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Studies of . I. The Pathway of Nucleic Ribose Synthesis in a Human Carcinoma in Culture

Howard H. Hiatt

J Clin Invest. 1957;36(10):1408-1415. https://doi.org/10.1172/JCI103540.

Research Article

Find the latest version: https://jci.me/103540/pdf STUDIES OF RII3OSE METABOLISM. I. THE PATHWAY OR RIBOSE SYNTHESIS IN A HUMAN CARCINOMA CELL IN TISSUE CULTURE' By HOWARD H. HIATT (From the Department of Medicine, ifeth Israel Hospital, and Harvard Medical School, Boston, Mass.) (Submitted for publication March 1, 1957; accepted June 11, 1957)

An impressive body of evidence is available day, together with additional radioactive substrate. The demonstrating the importance of the phos- period of growth varied from 7 to 14 days. The radio- phate pathway ( mnonophosphate shunt) in active compounds in the amounts employed had no dis- the cernible effect on cellular morphology or on rate of intermediary metabolism of (1). growth. At the termination of the growth period the cells The pathway is involved in a number of vital bio- were scraped from the wall of the bottle with a rubber logical processes, including (2), policeman into 5 ml. of cold 10 per cent trichloroacetic carbohydrate utilization by many (3), the acid (TCA). elaboration of the ribose moiety of nucleic in Isolation of ribose. The were isolated the chick (4), in certain by a modification (16) of the procedure of Schmidt and (5-8), Thannhauser (17). The harvested HeLa cells were and in the rat (9, 10), the synthesis of the pre- homogenized for two minutes in a Potter-Elvehjem teflon cursor of aromatic amino acids (11 ), and the gen- homogenizer in 10 per cent TCA at 00 C. After centri- eration of reduced triphosphopyridine , fugation the precipitate was washed with 5 ml. of cold 5 which are required for the synthesis of several sub- per cent TCA. The precipitate was then extracted con- stances including fatty acids and secutively with 8 ml. of cold ethanol, 8 ml. of ethanol at cholesterol (12). 250 C., and twice with 8 ml. of an ethanol: (3: 1) In addition, it is the route by which a substantial mixture. The cellular residue with 5 mg. each of ribo- fraction of is catabolized by rat slices nucleic acid and of deoxyribonucleic acid as carrier was (13). Recent work has demonstrated its role in then extracted with 1 ml. of 10 per cent sodium chloride in the in vivo conversion of ribose and a boiling bath for 20 minutes. After centrifugation to mouse liver (14). the residue was similarly extracted twice with 0.5 ml. of 10 per cent sodium chloride for 20 minutes. To the com- The present investigation was undertaken to bined extracts two volumes of ethanol were added. This study the mechanism of nucleic acid ribose syn- mixture was kept at 50 C. overnight. The white precipi- thesis by a human carcinoma cell (strain HeLa) tate was centrifuged, washed with 1 ml. of cold ethanol grown in tissue culture. The results are consistent and with 1 ml. of ether, and then dried in air. One ml. with ribose production via both the of 1 N KOH was added and the mixture incubated at oxidative and 300 C. for 22 hours. Deoxyribonucleic acid was precipi- nonoxidative steps of the pentose phosphate path- tated by acidification with sufficient HSO, to make a way, with the nonoxidative route predominating. final concentration of 0.5 N, and removed by centrifuga- tion. Dephosphorylation of the ribonucleotides, which METHODS remained in the supernatant solution, was carried out by adjusting the pH to 5.6, adding acetate buffer to make a Tissue culture. A human carcinoma cell (strain HeLa) final concentration of 0.15 M, and introducing 0.1 ml. of and a human liver cell (Henle, strain 407) were grown acid (Worthington germ in the amino phosphatase, acid--salt-glucose medium described 20 mg. per ml. of water). The mixture was incubated at by Eagle (15) supplemented with 5 per cent whole horse 300 C. for two hours, and the reaction stopped serum. by placing After two or three days of incubation, at which the tube in water for two minutes. time the inoculated boiling The pH of cells were firmly adherent to the wall the solution was then adjusted to 6.0 with KOH. The of the 1-liter Blake bottle in which they were grown, nucleosides were the hydrolyzed by incubation with 1 ml. of C14-labeled substrate was added in the amount de- riboside hydrolase (18) at 340 C. for three hours. scribed. The medium was renewed The every second or third reaction was stopped in boiling water, and the solution 1 studies were supported by grants from the centrifuged. The supernatant solution was deionized by Jane Coffin Childs Memorial Fund for Medical Research passage through a mixed bed resin (MB-3; Fisher Sci- and from the National Institute, United States entific Company). The effluent was lyophilized to dry- Service (C 2673), and, in part, by an ness and the residue taken up in methanol. The methanol American Cancer Society grant to Harvard University. solution was streaked on Whatman No. 1 paper and the 1408 RNA RIBOSE SYNTHESIS BY A HUMAN CARCINOMA CELL 1409 ribose purified by descending chromatography in N-buta- of a liver cell were generously provided by Dr. George nol-glacial -water (4: 1: 5) for 20 hours (19). Foley. A standard ribose solution was spotted on the paper Bicarbonate-Cl' was prepared by absorbing in NaOH lateral to both margins of the HeLa cell mixture. The C"'O liberated from BaC"O0, and then neutralizing to position of the ribose was located by streaking with ani- the phenolphthalein end point. Glucose-l-C' was pur- line phthalate reagent (20) the area of the paper over chased from Nuclear Chemical and Instrument Corpora- which the standard ribose solution had migrated and the tion, and glucose-2-C" and ribose-1-C" from the National adjacent margins of the incubation mixture. The strip Bureau of Standards. The glucose substrates were de- containing the ribose was cut out and the eluted graded as described previously (14) and the ribose-1-C" with water. by Lactobacillus pentosus in order to verify Degradation of ribose. The RNA ribose, to which was isotope distribution. added sufficient unlabeled ribose to make a total of 350 ;&M, was degraded to acetate ( 1 and,2 of ribose)- and lactate (carbons 3, 4, and 5) by fermentation with RESULTS Lactobacillus pentosus (21). The fermentation products Incorporation of radioactive substrates were isolated as described by Bernstein (22). Acetate was converted to barium carbonate (BaCO,) in stepwise Al of the C04-substrates were incorporated into fashion as described by Phares. (23). Lactate was de- HeLa cell (Table I). Since the rate and amount graded to CO, (carbon 3 of ribose) and acetate (carbons 4 and 5) by the method of Aronoff, Haas, and Fries (24), of varied considerably from one study and the acetate was subjected to the Phares procedure to another, quantitative conclusions cannot be (23). The microbiological and chemical degradation drawn from the incorporation data. Radioactivity techniques permitted the isolation of each carbon of was distributed in several cellular extracs (Table the ribose molecule as BaCO, When ribose-1-C" was I). When carrier glycogen was added to the cold degraded in this fashion, over 99 per cent of the total TCA extract of HeLa cell grown in the presence radioactivity was found in carbon 1. Determination of radioactivity. Bicarbonate-C14, the of bicarbonate-Cl' and the glycogen then recrystal- radioactive , and the cellular extracts were counted lized, no radioactivity was detected. In similar at "infinite" thinness with a gas flow counter, operating fashion, no glycogen formation could be detected at an efficiency of 40 per cent. BaC"O, was counted at in the human liver cell grown in tissue culture in "infinite" thickness. All determinations were continued the presence of NaHC1403. sufficiently long so as to reduce the counting error to less than 3 per cent. Chemical and enzymatic procedures. Phosphate was de- Isotope distribution in RNA ribose (Table II) termined by the method of Taussky and Shorr (25), and ribose by a modification (26) of the Mejbaum procedure The ribose from cells grown in the presence of (27). Riboside hydrolase was prepared from Lacto- uniformly labeled glucose was also uniformly la- bacillus delbruckii, according to the method of Takagi and beled. (This sample of ribose was isolated and Horecker (18). The activity of the preparation was as- kindly provided by Drs. B. L. Horecker and H. sayed with inosine as substrate and xanthine oxidase, kindly provided by Dr. Boris Magasanik. Eagle.) Ribose derived from glucose-i-C14 was Materials. The initial stock cultures of HeLa cell and predominantly labeled in carbon 1 with somewhat

BLE I Distribution of C14 in HeLa cell grown in presence of C4-substrates

Total c.p.m. in cell extracts Incubation and Experiment C14 Totaluc. pernod Cold TCA alcohol-ether number substrate added (days) soluble soluble Nucleotides 3 NaHCY"Os 470 10 28,000 6,600 21,000 11 NaHC"lOs 430 19 75,700 7,600 27,000 12 NaHC"O0, 200 7 35,200 21,000 14,700 31 Glucose-2-CM 14.8 6 18,900 68,000 27,000 38 Glucose-i-C4 11 6 18,000 43,000 13,800 39 Glucose-i-C14 22 10 12,200 52,000 59,200 41 Glucose-2-C4 13 10 12,100 23,000 8,650 43 Glucose-2-C4 13 6 15,800 38,000 21,900 44 Glucose-i-C14 11 6 18,500 20,000 25,900 42 Ribose-1-C4 10 4 10,400 19,500 39,700 1410 HOWARD H. HIATT

TABLE II C1' distribution in RNA ribose of HeLa cell grown in medium containing can-substrate

Per cent total activity* Experiment C14 number substrate C-1 C-2 C-3 Cw C-S 1 Glucose-U-C" 19.9 20.1 20.0 20.0 20.0 (32.1) 39 Glucose-i-C" 52.8 4.9 8.2 0.5 33.6 (6.55) 44 Glucose-1-C4 47.8 4.2 8.0 2.8 37.2 (0.99) 31 Glucose-2-C4 22.6 38.7 9.9 26.0 2.8 (1.76) 43 Glucose-2-C4 23.3 37.0 11.6 22.3 5.8 (0.37) 42 Ribose--C" 74.0 3.9 9.8 1.9 10.4 (5.80) * The specific activity (in millimicrocuries per millimole of carbon) of the most radioactive carbon atom is given inparentheses. less activity in carbon 5. The major sites of iso- former. Ribose from cells grown in the pres- tope localization in ribose synthesized from glu- ence of ribose-1-C14 was most heavily labeled in cose-2-C"4 were positions 2, 1, and 4, with the carbon 1, but some randomization of isotope had latter two carbon less active than the taken place, particularly to carbons 3 and 5.

C C C -i1--' C

c-P d-P Glucose 6-P 6-P -P

(Co2)

I ( 4-P)

ICP [-j Penose-P

DX

C C

-4 C t C C -7 .P + C-P Fructose 6-P Erythrose 4-P Sedobeptulose 7-P GlyceraldekWde-P FIG. 1. PENTOSE PHOSPHATE SYNTHESIS VIA THE OXIDATIVE (A) AND NONOXIDATIVE STEPS (B, C, D) OF THE PENTOSE PHOSPHATE PATHWAY The compounds in parentheses (steps A and B) are the reaction products other than pentose phosphate. RNA RIBOSE SYNTHESIS BY A HUMAN CARCINOMA CELL 1411 HCO

HC OH H2C* OH HC*O l HOCH C:O HCOH 1,2,3 4 HCOH HCOH HCOH HCOH HCOH HCOH H2 COPO3 H2 H 2COPO3 H 2 H2 COPO3 H 2

Glucose 6-P 5-P Ribose 5-P 1. Glucose 6-P dehydrogenase 2. Lactonase 3. 6-P gluconic dehydrogenase 4. Pentose phosphate FIG. 2. PRODUCTION OF RiBoSE-1-C" PHOSPHATE BY OXIDATm REMOVAL OF CARBON 1 OF GLUCOSE-2-C' PHOSPHATE C1' is indicated by the asterisk. To exclude the possibility that the RNA ribose tose phosphate pathway (28) (Figure 1). In the may have been contaminated by ribose-1-C4 sub- oxidative steps carbon 1 of glucose 6-phosphate is strate, 4 ,fc. of ribose-1-C" were added to HeLa converted to CO2 and carbon atoms 2 to 5 to ribu- cells grown in the absence of C14 and treated with lose phosphate (Figures 1 (A) and 2). The non- TCA. The fraction isolated following oxidative reactions are described in Figures 1 (B, the steps outlined above was found to be free of C, and D), 3, and 4. One nonoxidative reaction radioactivity. involves the transfer of the first two carbon atoms of a molecule of fructose DISCUSSION 6-phosphate to an ap- propriate acceptor, such as glyceraldehyde phos- Pentose phosphate may be generated by both phate (Figures 1 (B) and 3) (29). This reaction oxidative and nonoxidative reactions of the pen- is catalyzed by the , transketolase (30),

H2 COH

II O H2 COH HOCH C=O HCO I + I I HIOH Transketolase HOCH + HCOH I I HCOH HCOH HCOH HCOH I I H2 COPO3 H2 H2 COPO3 H2 2CoUOP3H2 E~ COPOr3 H2 Fructose 6-P Glyceraldehyde-P 5-P Erythrose 4-P FIG. 3. PRODUCTION OF XYLULOSE-2-C' PHOSPHATE FROM HExoSE-2-C" PHOSPHATE BY TRANSFER OF THE FIRST Two CARBONS OF HEXOSE TO GLYCER- PHOSPHATE The other product of this reaction, erythrose-4-phosphate, which is de- rived from carbons 3 to 6 of hexose, may then participate in the reactions shown in Figure 4. 1412 HOWARD H. HIATT

M2 COH N 2COH Iwo I- Mj30 cioo H2 COHI. HCO~~~~~~~~I HOCH HCO- HCOH CUO HCOH I I I I HCOH HCOH Transaldolas HCO HiOH Trawaketolase + HCOH I I HCOH + HCOH HCOH + HjOH HCOH HCOH I I I I H n 2%%pO3 H2 H2COPo3 n2 2 COPO3 2 K2 COPO3 H 2 H2COPO3KH2 H2CvOPv3 n2 Fructose 6-P Erythrose 4-P Glyceraldehyde P Ueoheptuiom 7-P xylulose s-P Riboue *-r FIG. 4. PRODUCTION OF XYLULOSE-2-C1' PHOSPHATE AND RiBnOS PHOSPHATE FROM HEXOsE-2-C4 PHOSPHATE AND ERYTHROSE PHOSPHATE In the transaldolase reaction the first three carbons of hexose are transferred to erythrose, resulting in the forma- tion of a 7-carbon sugar phosphate, -2-C4, and glyceraldehyde phosphate. The first two carbons of sedoheptulose are then cleaved in the transketolase reaction, and condense with to form a molecule of xylulose- 2-C14 phosphate, leaving a molecule of unlabeled ribose phosphate. and the products are a molecule of pentose phos- pathway are reversible, so that hexose monophos- phate, xylulose 5-phosphate, and one of a 4-carbon phate may be generated from pentose phosphate. sugar phosphate, erythrose 4-phosphate. The The responsible for the interconversion erythrose 4-phosphate may then act as acceptor of the three pentose phosphates, xylulose, ribulose, for a 3-carbon unit cleaved from a second molecule and ribose, which arise in these reactions (Figure of fructose 6-phosphate under the influence of 5) are widespread in nature (34). Hence, both transaldolase (Figures 1 (C) and 4) (31). The of the ketopentose esters, ribulose and xylulose, products of this reaction are a molecule of a may be converted to ribose 5-phosphate. It is the 7-carbon sugar phosphate, sedoheptulose 7-phos- latter which is involved in nucleotide synthesis phate, and one of triose phosphate. Transketolase (35). may then mediate the transfer of the first two car- Examination of the isotope distribution in HeLa bons of sedoheptulose phosphate to triose phos- cell ribose permits a recapitulation of the reactions phate, yielding two molecules of pentose phosphate, by which the pentose was synthesized. If HeLa one of xylulose 5-phosphate (32, 33) and one of cell ribose were generated via oxidative loss of ribose 5-phosphate (Figures 1 (D) and 4). To carbon 1 of hexose phosphate (Figure 2), the summarize these reactions (Figure 1), the oxida- pentose derived from glucose--C'4 would be un- tive steps in the pathway mediate the conversion labeled, and that obtained from glucose-2-C4 of a molecule of hexose phosphate to one each of would be labeled in carbon 1. On the other hand, pentose phosphate and of CO2. The transketolase- if the major pathway of ribose production involved transaldolase reactions, on the other hand, catalyze the transketolase-transaldolase reactions, then the conversion of two molecules of hexose phos- carbons 1 and 2 of ribose would be derived from phate and one of glyceraldehyde phosphate to carbons 1 and 2 of hexose (Figures 3 and 4). three molecules of pentose phosphate. All of the Thus, ribose synthesized via the nonoxidative se- nonoxidative reactions in the pentose phosphate quence of reactions from glucose-i-C14 would be

H 2COH Phosphoketopento- H2 COH Pentose phosphate HCO epimerase isomerase cIo Iso H; OH I ,f I le HOCH HCOH 7 HCOH HCOH I HCOH HC|OH H2 coPO3 H2 H2 Copo3 H 2 H2 COP03 H2

Xylulose 5-P Ribulose 5-P Ribose 5-P FIG. 5. INTERCONVERSION OF THE THREE PENTOSE PHOSPHATES INVOLVED IN THE PENTOSE PHOSPHATE PATHWAY RNA RIBOSE SYNTHESIS BY A HUMAN CARCINOMA CELL 1413 (a) H2COK03 H2 H2COP% H2 I% COFO9 "a - I* C?=o c*: 0 p HC OH Aldolase I lsomereTrisme HOCH H2 COH HCO HCOH HCOH HCO i COP. H2 HCOH 4 COP0C H2 Hexose di-P Dthydroxyacetone P Glyceraldehyde P Glyceraldebyde P

(b) H2 COH Tranaketolaae CuOIC I& H COH HOCH C0O HCO HCOH HCO HOCH HCOH HCOH + IH H HCd'OH I I I HCO1I

2 COP9 H2 COPJi , H2tCoi H 2 Hi COn H2 Fructose 6-P Glyceraldehyde P Pentose P Erythrose 4-P FIG. 6. (a) PRODUCTION OF GLYCERALDEHYDE-2-CO' PHOSPHATE FROM HEXOSE-2-C1' DIPHOSPHATE VIA THE ALDOLASE AND ISOMERASE REACTIONS (b) PRODUCTION OF PENTOSE4-C1' PHOSPHATE FROM GLYCERALDEHYDE-2-C' PHOSPHATE IN THE TRANSKETOLASE REACTION Isotope in carbon 2 of the two-carbon donor in this reaction, e.g., fructose- 2-C1' phosphate, would, of course, appear in carbon 2 of the pentose phosphate. labeled in position 1, and that obtained from glu- The participation of both oxidative and non- cose-2-C1' would have isotope in position 2. In oxidative steps of the pentose phosphate pathway the studies in which glucose-2-C14 was the HeLa in ribose synthesis by HeLa cell is similar to re- cell substrate considerable radioactivity was found ported mechanisms of nucleic acid ribose produc- in both positions 1 and 2 of ribose., Therefore, tion in vivo by rat liver (9, 10) and of synthesis both oxidative removal of the first carbon of hex- of the ribityl moiety of riboflavin in Ashbya gos- ose phosphate and the nonoxidative reactions may syppie (7). Ribose synthesis by be presumed to have been operative in ribose pro- (5, 8) and by Torula utilis (6), on the other hand, duction. The fact that ribose obtained from glu- occurs mainly via oxidative loss of carbon 1 of cose-2-C" had 50 per cent more activity in posi- glucose phosphate, while in the chick little pentose tion 2 than in 1 suggests that the nonoxidative is produced by this means (4). reactions are the predominant route of ribose syn- The substantial radioactivity in carbon 4 of thesis in HeLa cell. No quantitative conclusions ribose derived from glucose-2-C14 and in carbon concerning the relative roles of the oxidative and 5 of ribose synthesized from glucose-i-C14 is con- nonoxidative series of reactions can be drawn from sistent with the intense glycolytic activity of tu- the studies with glucose-l-Cl4. In these experi- mor cells (36, 37). As a result of aldolase cleav- ments only the participation of the transketolase age of hexose diphosphate and isomerization of reaction would be reflected in the isotope distribu- the triose phosphates, glucose-i-C14 and glucose- tion in ribose, for any pentose synthesized via loss 2-C14 would give rise to glyceraldehyde phosphate of carbon 1 of glucose-i-C14 would, of course, be labeled in positions 3 and 2, respectively [Figure unlabeled. Ribose synthesized via the transketo- 6(a)]. The condensation of a two-carbon frag- lase reaction, however, would be labeled in car- ment with triose-2-C14 phosphate in the transketo- bon 1. lase reaction would give rise to ribose with isotope 1414 HOWARD H. HIATT in carbon 4 [Figure 6(a)], while by the same tope distribution, however, cannot be ascertained mechanism radioactivity in position 3 of triose from our. data. For example, exchange via the phosphate would appear in carbon 5 of ribose. transketolase reaction of the first two carbons of Similar isotope distribution apparently via the a molecule of hexose-2-C14phosphate with the first aldolase reaction has been observed in the ribityl two carbons of a molecule of unlabeled pentose portion of riboflavin synthesized by Ashbya gos- phosphate, would give rise to a molecule of pen- syppie (7). Ribose has been shown to support the growth C C of HeLa cell in the absence of glucose (38). Our $14 C C studies, in which glucose was the principal carbo- C- c C 14 source and ribose-1-C14 was added in I tracer quantity, suggest that considerable ribose C U ~-P s-,I is incorporated directly into nucleotide, rather than following prior conversion to hexose. Ribose- A P C-P., 1-C14 has been shown to be converted to hexose- both in vitro (39) and in the mouse in tose-2-C14 phosphate without any net synthesis 1,3-C14, having taken place. Experiments now in progress vivo (14). If such a prior reaction had taken mole- place to an appreciable extent in our system, then suggest that such alterations in the pentose would have been synthesized from cule do not occur to a significant extent as com- some ribose from net the oxidative loss of carbon 1 of glucose-1,3-C"4 pared with isotope movement resulting and would have been labeled in position 2. Fur- synthetic reactions. ther, aldolase cleavage of hexose-1,3-C14 would have given rise to triose-1,3-C04. Condensation SUMMARY of a 2 carbon fragment in the transketolase reac- Nucleic acid ribose has been isolated from a tion with triose-1,3-C14 would have resulted in human carcinoma cell (strain HeLa) grown in radioactivity in positions 3 and 5 of ribose (Fig- an -vitamin-salt-glucose medium con- ure 6). The appearance of a relatively small con- taining C'4-labeled substrates. Isotope distribu- centration of isotope in those sites, particularly in tion in the ribose is consistent with its synthesis positions 3 and 5 (Table II), suggests that some via both oxidative and nonoxidative steps of the ribose-to-hexose conversion may have preceded pentose phosphate pathway. The nonoxidative ribose incorporation into nucleotide. The marked mechanism appears to predominate. No glycogen predominance of isotope in carbon 1, however, in- synthesis was detected in HeLa cell or in a liver dicates that the major fraction of ribose-C14 was cell grown in tissue culture. incorporated unchanged into nucleotide. From our studies one cannot determine whether ACKNOWLEDGMENTS ribose is incorporated directly into nucleotide fol- lowing its synthesis by HeLa cell. If incorpora- The valuable assistance of Misses Jacqueline Lareau and Arlene Guenzel is gratefully acknowledged. The tion is not direct, exchange reactions, as well as generous advice and help of Dr. George Foley greatly ribose to hexose to ribose recycling, could take assisted in the establishment of the tissue culture program. place, and isotope distribution would thereby be altered. As a result, the pattern of radioactivity REFERENCES in the ribose molecule would reflect not only the 1. Horecker, B. L., and Mehler, A. H., Carbohydrate reactions involved in net synthesis, but also those metabolism. Ann. Rev. Biochem., 1955, 24, 207. participating in exchange and recycling. From 2. Bassham, J. A., Benson, A. A., Kay, L. D., Harris, the experiment with ribose-1-C14 one may con- A. Z., Wilson, A. T., and Calvin, M., The path of clude that ribose to hexose to ribose recycling does carbon in photosynthesis. XXI. The cyclic re- not take place to a sufficient extent to alter mark- generation of acceptor. J. Am. Chem. Soc., 1954, 76, 1760. edly the isotope pattern in RNA ribose synthesized 3. 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