Pyrimidine Nuc leoside, Pseudouridine, and Modified Nucleoside Excretion by Growing and Resting Fibroblasts M A Y 0 UZIEL AND J. K. SELKIRK Biology Division, O a k Ridge National Laboratory, Oak Ridge, Tennessee 37830

ABSTRACT We are examining the relationship of RNA metabolism and de novo pyrimidine synthesis as parameters of malignant transformation. These initial experiments on normal hamster embryo fibroblasts have shown that excreted nucleosides are markers for intracellular RNA metabolism. We employed affinity chromatography to concentrate the nucleosides in the medium and sensitive column chromatographic procedures to quantitatively measure them. The excretion of pyrimidine nucleoside from hamster embryo fibroblasts in culture was found to be dependent on the growth stage of the cells, with the greatest accumulation occurring during cell quiescence. The major nucleoside excretion products, uridine and cytidine, were both normal end products of RNA metabolism and the major nucleoside excretion products from cultured cells. The modified nucleosides N-1-methylguanosine, N-2-methylguanosine, N2-dimethylguanosine, N-4-acetylcytidine, N-1-methylinosine, pseudouridine, N1-methyladenosine, N-3-methylcytidine, and 5-methylcytidine were found, as were several unidentified nucleosides. The relationships of RNA or RNA precursor content of cells to growth are a complex series of biochemical events that begins with precursor transport across membrane, polymerization in the nucleus, processing in the nucleoplasm and cytosol, and ends with hydrolysis regenerating the precursors plus other end products. The movement of nucleosides from the cell into the surrounding medium is inextricably linked to these biochemical processes. We are interested in using these excreted nucleosides as markers of RNA metabolism during malignant transformation. The known excreted nucleosides found in urine include the ordinary nucleosides in trace amounts, the modified nucleosides derived from RNA, and a variety of intermediates derived from de novo synthesis or enzymatic cofactors (review by Chheda, '71; Adler and Guttman, '59; Uziel and Taylor, '78). The regulation of the excretion of these compounds is not understood in the intact animal, although there is evidence t h a t they are excreted in increased quantities by patients and animals with proliferative diseases (Waalkes et al., '75; Uziel and Taylor, '78). Utilizing recent techniques for rapid separaJ. CELL. PHYSIOL. (1979) 99: 217-222

tion of nucleosides from tissue culture medium (Uziel e t al., '76) and coupling these to high performance liquid chromatographic systems, we have been able t o identify a number of excreted nucleosides (Uziel and &Ikirk, '78). We have begun studies of excretion in Syrian hamster embryo fibroblasts in culture, as these cells are widely used in studying carcinogenesis and they have been well characterized with respect to ribosome content (Becker et al., '71) and macromolecular metabolism (Stanners and Becker, '71). MATERIALS A N D METHODS

Primary hamster embryonic fibroblasts were prepared from whole Syrian golden hamster embryos and grown in Dulbecco's minimum essential medium supplemented with 0.45%glucose and 10%heat-inactivated fetal Received Aug. 29, '78. Accepted Dec. 12, '78. ' Research sponsored by the Division of Biomedical and Environmental Research, U S . Department of Energy, under contract W-7405-eng-26with the Union Carbide Corporation. By acceptance of this article, the publisher or recipient acknowb edge8 the right of the US. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. Direct inquiries to this author.

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MAY0 UZIEL AND J. K. SELKIRK

calf serum (Selkirk et al., '76). All experiments were performed on the third subculture grown a t 37°C in 8.5% GO, a t 98% relative humidity. Tertiary cultures were seeded a t 2 X lo6 cells per 100-mm plastic dishes (Corning) containing 9 ml of medium. Each dish was pulsed with 10 pM radioactive Urd in fresh medium for six hours beginning a t seven hours after seeding, and the medium was changed a t 2.5to 3-day intervals. No nonradioactive Urd was added after the pulse, but the cells were rinsed twice with fresh medium to dilute residual radioactive label before they were returned to the incubator. [2- OCIUrd, [6-3H1Urd, and L3H-CH31Met were purchased from Schwartz/Mann or New England Nuclear and had specific activities of 58 mCiimmole, 20 Ciimmole, or 200 mCil mmole, respectively. Samples were counted in a Beckman LS250 scintillation counter with Aquasol (New England Nuclear) as the counting medium. Nucleosides were isolated by affinity concentration of 3-ml samples of culture fluid on 2-ml columns of aminophenylboronate a t tached to polyacrylhydrazylsuccinate (Uziel et al., '76). Separations were done on octadecylsiliconate reversed-phase columns (Waters Associates C18 pbondapack) with solvent modifications of the procedures of Hartwick and Brown ('76). After sample injection and

passage of 10 ml of 2% acetonitrile in 0.02 M NH, phosphate buffer (pH 5.61, the solvent was changed to 5% acetonitrile in the same buffer for 26.3 ml and the column finally washed with 13.3 ml of 20%acetonitrile in the same buffer. Nucleosides were purchased from Pierce Chemical Co., Cyclo Chemical Co., and Sigma Chemical Co. RESULTS AND DISCUSSION

We have identified 11nucleosides in the medium from the hamster fibroblasts both during exponential growth and a t confluence (figs. 1, 2). The medium was treated with an immobilized boronate affinity gel (Uziel et al., '76) to recover ribosyl nucleosides with a free diol. Figure 1 illustrates the labeling pattern from medium with exponentially growing cells. The major radioactive products are q r d , Cyd, and Urd; there is some label in the 5MeCyd position. Figure 2 illustrates the nucleosides present in medium from confluent cells after the culture was pulse-labeled a t early log with both [2-I4C1Urd and L3H-CH31Met. Radioactivity was found in positions corresponding to those of authentic Trd, Cyd, 3MeCyd, Urd, 1-Me-Ado, 5-MeCyd, 1-MeIno, 1MeGuo, 4-AcCyd, 2-MeGuo, and 2-diMeGuo. The absorbancy ratios (280 nm/260 nm) of 'Prd, Urd, and Cyd were identical to those of authentic nucleosides. Three compounds contained both radioactive labels. The compound

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Fig. 1 The nucleosides from 1.5 ml of medium were separated and concentrated as described in MATE^ RIALS AND METHODS and analyzed as described in figure 3. The analysis in the figure was taken from medium obtained at 46 hours after seeding. Urd and other nucleosides were collected together from the borate gel before concentration. The major radioactive peaks eluted in positions corresponding to 4'. C, U, and mJC (in that order) with retention times of 6.4, 7.6, 10.0, and 13.2 minutes, respectively. The flow rate was 0.7 mllmin.

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NUCLEOSIDE EXCRETION BY GROWING OR RESTING CELLS

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FRACTION NO. Fig. 2 Methyl modified nucleosides excreted from hamster embryo fibroblasts. Cells were pulsed with 10 p M [2-'CIUrd (58 pCi/pmole) and 200 pMPH-CHJ methionine (5 pCi/pmole) as described in MATERIALS AND METHODS. After eight days in culture, the medium from each of the medium changes was pooled and 1.2

ml was processed to recover the nucleosides (Uziel et al., '76), and all of the sample was applied to the octadecylsiliconate column. Fractions were collected (0.2 ml per fraction) a t a flow rate of 0.67 ml/min; solvents were the same a s described in figure 3. There were eight 13H-CHJ-labeled peaks with retention times of 9.5 ( m V , 12 (mlA), 14 GnsC), 31 (mil), 34 (m'G), 36 (m@), and 47 (m$G) minutes. An additional peak a t 22 minutes (NJ is as yet unidentified. Five ['CIUrd regions were identified a t 6.7 ('W, 8 (C), 10.5 (U), 14 (msC), and 35 (ac'C) minutes. The remainder have not been identified. The flow rate was 0.67 ml/min. The counts are plotted with two scales hnax = 5,000, 500). The beginning of the 500 scales are indicated by vertical arrows. The labelled compounds were present in the 1.2 ml of medium in the following amounts (nmol): Yrd (2.5), Cyd (l.l), m v y d (0.0261, Urd (2.41, mlAdo (0.039), msCyd (0.0751, N (0.033), mIIno (0.005), a c C (0.009), m'Guo (0.032); m G u o (0.015) and m p u o (0.048). The amounts of the methyl labelled compounds were calculated from the total DPM and the original specific activity of the methyl group (5 pCi/pmole).

corresponding to 5-MeCyd was collected with carrier 5-MeCyd, and the mixture was rechromatographed on a cation exchange resin (Uziel et al., '68). The radioactive peak corresponding to the ultraviolet absorbancy eluted with the same curve shape and retention time (not shown). A similar experiment with the peak seen at 22 minutes (position of 5-MeUrd) did not show a correspondencebetween carrier and labeled compound, and the ratio of 3H to 'F was also different from that in 5-MeCyd (fig. 2). The remaining compounds are tentatively identified by their retention times and radioactivity. Chromatography of the reference authentic compounds on the reverse-phase column is illustrated in figure 3. To ensure that the excreted Urd was not an aberration of the added radioactive Urd, the cells were grown without the pulse of Urd. Figure 4 shows the accumulation of Yrd, Urd, and Cyd to concentrations

of 0.61, 2.2, and 2.7 pM, respectively, after growth corresponding to the 92-hour time point in figure 5. Thus the excreted nucleosides are derived from endogenous Urd. The time course of appearance of the modified nucleoside Yrd and the nucleosides Urd and Cyd is illustrated in figure 5. Since the medium was changed at 24 hours to remove the excess label, the initial concentration of these compounds is dependent on their concentration in the added serum. The serum used in these experiments contained 1pM Urd and less than 1 p M Yrd and no Cyd. The medium contains 10%FCS, so the added Urd and Yrd amounts to 0.1 pM or less, which is not a significant quantity in these experiments. Yrd continuously accumulates in the medium with a n increasing rate during growth, followed by a reduced rate, to a final concentration of 1.4 pM as the cells become confluent. Uridine and cytidine on the other hand

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M A Y 0 UZIEL AND J. K. SELKIRK

TIME (mid Fig. 3 Chromatographic separation of nucleoside standards by reversed-phase chromatography. The fol' (6.651, C (8.051, m'Y: (9.71, U (10.61, lowing nucleosides were separated from each other in the order listed: P m'A (11.61, mY! (13.71, m'G (16.91, m5U (20.51, G (23.31, m l I (32.51, mlG (34.51, ac'C (35.3), m% (36.41, A (40.2), m c (47.3), m6A (50.8), and m!A (54.61. The numbers in parentheses are the retention times (minutes) on a Waters 300 mm x 4 mm C18 pbondapack column. The column was equilibrated with 2%acetonitrile in 0.02 M ammonium phosphate (pH 5.11. This solvent was pumped for 15 minutes before the acetonitrile concentration was increased to 5% for 40 minutes and then to 20%for completion of the analysis. The flow rate was 0.67 ml/min a t room temperature.

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Fig. 4 Chromatographic analysis of nucleoside excretion products in medium from confluent hamster embryo fibroblast cultures grown in the absence of added Urd. Cells were grown in Dulbecco's minimum essential medium supplemented with 10%fetal calf serum without l3HIUrd. The nucleosides were collected from 3 ml of medium three days after seeding. The nucleosides from 1.5 ml of culture fluid were analyzed by reversed-phase chromatography as shown in figure 2. This sample contained 0.92 nmole Y ,3.3 nmole C, and 4.1 nmole U. The concentration of each in t h e medium was 0.61, 2.2, and 2.7 p M , respectively.

reached a minimal concentration during exponential growth, probably due to uptake and utilization, followed by a rapid accumulation in the medium beginning at one-half maximal growth and continuing at confluence t o a concentration of 2.4p M at 168 hours.

The excreted Trd serves as an internal marker for total R N A hydrolysis due to either cell lysis or normal turnover. This is due to its presence only in R N A (it is synthesized only on intact RNA),the absence of enzymatic degradation of free Yrd and its inherent high

NUCLEOSIDE EXCRETION BY GROWING OR RESTING CELLS

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chemical stability. The course of q r d appear- events occur: the asynchronous progression of ance begins a t early log growth and continues the cells from G , to Goas the cell number conexponentially until quiescence where the rate tinues to increase and the loss of rRNA levels off to a maximum of 5% increase per 30 (Becker et al., '71). This pattern of excretion is not consistent hours (140-170 hours) (fig. 5 ) . Thus 5% is the upper limit for lysis during the 140- to 170- with a stationary state existence where new hour growth time. Urd excretion however is cells are formed to balance the loss of lysed not coordinated with 9 r d excretion. During cells and maintain the cell number while lysis early log growth (up to 0.5 maximal cell num- products accumulate. Although some lysis ber) about 30%of the excreted q r d appears in cannot be ruled out, the major excretion the medium, while less than 5% of the ex- changes occur during late log growth when creted Urd is in the culture fluid. During lysis would be minimal. Further, the rates of growth from 72 to 80 hours (50 to 70 of max- excretion decrease during quiescence when imal cell number) another 30%of the q r d ap- lysis is expected to be maximal. Thus, nucleopears in the medium, however, almost 50% of side excretion is temporally linked to the the excreted Urd also appears during this time physiological processes governing progression period. As the cells enter quiescence RNA from G, to Go. breakdown ends, as measured by q r d excreTABLE 1 tion, with only 5%of the q r d being added to the medium during the 140- to 170-hour Nucleoside excretion per cell ' growth period. During this time, however, 25% Hours 24 45 72 80 140 170 of the total Urd is accumulated in the medium. This noncoordinated pattern of excre'Prd 0.04 0.08 0.15 0.16 0.12 tion is also reflected in the excretion rate per 0.19 0.25 Urd 0.025 0.014 0.18 lo6 cells (table 1).The highest rate of excre- ' The concentration of nucleoside (pM)divided by the number of tion occurs between 72 and 80 hours, the time cells ( x 10-9. 2These data are taken from figure 5. period when several other physiological

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MAY0 UZIEL AND J. K. SELKIRK

As a n independent measure of stability of the nondividing culture, the cell viability, as measured by trypan blue dye exclusion, was constant (90 f 5% free of dye) from late log phase through nine days in culture, indicating again, the cell loss, if any, was marginal and the excretion patterns were not significantly contaminated by cell lysis. In addition, on subculture, these cells show 60-80% plating efficiency. We have observed similar nucleoside profiles from the culture fluid of mouse lymphocytes and embryonic fibroblasts, as well as rat liver epithelial cells and monkey kidney cells (Uziel, unpublished observations). The ubiquitous accumulation of pyrimidine nucleosides in culture medium again suggests they arise during normal intracellular metabolism. Many modified bases as well as their corresponding nucleosides are usually present in urine (see Chheda, '71 for review) suggesting that normal mechanisms exist for the cleavage of the ribosyl-base linkage. Since little is known about these conversions in vivo any of several mechanisms may be present. These include nucleoside phosphorylase, phosphoribosyltransferase or possibly a direct hydrolytic cleavage. Recently Trewyn and Kerr ('78) have shown that 10 pM N-7-methylguanine or N-l-methylguanine can induce transformation of Chinese hamster embryo fibroblasts in culture. And one of the cell lines caused tumor formation in nude mice. There was no concentration dependence data shown so it is not known what the threshold concentration of modified base is needed to induce the transformation. We have not yet directly measured the concentration of these bases i n our cultures. However, it will be of interest to measure the levels of free bases in cultures of cells t h a t undergo high rates of spontaneous transformation to see if the free bases are of sufficient concentration to help explain the spontaneous rate. The accumulation of Urd in the medium also poses the technical problem of correlating cpm from radioactive Urd added to the medium with the real concentration of Urd or its derivative. This is resolved only by measuring the specific activities of the tracer. We have observed decreases in the specific radioactivity of extracellular Urd from growing cells

(Uziel and Selkirk, '78) that parallel the observations of Kramer et al. ('73) on intracellular UTP specific radioactivities in growing cells. These parallel changes are expected if the extracellular and intracellular pools are in equilibrium. These observations indicate that many uridine incorporation experiments with intact cells may require reevaluation, especially those where the tracer is present a t less than 10 pM. LITERATURE CITED Adler, M., and A. B. Gutman 1959 Uridine isomer (5 ribosyl uracil) in human uridine. Science, 230: 860-863. Becker, H., P. Stanners and J. E. Kudlow 1971 Control of macromolecular synthesis in proliferating and resting Syrian hamster cells. 11. Ribosome content in resting and early G, cells. J. Cell. Physiol., 77: 43-48. Chheda, G. B. 1971 Puridine and pyrimidine derivatives excreted in human urine. In: Handbook of Biochemistry. Second ed. H. Sober, ed. The Chemical Rubber Co., Cleveland, Ohio, pp, G106-Gl13. Hartwick, R. A,, and P. R. Brown 1976 Evaluation of microparticle chemically bonded reversed phase packings in the high pressure liquid chromatographic analysis of nucleosides and their bases. J. Chromatogr., 226: 679-691. Kramer, G.,U. Weigers and H. Hilz 1973 mRNA turnover studies applying labelled uridine requires an evaluation of specific activities of UTP and RNA-U. Biochem. Biophys. Res. Commun., 55: 273-281. Selkirk, J. K., R. G. Croy, F. J. Weibel and H. V. Gelboin 1976 Differences in benzo(afpyrene metabolism between rodent microsomes and embryonic cells. Cancer Res., 36: 4476-4479. Stanners, C. D., and H. Becker 1971 Control of macromolecular synthesis in proliferating and resting Syrian hamster cells in monolayer culture. I. Ribosome function. 3. Cell. Physiol., 77: 31-40. 'Treswyn, R. W., and S. Kerr 1978 Altered growth properties of Chinese hamster embryo cells exposed to 1 methylguanine and 7 methylguanine. Cancer Res., 38: 2285-2289. Uziel, M., C. Koh and W. E. Cohn 1968 Rapid ion-exchange chromatographic microanalysis of ultraviolet absorbing materials and its application to nucleosides and bases. Anal. Biochem., 25: 77-84. Uziel, M., and J. K. Selkirk 1978 Nucleoside excretion in growing and resting cells. Fed. Proc., 37: 1390. Uziel, M., and L. H. Smith 1976 Modified nucleosides in normal and tumor tissue. Fed. Proc., 35: 1675. Uziel, M.,L.H. Smith and S. A. Taylor 1976 Modified nucleosides in urine: Selective removal and analysis. Clin. Chem., 22: 1451-1455. Uziel, M., and S. A. Taylor 1978 3-Methylcytidine: A normal constituent of human and mouse urine. J. Carbohydr. Nucleosides Nucleotides, 5: 235-249. Waalkes, T.P., C. Gehrke, R. Zumwalt, S. Y. Chang, D. Lakings, D. Tormey, D. L. Ahmann and C. G. Moertel 1975 The urinary excretion of nucleosides of RNA by patients with advanced cancer. Cancer, 36: 390-398.

Pyrimidine nucleoside, pseudouridine, and modified nucleoside excretion by growing and resting fibroblasts.

Pyrimidine Nuc leoside, Pseudouridine, and Modified Nucleoside Excretion by Growing and Resting Fibroblasts M A Y 0 UZIEL AND J. K. SELKIRK Biology Di...
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