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Purine transport in cultured-normal and mouse sarcoma virus-transformed rat kidney cells
BIOCHEMICAL
17, 87-98
MEDICINE
(19771
Purine Transport in Cultured-Normal and Mouse Sarcoma Virus-Transformed Rat Kidney Cells Y. H. JOY YANG Department
of Biochemistry.
University Angeles. Received
AND D. W. VISSER
of Southern California August
California, 90033
School
of Medicine,
Los
5. 1976
INTRODUCTION Numerous studies have shown that purines are transported into mammalian cells by a saturable process which is rate-limiting relative to intracellular reactions, but the mechanism of transport is controversial. Studies with cultures of human fibroblasts which are deficient in hypoxanthine phosphoribosyltransferase activity (l), as well as studies with membrane vesicles of mammalian cells (2), have led to the conclusion that transport may involve phosphoribosylation as an integral part of the transport process. Recent studies by Zylka and Plagemann (3) on the transport of purines by Novikoff cells support the earlier conclusion of Hawkins and Berlin (4) and R. D. Berlin (5) from studies with polymorphonuclear metrophiles that phosphoribosylation of adenine occurs intracellularly subsequent to transport into the cell by facilitated diffusion processes. Zylka and Plagemann (3) demonstrated that hypoxanthine and guanine are transported in Novikoff cells by a single transport system which is distinct from that of adenine transport. A more precise knowledge of the characteristics of purine transport by mammalian cells is of practical importance for several reasons. The de FZOVOsynthesis of purines is absent or limited in certain cells (6) which must utilize preformed purines from the blood to satisfy their purine requirements. Much effort has been focused on specific intracellular enzymatic reactions which are affected by purine analogs. Little attention has been directed toward investigations of the transport of purine analogs into cells or the effects of analogs on the transport of naturally occurring substrates. The chemotherapeutic value of analogs is generally thought to be based primarily on their intracellular effects. However, the transport properties of purine analogs may be of considerable importance in relation to their chemotherapeutic efficiency, particularly since there is general agreement that the uptake of purines and, presumably, purine analogs is a rate-limiting process. 87 Copyright All rights
@ I977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN
ooO6-2944
88
JOY
‘r’ANG
AND
VISSEK
This investigation of the transport characteristics of purines in normal rat kidney cells (NRK) and NRK cells nonproductively transformed (TRK) by Kirsten mouse sarcoma virus (7) was initiated to gain information about the structural specificity of these transport processes in the two cell types. These objectives were approached by a careful delineation of the transport characteristics of adenine, guanine, and hypoxanthine in normal and transformed cells and by the determination of the effects of various analogs on the transport of these bases. The studies were carried out at low concentrations of substrate (below the apparent K, ) with short incubation periods (2 min), conditions at which the transport process is rate-limiting. MATERIALS
AND METHODS
Materials Normal rat kidney (NRK) cells and NRK cells nonproductively transformed (TRK) by Kirsten mouse sarcoma virus were a gift of Dr. P. Roy-Burman of the University of Southern California. Radioactive chemicals were obtained as follows: [2-3H]Adenine from New England Nuclear: [8-3H]guanine and [8-3H]hypoxanthine from Schwarz/Mann. The purity of the radioactive compounds was checked periodically by paper chromatography. Adenine, guanine, hypoxanthine, and 6-mercaptopurine were purchased from Calbiochem. Pm-me, 2-mercaptopurine, 2-amino6-methylmercaptopurine, 2-amino-6-mercaptopurine, 2,6-diaminopurine, 6-methylpurine, 6-amino-2-mercaptopurine, 6-methylmercaptopurine, 8-azaadenine, 8-azaguanine, 8azahypoxanthine, 8-bromoadenine. allopurinol, and 4-aminopyrazolo(3,4-d) pyrimidine were purchased from Sigma Chemical Co. 3-Deazaguanine was a gift from Dr. T. Khwaja. Serum-free MEM medium, fetal bovine serum, glutamine, and trypsin were obtained from Flow Laboratories. Gentamicin was purchased from Biological Associates. Other reagents were obtained from standard commercial sources. Growth of Cells NRK and TRK cells were routinely maintained in 75cm2 Falcon plastic tissue culture flasks at 37°C using MEM medium supplemented with 2 mM glutamine, 10% fetal bovine serum, 0.01% gentamicin, and 0.1 M Tricine at pH 7.3. The medium was changed every 3 days. Fetal bovine serum was heated at 60°C for 20 min just prior to use. Cultures were restarted from frozen stocks after about 50 passages for NRK cells and about 80 passages for TRK cells. Cultures were checked for mycoplasm by the use of a scanning electron microscope. Confluent cells were trypsinized and plated in 35mm Falcon plastic
PURINE
TRANSPORT
89
tissue culture dishes (usually 4.6 x lo5 cells/dish) in the same medium and incubated at 37°C in an atmosphere of 5% CO, in air. Fresh medium was supplied 4 hr later, and the cells were used for assays on the following day (20 hr). At these conditions, both NRK and TRK cells adhere to assay plates predominantly in the monolayer, and the cell density of both cell lines was the same at the time of assay (generation time 20-22 hr). The medium may be removed rapidly by aspiration, and the cells may be washed on the plates without appreciable cell loss. Transport
Studies
The transport studies in both cell types were carried out in parallel experiments on the same day. This procedure is desirable because transport characteristics were found to be more variable when the experiments were carried out at different times. The medium was removed from assay plates by aspiration, and the cells were rinsed with phosphate-buffered saline (PBS) twice at 37°C. The radioactive substrate in 1 ml of PBS was added immediately, and the cells were incubated on a slide warmer at 37°C for the designated time periods. At the end of the incubation period, the medium was removed by aspiration, and the cells were rinsed twice with 2 ml of PBS at 4°C. One milliliter of cold 10% TCA was added to each dish and maintained at 4°C for 30 min. An aliquot of TCA extract was placed in the vials containing 10 ml of scintillation fluid (0.8% PPO; 5% Bio-Solv in toluene), and radioactivity was determined in a Beckman LS-245 liquid scintillation counter. Control values were determined in the same manner except that the medium was aspirated from the cells immediately after addition. Purine analogs at the designated concentrations were added together with radioactive adenine, guanine, or hypoxanthine. The substrate concentrations (0.5 to 10 FM) used were below the K, for uptake of each put-me in each cell line. This procedure was followed to ensure maximal sensitivity to the competing substrate for transport. TCA-insoluble material was incubated in 1 ml of NaOH (1 .O N) at 37°C for 2 hr. An aliquot of the NaOH digests was used for protein determinations and, in some cases, for determinations of radioactivity. Incorporation of radioactivity into acid-insoluble material was negligible. Protein determinations were carried out by the method of Lowry et al. (8) using crystalline bovine albumin as the standard. Analysis of Radioactive Products in NRK and TRK Cells after Incubation with 3H-Labeled Adenine or Hypoxanthine
Analyses of the intracellular radioactive products were carried out as follows. Following a 2-min incubation with [2-3H]adenine or [g“Hlhypoxanthine, the cells were washed twice with 2 ml of PBS (4°C).
90
JOY
YANG
AND
VISSEK
One milliliter of ethanol at -20°C was added to the assay plates immediately after washing, and the plates were kept at -20°C for 2 hr. The ethanol was evaporated under a gentle stream of air. and the cells were extracted with I ml of HaO. The water extracts were lyophilized and redissolved in a small volume of water. The components in each of the extracts were separated by descending chromatography on Whatman 3MM paper using n-butanol-H,O (86:14, v/v) as the solvent system (9). Areas of the paper containing adenine or hypoxanthine and the corresponding nucleosides and nucleotides were located by ultraviolet light. Radioactivity in these areas was determined by the use of a liquid scintillation counter. RESULTS
Adenine Transport Intracellular radioactivity after a 2-min incubation in the presence of [3H]adenine was found predominantly (98%) in the TCA-soluble fraction of both NRK and TRK cells. Even after IO-min incubation periods, incorporation into acid-insoluble materials was less than 4% of the total radioactivity found in the cells. Therefore, TCA-soluble material was used as a measure of transport. Addition of glucose to the uptake medium did not increase the uptake of adenine by NRK cells, even though the cells were starved for 1 hr prior to uptake determinations. Uptake was linear with the time of incubation up to 3 min with 5 PM adenine, and the total incorporated was less than 2% of the substrate added after 3 min in the presence of 5 PM adenine. Similar results were observed for guanine and hypoxanthine uptake. Chromatographic analyses showed that over 95% of the radioactivity in the acid-soluble pool was associated with nucleotides after either NRK or TRK cells were incubated for 2 min in the presence of 5 PM [3H]adenine. The remaining radioactivity was associated with free adenine. Adenine uptake by the NRK and TRK cells at concentrations from 0.5 to 500 PM is presented in Fig. 1. The uptake studies in both cell types were carried out in parallel experiments on the same day and repeated at least twice. Figure 1 is representative of the differences in the uptake by the two cell lines. Adenine transport is greater in NRK cells than in TRK cells at each extracellular concentration of adenine below 30 PM. The reverse is true at concentrations above 30 ,u~ adenine. Adenine transport at concentrations above 50 PM for NRK and above 100 PM for TRK is proportional to the substrate concentration in the medium, indicating that, in addition to a facilitated diffusion process, adenine enters cells by a nonsaturable process which is presumed to be simple diffusion. Assuming the latter is simple diffusion, the rate can be estimated from the uptake
91
PURINE TRANSPORT
FIG. 1. Adenine uptake as a function of adenine concentration. Cultures of NRK and TRK cells were incubated with [2JH]adenine at 37°C for 2 min. as described in the text. NRK cells: (0 -0) TRK cells. (0 -0)
curve by drawing a line through the origin parallel to the linear portion of the uptake curves. A curve typical for a saturable transport process is obtained by subtraction of the amount transported by diffusion from the total uptake. Diffusion rates and the apparent K, and V,,, values for adenine and hypoxanthine transport in the two cell lines are summarized in Table 1. The diffusion rates of adenine in both cell lines are similar but the K, and V,,, values are more than twofold higher in TRK cells. TABLE KINETIC
CONSTANTS
OF ADENINE
1 AND
HYPOXANTHINE
UPTAKES
NRK cells
Substrate Adenine Hypoxanthine
Diffusion rate (pmolesimg of protein/min/~M)
pnl @M)
V max (pmoles/mg of proteinimin)
2.45 + 0.05 1.34 " 0.4
22.2 t 3 6.58 ? 0.3
290 5 40 223 k 15
TRK cells Adenine Hypoxanthine
2.65 2 0.25 1.34 -c 0.03
92 2 25 35.8 f 10
666 I? 166 445-t 13
a Data for uptake were obtained as described in Figs. 1 and 2. and K, and V,,, values were estimated from double reciprocal plots after correction for the diffusion rates. The values given in the table represent an average of two experiments.
92
JOY YANG AND VlSSER
The effects of inhibitors on adenine transport in NRK and TRK cells were determined at 5 and JO &&M adenine. respectively, These concentrations of adenine provide conditions at which the nonsaturable transport process is negligible relative to the facilitated transport process. The purines, 3-deazaguanine, 4-aminopyrazolo(3,4-d)pyrimidine, 2-mercaptopurine, 6-amino-2-mercaptopurine, 2,6-diaminopurine, 6-methylpurine, 6-methylmercaptopurine, 8-azaadenine. R-bromoadenine, 2-amino-6-methylmercaptopurine, 6-mercaptopurine, xanthine, uric acid. purine. allopurinol, and hypoxanthine had little (less than 10%) or no effect on adenine transport in either NRK or TRK cells at analog concentrations up to a 20-fold molar excess. Guanine, at a fivefold molar excess, inhibited adenine uptake about 20% in TRK cells. Higher concentrations of guanine are not only less inhibitory but, at very high concentrations of guanine, the uptake of adenine is stimulated. For example. at 500 PM guanine. adenine uptake is stimulated to a transport rate about six times that obtained in the absence of guanine. These effects of guanine are not observed in NRK cells. Hypoxanthine
and Glranine Transport
The characteristics of guanine and hypoxanthine uptake are shown in Table 1 and Figs. 2 and 3. As in the case of adenine, uptake by NRK cells is greater than uptake by TRK cells at a low extracellular concentration of hypoxanthine (Fig. 2), whereas the reverse is true at high extracellular concentrations of hypoxanthine. Hypoxanthine enters both cell types by saturable and nonsaturable processes, as was observed for adenine uptake. As indicated in Table 1, diffusion rates are similar in both cell lines
FIG. 2. Hypoxanthine uptake as a function of hypoxanthine concentration in NRK and TRK cells. Cells were incubated with [WHlhypoxanthine at 37°C for 2 min. as described in 0) NRK cells; (0 -0) TRK ceils. the text. (O-
PURINE TRANSPORT
93
but the apparent K, and V,,, values for the facilitated transport process are higher for TRK cells than the corresponding values for NRK cells. The average K, (6.1 PM) obtained for hypoxanthine transport in NRK cells is very similar to that reported previously for hypoxanthine transport in Novikoff cells (3). In contrast to the characteristics of adenine and hypoxanthine transport by the two cell types, guanine transport is slightly greater in NRK cells than in TRK cells (Fig. 3) at all concentrations tested. The kinetic constants for guanine are not included in Table 1 for reasons which will be given in the Discussion. The distribution of radioactivity in the free base and nucleotide fractions after incubation in the presence of 5 and 10 PM [3H]hypoxanthine showed that the major intracellular component (over 95%) in both NRK and TRK cells is the nucleotide fraction, and less than 5% is present as the free base. Compounds which inhibit the uptake of guanine and hypoxanthine are summarized in Table 2. 6-Thioguanine and 6-mercaptopurine inhibit the uptake of hypoxanthine and guanine in both NRK and TRK cells. Reciprocal plots of the data show that the inhibition is competitive and that the apparent K, values are lower than the corresponding K, values in each case. Purine is an effective inhibitor of the uptake of hypoxanthine and guanine in NRK cells but has no effect in TRK cells. At low guanine concentrations (0.4 to 5 FM), hypoxanthine inhibits guanine uptake competitively with apparent Ki values of 0.19 and 6 PM for NRK and TRK cells, respectively. Guanine, however, does not inhibit hypoxanthine uptake in either cell line, even at a 15-fold molar excess of guanine. Adenine inhibits hypoxanthine and guanine uptake in both cell lines. Maximum inhibition of guanine uptake is 60% in NRK cells and 30% in
GUANINE,
pk.4
FIG. 3. Rate of guanine uptake by NRK and TRK cells as a function of substrate concentration. Cells were incubated with [VH]guanine at 37°C for 2 min. as described in the text. (0 -0) NRK cells; (0 -0) TRK cells.
94
JOY
YANG
AND
VISSER
TRK cells at a 20-fold molar excess of adenine. Higher concentrations ot adenine do not increase these inhibitory effects. A 20-fold molar excess of adenine inhibits hypoxanthine uptake maximally (70%) in NRK cells and 40% in TRK cells. 8-Azaadenine and 3-deazaguanine are not inhibitory but, at high concentrations, these analogs stimulate the uptake of both guanine and hypoxanthine. For example, a 20-fold molar excess of 3-deazaguanine stimulates both guanine and hypoxanthine uptake in both cell types about 25%. Similarly, 8-azaadenine stimulates hypoxanthine uptake 20% and guanine uptake 75% in both cell types at a 20-fold molar excess of the analog. The analogs, 2-amino-6-amino-6-methylmercaptopurine, 2-mercaptopurine, 6-amino-2-mercaptopurine, 2,6-diaminopurine, 8-azaguanine, 6-methylmercaptopurine, 8-azahypoxanthine. xanthine, or allopurinol, do not inhibit or stimulate guanine or hypoxanthine uptake in either cell line at concentrations up to a 20-fold molar excess. DISCUSSION
The differences observed in the uptake characteristics of hypoxanthine, guanine, and adenine by NRK cells, as compared to their uptake by TRK cells (Figs. 1, 2, and 3), provide a clear demonstration that viral transformation induces changes in transport properties which are reflected in differences in V,,, and apparent K, values for transport. This conclusion is based on data obtained at conditions which allowed a direct comparison of the transport properties of the two cell types under identical conditions. The differences in transport properties of the two cell lines represent alterations in the transport systems rather than changes in intracellular enzymes since no difference in the intracellular concentration of free adenine or hypoxanthine was observed in the two cell lines after incubation under the assay conditions. This conclusion is also consistent with the generally accepted concept that the transport of purines by mammalian cells is rate-limiting relative to subsequent intracellular metabolic reactions (3, 4). Adenine is transported predominantly by a saturable transport system in TRK and NRK cells (Fig. 1) at low concentrations. In addition, transport occurs by a nonsaturable process in both cell lines, the predominant mode of transport at high concentrations. The latter process is similar in both cell lines and has the characteristics of simple diffusion, although a transport system with a very high K,, such as that reported for adenine transport by Hawkins and Berlin (4) in rabbit polymorphonuclear leukocytes (K, = 100 mM), cannot be eliminated as an explanation for the slower uptake process. Both the V,,, and the apparent K, values for adenine uptake are much higher in the transformed cells (Fig. I, Table 1). As a result of these
PURINE
TRANSPORT
95
differences in the transport characteristics of the two cell lines, the amount of adenine transported by NRK cells is greater than that transported by TRK cells at extracellular concentrations of adenine approximating the K, in NRK cells, whereas the reverse is true at extracellular concentrations above the V,,, in NRK cells. The structural requirements for the adenine transport system are very specific in both cell types as evidenced in the lack of transport inhibition by uric acid, xanthine, hypoxanthine, or any of the various purine analogs tested (Table 2). The one exception is the effect of guanine which, at a fivefold molar excess, inhibits adenine uptake in TRK cells about 20%, whereas, at a much higher concentration (500 FM), guanine stimulates adenine uptake about six times that of the control. These effects of guanine are not observed in NRK cells, indicating that the transport components in the two cells are different. An explanation for this stimulatory effect is not apparent, but may be related to a similar effect observed by Zylka and Plagemann (3). They found that high concentrations of uracil cause an increase in the V,,, for guanine and hypoxanthine uptake. The apparent alterations in the transport of hypoxanthine produced by viral transformation are similar to those observed for adenine (Table 1, Fig. 2). As in the case of adenine. hypoxanthine is transported by a nonsaturable process in both cell types, and the relative amount of hypoxanthine transported into the two cell lines is variably dependent upon the extracellular purine concentration. In contrast, the uptake of guanine by TRK cells is slightly less than that by NRK cells at all extracehular concentrations, and the apparent nonsaturable transport process contributes appreciably to total transport, even at low concentrations of guanine. Based on the data obtained with inhibitors as discussed below, this nonsaturable process for guanine uptake may represent a facilitated transport process with high K, and V,,, values. Viral transformation of NRK cells causes a change in the transport of guanine which is markedly different from the alterations in hypoxanthine and adenine transport. This result, as well as the changes in the kinetic constants for transport, indicate that transformation affects the transport processes directly. It has been reported (3) that guanine and hypoxanthine are transported by a single transport system in Novikoff cells and are mutually and competitively inhibitory. Our results, however, show that there are separate transport systems in NRK and TRK cells. Hypoxanthine inhibits guanine uptake competitively with a Ki value much lower than the corresponding K, values for guanine in both cell lines (Table 2), whereas guanine does not inhibit hypoxanthine uptake in either cell line, even at a ISfold molar excess of guanine. Separate transport systems for hypoxanthine and guanine are also indicated from the data of Dybing (10) who
None 6Mercaptopurine 6-Thioguanine Purine
Hypoxanthine
AND
HYPOXANTHINE
Competitive Competitive Competitive
Competitive Competitive Competitive Competitive
ANALOGS
or Ki
IN NRK
-
Competitive Competitive Noninhibitory
Competitive Competitive Competitive Noninhibitory
of
TRK
inhibition
Type
AND
TRK
CELLS”
cells
38.5 9.5 10.7
(/Lhl)
K”, or h /
The analogs and substrates were added simuitaneouzly and from double The apparent K,, and Ki values were estimated used were between 0.4 and 5 FM for NRK cells and 0.5 and IO
6.1 4.5 1.15 6.25
8.6 0.19 2.0 1.7 3.6
K,
-
cells
PURINE
b-4
of
2 BY THE
inhibition
Type
NRK
TABLE TRANSPORT
u The inhibition studies for NRK and TRK cells were carried out at different times. incubated at 37°C for 2 min. The values represent an average of two or more experiments. reciprocal plots without correction for the diffusion rates. The concentrations of substrates GM for TRK cells.
None Hypoxanthine 6-Mercaptopurine 6-Thioguanine Purine
Inhibitor
OF GUANINE
Guanine
Substrate
INHLBITION
PURINE TRANSPORT
97
found that actinomycin D reduces the uptake of hypoxanthine in a MH, C, strain of rat hepatoma cells, whereas guanine uptake is not significantly altered by the antibiotic. If hypoxanthine-guanine phosphoribosyltransferase were involved in the transport of guanine and hypoxanthine, it would be expected that these purines would be mutually inhibitory for their transport. Since this was not observed, the data also support the conclusion of Zylka and Plagemann (3) that phosphoribosyltransferase is not involved in the transport process for these purines. Differences in the transport systems of NRK and TRK cells are apparent in the effects of inhibitors on the transport systems. Although 6-mercaptopurine and 6-thioguanine behave similarly as potent competitive inhibitors of hypoxanthine and guanine uptake in both cell types, other inhibitors produce markedly different effects in the two cell lines (Table 2). Purine inhibits hypoxanthine and guanine uptake competitively in NRK cells but is without effect on the transport of either purine in TRK cells. Adenine inhibits uptake of hypoxanthine and guanine to a much greater extent in NRK cells than in TRK cells and, as previously discussed, the effects of guanine on adenine transport are markedly different. It may be concluded that the transport properties of purines are similar but not identical in the two cell types. Structural requirements for transport are rigid in both cell lines, particularly for adenine transport. These characteristics of the transport processes for purines may be predicted to be of importance in determining the selective toxicity of purine analogs in chemotherapy. In particular, the results emphasize that the extracellular concentrations of purines and purine analogs may have a strong influence on the relative amount of purines which are transported by different types of cells. SUMMARY
The characteristics of adenine, hypoxanthine, and guanine uptake in normal rat kidney (NRK) cells and NRK cells nonproductively transformed (TRK) by Kirsten mouse sarcoma virus have been studied. The data show that adenine and hypoxanthine are transported by saturable and nonsaturable processes in both cell types. The nonsaturable process for adenine uptake (about 2.5 pmoleslmg of protein/mm/FM external concentration) and hypoxanthine uptake (about 1.3 pmoles/mg of protein/mm/PM external concentration) is similar in both cell lines. The apparent K, and V,,, values for the facilitated transport process of both purines are more than twofold higher for TRK cells than the corresponding values for NRK cells. Uptake by NRK cells is greater than that by TRK cells at low extracellular concentration of adenine or hypoxanthine, whereas the reverse is true at high concentrations of adenine or hypoxan-
98
JOi’ ‘t’-\N(i AND VISSEK
thine. In contrast to the characteristics of adenine and hypoxanthine uptake by two cell types, guanine uptake is slightly greater in NRK cells than in TRK cells at all concentrations tested. The structural requirements for the adenine transport system are very specific in both cell types, as indicated by the inability of uric acid, hypoxanthine, and several purine analogs to inhibit adenine uptake. 6-Thioguanine and 6-mercaptopurine inhibit uptake of guanine and hypoxanthine in NRK and TRK cells. Inhibition is competitive with the apparent Kj values lower than the corresponding k,n values in each case. Hypoxanthine inhibits guanine uptake competitively with apparent Ki values of 0.19 and 6 PM for NRK and TRK cells, respectively. Guanine, however, does not inhibit hypoxanthine uptake in either cell line. Therefore, hypoxanthine and guanine have separate transport systems in both cell lines which do not involve hypoxanthine-guanine phosphoribosyltransferase. Purine inhibits guanine and hypoxanthine uptake competitively in NRK cells but is without effect in TRK cells, and adenine inhibits the uptake of guanine and hypoxanthine to a greater extent in NRK cells than in TRK cells. These results show that differences exist in the transport systems of NRK and TRK cells for these purines. ACKNOWLEDGMENTS This work was supported by Grants CA-02373 and CA- 14089 from the National Cancer Institute. U. S. National Institutes of Health. The authors wish to thank Miss Chia-Wei Wu for her able assistance.
REFERENCES I. 2. 3. 4. 5. 6.
Benke. P. J.. Herrick. N.. and Hebert. A.. Biochern. Med. 8, 309 (1973). Quinlan. D. C.. and Hockstadt, J.. Fed. Puoc,. 33, 1359 (1974). Zylka, J. M.. and Plagemann. P. G. W.. J. Bid. Chrm. 250, 5756 (1975). Hawkins. R. A.. and Berlin. R. D.. Biochinz. Biophy.~. Am 173. 324 (1969). Berlin, R. D., Science 168, 1539 ( 1970). Murray, A. W., Elliot, D. C.. and Atkinson, M. R.. Pro~r. Nucl. Acid Res. Mol. Bid. 10, 87 (1970). 7. Roy-Burman. P.. and Klement, V., J. G‘c,r~.I/ire/. 28, 193 (1975). 8. Lowry. 0. H., Rosebrough. N. J.. Fat-r. A. L., and Randell, R. J.. J. Bid. Cham. 193, 265 (1951).
9. Roy-Burman, S.. and Visser, D. W., J. Bid. Chum. IO. Dybing. E.. Biochenz. Phurn~crc~o/. 23, 3045 (1974).
2.50, 9270 (197.5).
17, 87-98
MEDICINE
(19771
Purine Transport in Cultured-Normal and Mouse Sarcoma Virus-Transformed Rat Kidney Cells Y. H. JOY YANG Department
of Biochemistry.
University Angeles. Received
AND D. W. VISSER
of Southern California August
California, 90033
School
of Medicine,
Los
5. 1976
INTRODUCTION Numerous studies have shown that purines are transported into mammalian cells by a saturable process which is rate-limiting relative to intracellular reactions, but the mechanism of transport is controversial. Studies with cultures of human fibroblasts which are deficient in hypoxanthine phosphoribosyltransferase activity (l), as well as studies with membrane vesicles of mammalian cells (2), have led to the conclusion that transport may involve phosphoribosylation as an integral part of the transport process. Recent studies by Zylka and Plagemann (3) on the transport of purines by Novikoff cells support the earlier conclusion of Hawkins and Berlin (4) and R. D. Berlin (5) from studies with polymorphonuclear metrophiles that phosphoribosylation of adenine occurs intracellularly subsequent to transport into the cell by facilitated diffusion processes. Zylka and Plagemann (3) demonstrated that hypoxanthine and guanine are transported in Novikoff cells by a single transport system which is distinct from that of adenine transport. A more precise knowledge of the characteristics of purine transport by mammalian cells is of practical importance for several reasons. The de FZOVOsynthesis of purines is absent or limited in certain cells (6) which must utilize preformed purines from the blood to satisfy their purine requirements. Much effort has been focused on specific intracellular enzymatic reactions which are affected by purine analogs. Little attention has been directed toward investigations of the transport of purine analogs into cells or the effects of analogs on the transport of naturally occurring substrates. The chemotherapeutic value of analogs is generally thought to be based primarily on their intracellular effects. However, the transport properties of purine analogs may be of considerable importance in relation to their chemotherapeutic efficiency, particularly since there is general agreement that the uptake of purines and, presumably, purine analogs is a rate-limiting process. 87 Copyright All rights
@ I977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN
ooO6-2944
88
JOY
‘r’ANG
AND
VISSEK
This investigation of the transport characteristics of purines in normal rat kidney cells (NRK) and NRK cells nonproductively transformed (TRK) by Kirsten mouse sarcoma virus (7) was initiated to gain information about the structural specificity of these transport processes in the two cell types. These objectives were approached by a careful delineation of the transport characteristics of adenine, guanine, and hypoxanthine in normal and transformed cells and by the determination of the effects of various analogs on the transport of these bases. The studies were carried out at low concentrations of substrate (below the apparent K, ) with short incubation periods (2 min), conditions at which the transport process is rate-limiting. MATERIALS
AND METHODS
Materials Normal rat kidney (NRK) cells and NRK cells nonproductively transformed (TRK) by Kirsten mouse sarcoma virus were a gift of Dr. P. Roy-Burman of the University of Southern California. Radioactive chemicals were obtained as follows: [2-3H]Adenine from New England Nuclear: [8-3H]guanine and [8-3H]hypoxanthine from Schwarz/Mann. The purity of the radioactive compounds was checked periodically by paper chromatography. Adenine, guanine, hypoxanthine, and 6-mercaptopurine were purchased from Calbiochem. Pm-me, 2-mercaptopurine, 2-amino6-methylmercaptopurine, 2-amino-6-mercaptopurine, 2,6-diaminopurine, 6-methylpurine, 6-amino-2-mercaptopurine, 6-methylmercaptopurine, 8-azaadenine, 8-azaguanine, 8azahypoxanthine, 8-bromoadenine. allopurinol, and 4-aminopyrazolo(3,4-d) pyrimidine were purchased from Sigma Chemical Co. 3-Deazaguanine was a gift from Dr. T. Khwaja. Serum-free MEM medium, fetal bovine serum, glutamine, and trypsin were obtained from Flow Laboratories. Gentamicin was purchased from Biological Associates. Other reagents were obtained from standard commercial sources. Growth of Cells NRK and TRK cells were routinely maintained in 75cm2 Falcon plastic tissue culture flasks at 37°C using MEM medium supplemented with 2 mM glutamine, 10% fetal bovine serum, 0.01% gentamicin, and 0.1 M Tricine at pH 7.3. The medium was changed every 3 days. Fetal bovine serum was heated at 60°C for 20 min just prior to use. Cultures were restarted from frozen stocks after about 50 passages for NRK cells and about 80 passages for TRK cells. Cultures were checked for mycoplasm by the use of a scanning electron microscope. Confluent cells were trypsinized and plated in 35mm Falcon plastic
PURINE
TRANSPORT
89
tissue culture dishes (usually 4.6 x lo5 cells/dish) in the same medium and incubated at 37°C in an atmosphere of 5% CO, in air. Fresh medium was supplied 4 hr later, and the cells were used for assays on the following day (20 hr). At these conditions, both NRK and TRK cells adhere to assay plates predominantly in the monolayer, and the cell density of both cell lines was the same at the time of assay (generation time 20-22 hr). The medium may be removed rapidly by aspiration, and the cells may be washed on the plates without appreciable cell loss. Transport
Studies
The transport studies in both cell types were carried out in parallel experiments on the same day. This procedure is desirable because transport characteristics were found to be more variable when the experiments were carried out at different times. The medium was removed from assay plates by aspiration, and the cells were rinsed with phosphate-buffered saline (PBS) twice at 37°C. The radioactive substrate in 1 ml of PBS was added immediately, and the cells were incubated on a slide warmer at 37°C for the designated time periods. At the end of the incubation period, the medium was removed by aspiration, and the cells were rinsed twice with 2 ml of PBS at 4°C. One milliliter of cold 10% TCA was added to each dish and maintained at 4°C for 30 min. An aliquot of TCA extract was placed in the vials containing 10 ml of scintillation fluid (0.8% PPO; 5% Bio-Solv in toluene), and radioactivity was determined in a Beckman LS-245 liquid scintillation counter. Control values were determined in the same manner except that the medium was aspirated from the cells immediately after addition. Purine analogs at the designated concentrations were added together with radioactive adenine, guanine, or hypoxanthine. The substrate concentrations (0.5 to 10 FM) used were below the K, for uptake of each put-me in each cell line. This procedure was followed to ensure maximal sensitivity to the competing substrate for transport. TCA-insoluble material was incubated in 1 ml of NaOH (1 .O N) at 37°C for 2 hr. An aliquot of the NaOH digests was used for protein determinations and, in some cases, for determinations of radioactivity. Incorporation of radioactivity into acid-insoluble material was negligible. Protein determinations were carried out by the method of Lowry et al. (8) using crystalline bovine albumin as the standard. Analysis of Radioactive Products in NRK and TRK Cells after Incubation with 3H-Labeled Adenine or Hypoxanthine
Analyses of the intracellular radioactive products were carried out as follows. Following a 2-min incubation with [2-3H]adenine or [g“Hlhypoxanthine, the cells were washed twice with 2 ml of PBS (4°C).
90
JOY
YANG
AND
VISSEK
One milliliter of ethanol at -20°C was added to the assay plates immediately after washing, and the plates were kept at -20°C for 2 hr. The ethanol was evaporated under a gentle stream of air. and the cells were extracted with I ml of HaO. The water extracts were lyophilized and redissolved in a small volume of water. The components in each of the extracts were separated by descending chromatography on Whatman 3MM paper using n-butanol-H,O (86:14, v/v) as the solvent system (9). Areas of the paper containing adenine or hypoxanthine and the corresponding nucleosides and nucleotides were located by ultraviolet light. Radioactivity in these areas was determined by the use of a liquid scintillation counter. RESULTS
Adenine Transport Intracellular radioactivity after a 2-min incubation in the presence of [3H]adenine was found predominantly (98%) in the TCA-soluble fraction of both NRK and TRK cells. Even after IO-min incubation periods, incorporation into acid-insoluble materials was less than 4% of the total radioactivity found in the cells. Therefore, TCA-soluble material was used as a measure of transport. Addition of glucose to the uptake medium did not increase the uptake of adenine by NRK cells, even though the cells were starved for 1 hr prior to uptake determinations. Uptake was linear with the time of incubation up to 3 min with 5 PM adenine, and the total incorporated was less than 2% of the substrate added after 3 min in the presence of 5 PM adenine. Similar results were observed for guanine and hypoxanthine uptake. Chromatographic analyses showed that over 95% of the radioactivity in the acid-soluble pool was associated with nucleotides after either NRK or TRK cells were incubated for 2 min in the presence of 5 PM [3H]adenine. The remaining radioactivity was associated with free adenine. Adenine uptake by the NRK and TRK cells at concentrations from 0.5 to 500 PM is presented in Fig. 1. The uptake studies in both cell types were carried out in parallel experiments on the same day and repeated at least twice. Figure 1 is representative of the differences in the uptake by the two cell lines. Adenine transport is greater in NRK cells than in TRK cells at each extracellular concentration of adenine below 30 PM. The reverse is true at concentrations above 30 ,u~ adenine. Adenine transport at concentrations above 50 PM for NRK and above 100 PM for TRK is proportional to the substrate concentration in the medium, indicating that, in addition to a facilitated diffusion process, adenine enters cells by a nonsaturable process which is presumed to be simple diffusion. Assuming the latter is simple diffusion, the rate can be estimated from the uptake
91
PURINE TRANSPORT
FIG. 1. Adenine uptake as a function of adenine concentration. Cultures of NRK and TRK cells were incubated with [2JH]adenine at 37°C for 2 min. as described in the text. NRK cells: (0 -0) TRK cells. (0 -0)
curve by drawing a line through the origin parallel to the linear portion of the uptake curves. A curve typical for a saturable transport process is obtained by subtraction of the amount transported by diffusion from the total uptake. Diffusion rates and the apparent K, and V,,, values for adenine and hypoxanthine transport in the two cell lines are summarized in Table 1. The diffusion rates of adenine in both cell lines are similar but the K, and V,,, values are more than twofold higher in TRK cells. TABLE KINETIC
CONSTANTS
OF ADENINE
1 AND
HYPOXANTHINE
UPTAKES
NRK cells
Substrate Adenine Hypoxanthine
Diffusion rate (pmolesimg of protein/min/~M)
pnl @M)
V max (pmoles/mg of proteinimin)
2.45 + 0.05 1.34 " 0.4
22.2 t 3 6.58 ? 0.3
290 5 40 223 k 15
TRK cells Adenine Hypoxanthine
2.65 2 0.25 1.34 -c 0.03
92 2 25 35.8 f 10
666 I? 166 445-t 13
a Data for uptake were obtained as described in Figs. 1 and 2. and K, and V,,, values were estimated from double reciprocal plots after correction for the diffusion rates. The values given in the table represent an average of two experiments.
92
JOY YANG AND VlSSER
The effects of inhibitors on adenine transport in NRK and TRK cells were determined at 5 and JO &&M adenine. respectively, These concentrations of adenine provide conditions at which the nonsaturable transport process is negligible relative to the facilitated transport process. The purines, 3-deazaguanine, 4-aminopyrazolo(3,4-d)pyrimidine, 2-mercaptopurine, 6-amino-2-mercaptopurine, 2,6-diaminopurine, 6-methylpurine, 6-methylmercaptopurine, 8-azaadenine. R-bromoadenine, 2-amino-6-methylmercaptopurine, 6-mercaptopurine, xanthine, uric acid. purine. allopurinol, and hypoxanthine had little (less than 10%) or no effect on adenine transport in either NRK or TRK cells at analog concentrations up to a 20-fold molar excess. Guanine, at a fivefold molar excess, inhibited adenine uptake about 20% in TRK cells. Higher concentrations of guanine are not only less inhibitory but, at very high concentrations of guanine, the uptake of adenine is stimulated. For example. at 500 PM guanine. adenine uptake is stimulated to a transport rate about six times that obtained in the absence of guanine. These effects of guanine are not observed in NRK cells. Hypoxanthine
and Glranine Transport
The characteristics of guanine and hypoxanthine uptake are shown in Table 1 and Figs. 2 and 3. As in the case of adenine, uptake by NRK cells is greater than uptake by TRK cells at a low extracellular concentration of hypoxanthine (Fig. 2), whereas the reverse is true at high extracellular concentrations of hypoxanthine. Hypoxanthine enters both cell types by saturable and nonsaturable processes, as was observed for adenine uptake. As indicated in Table 1, diffusion rates are similar in both cell lines
FIG. 2. Hypoxanthine uptake as a function of hypoxanthine concentration in NRK and TRK cells. Cells were incubated with [WHlhypoxanthine at 37°C for 2 min. as described in 0) NRK cells; (0 -0) TRK ceils. the text. (O-
PURINE TRANSPORT
93
but the apparent K, and V,,, values for the facilitated transport process are higher for TRK cells than the corresponding values for NRK cells. The average K, (6.1 PM) obtained for hypoxanthine transport in NRK cells is very similar to that reported previously for hypoxanthine transport in Novikoff cells (3). In contrast to the characteristics of adenine and hypoxanthine transport by the two cell types, guanine transport is slightly greater in NRK cells than in TRK cells (Fig. 3) at all concentrations tested. The kinetic constants for guanine are not included in Table 1 for reasons which will be given in the Discussion. The distribution of radioactivity in the free base and nucleotide fractions after incubation in the presence of 5 and 10 PM [3H]hypoxanthine showed that the major intracellular component (over 95%) in both NRK and TRK cells is the nucleotide fraction, and less than 5% is present as the free base. Compounds which inhibit the uptake of guanine and hypoxanthine are summarized in Table 2. 6-Thioguanine and 6-mercaptopurine inhibit the uptake of hypoxanthine and guanine in both NRK and TRK cells. Reciprocal plots of the data show that the inhibition is competitive and that the apparent K, values are lower than the corresponding K, values in each case. Purine is an effective inhibitor of the uptake of hypoxanthine and guanine in NRK cells but has no effect in TRK cells. At low guanine concentrations (0.4 to 5 FM), hypoxanthine inhibits guanine uptake competitively with apparent Ki values of 0.19 and 6 PM for NRK and TRK cells, respectively. Guanine, however, does not inhibit hypoxanthine uptake in either cell line, even at a 15-fold molar excess of guanine. Adenine inhibits hypoxanthine and guanine uptake in both cell lines. Maximum inhibition of guanine uptake is 60% in NRK cells and 30% in
GUANINE,
pk.4
FIG. 3. Rate of guanine uptake by NRK and TRK cells as a function of substrate concentration. Cells were incubated with [VH]guanine at 37°C for 2 min. as described in the text. (0 -0) NRK cells; (0 -0) TRK cells.
94
JOY
YANG
AND
VISSER
TRK cells at a 20-fold molar excess of adenine. Higher concentrations ot adenine do not increase these inhibitory effects. A 20-fold molar excess of adenine inhibits hypoxanthine uptake maximally (70%) in NRK cells and 40% in TRK cells. 8-Azaadenine and 3-deazaguanine are not inhibitory but, at high concentrations, these analogs stimulate the uptake of both guanine and hypoxanthine. For example, a 20-fold molar excess of 3-deazaguanine stimulates both guanine and hypoxanthine uptake in both cell types about 25%. Similarly, 8-azaadenine stimulates hypoxanthine uptake 20% and guanine uptake 75% in both cell types at a 20-fold molar excess of the analog. The analogs, 2-amino-6-amino-6-methylmercaptopurine, 2-mercaptopurine, 6-amino-2-mercaptopurine, 2,6-diaminopurine, 8-azaguanine, 6-methylmercaptopurine, 8-azahypoxanthine. xanthine, or allopurinol, do not inhibit or stimulate guanine or hypoxanthine uptake in either cell line at concentrations up to a 20-fold molar excess. DISCUSSION
The differences observed in the uptake characteristics of hypoxanthine, guanine, and adenine by NRK cells, as compared to their uptake by TRK cells (Figs. 1, 2, and 3), provide a clear demonstration that viral transformation induces changes in transport properties which are reflected in differences in V,,, and apparent K, values for transport. This conclusion is based on data obtained at conditions which allowed a direct comparison of the transport properties of the two cell types under identical conditions. The differences in transport properties of the two cell lines represent alterations in the transport systems rather than changes in intracellular enzymes since no difference in the intracellular concentration of free adenine or hypoxanthine was observed in the two cell lines after incubation under the assay conditions. This conclusion is also consistent with the generally accepted concept that the transport of purines by mammalian cells is rate-limiting relative to subsequent intracellular metabolic reactions (3, 4). Adenine is transported predominantly by a saturable transport system in TRK and NRK cells (Fig. 1) at low concentrations. In addition, transport occurs by a nonsaturable process in both cell lines, the predominant mode of transport at high concentrations. The latter process is similar in both cell lines and has the characteristics of simple diffusion, although a transport system with a very high K,, such as that reported for adenine transport by Hawkins and Berlin (4) in rabbit polymorphonuclear leukocytes (K, = 100 mM), cannot be eliminated as an explanation for the slower uptake process. Both the V,,, and the apparent K, values for adenine uptake are much higher in the transformed cells (Fig. I, Table 1). As a result of these
PURINE
TRANSPORT
95
differences in the transport characteristics of the two cell lines, the amount of adenine transported by NRK cells is greater than that transported by TRK cells at extracellular concentrations of adenine approximating the K, in NRK cells, whereas the reverse is true at extracellular concentrations above the V,,, in NRK cells. The structural requirements for the adenine transport system are very specific in both cell types as evidenced in the lack of transport inhibition by uric acid, xanthine, hypoxanthine, or any of the various purine analogs tested (Table 2). The one exception is the effect of guanine which, at a fivefold molar excess, inhibits adenine uptake in TRK cells about 20%, whereas, at a much higher concentration (500 FM), guanine stimulates adenine uptake about six times that of the control. These effects of guanine are not observed in NRK cells, indicating that the transport components in the two cells are different. An explanation for this stimulatory effect is not apparent, but may be related to a similar effect observed by Zylka and Plagemann (3). They found that high concentrations of uracil cause an increase in the V,,, for guanine and hypoxanthine uptake. The apparent alterations in the transport of hypoxanthine produced by viral transformation are similar to those observed for adenine (Table 1, Fig. 2). As in the case of adenine. hypoxanthine is transported by a nonsaturable process in both cell types, and the relative amount of hypoxanthine transported into the two cell lines is variably dependent upon the extracellular purine concentration. In contrast, the uptake of guanine by TRK cells is slightly less than that by NRK cells at all extracehular concentrations, and the apparent nonsaturable transport process contributes appreciably to total transport, even at low concentrations of guanine. Based on the data obtained with inhibitors as discussed below, this nonsaturable process for guanine uptake may represent a facilitated transport process with high K, and V,,, values. Viral transformation of NRK cells causes a change in the transport of guanine which is markedly different from the alterations in hypoxanthine and adenine transport. This result, as well as the changes in the kinetic constants for transport, indicate that transformation affects the transport processes directly. It has been reported (3) that guanine and hypoxanthine are transported by a single transport system in Novikoff cells and are mutually and competitively inhibitory. Our results, however, show that there are separate transport systems in NRK and TRK cells. Hypoxanthine inhibits guanine uptake competitively with a Ki value much lower than the corresponding K, values for guanine in both cell lines (Table 2), whereas guanine does not inhibit hypoxanthine uptake in either cell line, even at a ISfold molar excess of guanine. Separate transport systems for hypoxanthine and guanine are also indicated from the data of Dybing (10) who
None 6Mercaptopurine 6-Thioguanine Purine
Hypoxanthine
AND
HYPOXANTHINE
Competitive Competitive Competitive
Competitive Competitive Competitive Competitive
ANALOGS
or Ki
IN NRK
-
Competitive Competitive Noninhibitory
Competitive Competitive Competitive Noninhibitory
of
TRK
inhibition
Type
AND
TRK
CELLS”
cells
38.5 9.5 10.7
(/Lhl)
K”, or h /
The analogs and substrates were added simuitaneouzly and from double The apparent K,, and Ki values were estimated used were between 0.4 and 5 FM for NRK cells and 0.5 and IO
6.1 4.5 1.15 6.25
8.6 0.19 2.0 1.7 3.6
K,
-
cells
PURINE
b-4
of
2 BY THE
inhibition
Type
NRK
TABLE TRANSPORT
u The inhibition studies for NRK and TRK cells were carried out at different times. incubated at 37°C for 2 min. The values represent an average of two or more experiments. reciprocal plots without correction for the diffusion rates. The concentrations of substrates GM for TRK cells.
None Hypoxanthine 6-Mercaptopurine 6-Thioguanine Purine
Inhibitor
OF GUANINE
Guanine
Substrate
INHLBITION
PURINE TRANSPORT
97
found that actinomycin D reduces the uptake of hypoxanthine in a MH, C, strain of rat hepatoma cells, whereas guanine uptake is not significantly altered by the antibiotic. If hypoxanthine-guanine phosphoribosyltransferase were involved in the transport of guanine and hypoxanthine, it would be expected that these purines would be mutually inhibitory for their transport. Since this was not observed, the data also support the conclusion of Zylka and Plagemann (3) that phosphoribosyltransferase is not involved in the transport process for these purines. Differences in the transport systems of NRK and TRK cells are apparent in the effects of inhibitors on the transport systems. Although 6-mercaptopurine and 6-thioguanine behave similarly as potent competitive inhibitors of hypoxanthine and guanine uptake in both cell types, other inhibitors produce markedly different effects in the two cell lines (Table 2). Purine inhibits hypoxanthine and guanine uptake competitively in NRK cells but is without effect on the transport of either purine in TRK cells. Adenine inhibits uptake of hypoxanthine and guanine to a much greater extent in NRK cells than in TRK cells and, as previously discussed, the effects of guanine on adenine transport are markedly different. It may be concluded that the transport properties of purines are similar but not identical in the two cell types. Structural requirements for transport are rigid in both cell lines, particularly for adenine transport. These characteristics of the transport processes for purines may be predicted to be of importance in determining the selective toxicity of purine analogs in chemotherapy. In particular, the results emphasize that the extracellular concentrations of purines and purine analogs may have a strong influence on the relative amount of purines which are transported by different types of cells. SUMMARY
The characteristics of adenine, hypoxanthine, and guanine uptake in normal rat kidney (NRK) cells and NRK cells nonproductively transformed (TRK) by Kirsten mouse sarcoma virus have been studied. The data show that adenine and hypoxanthine are transported by saturable and nonsaturable processes in both cell types. The nonsaturable process for adenine uptake (about 2.5 pmoleslmg of protein/mm/FM external concentration) and hypoxanthine uptake (about 1.3 pmoles/mg of protein/mm/PM external concentration) is similar in both cell lines. The apparent K, and V,,, values for the facilitated transport process of both purines are more than twofold higher for TRK cells than the corresponding values for NRK cells. Uptake by NRK cells is greater than that by TRK cells at low extracellular concentration of adenine or hypoxanthine, whereas the reverse is true at high concentrations of adenine or hypoxan-
98
JOi’ ‘t’-\N(i AND VISSEK
thine. In contrast to the characteristics of adenine and hypoxanthine uptake by two cell types, guanine uptake is slightly greater in NRK cells than in TRK cells at all concentrations tested. The structural requirements for the adenine transport system are very specific in both cell types, as indicated by the inability of uric acid, hypoxanthine, and several purine analogs to inhibit adenine uptake. 6-Thioguanine and 6-mercaptopurine inhibit uptake of guanine and hypoxanthine in NRK and TRK cells. Inhibition is competitive with the apparent Kj values lower than the corresponding k,n values in each case. Hypoxanthine inhibits guanine uptake competitively with apparent Ki values of 0.19 and 6 PM for NRK and TRK cells, respectively. Guanine, however, does not inhibit hypoxanthine uptake in either cell line. Therefore, hypoxanthine and guanine have separate transport systems in both cell lines which do not involve hypoxanthine-guanine phosphoribosyltransferase. Purine inhibits guanine and hypoxanthine uptake competitively in NRK cells but is without effect in TRK cells, and adenine inhibits the uptake of guanine and hypoxanthine to a greater extent in NRK cells than in TRK cells. These results show that differences exist in the transport systems of NRK and TRK cells for these purines. ACKNOWLEDGMENTS This work was supported by Grants CA-02373 and CA- 14089 from the National Cancer Institute. U. S. National Institutes of Health. The authors wish to thank Miss Chia-Wei Wu for her able assistance.
REFERENCES I. 2. 3. 4. 5. 6.
Benke. P. J.. Herrick. N.. and Hebert. A.. Biochern. Med. 8, 309 (1973). Quinlan. D. C.. and Hockstadt, J.. Fed. Puoc,. 33, 1359 (1974). Zylka, J. M.. and Plagemann. P. G. W.. J. Bid. Chrm. 250, 5756 (1975). Hawkins. R. A.. and Berlin. R. D.. Biochinz. Biophy.~. Am 173. 324 (1969). Berlin, R. D., Science 168, 1539 ( 1970). Murray, A. W., Elliot, D. C.. and Atkinson, M. R.. Pro~r. Nucl. Acid Res. Mol. Bid. 10, 87 (1970). 7. Roy-Burman. P.. and Klement, V., J. G‘c,r~.I/ire/. 28, 193 (1975). 8. Lowry. 0. H., Rosebrough. N. J.. Fat-r. A. L., and Randell, R. J.. J. Bid. Cham. 193, 265 (1951).
9. Roy-Burman, S.. and Visser, D. W., J. Bid. Chum. IO. Dybing. E.. Biochenz. Phurn~crc~o/. 23, 3045 (1974).
2.50, 9270 (197.5).