Purine Uptake by Azaguanine-resistant Chinese Hamster Cells MARILYN PARSONS,' JOHN MORROW,' 4 DOUGLAS STOCCO 2 AND PAUL KITOS3 1 Department of Genetics, Stanford Unioet-sity School of Medicine, Stanford, California; 2 Department of Biochemistry, Texas Tech University School of Medicine, Lubbock, Texas 79409 and 3 Department of Biochemistry, The Uniuersity of Kansas, Lawrence. Kansas 66045
ABSTRACT In this study the resistance of a numher of lines of Chinese hamster ovary cells to azaguanine is examined. Those which are drug resistant by virtue of a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) fail to take up any exogenous hypoxanthine or azaguanine. A second class of drug resistant cells which grow in the reverse selective HAT medium and have levels of HPRT in the range of the wild type parent line take up these purines at lower rates than the nonresistant cells and incorporate smaller amounts of them into trichloracetic acidinsoluble constituents. The results suggest that their basis for resistance resides in lowered incorporation of azaguanine into DNA and RNA, possibly due to a mofified HPRT molecule which accepts hypoxanthine, hut not azaguanine as a substrate.
8-Azaguanine (AG), an analogue of guanine, inhibits the growth of animal cells in part, at least, by interfering with the formation of GMP (Davidson, '72) and with normal maturation of RNA (Weiss and Pitot, '74).In general, cells which are resistant to the toxic effects of AG are thought to owe their immunity to the absence of functional hypoxanthine-guanine phosphoribosyltransferase (HPRT: EC 2.4.2.8). This enzyme catalyzes the synthesis of azaguanylic acid, derivatives of which are responsible for the inhibitory effects. Because HPRT-deficient cells are unable to form the aberrant nucleotides they are able to thrive in the presence of AG. Several recent reports suggest that the HPRT deficiencies of some AG-resistant cell lines are due to mutations within the structural gene for that enzyme. Beaudet, et al. ('73) showed that deficient Chinese hamster cells fall into two general categories: those which produce a product which cross reacts with antibody to HPRT, and those which do not. From either class of mutants revertants to HPRT-positive forms can be obtained. Sekiguchi and Sekiguchi ('73) reported that allelic complementaJ. CELL. PHYSIOL.,89: 209-218
tion, accomplished by fusing cells of two different HPRT-deficient hamster lines, generates HPRT-positive synkaryons. Bakay and Nyhan ('72) showed that nonfunctional HPRT from Lesch-Nyhan patients becomes functional if it is mixed with the normal human enzyme and then separated from the mixture by electrophoresis. They ascribe the activation to the transfer of a polypeptide from the active to the inactive catalytic partner. The human enzyme was reported by Arnold and Kelley ('71) to be composed of two identical subunits of 34,000 daltons and the same net charge and to resolve into three electrophoretically distinguishable isozymes. Hughes et al. ('75)provide evidence to indicate that the enzyme is a trimer of 27,000 dalton subunits, the same as mouse HPRT. Olson and Milman ('74) have shown that Chinese hamster HPRT exists in at least three isozymic forms and that the enzymes isolated from the hamster brain, liver and V79 tissue culture cells appear to be structurally and catalytically identical. InconsisReceived April 10, '75. Accepted Feh. 6, '76. Reprint requests should be directed to Dr. John Morrow.
209
210
M. PARSONS, J. MORROW, D. STOCCO AND P. KITOS
tent with the concept that a structural gene defect is responsible for AG-resistance, is the report of Harris ('71) in which he noted that ploidy levels do not affect the mutation rate to AG-resistance. Azaguanine-resistance has been used as a phenotypic marker in assessing mutation rates (Morrow, '701, identifying and selecting interspecific cell hybrids (Littlefield, '64) and characterizing the Lesch-Nyhan condition in vitro (Albertini and DeMars, '73). In their studies of AG-resistant hamster cells, Morrow and his colleagues ('73) noted a not-infrequent incidence of clones which exhibit high levels of HPRT. Similar results have also been obtained by other investigators (Harris and Whitmore, '74; Gillin, et al., '72). Unlike their HPRTdeficient counterparts these mutants do grow in a medium supplemented with HAT 5 ; the exogenous hypoxanthine satisfied all their purine requirements. In the present report we examine both HPRT-sufficient and -deficient azaguanine-resistant Chinese hamster cells. The results depict principal differences between these two classes of mutants and implicate reduced incorporation of azaguanine as the basis of resistance. MATERIALS AND METHODS
Cell culture For these studies a proline-requiring line of Chinese hamster ovary cells, CHO/pro(Kao and Puck, '671, kindly provided by Dr. M. W. Taylor of the Department of Microbiology, Indiana University, was used. Sublines resistant to AG were isolated by plating them in a medium which contained 30 pg per ml of AG. Within two weeks most cells died and clones of viable cells arose. Several clones were isolated and the remainder were incubated in "HAT" medium. In HAT approximately 85% of the AG-resistant clones died, confirming that they were deficient in HPRT. Several HAT-resistant clones were then isolated (Morrow et al., '73). These clones were grown for extended periods in the absence of azaguanine and retested frequently, during which time they retained their phe-
notype, in some cases for up to two years. All clones which were HAT-resistant had levels of HPRT in 1 he range of that found in the CHO/pro- parent. Cells were cultivated in Falcon plastic tissue culture vessels or 250 ml milk dilution bottles. Eagle's autoclavable minimal medium (Flow Laboratories, Rockville, Maryland) Supplemented with 5% calf serum (Colorado Serum Company, Denver, Colorado), non-essential amino acids (Eagle, '591, antibiotics and glutamine was used for cloning as previously described (Morrow et al., '73) For most other studies medium MD705/1 (Kitos, Sinclair and Waymouth, '62) plus 5% calf serum (Grand Island Biological Company, Grand Island, New York) was used. The cells were transferred regularly by trypsinization or mechanical scraping (2nd between transfers the medium was changed every three days.
Hypoxanthins and azaguanine uptake studies The cells were plated in complete medium and allowed to attach overnight. Depending on the cloning efficiency of the subline, densities of 106to 2 x 106 cells per milk dilution bottle were used (30cells per mm2).The medium was then decanted and fresh medium containing either 5-PHluridine (ICN Corp., 0.833 pCi per ml, 3.33 x pM) and 8-ClClhypoxanthine (New England Nuclear Corp., 0.167 pCi per ml, 16.5 pM) or 5-C3H[luridine and 2-C14C1azaguanine (ICN Corp., 0.167 pCi per ml, 16.5 pM) was added. For certain experiments, unlabeled guanine (33. pM) was added. The cells were incubated in the labeled medium foir three hours, at which time the medium vvas decanted. The cultures were then washed with approximately 200 ml of flowing isotonic saline by directing the flow at the inner end of the inverted milk-dilution bottle. The saline flowed out of the bottle, passing over the cell monolayer, and collected in a large receptacle. The excess saline was drained from the culture vessels, and 4 ml of 0.5 5 5 pg per ml thymidints and hypoxanthine, 0 1 pg ml ammopterm
PUHINE UPTAKE IN CHO CELLS
per cent sodium dodecylsulfate (SDS) was added. The cells were disrupted by the SDS and then 1 ml of 50 per cent (W/VI cold trichloroacetic acid (TCA)was added to precipitate the acid insoluble material. A representative 2 ml portion of the resulting suspension was filtered through a 25 mm diameter Millipore membrane (type HA, 0.45 p pore size) to separate the soluble constituents from the insoluble. A 200 p1 portion of the soluble fraction was spotted on a 25 mm diameter GF/A Whatman glass-fiber filter disc and the membranes and glass-fiber discs were dried under a heat lamp. They were placed in plastic minivials containing 5 ml of scintillation cocktail (4 g Omnifluor [New England Nuclear Corp.1 per liter of toluene) and the radioactivity was determined by scintillation spectrometry. Pilot studies indicated that incorporation of hypoxanthine into the cells proceeded linearly over a three hour period.
Amino acid uptake studies The procedure used for amino acid uptake studies is the same as that described above except that the medium contained a mixture of [I4C7-labeled L amino acids (New England Nuclear Corp., 0.1 pCi per ml, 1.34 pg per ml) and the incubation period was two hours.
211
mCi/mmole,4 mCi/mgm, Amersham/Searle Corp., Arlington Heights, Illinois) or [8-'4C1 -hypoxanthine (48.6 mCi/mMole, New England Nuclear, Boston, Massachusetts) were added to the cells at a concentration of 10 pCi/ml for the 3H-isotope and 0.2 pCi/ml for the 14C-radiolabel. Following ten minutes of incubation in the presence of radioisotope the cells were rapidly collected by centrifugation in a clinical centrifuge and washed by resuspending the pelleted cells in Hanks solution. After a second centrifugation the cells were resuspended in N perchloric acid and quantitatively transferred to a Dounce glass homogenizer and thoroughly homogenized. Acid insoluble material was pelleted by centrifugation at 12,000 x g for ten minutes. The acid soluble supernatant was carefully removed and neutralized with an equal volume of Alamine in chloroform according to Stocco et al. ('72). All operations were performed at 4°C. The neutralized acid soluble fraction was then fractionated by means of anion exchange chromatography on columns of DEAE-cellulose (DE 23 Whatman Co.). Marker hypoxanthine (50 p1 of a 1 mgmlml solution) was added to the samples in order to determine the position of free hypoxanthine in the elution profile. Nucleotides were then eluted with a linear gradient of NbHCO, (0.002 to 0.250 M, pH 8.6).Fractions were collected in 10 ml aliquots at a rate of approximately 1ml/min. Each fraction was assayed for radioactivity by placing 0.5 ml in polyethylene scintillation minivials and dispersing in scintillation fluid consisting of 75% toluene-25% triton X-114 and 4.0 gm/litre Omnifluor (New England Nuclear, Boston, Massachusetts). Samples were then counted in a Beckman LS 230 liquid scintillation counter. In addition each fraction was analyzed at 260 nm to determine the position of the hypoxanthine marker.
Determination of free hypoxanthine in the cells For these experiments both CHO/procells and V79 cells (a Chinese hamster cell line derived from the lung) were used. Cells were grown in milk dilution bottles in F-10 media plus 5% calf serum (Kansas City Biological) and harvested with Viocase (GIBCO). Detached cells were washed and resuspended in Hanks balanced salt solution. Aliquots of cells were counted with an electronic particle counter (Particle Data Inc., Elmwood, 11linois). The remainder of the cells were RESULTS transferred to 50 ml erlenmeyer flasks and Purine uptake by CHO cells placed in a water bath at 37°C and shaken As shown in tables 1 and 2, Chinese gently throughout the course of the experiment. Either 3H-(G)-hypoxanthine (570 hamster cells which are resistant to
212
hl PARSONS. J. MORROW. 1). STOCCO AND P. KITOS
TABLE 1
Hypoxanthine and uridine uptake by azuguanine-sensitive and resistant lines of Chinese hamster cells 3H iiridine uptake
"C hypoxanthine uptake Cell line
Total % of control
~
TCA insolulde % of total
Total % of control
TCA insoluble % of total
~
100 (14,361 cpm) 5 4 7 9 62 44 30
31
CHO/proCHO/2 CHO/E CHO/F CH0/23 CH0/28 CH0/29 CHO/M
-
-
16 54 29 17
44
30 13 20 30 34 45 42
The parental cell line, CHO/pro-, is uaguanine sensitive. All other lines are azagrianine-resistant derivatives of it. Of them, lines CHO/&.CHO/E and CHO/F are HAT-sensitiveand lines CH0123, CHO/28, CHOP29 and CHO/M are HAT-resistant. I The number of separate experiments from which these data are calculated is provided in parentheses along with the uptake data. TABLE 2
Azuguanine and uridine uptake by azaguanine-sensitive and resistant lines of Chinese hamster cells '4C hypoxanthine uptake
Cell line
Total % of control
CHO/proCH0/2 CHO/E CHO/F CH0/23 CH0/28 CH0/29 CHO/M
100 (4) I (1,969cpm) 0 (4) 0 (1) 3 (1) 30 (4) 8 (4) 12 (2) 20 (2)
uridine uptake
TCA insoluble 96 of total
27
0 18 18 13 7
Total % of control
100 (1,484cpm) 40 30 53 54 29 19 46
TCA insolulde % of total
30 32 27 25 29 35 33 11
Cell line descriptions are given in ttlble 1. I The number of separate experiments from which these data are calculated is provided in parentheses along with the uptake data.
azaguanine can be divided into two general classes: those which grow in HAT medium (lines CH0/23, CH0/28, CH0/29 and CHON) take up less hypoxanthine and azaguanine than t h e parental line, CHO/pro-; those which do not grow in HAT medium (lines CH0/2, CHO/E, and CHO/F) take up virtually none of these bases. These findings are similar to those reported by Benke, Herrich and Hebert ('73a,b) on fibroblasts of Lesch-Nyhan and hyperuricemic patients. The results show that in the CHO cells the extent of
azaguanine uptake i,s much less than that of hypoxanthine. To facilitate comparisons of the data the specific activities of the radioactive hypoxanthine and azaguanine w e r e prestandartlized and identical amounts of each were used. Comparing tables 1 and 2, it can be seen that the CHO/pro- cells incorporate about 30 times as much hypoxanthine as azaguanine and that even the most efficient of the azaguanine resistant cell lines (CH0/23) incorpor ates less than one third as much azaguanine as the control culture. During the 3-hour
213
PURINE UPTAKE IN CliO CELLS
period of incubation less than half of the ingested 1% azaguanine or hypoxanthine is incorporated into TCA-insoluble material of the cells. The bulk of the intracellular radioactive material still exists as acidsoluble constituents (either the free base or non-polymeric nucleotide derivatives of it). In each of these studies 3H-labeled uridine was included in the medium as an internal control. If pyrimidine nucleoside uptake is functionally distinct from that of the purine, its pattern of incorporation should be independent of variations in purine uptake. As can be seen in table 1, there appears to be a correlation between the uptake of uridine and hypoxanthine. The correlation is less evident between uridine and azaguanine (table 2). Since the purine concentration of the medium is about 500 times that of the uridine, the significance of the observation is moot. Recent work in our laboratory indicates that the decreased uridine uptake in the mutant lines may be an effect induced by the hypoxanthine present in the medium. When the mutant cells are incubated in medium containing no hypoxanthine, their uridine uptake is not depressed relative to that of the wild type and in some cases it is elevated slightly. In all cell lines azaguanine severely depresses uridine uptake (compare tables 1 and 2: decrease from 14,361 to 1,484 cpm).
The effects of guanine on hypoxanthine and azaguanine uptake The utilization of hypoxanthine and guanine in animal cells is known to occur via the action of HPRT. Consequently, excess guanine should compete with hypoxanthine in its conversion to IMP. The azaguanine-sensitive cell line (CHO/pro-1 and representative azaguanine-resistant, HATsensitive (CH0/2) and HAT-insensitive (CH0/23, CH0/28 and CHO/M) cell lines were examined to determine the effects of excess (33 pM) guanine on the uptake of either 16.5 pM hypoxanthine or azaguanine. Guanine essentially eliminates the uptake of azaguanine (table 4) and it lowers the uptake of hypoxanthine (table 3). The CHO/pro- line takes up less than one fifth as much hypoxanthine in the presence of guanine as in its absence. Thus it seems that these bases are modified to form phosphorylated derivatives by a common mobilizing enzyme. The addition of guanine to hypoxanthine-containing rnedium does not depress uridine uptake (compare tables 1 and 3) but its addition to azaguanine-containing medium does (compare tables 2 and 4). Amino acid uptake by CHO cells The azaguanine-sensitive and -resistant lines were also tested for their abilities to take up a mixture of 14C-labeled L amino acids and incorporate them into TCA-in-
TAHLE 3
Hypoxanthine and uridine uptake by azaguanine-sensitiue and resistant Chinese hamster cells in the presence of guanine IJC hypoxanthine uptake
Cell line
Total % of control
CHOiproCH0/2 CH0/23 CH0/28 CHO/M ~
~
100 (2) I 110,530 cpm) 2 (2) 3 (21 44 (2) 47 (1)
TCA insolnhle % of total
18 0 0 93 6
'H uridine nptake Total % of control
100 114,480 cpm) 12 13 46 47
TCA mwlul~le % of total
21 23 11 80 8
~~
Information concerning cell lines is provided in table 1 . 1 The numl)er of separate experiments from which these data are calculated is provided in parentheses along with the uptake data.
214
M . PARSONS,
I.
MORROW, D. STOCCO AND P. KITOS
TABLE 4
Azaguanine and uridine uptake by azaguanine-sensitioe and resistant line9 of Chinese hamster cells in the presence of guanine 3H uridine uptake
"C hypoxanthine uptake Cell line
Total % of control
CHO/proCHO/2 CH0/23 CH0/28 CHO/M
TCA insoluble % of total
100 (2) ' (70 cpm) -(2) -(2) -(2) -(1)
Total % of control
-
100 (519cpm) 85 93 100 100
-
TCA insoluble % of total
40
30 30 34 30
Information on the cell lines is provided in table 1. ' The number of separate experiments from which these data are calculated is provided in parentheses along with the uptake data.
soluble cell constituents. Some variations in uptake can be seen (table 5) but they do not reflect the differences in purine transport which are evident in tables 1and 2. In the two hour labeling period used here slightly more than half of the incorporated radioactivity was found in the TCA-insoluble fraction of all cell lines. These observed differences in rates of amino acid transport and protein synthesis do not correlate with the differences in uptake and incorporation of hypoxanthine and azaguanine reported in tables 1 and 2. Thus deviations in the rates of purine uptake are not due to grossly dissimilar rates of metabolism among the mutant cell lines.
The metabolism of endogenous hypoxanthine In order to determine the intracellular conversion of hypoxanthine to IMP and other nucleotide derivatives, both CHO/pro- and V79 cells were incubated in the presence of 3H-hypoxanthine or 14Chypoxanthine. The results indicate that the amount of free hypoxanthine in the intracellular pool after 10 minutes is quite small. In figure 1is shown a representative profile of the nucleotide derivatives obtained following a 10 minute incubation of CHO/pro- cells in 14C-hypoxanthine.In all experiments employing CHO/pro- or V79 cells and radiolabeling with either I4Chypoxanthine or 3H-hypoxanthine, the amount of free hypoxanthine was found to
TABLE 5
Relative amino acid uptake and protein synthesis capabilities of the Chinese hamster mutant cell lines Cell line
CHO/ProCH0/2 CHO/E CHO/F CH0/23 CH0/28 CHO/M
(:< 1 0 3 )
Percent CPM found in TCA insoluble form
4.38 2.35 3.80 3.68 !!.70 3.80 4.90
58 55 62 58 57 55 61
CF'M/cell
be approximately 5% of the total radioactivity recovered. These data indicate that the intracellular conversion of hypoxanthine to its various nucleotide derivatives is both rapid and complete in the cells studied.
Axaguanine resistance in the presence of aminopterin A possible explanation for the existence of the doubly resistant lines would be an overly active system for the endogenous synthesis of purines. This could result in lowered purine u.ptake and thus azaguanine resistance. As HPRT would remain intact, the cells would be HAT resistant. Since aminopterin inhibits de nouo purine biosynthesis, incubation of such cells in aminopterin should force purine uptake and render the cells more sensitive
215
PURINE UPTAKE IN CHO CELLS
20 -
I
0-0
0-0
71.9%
. h
I
CPM O D 260 E = 0
a N c3 0
:.0
1.5
10
20
30
40
50
FRACTION NUMBER
Fig. 1 Acid soluble hypoxanthine derivatives in CHO/Pro- cells. Cells were incubated in the presence of '4C-hypoxanthine for 10 min and the acid soluble pool was extracted, neutralized and fractionated on DEAE cellulose columns. The numbers over the peaks represent the percentage of CPM present in each derivative of hypoxanthine
to azaguanine. In order to determine the extent to which aminopterin influences the growth of both azaguanine-sensitive cells (CHO/pro-) and doubly-resistant cells (i.e., resistant to both azaguanine and HAT) the cultures were exposed to varying concentrations of azaguanine in HAT medium or in HT medium (medium containing hypoxanthine and thymidine but lacking aminopterin). 105 cells were plated in 5 ml of appropriate medium in 60 mm Falcon plastic dishes. After five days the cells were harvested with two ml of 0.25 per cent trypsin and counted. As can be seen in figure 2, added aminopterin has no effect on the resistance of the cells of azaguanine. Evidently the toxicity due to azaguanine is neither augmented nor suppressed by aminopterin.
of azaguanine and hypoxanthine, but only sometimes by a complete lack of HPRT activity. Cells which are totally deficient in HPRT (azaguanine-resistant, HAT-sensitive) are consistently unable to ingest hypoxanthine or azaguanine while those which are not deficient in the enzyme (azaguanine-resistant, HAT-resistant1 re tain some ability to incorporate the two bases. The results of the amino-acid uptake studies indicate that the lowered uptake of purines is not due to a general depression of growth rate, but is rather specific for hypoxanthine and related molecules. In the HAT-resistant lines the slow purine uptake is not due to an endogenous over-production of purines because the profiles of resistance to azaguanine in the doublyresistant mutants are not altered by the presence of aminopterin (fig. 21. DISCUSSION Other authors have reported azaguanine Azaguanine resistance of CHO cells is al- resistant cell lines which are not deficient ways accompanied by lower incorporation in HPRT. Harris and Whitmore ('73) ob-
2 16
hl PARSONS, J . MORROW, I). STOCCO AND P. KlTOS
40
20
40
60
80
100
Fig. 2 Effect of aminopterin on inhibition of cell growth. Hypoxanthine and thyinidine were supplied to counteract the effect of aminopterin on cell growth. Ordinate: growth as percent of control: ahscissaazagrianine concentration pg per ml. Dotted line: minus aminopterin; solid line: plus aminopterin. On day zero each culture dish was seeded with 105 cells. The plating efficiency of the three cell lines was approximately 50%.Five days later the cells were harvested and counted. Growth is reported as per cent of the control cell population. The number of cell5 in the CHOiPro- and CHOi28 control cultures increased al)out %-fold during this period. The nrimher in CH0/23 increased approximately 8-fold in HAT and 12-fold in HT.
served a temperature sensitive mutation in cells which were resistant to azaguanine, but which seemed to have unaltered HPRT activity. Recently, Fenwick, et al. ('751have studied a class of mutants similar to those described here, in which the basis of resistance lies in alterations in the molecule such that it no longer accepts azaguanine as a substrate, but is still active with hypoxanthine. They suggest that this condition results from a missense mutation in the structural gene for the enzyme. Thus, our results can be conceived as resulting from genetic modifications in the HPRT molecule which are reflected in the lowered incorporation of azaguanine and related purines, while still allowing adequate hypoxanthine incorporation to fulfill the needs of the cells when cultivated in the presence of aminopterin. Although our results do not allow one to judge the role of HPRT in transport of purines, Plagemann and Richey ('74) have argued that phosphorylation is a process separate from
transport in mammalian cells. More recently, Alford and Barnes (personal communication) have shown that HPRT mutants of Chinese hamster lung fibroblasts still maintain facilitated diffusion in the complete absence of the enzyme. Thus, they conclude that the transport and phosphorylation of hypoxanthine are separate and distinct process'es in these cells. Since the pool of free hypoxanthine is quite small in the cells studied (fig. 1) and conversion to phosphorylated derivatives is extremely rapid, this might lead to the erroneous conclusion that the two processes are brought about by the same enzyme. ACKNOWLEDGMENTS
This work was supported in part by a grant from the University of Kansas General Research Fund, Grant Number CA-12310-03 from The National Cancer Institute and Grant Number DRG 1088 from the Damon Runyon Fund.
PURINE UPTAKE IN CHO CELLS
217
alteration in purine transport. J. Cell Physiol., 83: 43-51, Albertini, R. J,, and R. DeMars 1973 Somatic cell Hughes, S. H., G. M. Wahl and M. R. Capecchi 1975 Purification and characterization of mouse hypomutation: detection and quantitation of x-ray inxanthine-guanine phosphoribosyltransferase. J. duced mutation in cultured, diploid human Biol. Chem., 250: 120-126. fibroblasts. Mut. Res., 18: 199-224. Arnold, W. J., and W. N. Kelley 1971 Human hy- Kao, F. T., and T. T. Puck 1967 Genetics of somatic mammalian cells. IV. Properties of poxanthine-guanine phosphoril)osyltransferase. Chinese hamster cell mutants with respect to the Purification and subunit structure. J. Biol. Chem., requirement for proline. Genetics, 55: 5 13-524. 246: 7398-7404. Bakay, B., and W. L. Nyhan 1972 Activation of Kitos, P. A., R. Sinclair and C. Waymouth 1962 Glutamine metabolism by animal cells growing in a variants of hypoxanthine-guanine phosphosynthetic medium. Expl. Cell. Res., 27: 307-316. ribosyltransferase hy the normal enzyme. Proc. Nat. Littlefield, J. W. 1964 Selection of hybrids from Acad. Sci. (U.S.A.) 69: 2523-2526. Heaudet, A. L., D. J. Rouh and C. T. Caskey 1973 matings of fibro-blasts in rjitro and their presumed recombinants. Science, 145: 709-710. Mutations affecting the structure of hypoxanthine: guanine phosphorihosyltransferase in cultured Morrow, J. 1970 Genetic analysis of azaguanine reChinese hamster cells. Proc. Nat. Sci. (U.S.A.), 70: sistance in an established mouse cell line. Genetics, 65: 279-287. 320-324. Benke, P. J.. N. Herrick and A. Hebert 1973a Hypo- Morrow, J., J. Colofiore and D. Rintoul 1973 Azaguanine resistant hamster cell lines not defixanthine-guanine phosphoribosyltransferase cient in hypoxanthine-guanine phosphoribosylvariant associated with accelerated purine syntransferase. J. Cell. Physiol., 81; 97-100. thesis. J. Clin. Invest. 52: 2234-2240. Henke, P. J., N. Herrick and A. Hebert 197311 Trans- Olson, A. S., and G. Milman 1974 Chinese hamster port of hypoxanthine in fibroblasts with a normal HPRT: purification, structural and catalytic properand mutant hypoxanthine-guanine phosphoties. J. Biol. Chem., 249: 4030-4037. ribosyltransferase. Hiochem. Med., 8: 309-323. P l a g e m a n n , P. G. W., a n d D. P . Richey Davidson, J. N. 1972 The Biochemistry of Nucleic 1974 Transport of nucleosides, nucleic acid bases, Acids. Academic Press, New York, pp. 274. choline and glucose by animal cells in culture. Eagle, H. 1959 Amino acid metabolism in mamBiochim. Biophys. Acta, 344: 263-305. malian cell cultures. Science, 130: 432-438. Sekiguchi, T., and F. Sekiguchi 1973 Interallelic Fenwick, R. G., G. Kruh and C. T. Caskey 1975 Alcomplementation in hybrid cells derived from tered properties of hypoxanthine-guanine phosphoChinese hamster diploid clones deficient in hyporibosyltransferase from mutant Chinese hamster xanthine-guanine phosphoribosyltransferase accells. J. Cell Hiol., 67: 115a. tivity. Expl. Cell. Res., 77: 391-403. Gillin, F. D., D. J. Roufa, A. L. Beaudet and C. T. Stocco, D. M., P. C. Beers and A. H. Warner 1972 Caskey 1972 8-Azaguanine resistance in mamEffect of anoxia on nucleotide metabolism in enmalian cells. I. Hypoxanthine-guanine phosphorihocysted embryos of the brine shrimp. Dev. Hiol., 27: syltransferase. Genetics, 72: 239-252. 479-493. Harris, M. 1971 Mutation rates in cells at different Weiss, J. W., and H. C. Pitot 1974 Inhibition of riploidy levels. J. Cell Physiol., 78: 177-184. bosomal ribonucleic acid maturation by S-azacytidine and 8-azaguanine in Novikoff hepatoma cells. Harris, J. F., and G. F. Whitmore 1974 Chinese hamster cells exhihiting a temperature dependent Arch. Biochem. Biophys., 160: 119-129. LITERATURE CITED
ABSTRACT In this study the resistance of a numher of lines of Chinese hamster ovary cells to azaguanine is examined. Those which are drug resistant by virtue of a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) fail to take up any exogenous hypoxanthine or azaguanine. A second class of drug resistant cells which grow in the reverse selective HAT medium and have levels of HPRT in the range of the wild type parent line take up these purines at lower rates than the nonresistant cells and incorporate smaller amounts of them into trichloracetic acidinsoluble constituents. The results suggest that their basis for resistance resides in lowered incorporation of azaguanine into DNA and RNA, possibly due to a mofified HPRT molecule which accepts hypoxanthine, hut not azaguanine as a substrate.
8-Azaguanine (AG), an analogue of guanine, inhibits the growth of animal cells in part, at least, by interfering with the formation of GMP (Davidson, '72) and with normal maturation of RNA (Weiss and Pitot, '74).In general, cells which are resistant to the toxic effects of AG are thought to owe their immunity to the absence of functional hypoxanthine-guanine phosphoribosyltransferase (HPRT: EC 2.4.2.8). This enzyme catalyzes the synthesis of azaguanylic acid, derivatives of which are responsible for the inhibitory effects. Because HPRT-deficient cells are unable to form the aberrant nucleotides they are able to thrive in the presence of AG. Several recent reports suggest that the HPRT deficiencies of some AG-resistant cell lines are due to mutations within the structural gene for that enzyme. Beaudet, et al. ('73) showed that deficient Chinese hamster cells fall into two general categories: those which produce a product which cross reacts with antibody to HPRT, and those which do not. From either class of mutants revertants to HPRT-positive forms can be obtained. Sekiguchi and Sekiguchi ('73) reported that allelic complementaJ. CELL. PHYSIOL.,89: 209-218
tion, accomplished by fusing cells of two different HPRT-deficient hamster lines, generates HPRT-positive synkaryons. Bakay and Nyhan ('72) showed that nonfunctional HPRT from Lesch-Nyhan patients becomes functional if it is mixed with the normal human enzyme and then separated from the mixture by electrophoresis. They ascribe the activation to the transfer of a polypeptide from the active to the inactive catalytic partner. The human enzyme was reported by Arnold and Kelley ('71) to be composed of two identical subunits of 34,000 daltons and the same net charge and to resolve into three electrophoretically distinguishable isozymes. Hughes et al. ('75)provide evidence to indicate that the enzyme is a trimer of 27,000 dalton subunits, the same as mouse HPRT. Olson and Milman ('74) have shown that Chinese hamster HPRT exists in at least three isozymic forms and that the enzymes isolated from the hamster brain, liver and V79 tissue culture cells appear to be structurally and catalytically identical. InconsisReceived April 10, '75. Accepted Feh. 6, '76. Reprint requests should be directed to Dr. John Morrow.
209
210
M. PARSONS, J. MORROW, D. STOCCO AND P. KITOS
tent with the concept that a structural gene defect is responsible for AG-resistance, is the report of Harris ('71) in which he noted that ploidy levels do not affect the mutation rate to AG-resistance. Azaguanine-resistance has been used as a phenotypic marker in assessing mutation rates (Morrow, '701, identifying and selecting interspecific cell hybrids (Littlefield, '64) and characterizing the Lesch-Nyhan condition in vitro (Albertini and DeMars, '73). In their studies of AG-resistant hamster cells, Morrow and his colleagues ('73) noted a not-infrequent incidence of clones which exhibit high levels of HPRT. Similar results have also been obtained by other investigators (Harris and Whitmore, '74; Gillin, et al., '72). Unlike their HPRTdeficient counterparts these mutants do grow in a medium supplemented with HAT 5 ; the exogenous hypoxanthine satisfied all their purine requirements. In the present report we examine both HPRT-sufficient and -deficient azaguanine-resistant Chinese hamster cells. The results depict principal differences between these two classes of mutants and implicate reduced incorporation of azaguanine as the basis of resistance. MATERIALS AND METHODS
Cell culture For these studies a proline-requiring line of Chinese hamster ovary cells, CHO/pro(Kao and Puck, '671, kindly provided by Dr. M. W. Taylor of the Department of Microbiology, Indiana University, was used. Sublines resistant to AG were isolated by plating them in a medium which contained 30 pg per ml of AG. Within two weeks most cells died and clones of viable cells arose. Several clones were isolated and the remainder were incubated in "HAT" medium. In HAT approximately 85% of the AG-resistant clones died, confirming that they were deficient in HPRT. Several HAT-resistant clones were then isolated (Morrow et al., '73). These clones were grown for extended periods in the absence of azaguanine and retested frequently, during which time they retained their phe-
notype, in some cases for up to two years. All clones which were HAT-resistant had levels of HPRT in 1 he range of that found in the CHO/pro- parent. Cells were cultivated in Falcon plastic tissue culture vessels or 250 ml milk dilution bottles. Eagle's autoclavable minimal medium (Flow Laboratories, Rockville, Maryland) Supplemented with 5% calf serum (Colorado Serum Company, Denver, Colorado), non-essential amino acids (Eagle, '591, antibiotics and glutamine was used for cloning as previously described (Morrow et al., '73) For most other studies medium MD705/1 (Kitos, Sinclair and Waymouth, '62) plus 5% calf serum (Grand Island Biological Company, Grand Island, New York) was used. The cells were transferred regularly by trypsinization or mechanical scraping (2nd between transfers the medium was changed every three days.
Hypoxanthins and azaguanine uptake studies The cells were plated in complete medium and allowed to attach overnight. Depending on the cloning efficiency of the subline, densities of 106to 2 x 106 cells per milk dilution bottle were used (30cells per mm2).The medium was then decanted and fresh medium containing either 5-PHluridine (ICN Corp., 0.833 pCi per ml, 3.33 x pM) and 8-ClClhypoxanthine (New England Nuclear Corp., 0.167 pCi per ml, 16.5 pM) or 5-C3H[luridine and 2-C14C1azaguanine (ICN Corp., 0.167 pCi per ml, 16.5 pM) was added. For certain experiments, unlabeled guanine (33. pM) was added. The cells were incubated in the labeled medium foir three hours, at which time the medium vvas decanted. The cultures were then washed with approximately 200 ml of flowing isotonic saline by directing the flow at the inner end of the inverted milk-dilution bottle. The saline flowed out of the bottle, passing over the cell monolayer, and collected in a large receptacle. The excess saline was drained from the culture vessels, and 4 ml of 0.5 5 5 pg per ml thymidints and hypoxanthine, 0 1 pg ml ammopterm
PUHINE UPTAKE IN CHO CELLS
per cent sodium dodecylsulfate (SDS) was added. The cells were disrupted by the SDS and then 1 ml of 50 per cent (W/VI cold trichloroacetic acid (TCA)was added to precipitate the acid insoluble material. A representative 2 ml portion of the resulting suspension was filtered through a 25 mm diameter Millipore membrane (type HA, 0.45 p pore size) to separate the soluble constituents from the insoluble. A 200 p1 portion of the soluble fraction was spotted on a 25 mm diameter GF/A Whatman glass-fiber filter disc and the membranes and glass-fiber discs were dried under a heat lamp. They were placed in plastic minivials containing 5 ml of scintillation cocktail (4 g Omnifluor [New England Nuclear Corp.1 per liter of toluene) and the radioactivity was determined by scintillation spectrometry. Pilot studies indicated that incorporation of hypoxanthine into the cells proceeded linearly over a three hour period.
Amino acid uptake studies The procedure used for amino acid uptake studies is the same as that described above except that the medium contained a mixture of [I4C7-labeled L amino acids (New England Nuclear Corp., 0.1 pCi per ml, 1.34 pg per ml) and the incubation period was two hours.
211
mCi/mmole,4 mCi/mgm, Amersham/Searle Corp., Arlington Heights, Illinois) or [8-'4C1 -hypoxanthine (48.6 mCi/mMole, New England Nuclear, Boston, Massachusetts) were added to the cells at a concentration of 10 pCi/ml for the 3H-isotope and 0.2 pCi/ml for the 14C-radiolabel. Following ten minutes of incubation in the presence of radioisotope the cells were rapidly collected by centrifugation in a clinical centrifuge and washed by resuspending the pelleted cells in Hanks solution. After a second centrifugation the cells were resuspended in N perchloric acid and quantitatively transferred to a Dounce glass homogenizer and thoroughly homogenized. Acid insoluble material was pelleted by centrifugation at 12,000 x g for ten minutes. The acid soluble supernatant was carefully removed and neutralized with an equal volume of Alamine in chloroform according to Stocco et al. ('72). All operations were performed at 4°C. The neutralized acid soluble fraction was then fractionated by means of anion exchange chromatography on columns of DEAE-cellulose (DE 23 Whatman Co.). Marker hypoxanthine (50 p1 of a 1 mgmlml solution) was added to the samples in order to determine the position of free hypoxanthine in the elution profile. Nucleotides were then eluted with a linear gradient of NbHCO, (0.002 to 0.250 M, pH 8.6).Fractions were collected in 10 ml aliquots at a rate of approximately 1ml/min. Each fraction was assayed for radioactivity by placing 0.5 ml in polyethylene scintillation minivials and dispersing in scintillation fluid consisting of 75% toluene-25% triton X-114 and 4.0 gm/litre Omnifluor (New England Nuclear, Boston, Massachusetts). Samples were then counted in a Beckman LS 230 liquid scintillation counter. In addition each fraction was analyzed at 260 nm to determine the position of the hypoxanthine marker.
Determination of free hypoxanthine in the cells For these experiments both CHO/procells and V79 cells (a Chinese hamster cell line derived from the lung) were used. Cells were grown in milk dilution bottles in F-10 media plus 5% calf serum (Kansas City Biological) and harvested with Viocase (GIBCO). Detached cells were washed and resuspended in Hanks balanced salt solution. Aliquots of cells were counted with an electronic particle counter (Particle Data Inc., Elmwood, 11linois). The remainder of the cells were RESULTS transferred to 50 ml erlenmeyer flasks and Purine uptake by CHO cells placed in a water bath at 37°C and shaken As shown in tables 1 and 2, Chinese gently throughout the course of the experiment. Either 3H-(G)-hypoxanthine (570 hamster cells which are resistant to
212
hl PARSONS. J. MORROW. 1). STOCCO AND P. KITOS
TABLE 1
Hypoxanthine and uridine uptake by azuguanine-sensitive and resistant lines of Chinese hamster cells 3H iiridine uptake
"C hypoxanthine uptake Cell line
Total % of control
~
TCA insolulde % of total
Total % of control
TCA insoluble % of total
~
100 (14,361 cpm) 5 4 7 9 62 44 30
31
CHO/proCHO/2 CHO/E CHO/F CH0/23 CH0/28 CH0/29 CHO/M
-
-
16 54 29 17
44
30 13 20 30 34 45 42
The parental cell line, CHO/pro-, is uaguanine sensitive. All other lines are azagrianine-resistant derivatives of it. Of them, lines CHO/&.CHO/E and CHO/F are HAT-sensitiveand lines CH0123, CHO/28, CHOP29 and CHO/M are HAT-resistant. I The number of separate experiments from which these data are calculated is provided in parentheses along with the uptake data. TABLE 2
Azuguanine and uridine uptake by azaguanine-sensitive and resistant lines of Chinese hamster cells '4C hypoxanthine uptake
Cell line
Total % of control
CHO/proCH0/2 CHO/E CHO/F CH0/23 CH0/28 CH0/29 CHO/M
100 (4) I (1,969cpm) 0 (4) 0 (1) 3 (1) 30 (4) 8 (4) 12 (2) 20 (2)
uridine uptake
TCA insoluble 96 of total
27
0 18 18 13 7
Total % of control
100 (1,484cpm) 40 30 53 54 29 19 46
TCA insolulde % of total
30 32 27 25 29 35 33 11
Cell line descriptions are given in ttlble 1. I The number of separate experiments from which these data are calculated is provided in parentheses along with the uptake data.
azaguanine can be divided into two general classes: those which grow in HAT medium (lines CH0/23, CH0/28, CH0/29 and CHON) take up less hypoxanthine and azaguanine than t h e parental line, CHO/pro-; those which do not grow in HAT medium (lines CH0/2, CHO/E, and CHO/F) take up virtually none of these bases. These findings are similar to those reported by Benke, Herrich and Hebert ('73a,b) on fibroblasts of Lesch-Nyhan and hyperuricemic patients. The results show that in the CHO cells the extent of
azaguanine uptake i,s much less than that of hypoxanthine. To facilitate comparisons of the data the specific activities of the radioactive hypoxanthine and azaguanine w e r e prestandartlized and identical amounts of each were used. Comparing tables 1 and 2, it can be seen that the CHO/pro- cells incorporate about 30 times as much hypoxanthine as azaguanine and that even the most efficient of the azaguanine resistant cell lines (CH0/23) incorpor ates less than one third as much azaguanine as the control culture. During the 3-hour
213
PURINE UPTAKE IN CliO CELLS
period of incubation less than half of the ingested 1% azaguanine or hypoxanthine is incorporated into TCA-insoluble material of the cells. The bulk of the intracellular radioactive material still exists as acidsoluble constituents (either the free base or non-polymeric nucleotide derivatives of it). In each of these studies 3H-labeled uridine was included in the medium as an internal control. If pyrimidine nucleoside uptake is functionally distinct from that of the purine, its pattern of incorporation should be independent of variations in purine uptake. As can be seen in table 1, there appears to be a correlation between the uptake of uridine and hypoxanthine. The correlation is less evident between uridine and azaguanine (table 2). Since the purine concentration of the medium is about 500 times that of the uridine, the significance of the observation is moot. Recent work in our laboratory indicates that the decreased uridine uptake in the mutant lines may be an effect induced by the hypoxanthine present in the medium. When the mutant cells are incubated in medium containing no hypoxanthine, their uridine uptake is not depressed relative to that of the wild type and in some cases it is elevated slightly. In all cell lines azaguanine severely depresses uridine uptake (compare tables 1 and 2: decrease from 14,361 to 1,484 cpm).
The effects of guanine on hypoxanthine and azaguanine uptake The utilization of hypoxanthine and guanine in animal cells is known to occur via the action of HPRT. Consequently, excess guanine should compete with hypoxanthine in its conversion to IMP. The azaguanine-sensitive cell line (CHO/pro-1 and representative azaguanine-resistant, HATsensitive (CH0/2) and HAT-insensitive (CH0/23, CH0/28 and CHO/M) cell lines were examined to determine the effects of excess (33 pM) guanine on the uptake of either 16.5 pM hypoxanthine or azaguanine. Guanine essentially eliminates the uptake of azaguanine (table 4) and it lowers the uptake of hypoxanthine (table 3). The CHO/pro- line takes up less than one fifth as much hypoxanthine in the presence of guanine as in its absence. Thus it seems that these bases are modified to form phosphorylated derivatives by a common mobilizing enzyme. The addition of guanine to hypoxanthine-containing rnedium does not depress uridine uptake (compare tables 1 and 3) but its addition to azaguanine-containing medium does (compare tables 2 and 4). Amino acid uptake by CHO cells The azaguanine-sensitive and -resistant lines were also tested for their abilities to take up a mixture of 14C-labeled L amino acids and incorporate them into TCA-in-
TAHLE 3
Hypoxanthine and uridine uptake by azaguanine-sensitiue and resistant Chinese hamster cells in the presence of guanine IJC hypoxanthine uptake
Cell line
Total % of control
CHOiproCH0/2 CH0/23 CH0/28 CHO/M ~
~
100 (2) I 110,530 cpm) 2 (2) 3 (21 44 (2) 47 (1)
TCA insolnhle % of total
18 0 0 93 6
'H uridine nptake Total % of control
100 114,480 cpm) 12 13 46 47
TCA mwlul~le % of total
21 23 11 80 8
~~
Information concerning cell lines is provided in table 1 . 1 The numl)er of separate experiments from which these data are calculated is provided in parentheses along with the uptake data.
214
M . PARSONS,
I.
MORROW, D. STOCCO AND P. KITOS
TABLE 4
Azaguanine and uridine uptake by azaguanine-sensitioe and resistant line9 of Chinese hamster cells in the presence of guanine 3H uridine uptake
"C hypoxanthine uptake Cell line
Total % of control
CHO/proCHO/2 CH0/23 CH0/28 CHO/M
TCA insoluble % of total
100 (2) ' (70 cpm) -(2) -(2) -(2) -(1)
Total % of control
-
100 (519cpm) 85 93 100 100
-
TCA insoluble % of total
40
30 30 34 30
Information on the cell lines is provided in table 1. ' The number of separate experiments from which these data are calculated is provided in parentheses along with the uptake data.
soluble cell constituents. Some variations in uptake can be seen (table 5) but they do not reflect the differences in purine transport which are evident in tables 1and 2. In the two hour labeling period used here slightly more than half of the incorporated radioactivity was found in the TCA-insoluble fraction of all cell lines. These observed differences in rates of amino acid transport and protein synthesis do not correlate with the differences in uptake and incorporation of hypoxanthine and azaguanine reported in tables 1 and 2. Thus deviations in the rates of purine uptake are not due to grossly dissimilar rates of metabolism among the mutant cell lines.
The metabolism of endogenous hypoxanthine In order to determine the intracellular conversion of hypoxanthine to IMP and other nucleotide derivatives, both CHO/pro- and V79 cells were incubated in the presence of 3H-hypoxanthine or 14Chypoxanthine. The results indicate that the amount of free hypoxanthine in the intracellular pool after 10 minutes is quite small. In figure 1is shown a representative profile of the nucleotide derivatives obtained following a 10 minute incubation of CHO/pro- cells in 14C-hypoxanthine.In all experiments employing CHO/pro- or V79 cells and radiolabeling with either I4Chypoxanthine or 3H-hypoxanthine, the amount of free hypoxanthine was found to
TABLE 5
Relative amino acid uptake and protein synthesis capabilities of the Chinese hamster mutant cell lines Cell line
CHO/ProCH0/2 CHO/E CHO/F CH0/23 CH0/28 CHO/M
(:< 1 0 3 )
Percent CPM found in TCA insoluble form
4.38 2.35 3.80 3.68 !!.70 3.80 4.90
58 55 62 58 57 55 61
CF'M/cell
be approximately 5% of the total radioactivity recovered. These data indicate that the intracellular conversion of hypoxanthine to its various nucleotide derivatives is both rapid and complete in the cells studied.
Axaguanine resistance in the presence of aminopterin A possible explanation for the existence of the doubly resistant lines would be an overly active system for the endogenous synthesis of purines. This could result in lowered purine u.ptake and thus azaguanine resistance. As HPRT would remain intact, the cells would be HAT resistant. Since aminopterin inhibits de nouo purine biosynthesis, incubation of such cells in aminopterin should force purine uptake and render the cells more sensitive
215
PURINE UPTAKE IN CHO CELLS
20 -
I
0-0
0-0
71.9%
. h
I
CPM O D 260 E = 0
a N c3 0
:.0
1.5
10
20
30
40
50
FRACTION NUMBER
Fig. 1 Acid soluble hypoxanthine derivatives in CHO/Pro- cells. Cells were incubated in the presence of '4C-hypoxanthine for 10 min and the acid soluble pool was extracted, neutralized and fractionated on DEAE cellulose columns. The numbers over the peaks represent the percentage of CPM present in each derivative of hypoxanthine
to azaguanine. In order to determine the extent to which aminopterin influences the growth of both azaguanine-sensitive cells (CHO/pro-) and doubly-resistant cells (i.e., resistant to both azaguanine and HAT) the cultures were exposed to varying concentrations of azaguanine in HAT medium or in HT medium (medium containing hypoxanthine and thymidine but lacking aminopterin). 105 cells were plated in 5 ml of appropriate medium in 60 mm Falcon plastic dishes. After five days the cells were harvested with two ml of 0.25 per cent trypsin and counted. As can be seen in figure 2, added aminopterin has no effect on the resistance of the cells of azaguanine. Evidently the toxicity due to azaguanine is neither augmented nor suppressed by aminopterin.
of azaguanine and hypoxanthine, but only sometimes by a complete lack of HPRT activity. Cells which are totally deficient in HPRT (azaguanine-resistant, HAT-sensitive) are consistently unable to ingest hypoxanthine or azaguanine while those which are not deficient in the enzyme (azaguanine-resistant, HAT-resistant1 re tain some ability to incorporate the two bases. The results of the amino-acid uptake studies indicate that the lowered uptake of purines is not due to a general depression of growth rate, but is rather specific for hypoxanthine and related molecules. In the HAT-resistant lines the slow purine uptake is not due to an endogenous over-production of purines because the profiles of resistance to azaguanine in the doublyresistant mutants are not altered by the presence of aminopterin (fig. 21. DISCUSSION Other authors have reported azaguanine Azaguanine resistance of CHO cells is al- resistant cell lines which are not deficient ways accompanied by lower incorporation in HPRT. Harris and Whitmore ('73) ob-
2 16
hl PARSONS, J . MORROW, I). STOCCO AND P. KlTOS
40
20
40
60
80
100
Fig. 2 Effect of aminopterin on inhibition of cell growth. Hypoxanthine and thyinidine were supplied to counteract the effect of aminopterin on cell growth. Ordinate: growth as percent of control: ahscissaazagrianine concentration pg per ml. Dotted line: minus aminopterin; solid line: plus aminopterin. On day zero each culture dish was seeded with 105 cells. The plating efficiency of the three cell lines was approximately 50%.Five days later the cells were harvested and counted. Growth is reported as per cent of the control cell population. The number of cell5 in the CHOiPro- and CHOi28 control cultures increased al)out %-fold during this period. The nrimher in CH0/23 increased approximately 8-fold in HAT and 12-fold in HT.
served a temperature sensitive mutation in cells which were resistant to azaguanine, but which seemed to have unaltered HPRT activity. Recently, Fenwick, et al. ('751have studied a class of mutants similar to those described here, in which the basis of resistance lies in alterations in the molecule such that it no longer accepts azaguanine as a substrate, but is still active with hypoxanthine. They suggest that this condition results from a missense mutation in the structural gene for the enzyme. Thus, our results can be conceived as resulting from genetic modifications in the HPRT molecule which are reflected in the lowered incorporation of azaguanine and related purines, while still allowing adequate hypoxanthine incorporation to fulfill the needs of the cells when cultivated in the presence of aminopterin. Although our results do not allow one to judge the role of HPRT in transport of purines, Plagemann and Richey ('74) have argued that phosphorylation is a process separate from
transport in mammalian cells. More recently, Alford and Barnes (personal communication) have shown that HPRT mutants of Chinese hamster lung fibroblasts still maintain facilitated diffusion in the complete absence of the enzyme. Thus, they conclude that the transport and phosphorylation of hypoxanthine are separate and distinct process'es in these cells. Since the pool of free hypoxanthine is quite small in the cells studied (fig. 1) and conversion to phosphorylated derivatives is extremely rapid, this might lead to the erroneous conclusion that the two processes are brought about by the same enzyme. ACKNOWLEDGMENTS
This work was supported in part by a grant from the University of Kansas General Research Fund, Grant Number CA-12310-03 from The National Cancer Institute and Grant Number DRG 1088 from the Damon Runyon Fund.
PURINE UPTAKE IN CHO CELLS
217
alteration in purine transport. J. Cell Physiol., 83: 43-51, Albertini, R. J,, and R. DeMars 1973 Somatic cell Hughes, S. H., G. M. Wahl and M. R. Capecchi 1975 Purification and characterization of mouse hypomutation: detection and quantitation of x-ray inxanthine-guanine phosphoribosyltransferase. J. duced mutation in cultured, diploid human Biol. Chem., 250: 120-126. fibroblasts. Mut. Res., 18: 199-224. Arnold, W. J., and W. N. Kelley 1971 Human hy- Kao, F. T., and T. T. Puck 1967 Genetics of somatic mammalian cells. IV. Properties of poxanthine-guanine phosphoril)osyltransferase. Chinese hamster cell mutants with respect to the Purification and subunit structure. J. Biol. Chem., requirement for proline. Genetics, 55: 5 13-524. 246: 7398-7404. Bakay, B., and W. L. Nyhan 1972 Activation of Kitos, P. A., R. Sinclair and C. Waymouth 1962 Glutamine metabolism by animal cells growing in a variants of hypoxanthine-guanine phosphosynthetic medium. Expl. Cell. Res., 27: 307-316. ribosyltransferase hy the normal enzyme. Proc. Nat. Littlefield, J. W. 1964 Selection of hybrids from Acad. Sci. (U.S.A.) 69: 2523-2526. Heaudet, A. L., D. J. Rouh and C. T. Caskey 1973 matings of fibro-blasts in rjitro and their presumed recombinants. Science, 145: 709-710. Mutations affecting the structure of hypoxanthine: guanine phosphorihosyltransferase in cultured Morrow, J. 1970 Genetic analysis of azaguanine reChinese hamster cells. Proc. Nat. Sci. (U.S.A.), 70: sistance in an established mouse cell line. Genetics, 65: 279-287. 320-324. Benke, P. J.. N. Herrick and A. Hebert 1973a Hypo- Morrow, J., J. Colofiore and D. Rintoul 1973 Azaguanine resistant hamster cell lines not defixanthine-guanine phosphoribosyltransferase cient in hypoxanthine-guanine phosphoribosylvariant associated with accelerated purine syntransferase. J. Cell. Physiol., 81; 97-100. thesis. J. Clin. Invest. 52: 2234-2240. Henke, P. J., N. Herrick and A. Hebert 197311 Trans- Olson, A. S., and G. Milman 1974 Chinese hamster port of hypoxanthine in fibroblasts with a normal HPRT: purification, structural and catalytic properand mutant hypoxanthine-guanine phosphoties. J. Biol. Chem., 249: 4030-4037. ribosyltransferase. Hiochem. Med., 8: 309-323. P l a g e m a n n , P. G. W., a n d D. P . Richey Davidson, J. N. 1972 The Biochemistry of Nucleic 1974 Transport of nucleosides, nucleic acid bases, Acids. Academic Press, New York, pp. 274. choline and glucose by animal cells in culture. Eagle, H. 1959 Amino acid metabolism in mamBiochim. Biophys. Acta, 344: 263-305. malian cell cultures. Science, 130: 432-438. Sekiguchi, T., and F. Sekiguchi 1973 Interallelic Fenwick, R. G., G. Kruh and C. T. Caskey 1975 Alcomplementation in hybrid cells derived from tered properties of hypoxanthine-guanine phosphoChinese hamster diploid clones deficient in hyporibosyltransferase from mutant Chinese hamster xanthine-guanine phosphoribosyltransferase accells. J. Cell Hiol., 67: 115a. tivity. Expl. Cell. Res., 77: 391-403. Gillin, F. D., D. J. Roufa, A. L. Beaudet and C. T. Stocco, D. M., P. C. Beers and A. H. Warner 1972 Caskey 1972 8-Azaguanine resistance in mamEffect of anoxia on nucleotide metabolism in enmalian cells. I. Hypoxanthine-guanine phosphorihocysted embryos of the brine shrimp. Dev. Hiol., 27: syltransferase. Genetics, 72: 239-252. 479-493. Harris, M. 1971 Mutation rates in cells at different Weiss, J. W., and H. C. Pitot 1974 Inhibition of riploidy levels. J. Cell Physiol., 78: 177-184. bosomal ribonucleic acid maturation by S-azacytidine and 8-azaguanine in Novikoff hepatoma cells. Harris, J. F., and G. F. Whitmore 1974 Chinese hamster cells exhihiting a temperature dependent Arch. Biochem. Biophys., 160: 119-129. LITERATURE CITED
