoring the effects of arabinosyl nucleo-
sides on DNA synthesis. Correlation of Cellular Nucleotide Analog Levels with In
hibition of DNA Synthesis. The foregoing experiments and those from other studies (6) indicate that, as is the case with its congener ara-A, the active metabolite of F-ara-A is the nucleo- side triphosphate, F-ara-ATP, and that its major metabolic
target is cellular DNA synthesis. Therefore, to evaluate the relative inhibitory potency of ara-ATP and F-ara-ATP in vivo, it
w 0.8 "öj O o 0.6
E 0.4 C
Hours 02
Chart 4 Effect of F-ara-A (100 nmol/ml) or ara-A (100 nmol/ml) plus dCF (10 nmol/ml) on the incorporation of [3H]thymidine, [3H]uridine. or (3H]leucine
into acid-insoluble material of CCRF-CEM cells. A, control; O. F-ara-A; •.ara-A plus dCF.
Table 1 Effect of F-ara-A or ara-A plus dCF on the specific activity of cellular /3H/o7TP
and its incorporation into the DNA of CCRF-CEM cells
CCRF-CEM cells were incubated in the presence of pHJthymidine and either 100 fiM F-ara-A or 100 /IM ara-A. plus 1 fiM dCF for 4 hr. The effect of these compounds on the specific activity of cellular [3H]dTTP and its incorporation into DNA was determined as described in "Materials and Methods."
TreatmentControl
specific activity (104dpm/nmol)4.83(100)a
incorpora tion into DNA(nmol/107
Numbers in parentheses, percentage of control
Chart 5. Accumulation of F-ara-ATP and ara-ATP by CCRF-CEM cells incu bated with various concentrations of F-ara-A or ara-A plus dCF (10 nmol/ml) for 1 hr. Cellular nucleotide levels were determined as described in "Materials and Methods." O, F-ara-ATP; •.ara-ATP. The mean cell volume was 1084 cu /im or 9.24 x 10" cells/ml. The concentration of nucleotides in the cells, in nmol/ml.
may be calculated by multiplying the ordinate values by 0 924.
was necessary to determine and compare the effect of known cellular concentrations of each nucleotide on the cellular DNA- synthetic capacity. Cells were incubated with various concen trations of F-ara-A or ara-A plus dCF (10 nmol/ml) for 50 min before addition of [3H]thymidine for the remaining 10 min of
the incubation. The acid-soluble material was analyzed for nucleotide analogs and the incorporation of [3H]thymidine into
acid-insoluble material was used as an indicator of DNA syn thesis. Chart 5 illustrates that each nucleotide accumulates to similar levels in proportion to the exogenous nucleoside con centration. Calculations indicated that the cellular concentra tion of each nucleotide analog exceeded that of the exoge-
nously added nucleoside at each point, although this was more pronounced at lower concentrations of F-ara-A. This relatively
brief incubation did not detectably alter the mean cell volume of either culture.
In this same experiment, after cells were allowed to accu mulate F-ara-ATP and ara-ATP for 50 min, [3H]thymidine was
added for 10 min to determine the effects of the nucleotide analogs on DNA synthesis. The results of these determinations (Chart 6) indicate that F-ara-A was more inhibitory to thymidine
incorporation than were equal concentrations of exogenously added ara-A in the presence of dCF. This was particularly evident at lower nucleoside concentrations where F-ara-ATP
was found to be at higher concentrations in the cell (Chart 5). The findings from 2 different analyses of the same experi
ment, presented in Charts 5 and 6, are combined in Chart 7 to facilitate comparison of the effect of different cellular concen trations of F-ara-ATP and ara-ATP on DNA synthesis. It appears that, at equal cellular concentrations of nucleotide, F-ara-ATP
was slightly more inhibitory to thymidine incorporation than was ara-ATP. The sensitivity of our assay for F-ara-ATP did not permit determination of the cellular nucleotide concentration when thymidine incorporation was inhibited by 50%. However, the calculated cellular ara-ATP concentration was 3 nmol/1 x 106 cells when thymidine incorporation was inhibited by
50%. Cellular Flux of F-ara-ATP and ara-ATP and the Effect on
DNA Synthesis. The previous experiments were designed to provide a static view of the DNA-synthetic capacity of cells
during a short time interval when the change in cellular nucleo tide analog levels was minimal. The following experiments give
2352 CANCER RESEARCH VOL. 40
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100
50
u'S
100
Chart 6. Effect of various concentrations of F-ara-A or ara-A plus dCF (10 nmol/ml) on the incorporation of [3H)thymidine (10 nmol/ml; 2.86 x 10s dpm/
nmol) by CCRF-CEM cells. Cells were incubated with the drugs for 50 min before addition of [JH]thymidine for 10 min. Acid-insoluble material was then extracted from the cells as described in 'Materials and Methods. The control value was 352 pmol (3H]thymidine incorporated per IO6 cells per 10 min. O. F-ara-A; •
ara-A plus dCF.
pmol ara-ATP or F-ara-ATP / IxlO6 Cells
Chart 7. Effect of cellular levels of F-ara-ATP and ara-ATP on DMA synthesis by CCRF-CEM cells. The data presented in Charts 5 and 6 have been replotted to facilitate comparison O. F-ara-ATP; •.ara-ATP
a dynamic view of these ongoing processes. Cells were incu bated for 2 hr with F-ara-A or ara-A plus dCF and allowed to accumulate the respective triphosphates before being washed into drug-free media. The cellular nucleotide analog levels were determined at the indicated times (Chart 8). It is evident that F- ara-ATP disappears from the cells at several times the rate at which ara-ATP decays. In 4 experiments, the half-life of F-ara- ATP ranged from 2.5 to 4.7 hr, whereas that of ara-ATP ranged
from 12.9 to 15.3 hr. Knowing that F-ara-ATP may be more inhibitory to DMA
synthesis than is ara-ATP (Chart 7) but disappears more rapidly
from the cell in the absence of exogenous nucleoside (Chart 8), it was of interest to compare the rates at which cells treated with a pulse incubation of F-ara-A and ara-A recover their ability to synthesize DNA. Cells were incubated with F-ara-A or ara-A plus dCF for 1 hr and washed into fresh medium, and
their ability to incorporate [3H]thymidine during a 15 min pulse
was determined at the indicated times (Chart 9). Both cultures display similar kinetics of recovery of DNA-synthetic capacity,
being maximally inhibited for 8 hr and recovering to approxi mately 50% of control values after 14 hr. Cellular nucleotide analog concentrations, determined in the cells taken from each culture immediately after washing into drug-free medium, were 16.7 pmol of F-ara-ATP per 106 cells and 9.6 pmol of ara-ATP per 106 cells. Using the rates of disappearance of each nu
cleotide analog from Chart 8, the cellular concentrations of F- ara-ATP and ara-ATP 14 hr after washing were calculated to be 3.2 and 5.1 pmol/106 cells, respectively. The mean cell
volume of cells treated with each nucleoside analog increased 35% from 882 to 1194 cu ¿imduring the 14-hr incubation in drug-free medium, thus further diluting the concentration of the
intracellular nucleotide analogs. The mean volume of control cells increased slightly to 955 cu /im.
20\
\i>
8Shr16Hours24
Chart 8. Retention of F-ara-ATP and ara-ATP by CCRF-CEM cells. Cells were incubated for 2 hr with F-ara-A (100 nmol/ml) or ara-A (100 nmol/ml) plus dCF (10 nmol/ml) and washed into prewarmed (37°)drug-free media, and the levels
of cellular nucleotide analogs were determined at the indicated times as de scribed in 'Materials and Methods." O. F-ara-ATP; •.ara-ATP
70r
Hours
Chart 9. Recovery of the DNA-synthetic capacity of CCRF-CEM cells after a pulse with F-ara-A (100 nmol/ml) of ara-A (100 nmol/ml) plus dCF (2 nmol/ml). Following incubation for 1 hr, cells were washed into prewarmed (37°)drug-free
media, and the DNA-synthetic capacity was determined as the ability of a portion of the culture to incorporate (3H]thymidine (10 nmol/ml; 2.91 x 105 dpm/nmol)
into acid-insoluble material during a 15-min pulse. This was expressed as a percentage of the control value at each point. The mean control value of the 7 time points was 518 ±34 pmol [3H]thymidine incorporated per 106 cells per 15
min. O. F-ara-A; •.ara-A plus dCF.
JULY 1980 2353
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DISCUSSION
The objective of this study was to compare the growth- inhibitory activity and the cellular metabolism of F-ara-A with those of ara-A. Continuous incubation of human lymphoblastoid
cells with these compounds indicated that they inhibited cell growth equally when deaminative detoxification of ara-A was
eliminated by addition of dCF to the cultures (Chart 1). How ever, F-ara-A, which is not a substrate for adenosine deaminase
and the toxicity of which is not augmented by addition of dCF, was equally toxic at one-tenth the concentration of ara-A in the
absence of dCF. Although these compounds are equally potent in cell culture in the presence of dCF, it has been reported that inhibitors of adenosine deaminase do not completely inhibit host tissue enzyme activity in vivo (29, 30) and that substantial deamination of ara-A occurs even after high doses of deami nase inhibitors (20). Since host tissue enzyme comprises the bulk of the deaminase activity in vivo, this residual activity may in part account for the observed differences in therapeutic activity between F-ara-A and ara-A plus dCF in vivo (6). The fact that F-ara-A retains its cytotoxicity in the absence of
inhibitors of adenosine deaminase provides a basis for further investigations of its antitumor properties.
Both F-ara-A and ara-A were concentratively phosphorylated to the respective 5'-triphosphates, the presumed active metab
olite of each compound. F-ara-ATP accumulated to higher cellular concentrations than did ara-ATP after 1 hr of incubation
with low exogenous levels of nucleoside (Chart 5). Cells incu bated with the analogs at higher concentrations accumulated F-ara-ATP at a nearly linear rate for 5 hr, whereas ara-ATP accumulation reached a plateau value of about one-third the 5- hr F-ara-ATP concentration by 3 hr (Chart 3). We have ob
served similar ratios for the accumulation of each nucleotide in phytohemagglutinin-stimulated human lymphocytes (22), but the value for F-ara-ATP:ara-ATP accumulated by normal hu man bone marrow cells approaches 9.4 Differences in the
amount of each nucleotide accumulated may reflect the pos sibility that the 2 nucleosides are initially phosphorylated by separate enzymes (5), as well as the observed differences in the rates of degradation (Chart 8).
The major cnetabolic effect of each nucleoside analog that we have observed is directed at DNA synthesis (Chart 4), upon which F-ara-A is demonstrably more inhibitory than are equal exogenous concentrations of ara-A plus dCF (Chart 6). While the action of ara-A appeared relatively specific, 100 ¡IMF-ara-
A did inhibit the incorporation of leucine into protein (Chart 4), an effect seen at lower drug concentrations in L1210 cells (6). The nature of this effect is not known, although the similar apparent inhibition of RNA synthesis was due to a decrease in the specific activity of [3H]UTP in cells labeled with [3H]uridine.
However, we have not detected a significant effect on the specific activity of [3H]dTTP in these (Table 1) or other cells
(20) that have been treated with arabinosyl nucleosides and subsequently labeled with [3H]thymidine.
The potency of inhibition of DNA synthesis by F-ara-ATP and ara-ATP in whole cells has been determined by 2 different methods. In the first, the F-ara-ATP and ara-ATP levels were determined in cells incubated with the respective arabinosyl nucleosides and compared to the DNA-synthetic capacity of
4 W. Plunkett. S. Chubb, and G. Spitzer, manuscript in preparation.
the same cells (Chart 7). The results indicated that, at intracel-
lular concentrations of 25 to 100 pmol of arabinosyl nucleotide per 1 x 106 cells, F-ara-ATP was slightly more inhibitory to
DNA synthesis than was ara-ATP. The cellular ara-ATP con centration was 3 nmol/1 x 106 cells in cultures inhibited by
50% in DNA-synthetic capacity, but our procedures were not sensitive enough to make a similar calculation for F-ara-ATP. In the second method, cells were allowed to accumulate F-ara- ATP and ara-ATP, the levels of which were determined imme diately after the cells were washed into drug-free media. The
cellular concentration of each nucleotide analog was calcu lated, using previously determined values for intracellular nu cleotide disappearance (Chart 8), as cells in each culture recovered their DNA-synthetic capacity. The calculations indi cate that the cellular concentrations of F-ara-ATP and ara-ATP were 3.2 and 5.1 nmol/106 cells, respectively, at 14 hr, a time
when the cultures had recovered about one-half of their DNA-
synthetic capacity. The values determined by these different methods for the
concentration of ara-ATP present in cells in which DNA synthe
sis was inhibited by 50% are in good agreement. Furthermore, although not directly comparable, these values are similar to the K,'s of isolated mammalian DNA polymerases for ara-ATP,
assuming a mean cell volume of 1000 cu jum (8, 10, 18). The results in Chart 7 and those calculated from Chart 9 suggest that F-ara-ATP may inhibit the processes measured by thymi- dine incorporation slightly more than ara-ATP. It will be of interest to compare the value's that we have obtained for the
inhibition of DNA synthesis by F-ara-ATP and ara-ATP in whole
cells to similar measurements in both permeabilized cells and to the effects of these nucleotides on various isolated DNA polymerases. Although arabinosyl nucleotides derived from ara-A are incorporated into the DNA of whole cells (23), it is not known if this is also true of F-ara-A nucleotides or if this is
part of the mechanism of toxicity of either compound. Finally, the experiments presented here illustrate how deter
minants of the action of nucleotide analogs, such as the rates of nucleotide accumulation, degradation, and inhibitory effi cacy, can interact in a complex manner so that similar inhibition of DNA synthesis and cell growth is evoked by nucleosides that vary widely in these individual determinants. The interactions of these cellular determinants, which are known to differ in other cell types as mediated by pharmacological parameters, will ultimately determine the cytotoxicity and therapeutic effi cacy of these nucleoside analogs.
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β-d-Arabinofuranosyl-2-fluoroadenine and 9- βComparison of the Toxicity and Metabolism of 9-
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