Purine and Pyrimidine Nucleotide Patterns of Normal, Differentiating, and Regenerating Liver and of Hepatomas in Rats

[CANCER RESEARCH 40, 1286-i 291 . April 1980] 0008-5472 /80/0040-0000$02.00 Purine and Pyrimidine Nucleotide Patterns of Normal, Differentiating, and Regenerating Liver and of Hepatomas in Rats

Robert C. Jackson,1 May S. Lui, Theodore J. Boritzki, Harold P. Morris,2 and George Weber3

Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46223 (R. C. J.. M. S. L., T. J. B., G. W.J,and the Department of Biochemistry, Howard University Medical College. Washington, 0. C. 20001 (H. P. M.J

ABSTRACT nucleotide pattern are linked with transformation on pnogres sion in hepatomas of different growth mates.The pattern of The pattern of punine and pynimidine nucleotides was eluci deoxynucleoside tniphosphate pools in rapidly growing differ dated in freeze-clamped preparations of normal liven; in hepa entiating and regenerating liver and in hepatoma 3924A shows tomas of very slow (961 8A), intermediate (8999), and rapid that the concentrations of these key precursors of DNA biosyn (3924A) growth rates; in regenerating liven; and in liven of thesis are particularly increased in the tumor, and this may neonatal rats. The nibonucleotide content was measured by confer selective advantages on the neoplastic cells. high-pressure liquid chromatography, and that of the deoxyni bonucleoside tniphosphates was determined by enzymic as INTRODUCTION says. In the liver, the contents of deoxythymidine tniphosphate, In neoplastic transformation and progression, an integrated deoxycytidine tniphosphate, deoxyadenosine tniphosphate, and program of increases in enzyme activities has been observed deoxyguanosine tniphosphate were very low (1 to 5.1 nmol/g (21 , 22). Particularly striking elevations were found in the wet weight). The concentrations of deoxynibonucleoside tn activities of nibonucleotide reductase (3), IMP dehydnogenase phosphates were increased relative to liver in all hepatomas, (1 1), CTP synthetase (26), and thymidine kinase (21 , 22). and the rise correlated with the tumor growth rates. Adenosine However, not all the enzymes of nucleotide biosynthesis have tniphosphate (ATP) concentration and total adenine nucleotide increased activity after transformation; adenylate kinase, for amounts gave a negative correlation with tumor growth rate. example, is decreased (2). The regulation of nucleotide biosyn The ATP level in hepatoma 3924A was decreased to 43% of thesis is so complex that it cannot be expected that a simple the normal livenconcentration. By contrast, contents of cytidine correlation should exist between enzyme activities and the pool tniphosphate and of total cytosine nucleotides were increased levels of the products of the enzymes. Because of the many in all 3 liver tumors. The xanthosine monophosphate content of feedback-regulatory effects, an understanding of nucleotide hepatoma 3924A was 233% of that of normal liver and gua metabolism requires data concerning cellular nucleotide con nosine monophosphate concentration in all three hepatomas centrations under various metabolic conditions. The most ef was above the liven values. There were no other consistent fective use of antimetabolite drugs will likewise require infor changes in the guanine and unacil nucleotide concentrations in mation concerning concentrations of competing substrates and the hepatomas. feedback effectors. A preliminary report from this laboratory In the 24-hr regenerating liver, the cytidine tniphosphate and (8) compared nibonucleotide contents of normal rat liven and xanthosine monophosphate levels were higher than normal the rapidly growing hepatoma 3924A. The present study has liven values. Unlike the liven tumors, the regenerating liver had extended this work to hepatomas of slow and medium growth increased unidine nucleotides and normal ATP concentration. rate and to the nonmalignant proliferating hepatic cell popula In the liver of 6-day-old rats, with the exception of a 27% tions of regenerating and neonatal liven.The pattern of deoxy increase in cytidine tniphosphate level, the nibonucleotide con nibonucleotides has also been elucidated. Punine and pynimi tents did not differ from those of adult rat liver. dine nucleotides have numerous metabolic roles apart from In normal rat liver, a period of ischemia of up to 10 mm their function as substrates for nucleic acid biosynthesis. In resulted in extensive dephosphorylation of ATP and other particular, the adenine nucleotides act as the ubiquitous donors nucleoside tniphosphates, with the degraded material accu and acceptors of phosphate groups in energy metabolism. mulating as monophosphates and nucleosides. However, hep Malignant tissues have long been known to have abnormal atoma 3924A (and the other hepatomas to a much lesser energy metabolism, characterized by decreased respiration extent) possessed the ability to maintain ATP levels near normal and increased dependence upon glycolysis (25). In the present values for 10 mm of ischemia. This capability was lost following study, we have used the ischemic condition to examine the iodoacetate treatment, suggesting that the maintenance of a relationship between the altered energy metabolism of tumors normal ATP level in hepatoma 3924A during ischemia was and the behavior of the nucleotide pools. associated with the high activity of anaerobic glycolysis in this tumor. MATERIALS AND METHODS The results indicate that aspects of the pynimidineand pumine Punine and pynimidine nucleotides, calf thymus DNA, and

â€˜Supported by Grant CA-i 81 29 from the National Cancer Institute, NIH. poly(deoxyadenylate-deoxythymidylate) were purchased from 2 Supported by Grant CA-i 0729 from the National Cancer Institute, NIH. Sigma Chemical Co., St. Louis, Mo. Tnitium-labeled dATP and 3 Supported by Grants CA-13526 and CA-05034 from the National Cancer Institute, NIH. To whom requests for reprints should be addressed. dGTP were obtained from the Radiochemical Centre, Amen Received October 8, 1979: accepted January 10, 1980. sham, England and DNA polymenase(Micrococcus luteus) from

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Miles Laboratories, Elkhart, Ind. High-pressure liquid chroma detectors at 254 and 280 nm and electronically integrated tognaphy columns were purchased from Whatman Inc., Clifton, using a Vanian Model CDS-111C integrator which had been N. J. Reagent chemicals were from Fisher Scientific Co., Fain calibrated against known quantities of standard nucleotides. Lawn, N. J., or from J. T. Baker Chemical Co., Phillipsburg, N. J., analytical grades being used where available. RESULTS Animals and Tumors. Hepatoma 3924A was maintained as a bilateral s.c. transplant in male ACI/N rats and was trans Ribonucleotides in Hepatomas. In these experiments, we planted at 1-month intervals. Hepatomas 96i 8A and 8999 used the freeze-clamp procedure to prepare nucleotide pool weresimilarlymaintainedinmaleBuffalorats;hepatoma96i 8A extracts from the livers of Buffalo and ACI/N rats, and from 3 was transplantedat 10-month intervals,and hepatoma8999 transplanted hepatoma lines: 96i 8A, a slowly growing tumor wastransplantedat 3.5-monthintervals.Forregeneratingliven (transplant interval, 10.5 months) grown in Buffalo rats; 8999, experiments, 200-g male ACI/N rats were used, and partial a hepatoma of medium growth rate (transplant interval, 3.5 hepatectomy was performed by the standard procedure (6). months), also grown in Buffalo rats; and 3924A, a rapidly For experiments with neonatal liven, Wistar matswere used. growing hepatoma (transplant interval, 1 month).maintained in Animals were kept in separate cages with water and Purina ACI/N rats. Ribonucleotide contents of these tissues are shown Laboratory chow available ad libitum. The animals were kept in in Table 1. Certain systematic trends are apparent in these a 12-hr light-i 2-hr dark cycle, and all experimental samples data. The concentrations of ATP, and of total adenine nucleo weretaken between9:30 and 10:30 a.m. tides, were significantly lower in all the tumors than in the Tissue Extraction. Rats were lightly anesthetized with ether; normal liver samples. Moreover, a negative correlation with livers and hepatomas were exposed and freeze-clamped tumor growth matewasapparent. The ATP content of hepatoma (within 2 sec of severing the blood vessles) between liquid 961 8A was 73% control, decreasing to 57% in 8999, and 43% nitrogen-cooled tongs (24, 27, 28). The frozen tissue was in 3924A. The contents of GTP and GDP were not significantly pulverized in a liquid nitrogen-cooled mortar, and 0.5 to 2.0 g different in the hepatomas from the control liven values, but of powder were extracted in aqueous perchlonic acid (0.7 M) increased levels of GMP and its immediate precursor, XMP, or in 60% aqueous methanol for nucleotide assays. were found in all 3 tumors. These increases, however, did not Deoxyribonucleoside Triphosphate Assay. Freeze appear to correlate with hepatoma growth rate. Unidinenucleo clamped powders were extracted in 60% aqueous methanol, tides were present in the hepatomas in about the same con and extracts were worked up by the procedure of Tynsted(20), centrations as in normal rat liven. CTP, however, showed sig as modified by Hamnapand Paine (5). dCTP and dGTP were nificantly elevated concentration in all 3 tumor lines, and the assayed using the enzymatic method of Solten and Handschu CTP content gave a positive correlation with hepatoma growth macher (17) with calf thymus DNA acting as template primer. rate, ranging from a 30% increase in 961 8A to a 306% increase dATP and dTTP were measured with DNA polymenase, modi in3924A. fied according to the method of Lindbeng and Skoog (13), Other aspects of the nibonucleotide pool composition not using poly(deoxyadenylate-deoxythymidylate) as template shown in Table 1 are worthy of note. ITP, lOP, XTP, and XDP, primer. all of which were resolvable in our analytical system, were Ribonucleotide Analysis. Freeze-clamped powders (0.5 g) never seen in any of these rat tissues. Recoveries of these were extractedin 5 ml of 0.7 Mperchlonicacid.Extractswere compounds when worked through the extraction procedure centrifuged to remove precipitated protein and then neutralized were better than 90%; it may be concluded that none of these with solid potassium bicarbonate (70 mg/mI). After centnifu 4 nucleotides is present in nat liver or hepatoma at a concen gation to remove potassium perchlonate, the pH of the samples tnation of greater than 1 nmol/g, wet weight. Small amounts of was brought to 3.0 by addition of 10 @.dof4 N hydrochloric material chromatographing in the onotidine 5'-monophosphate acid. Aliquots of these extracts (usually 25 or 50 @cl)were position were sometimes seen. This pynimidine precursor could analyzed by anion-exchange chromatography. A VanianModel not be consistently resolved from lange nearby peaks of ADP 8520 high-pressure liquid chromatognaph, fitted with a Vydac nibose and flavin adenine dinucleotide. Nucleosides and free AX pnecolumn (0.2 x 8 cm) and a Whatman Partisil PSX 10/25 punineand pynimidine bases were also seen in all these tissues. SAX column (0.46 x 25 cm) was used, and buffer conditions Preliminary results of our analyses of nucleosides and bases in were as described by Lui et a!. (14). For someanalyses,the rat liver have been published (9), and more detailed results will separating conditions were modified to improve resolution of form the basis of a future study. Other compounds not shown monophosphates, as follows. Starting buffer was 5 m@iam in Table i were also regularly seen in the chromatograms, monium phosphate, pH 2.8, and final buffer was 0.5 M ammo including flavin and pynidine nucleotides, and compounds be nium phosphate, pH 4.8. After injection of the sample, the lieved on the basis of their spectral properties to be UDP column was eluted with starting buffer for 5 mm, then the saccharides. proportion of high-ionic-strength buffer in the elution mix was Deoxyribonucleotides in Hepatomas. The buffer systems increased to 5%, oven a i -mm period. The elution buffer used in our high-pressure liquid chromatography separation composition was held constant for 5 mm, and then the ratio of did not distinguish between nibonucleotides and deoxynibonu high-ionic-strength buffer in the mixture was increased at a cleotides. Since the deoxynucleotides do not normally exceed rate of 5% pen mm until 100% final buffer was being used. about 5% of the concentration of the analogous nibonucleotide, Under these conditions, retention times of nucleoside mono the resulting error in the nibonucleotide estimate was not great, phosphates, at 23Â°,were as follows: CMP, 4.0 mm; NAD, 6.5 but it became necessary to use another analytical technique mm; AMP, 7.5 mm; UMP, 9.0 mm; IMP, 10.5 mm; GMP, 11.3 for deoxynucleotides. Table 2 shows deoxynucleoside tniphos mm; and XMP, 13.0 mm. Peaks were detected using optical phate contents of rat livenand hepatomas as measured by the

APRIL1980 1287

Table 1 Ribonucleotides in rat liver and hepatomas Extraction and assay procedures were as described in â€˜â€˜MaterialsandMethods.â€• wt)Liver Nucleotide contents (nmol/g wet

BA89993924AATP2470 (Buffalo)Liver (ACI)961 (43)CADP786 Â±2iOa2290 Â±851805 Â± 73 (73)L@c1410 Â± 58 (57)C965 Â±75 (76)CAMP Â± 281 020 Â±40972 Â±105 (1 24)858 Â± 69 (1 09)778 Â±75 Â± 27 Â±30 Â± 45 (115) Â± 50 (132) Â±43 (81) Total(56)cGTP312 adenine nucleotides339 3600 Â±250340 3610 Â±30389 3160 Â±130 (88)c449 2720 Â±220 (76)c274 2020 Â±70 (87)GOP Â± 34371 Â±58250 Â± 27 (80)259 Â± 13 (83)324 Â±10 ii 28(i3i) 15(122) 15(104) (325)cTotalGMP97Â± 33 Â± 9132Â±24 16 Â± 4127Â± 67 Â± 3 (203)c118Â± 5@Â± 7 (173)C137Â± 52 Â± 7 guanine nucleotides Â± 80 Â±18 Â± 64 (100) Â± 46 (98) Â± 9 (99) @ (406)CCOP35CTP442 57 Â± 5519 51 Â± 8444 75 Â± 10 (131)C434 77 ii (135)C513 207 Â± 12 (139)CMP35 Â± 1536 Â± 543 Â± 27 (123)42 Â± 13 (120)50 Â±13 (167)CTotalcytosinenucleotides127 Â± 1533 Â±1229 Â± ii (83)40 Â± 4 (114)55 Â± 8 (240)CUTP296 Â± 12120 Â±14146 Â± i7 (115)159 Â± 16 (125)C312 Â±ii (85)UDP112 Â± 24303 Â±28213 Â± 15 (72)228 Â± 16 (77)257 Â±52 (193)@MP72 Â± 21129 Â±30105 Â± 9 (94)128 Â± ii (114)249 Â±90 (126)uDP-glucose384 Â± 34125 Â±19136 Â± 57 (189)291 Â± 18 (404)'157 Â± 14 (91)UDP-N-acetylglucosamine173 Â± 48362 Â±28295 Â± 4 (77)326 Â± 30 (85)329 Â±26 (76)Total Â± 51224 Â±35267 Â± 14 (154)232 Â± 27 (134)170 Â±18 uracil nucleotides 037 Â± 47 143 Â±40 01 6 Â± 53 (98) 205 Â± 39 (116) 162 Â±48 (102) (70)XMP3IMP1 31 Â± 111 27 Â± 71 63 Â± 3 (203)c1 32 Â± 10 (103)1 19 Â± 5 Â± 23 Â± 24 Â± 2 (133)10 Â± 7 (333)7 Â± 2 (233)

a Mean Â± SE. b Numbers in parentheses, tumor values as percentages of appropriate liver control.

C Significant differences from control liver (p < 0.05 in Student's t test).

Table 2 Deoxyribonucleoside triphosphates in rat liver and hepatomas Extraction and assay procedures were as described in â€˜â€˜MaterialsandMethods. wt)Liver Deoxyribonucleotide triphosphate contents (nmol/g wet

BA89993924AdATP1.07 (Buffalo)Liver (ACI)961 Â±043aÂ± 0.353.74 Â±0.94 Â±0.60 Â±3.93 748)cdGTP1.08 (350)b. c4.73 (442)c17.65 (1 Â±0.170.89 Â±Oh1.33 Â±0.6 Â±0.18 Â±0.88 (482)'dCTP5.12 (123)2.18 (202)c4.29 Â±0.576.62 Â±0.587.30 Â±0.60 Â±3.33 Â±7.56 (793)CdTTP1.28 (i43)a18.56 (362)c52.5 Â±0.280.70 Â±0.241.46 Â±0.37 Â±0.94 Â±1.18 (1214)CNo.ofsamples1287710 (114)3.20 (250)'8.50

a Mean Â± SE. b Numbers in parentheses, tumor values as a percentage of the appropriate liver control. @ Significant difference from control liver (p < 0.05 in Student's t test).

DNA polymerase method (5, 13, 17, 20). Normal rat liven livenwere comparatively small. ATP was slightly decreased, to always contained small amounts of dCTP, but dTTP, dATP, 92% of the normal ACI/N liven value (Table i), but a similar and dGTP were often below limits of detection. However, all of change was found in livers of sham-operated rats; thus, this these deoxynucleotides were found in some of the samples, minor change may have been a response to surgical manipu and the values given in Table 2 are means for 8 on 12 samples. lation. Regenerating liver resembled the hepatomas in that All 4 deoxynucleoside tniphosphates were increased in amount significant increases were seen in CTP and XMP. Unlike the in all 3 hepatomas and, in all 4 cases, the increase correlated hepatomas, however, regenerating liver had a rather higher with hepatoma growth nate. In most of the hepatoma samples, total unidine nucleotide content than did control liver. By 48 hr the most abundant deoxynucleotide was dCTP. This contrasts after hepatectomy, all values had returned to the control levels with previous results obtained with tissue culture cells where (data not shown). Table 3 also shows the nibonucleotide con dTTP is normally predominant (i 9). However, the hepatomas tent of 6-day-old Wistar natlivenin comparison with the liver of resembled the tissue culture cells in that dGTP was the least adult Wistar rats. Six-day-old rats were chosen for this exper abundant of the 4 deoxynucleoside tniphosphates (Table 2). iment because by this stage of development liven no longer Nucleotides in Regenerating and Neonatal Liver. The ques contains the hemopoietic cells found in fetal liven, but the tion arises whether the changes seen in the nucleotide pool of hepatocytes are still proliferating, with a doubling time of 7 hepatomas are absolute requirements for all rapid cell pnolif days (23). The nibonucleotide profile of the neonatal liven eration, or whether they are specific to the neoplastic state. resembled the adult liver closely, although the ATP content Table 3 compares nibonucleotide contents of regenerating was slightly decreased (84%). Deoxynibonucleoside tniphos ACI/N rat liven 24 hr after partial hepatectomy and liven of phate concentrations in regenerating and neonatal liver are sham-operated ACI/N rats. The changes seen in regenerating summarized in Table 4. The levels of all 4 deoxynucleotides in

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Table 3 Ribonucleotides in neonatal and regenerating liver Extraction and assay procedures were as described in â€˜â€˜MaterialsandMethods. wt)Wistar Ribonucleotide contents (nmol/g wet

liverAdult rat liverACI/N rat

hepatectomizedATP2100 (6-mo.)Neonatal (6-day)Sham-operatedPartially (98)ADP845 Â±i5Oa1890 Â±230 (90)b2150 Â±782100 Â±122 (87)AMP465 Â± 82926 Â± i72 (110)1040 Â±42902 Â± 47 (133)Total Â± 42503 Â±i35 (108)270 Â±35358 Â± 25 (97)GTP353adenine nucleotides3410 Â±2013320 Â±156 (97)3460 Â±323360 Â± 87 (96)GOP167 Â± 21388 Â± 37 (110)407 Â±43389 Â± 24 (96)GMP33 Â± 28133 Â± 32 (80)169 Â±21162 Â± 22 (138)Total Â± 916 Â± 5 (48)24 Â± 233 Â± 11 guanine nucleotides Â± 16 Â± 90 (97) Â±30 Â± 11 (97) CTP553(i43)'CDP 61 Â± ii537 78 Â± 2 (127)600 58 Â± 6584 83 Â± 12 Â± 7 Â± 9 (107) Â±18 Â± 16 (144) (127)TotalCMP41 19 Â± 544 N.D.d33 22 Â± 848 28 Â± 2 )CUTP266cytosine nucleotides1 21 Â± 171 22 Â± 8 (1 01)1 13 Â±161 59 Â± 12 (141 (109)IJDP94 Â± 24231 Â± 26 (87)321 Â±51349 Â± 37 (88)UMP146 Â± 151 08 Â± 18 (1 15)1 36 Â±541 20 Â± 30 (108)UDP-glucose440 Â± 26160 Â± 32 (110)205 Â±36221 Â± 31 (137)UDP-N-acetylglucosamine214 Â± 52377 Â± 57 (86)346 Â±82474 Â± 11 (136)Total Â± 35199 Â± 43 (93)232 Â±27315 Â± 12 19)IMP35uracil nucleotides1 160 Â± 841 075 Â±186 (93)1 240 Â±911 479 Â± 42 (1 (137)XMP2 Â± 1018 Â± 6 (51)30 Â±1041 Â± 16 Â± 23 Â± 2 (150)5 Â± 313 Â± 4 (260)c a Mean Â± S.D. b Numbers in parentheses, neonatal value as a percentage of the adult liver control or the regenerating liver value as a percentage of the sham-operated control. C Significant difference from control (p < 0.05 in Student's t test). d ND, not detected.

Table 4 Deoxyribonucleoside triphosphates in neonatal and regenerating liver Extraction and assay procedures as described in â€˜â€˜MaterialsandMethods. wt)WistarDeoxyribonucleoside triphosphate (nmol/g wet

liverAdult rat liverACI/Nrat

hr)dATP (6-mo.)Neonatal (6-day)Sham-operatedRegenerating (24 Â±0.82a Â±1.30 (248)b c Â±0.09 Â±0.69 (293)c dGTP 0.65 Â±0.23 1.15 Â±0.14 (177) 0.78 Â±0.10 1.64 Â±0.21 (2i0)c dCTP 3.29 Â±0.56 6.80 Â±0.25 (207)c 2.40 Â±0.83 7.92 Â±1.89 (232)' dTTP2.88 1.03 Â±0.367.14 2.06 Â±0.69 (200)c.66 1.86 Â±0.344.87 10.82 Â±1.68 (582)'

a Mean Â± S.D. b Numbers in parentheses indicate the neonatal value as a percentage of the adult liver control or the regenerating liver value as a percentage of the sham-operated control. C Significant difference from control (p < 0.05 in Student's t test). the regenerating liver were markedly increased above the shown). Hepatoma 3924A possessed the ability to maintain normal or sham-operated liver values and were close to the ATP concentration at 88% of control over a i 0-mm period of concentrations detected in rapidly growing hepatoma 3924A. ischemia. The more slowly growing hepatomas 96i 8A and In the neonatal liven samples, all 4 deoxynucleoside tniphos 8999 occupied a position intermediate between liver and hep phates had concentrations above the control values, but not so atoma 3924A in their ability to maintain ATP through a period greatly elevated as in regenerating liven. of ischemia. A similar situation was observed with nucleotides Effects of Ischemia on Hepatic Nucleotide Levels. The of guanine, cytosine, and uracil; i.e. tniphosphate concentra study of effects of ischemia on tissue nucleotide contents was tions decreased during ischemia, diphosphates were not undertaken for 2 reasons: (a) to allow an estimate of the degree greatly changed, and monophosphate concentrations in of alteration in nucleoside tniphosphate levels caused by the creased. In all cases, hepatoma 3924A showed only rather brief time lapse between severing the tissue blood supply and small changes, and the other 2 tumors possessed intermediate the cooling of the sample to liquid nitrogen temperature; (b) to properties. To establish the need for rapid sampling, a series study how the enzymic changes found in tumors affect the of experiments on the effect of 30 sec ischemia was conducted ability of the cell to maintain normal metabolism at low oxygen with Buffalo rat liven. Over this time period, levels of the availability. The consequences of a i 0-mm period of ischemia nucleoside tniphosphates decreased to the following percent prior to freeze-clampingare shownin Table 5. In normalliver ages of control: ATP, 89%; GTP, 92%; UTP, 95%; CTP, 77%. samples, we observed a pronounced decrease in ATP. Much We therefore concluded that our normal procedure, in which of the missing ATP was recovered as AMP and IMP (Table 5) the delay between tissue excision and freeze-clamping was 2 with the remainder accounted for as nucleosides (data not sec or less, was sufficiently rapid that nucleoside tniphosphate

APRIL 1980 1289

Table 5 hepatomas.Livers Ribonucleotides in ischemic liver and

wt)Buffalo (nmol/g wet wt)Hepatoma5 (nmol/g wet

8A89993924A0ratACI/N rat961 mmATP2250613 mina1 0 mine'0 mm i o mmo mmi o mmo mmi o mm0 mmi 0 (88)ADP760809 (27)c2230 530 (24)1940990 (51)15101096 (73)965846 (102)AMP3561364 (106)1060 i360 (128)693742 (107)713586 (82)778796 (103)Total (383)360 890 (247)390930 (238)392898 (229)27428i (95)cleotidesIMP10460adenine nu 33702790 (83)3650 2780 (76)30202660 (88)26202580 (98)20201 920

(105)Total (4600)20 602 (3010)34296 (870)3288 (275)1920 (95)cleotidesadenine nu 33803250 (96)3670 3380 (92)30502960 (97)26902670 (99)20401 940 IMPGTP2411+ (70)GOP791 21 (50)371 153 (41)2281 27 (56)2491 52 (61)324228 (98)GMP33108 26 (i 59)1 12 254 (227)1 271 78 (140)9082 (91 )1 371 34 (167)Total (327)16 i78(iii3)6788(130)48130(271)5287 (88)cleotidesXMP31guanine nu 353355 (i 01)499 585 (i i 7)422392 (93)387364 (94)51 3449

(57)CTP571 2 (400)3 15 (500)47 (1 75)1 09 (90)74 (99)COP3556 9 (33)51 5 (10)7441 (55)7740 (52)207203 (138)CMP3555 (160)36 40 (110)4373 (170)4275 (179)5069 (162)Total (157)33 50 (152)2945 (155)4043 (108)5589 (116)cleotides@TP296121cytosine nu 1271 30 (1 02)1 20 95 (79)1 461 59 (1 09)1 591 58 (1 00)31 2361

(77)uOP1 (41)303 124 (49)213122 (57)228148 (65)257197 (139)UMP72228 121 19 (106)129 141 (109)105i04 (99)128139 (109)249347 (58)UDP-sugars557496 (31 7)1 25 31 1 (249)1 36401 (295)291352 (1 21 )1 5791 (99)Total (89)586 533 (91)562430 (77)558518 (93)499504 (98)tidesuracil nucleo 1037964 (93)1 143 1109 (97)1 0161 057 (104)1 2051 157 (96)1 1621 139

a Freeze-clamped immediately after severing blood supply. b Freeze-clamped after a 1 0-mm period of ischemia. C Numbers in parentheses, value after 1 0 mm ischemia expressed as percentage of zero time value. breakdown would not be appreciable. In the regenerating and Table 6 neonatal liver, the course of events in a 10-mm ischemic period Effect of iodoacetamide injection on ribonucleoside triphosphate concentrations closely resembled the behavior of normal liver (data not shown). of hepatomaischemiaNucleotide 3924A during We speculated that the remarkable ability of hepatoma wt)0.9% concentrations (nmoi/g wet 3924A to maintain ATP levels in the absence of normal circu lodoacetamide0NaCI solution lation might be related to the rapid glycolytic activity of this tumor. To test this hypothesis, we injected tumors in situ with mm is- 10 mm is- 0 mm is- 10 mm is chemiaATP chemia chemia chemia 1 ml of i M iodoacetamide and, after 2 mm, took samples 945 (86)a 841 207 (25)b which were either freeze-clamped immediately or after a period GTP 274 242 (88) 180 64 (36t' of 10-mm ischemia. Under these conditions, the lactate pro @TP 283 277 (98) 137 64 (47t' duction was inhibited by 67% (mean for 6 iodoacetate-injected CTP 110 134 (122) 62 29 Lactate-pro 2100c (100) 700c (33) samples compared with four 0.9% NaCI solution-injected con duced1100 trols). Nucleoside tniphosphate contents of the hepatomas un a Numbers in parentheses, nucleotide levels after 1 0 mm ischemia as a denthese conditions are shown in Table 6. The results indicated percentage of zero time values, except in the last row where they indicate lactate that inhibition of glycolysis caused the response of the tumor production in the iodoacetamide-treated samples as a percentage of the 0.9% NaCI solution. to ischemia to resemble that of normal liven. b Significantly different from 0-mm value of the iodoacetamide group (p < 0.05 in Student's t test). DISCUSSION C Values are concentrations produced during 1 0-mm period of ischemia.

The changes in the nucleotide pool composition of hepato tissues. These results confirm and extend the previous report mas, in comparison with the normal adult rat liver, appear to be of elevated dTTP in regenerating liven (i ). The concentrations of 2 kinds: alterations which are concomitant with an increased of dATP, dCTP, and dGTP in regenerating liver, although rate of cell division; and alterations which appear to character significantly above levels in sham-operated liven, were not as ize more specifically the neoplastic state. In the essentially high as were the concentrations in hepatoma 3924A. The nonprolifenating cell population of adult rat liven, the deoxynu absolute level of dTTP in regenerating liver, however, was cleotides are present in low concentration, on completely ab greaten than the 3924A value. Of the 4 deoxynibonucleoside sent. Readily measurable amounts, however, are found in tniphosphates, dTTP showed the greatest sample-to-sample neonatal and regenerating liver and in all 3 hepatomas. The variation. Previous work (7) indicated that cellular dTTP levels concentrations of all 4 deoxynucleotides were higher in negen were critically dependent upon availability of free thymidine, erating liventhan in the more slowly proliferating neonatal liver and it may be that the observed variation in dTTP reflected and higher in rapidly-growing hepatoma 3924A than in the variation in circulating thymidine, on differences in rates of more slowly growing hepatomas, indicating a positive comnela thymidine catabolism. These aspects are presently under in tion with growth rate in both malignant and nonmalignant vestigation. i290 CANCERRESEARCHVOL. 40

2. Criss, W. E., Litwack, G.. Morris, H. P., and Weinhouse, S. Adenosine In the nbonucleotide pools, CTP provided the most conspic triphosphate phosphotransferase isozymes in rat liver and hepatomas. Can uous example of an increased level compared to control liven. cer Res., 30: 370-375, 1970. Small increases were found in the CTP content of regenerating 3. Elford, H. L., Freese, M.. Passamani, G., and Morris, H. P. Ribonucleotide reductase and cell proliferation. I. Variations of ribonucleotide reductase liver and the slow and medium growth rate hepatomas, but the activity with tumor growth rate in a series of rat hepatomas. J. Biol. Chem., CTP in hepatoma 3924A was 406% of control, a significant 245: 5228â€”5233.1970. quantitative difference from regenerating liver. The increases 4, Glazer, A. I., and Weber, G. bodoacetateinhibition of lactate production and lipid, protein, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) in the CTP pool are probably a direct consequence of increased synthesis in newborn rat brain cortex slices. Biochem. Pharmacol. , 20: CTP synthetase in regenerating liver and hepatomas (26). 2121â€”2123,1971. These observations are in line with findings indicating high CTP 5, Harrap, K. A., and Paine, A. M. Adenosine metabolism in cultured lymphoid cells. Adv. Enzyme Regul., 15: 169â€”193,1977. concentration in ascites hepatomacells (15). Similarly, the 6. Higgins, G. M., and Anderson, A. M. Experimental pathology of the liver. I. elevated XMP pool in hepatomas may reflect their high IMP Restoration of the liver of the white rat following partial surgical removal. Arch. Pathol., 12: 186-202, 1931. dehydrogenase activity (11). 7. Jackson, A. C. The regulation of thymidylate biosynthesis in Novikoft hepa An early report by Nodes and Reid (i 6) claimed that the ratio toma cells and the effects of amethopterin, 5-fluorodeoxyuridine, and 3- of ATP to AMP was decreased in primary hepatomas and that deaza'uridine. J, Biol. Chem., 253: 7440â€”7446, 1978. 8. Jackson, A. C., Boritzki, T. J., Morris, H. P., and Weber, G. Purine and the total content of punine nucleotides (but not of unidine pyrimidine ribonucleotide contents of rat liver and hepatoma 3924A and the nucleotides) was decreased in hepatomas. In the first applica effect of ischemia. Life Sci., 18: 1531â€”1536, 1976. tion of the freeze-clamp technique to solid tumors (24, 27), 9, Jackson, A. C., Boritzki, T. J., and Weber, G. Thymidmnerescue from methotrexate or 5-fluorodeoxyuridine: enhancement by 5-diazouracil. In: W. Weber et a!. reported a decrease in ATP and in the ATP:AMP Siegenthaler and A. LUthy (eds.), Current Chemotherapy, pp. 1264â€”1266. ratioof hepatomas,andtheypointedoutthatthis wasa growth Washington, 0. C.: American Society for Microbiology, 1978. rate-linked effect. The present work has shown that this de 10. Jackson, A. C., Morris, H. P., and Weber, G. Enzymes of the purine ribonucleotide cycle in rat hepatomas and kidney tumors. Cancer Res., 37: crease in punne nucleotideswas specific to the adeninenu 3057â€”3065,1977. cleotides, in that GTP and GDP were in the same range in 11. Jackson, R. C., Weber, G., and Morris, H. P. IMP dehydrogenase: an enzyme linked with proliferation and malignancy. Nature (Lond.), 256: 331 â€” hepatomas as in normal liven,and the GMP content was some 333, 1975. what higher. Unlike the changes in deoxynucleotides and in 12. Kizer, 0. E. , Lovig, C. A. . Howell, B. A. , and Cox, B. Changes in the adenylic CTP, the decreased ATP content of hepatomas appeared to acid deaminase activity of rat liver during carcinogenesis. Cancer Res., 24: 1050-1055, 1964. be specific to neoplasia and was not found in regenerating on 13. Lindberg, V., and Skoog, L. A method for the determination of dATP and neonatal rat liver. This decrease may be attributed to the low dTTP in picomole amounts. Anal. Biochem., 34: 152-160, 1970. activity of adenylate kinase and the high activity of AMP de 14. Lui, M. S., Jackson, R. C., and Weber, G. Enzyme pattern-directed chemo therapy. Effects of antipyrimidine combinations on the ribonucleotide content aminase in hepatomas (2, 10, 12) both of which are neoplastic of hepatomas. Biochem. Pharmacol., 28: 1189â€”1195,1979. transformation-linked alterations. 15. Mandel, P. Quelques aspects de Ia regulation des biosynthÃ¨sesdes acides The changes discussed above all concern steady-state pools ribonucleIques chez les animaux supÃ©rieurs.Bull.Soc. Chim. Biol., 46: 43â€” 70, 1964. of nucleotides;the rapidly growinghepatoma3924A also dif 16. Nodes, J. T., and Reid, E. Azo-dye carcinogenesis: ribonucleotides and fers from normal liver in the transient-stateproperties of its ribonucleases. Br. J. Cancer, 17: 745-774, 1963. 17. Solter, A. W., and Handschumacher, R. E. A rapid quantitative determination nucleotide pathways, notably in its ability to retain most of its of deoxyribonucleoside triphosphate based on the enzymatic synthesis of ATP over a 10-mm period of ischemia. This property was DNA. Biochim. Biophys. Acta, 174: 585-590, 1969. attributed to the rapid glycolytic mateofthis hepatoma (18, 25). i 8. Sweeney, M. J., Ashmore, J., Morris, H. P., and Weber, G. Comparative biochemistry of hepatomas. IV. Isotope studies of glucose and fructose Table 5 shows that GTP, UTP, and CTP could be similarly metabolism in liver tumors of different growth rates. Cancer Res., 23: 995â€” maintained during ischemia and that the more slowly growing 1002, 1963. hepatomas 961 8A and 8999 showed behavior intermediate 19. Tattersall, M. H. N., Jackson, A. C., Jackson, S. T. M., and Harrap, K. A. Factors determining cell sensitivity to methotrexate: studies of folate and between that of normal liven and of hepatoma 3924A. The deoxyribonucleoside triphosphate pools in 5 mammalian cell lines. Eur. J. experiment summarized in Table 6 was an attempt to quantitate Cancer, 10: 819â€”826,1974. 20. Tyrsted, G. The pool size of deoxyguanosine 5'-triphosphate and deoxycy the role of glycolysis in this phenomenon. lodoacetamide has tidine 5'-triphosphate in phytohemagglutinin stimulated and non-stimulated numerousmetaboliceffects but acts primarilyas a glycolytic human lymphocytes. Exp. Cell. Res., 91: 429â€”440, 1975. inhibitor (4), and under the conditions used in our experiments, 21. Weber, G. Enzymobogyof cancer cells, part 1. N. EngI. J. Med. 296: 486â€” 493, 1977. iodoacetamide caused about 65% inhibition of lactate produc 22. Weber, G. Enzymology of cancer cells, part 2. N. EngI. J. Med. 296: 541â€” tion (Table 6). Following iodoacetamide treatment, the baseline 551, 1977. levels of nucleoside tniphosphates were somewhat decreased. 23. Weber, G., Queener, S. F., and Ferdinandus, J. A. Control of gene expres sion in carbohydrate, pyrimidine and DNA metabolism. Adv. Enzyme Regul., During ischemia, the tniphosphate concentration in hepatomas 9: 63-95, 1971. declined rapidly, as in normal liver, proving that the ability of 24. Weber, G., Stubbs, M., and Morris, H. P. Metabolism of hepatomas of different growth rates in situ and during ischemia. Cancer Res., 31: 2177â€” hepatoma 3924A to maintain nucleoside tniphosphate levels 2183, 1971. during ischemia is dependent upon rapid glycolysis. These 25. Weinhouse, S. Oxidative metabolism of neoplastic tissues. Adv. in Cancer results are in good agreement with those observed in natkidney Res., 3: 269-325, 1955. 26. Williams, J. C., Kizaki, H., Weber, G., and Morris, H. P. Increased CTP @ tumors(27). synthetase activity in cancer cells. Nature (Lond.), 271: 71â€”73,1978. 27. Williamson, 0. H., Krebs, H. A., Stubbs, M., Page, M. A., Morris, H. P., and Weber, G. Metabolism of renal tumors in situ and during ischemia. Cancer REFERENCES Res., 30: 2049-2054, 1970. 28. Wollenberger, A., Ristau, 0., and Schoffa, G. Eine einfache Technik des 1. Bucher, N. L. R., and Oakman, N. J. Thymidmnetriphosphate content of extrem schnellen Abkuhlung grosserer GewebestUcke.Arch. gesamte Phys regenerating rat liver. Biochim. Biophys. Acta, 186: 13-20, 1969. iob.Mens. Tiere (PflUegers), 270: 399â€”412,1960.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1980 American Association for Cancer Research. Purine and Pyrimidine Nucleotide Patterns of Normal, Differentiating, and Regenerating Liver and of Hepatomas in Rats

Robert C. Jackson, May S. Lui, Theodore J. Boritzki, et al.

Cancer Res 1980;40:1286-1291.

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