Behavior of Opposing Pathways of Thymidine Utilization in Differentiating, Regenerating, and Neoplastic Liver1

CANCER RESEARCH 31, 550-556, May 1971)

John A. Ferdinandus, Harold P. Morris, and George Weber2

Department of Pharmacology, Indiana University School of Medicine, Indianapolis, Indiana 46202 /J. A. F., G. W.Â¡,and Department of Biochemistry, Howard University College of Medicine, Washington, D. C. 2000 ÃŒ[H.P. M.Â¡

SUMMARY expression in cancer cells (20, 24). The identification of the pattern should provide the investigator with an understanding The behavior of opposing pathways of thymidine utilization of the progressive metabolic changes that relate to neoplastic was compared in proliferating normal liver (from developing proliferation rate. The Molecular Correlation Concept rats and from partially hepatectomized rats) and in neoplastic developed in this laboratory postulated the existence of a liver (spectrum of hepatomas of different growth rates). The meaningful biochemical pattern, which may be understood studies were carried out by assaying simultaneously the when a number of preconditions are satisfied (21). Among incorporation of labeled thymidine into DNA (synthetic these preconditions is the need to obtain an insight into the pathway) and the degradation to C02 (catabolic pathway) in regulation of opposing synthetic and catabolic pathways and tissue slices in an in vitro system. key enzymes and to elucidate their relationship with tumor In the liver of newborn rats, the incorporation of thymidine growth rate. For example, it was recognized that the activity into DNA is very high and gradually decreases during of the key glycolytic enzymes increased and concurrently the differentiation to very low levels in the adult. The catabolic activity of the key gluconeogenic enzymes decreased in utilization of thymidine is high in the newborn rat liver, and it hepatomas parallel with the increase in the hepatoma growth further increases during differentiation, being approximately rate (20-25). twice as high in the adult as in the newborn. The rise in the This paper describes the behavior of the opposing pathways activity of the catabolic pathway is blocked by administration of synthetic utilization and catabolism of thymidine in of actinomycin. differentiating, regenerating, and neoplastic liver. The results Partial hepatectomy results in marked changes in the demonstrate that the incorporation of thymidine into DNA behavior of thymidine metabolism. The activity of the increases and concurrently the degradation to CO2 decreases synthetic pathway increases, while the activity of the catabolic parallel with hepatoma growth rate. The ratio of the activities pathway decreases. The increased activity of the synthetic of the synthetic:catabolic pathways correlates closely with pathway decreases to normal range and the decreased activity hepatoma growth rate. These observations are further evidence of the catabolic pathway returns to high levels about 96 hr in support of the Molecular Correlation Concept. after the operation. In the hepatoma spectrum, the activity of the synthetic pathway increases and that of the catabolic pathway decreases parallel with the increase in tumor growth rate. As a result, MATERIALS AND METHODS there is a pronounced increase in the ratio of synthetic: catabolic utilization of thymidine, which shows a close The animals were kept in separate cages and illuminated correlation with hepatoma growth rate. The close linking of daily from 6 a.m. to 7 p.m. Purina laboratory chow and water hepatoma growth rate with the behavior of the activities of were available ad libitum to the experimental animals. opposing pathways of thymidine utilization and of the key Studies on Regenerating Liver. For studies on regenerating enzymes of the pathways provides further evidence in support purchased from HarÃan Industries, Cumberland, Ind. The of the Molecular Correlation Concept. litters were allowed to stay in the same cage with the mother for 18 days after birth; then they were placed in individual cages. INTRODUCTION Studies on Regenerating Liver. For studies on regenerating Investigations in this laboratory have been directed to liver, male, albino Wistar rats weighing 180 to 200 g were uncovering the underlying pattern in the alteration of gene obtained from HarÃan Industries. The rats were partially hepatectomized under light ether anesthesia by removal of 1Supported by USPHS Grant CA-05034, an American Cancer Society 66% of the liver (3). The remaining liver lobes were examined Grant, a Damon Runyon Memorial Fund, Inc., grant (G. W.), and at different time intervals after operation. Sham-operated USPHS Grant CA-10729 (H. P. M.). 2To whom requests for reprints should be sent, at the Department of animals were used as controls. Tumor-bearing and Control Animals. Male Buffalo and Pharmacology, Indiana University School of Medicine, Indianapolis, ACI/N rats were used in these experiments. The tumor-bearing Ind. 46202. Received September 2, 1970; accepted January 4, 1971. and control rats were shipped by air express from Dr. H. P.

550 CANCER RESEARCH VOL.31

Morris, Washington, D. C., to our laboratories at Indiana Experiments with Actinomycin. For these studies, University School of Medicine, Indianapolis, Ind. Normal rats actinomycin D was purchased as dactinomycin in vials of the same strain, sex, age, and weight were used for controls containing 500 ^g (Merck Sharp and Dohme, West Point, Pa.). and were sacrificed along with the tumor-bearing rats under Further conditions are described in "Results and Discussion" the same conditions. We used a number of tumor lines but and in Table 3, where the data obtained with this drug are concentrated on hepatomas, which included the slow-growing presented. 9618-A and 9618-B; 7800 and 5123-D, which now are Expression and Evaluation of Results. The incorporation of considered of intermediate growth rate; and rapidly growing thymidine into DNA and the degradation of thymidine to CO2 3924-A, 7288-C, 7777, 3683-F, and 9618-A2. The tumors were expressed as dpm/g of tissue/hr. The counts obtained for were transplanted bilaterally in a s.c. position, and they were incorporation of thymidine into DNA were corrected for allowed to grow to a diameter of about 1 inch before they self-absorption by extrapolating cpm/g of tissue to infinite were harvested. The biological and growth properties of the dilution (13). hepatoma spectrum were previously described (9). The results were subjected to statistical evaluation by means Experimental Procedures. The rats were stunned, of the t test for small samples. Differences between means decapitated, and exsanguinated. Livers and tumors were giving a probability of less than 5% were considered to be rapidly removed and placed in beakers, which stood on significant. crushed ice. Tissues were carefully dissected free of necrotic, hemorrhagic, and nontumorous material. For routine studies on thymidine metabolism, the tissues RESULTS AND DISCUSSION were sectioned into 1- to 2-cm cubes, and slices were cut to a uniform thickness of about 1 to 2 mm by a Stadie-Riggs slicer. Behavior of the Activity of Opposing Pathways of Synthetic Tissue slices, 50 mg, were placed in separate 25-ml Erlenmeyer and Degradative Utilization of Thymidine in Differentiation. flasks, each containing 5 ml of Krebs-Ringer phosphate buffer For an understanding of the pattern of thymidine metabolism (pH 7.4), 12.5 mM glucose, 20 mM glycylglycine, and 0.5 Â¡id during development, the behavior of the activity of the of thymidine-2-14C (specific activity, 43.7 mCi/mmole; New synthetic and degradative pathways was examined in the liver England Nuclear, Boston, Mass.). Each flask was capped with a of rats of different age groups from the early postnatal time to rubber stopper (No. 8826 rubber stoppers, 13 mm; Arthur H. adulthood. The results in Table I indicate that the Thomas Co., Philadelphia, Pa.) from which was suspended, by incorporation of thymidine into DNA is high in the liver of a straight pin, a glass fiber filter strip (934AH glass fiber filter. newborn rats, and during subsequent development the activity 2.4 cm; H. Reeve Angel and Co., Inc., Clifton, N. J.) cut to 1 x of the thymidine biosynthetic pathway decreases to very low 2.4 cm. levels in the liver of the adult. The incorporation of thymidine Flasks were incubated at 37Â°and shaken at 72 rpm in a into DNA is 30 times higher in the 1-day-old rat than in the Dubnoff metabolic shaker. The reaction was terminated after adult. incubations of 0, 30, and 60 min by injections of 50 jul of The behavior of the activity of the degradative pathway Hyamine hydroxide (hydroxide of Hyamine 10-X, Packard contrasts with that of the synthetic one. The catabolism of Instrument Co., Downers Grove, 111.) through the rubber thymidine to C02 is active in the newborn rat liver, and in stopper onto the glass fiber filter strip and then injection of 1 subsequent development the activity increases to the high ml of 50% TCA3 into the bottom of the flask. The flasks were levels observed in adult liver (Table 1). As a consequence of shaken an additional 30 min at 37Â°,after which time the the opposing behavior of the activity of the synthetic and rubber stoppers were removed and the filter strips were placed catabolic pathways, the ratios of the activity of the opposing in scintillation vials. Immediately, 10 ml of scintillator fluid pathways are of interest. The ratios are near 1 in the newborn (0.03% POPOP and 0.5% PPO in toluene from New England rat liver, and subsequently they gradually decrease, reaching a Nuclear) were added to each vial. The vials were counted for very small fraction in the adult liver. Thus, in the adult liver radioactive C02 on a Packard Tri-Carb liquid scintillation the activity of the degradative pathway is overwhelmingly spectrometer (Model 314 EX). higher than the activity of the synthetic one. For determination of thymidine incorporation into DNA, These results might be interpreted as suggesting a sequential the remaining contents of the flasks were homogenized, derepression of the genes governing the enzymes of the transferred to 10-ml volumetric flasks, and brought to volume degradative pathway and a gradual repression of those for the with distilled water. The volumetric flasks were shaken biosynthetic pathway of thymidine during differentiation in vigorously for 30 sec, and aliquots of 0.3 and 0.6 ml were rat liver. For examination of the validity of this interpretation, pipetted into separate Millipore filter apparatuses that a comparison is given in Table 2 of the behavior of activities of contained glass fiber filter discs and 5 ml of ice-cold 10% TCA. opposing pathways and key enzymes of the synthetic and Suction was applied, and the filter discs were washed degradative utilization of thymidine in differentiating rat liver. successively with cold 10-ml portions of 10% TCA, The left side of Table 2 assembles the results on the alcohol :ether (1:1), and ether. The filters were air-dried for 1 behavior of thymidine incorporation into DNA and on the min by suction and then placed in scintillation vials, to which activities of thymidine kinase, dTMP kinase, dCMP deaminase, were added 10 ml of scintillator fluid. dTMP synthetase, and DNA polymerase as percentages of the

3The abbreviation used is: TCA, trichloroacetic acid.

MAY 1971 551

Table 1 Thvmidine metabolism in rat liver during differentiation Means and S.E. of experiments from 3 or more rats are given for each age group. All values are to be multiplied by the exponential given to arrive at the original counts. Thymidine incorporation into DNA and the degradation of thymidine to CO2 in liver slices were studied as a function of age. Liver slices (50 mg) were shaken at 37Â°in 5 ml of Krebs-Ringer phosphate buffer (pH 7.4) containing 0.5 Â¿iCiof thymidine-2-14C (specific activity, 43.7 mCi/mmole). ' 4CO7 was collected on glass fiber filter strips impregnated with Hyamine, while incorporation of thymidine into DNA was measured by filtering homogenized liver on glass fiber filter discs. The filter strips and discs were counted for radioactivity. Complete experimental details are given in "Materials and Methods."

Age(days)123461822242530Adultinto DNA to CO2 Thymidine into DNA: (dpm/g/hr XIO3)598 (dpm/g/hr3844 x IO3) thymidine to CO2 X 10

Â±50384 Â±661123Â±581128 Â±350 Â±24306 +260 Â±19273 Â±631185Â± +230 Â±19208 1311273 +160 Â±1984 Â±73157316471655168016907213449501841859+54 Â±370 Â±42 Â±1551 Â±34 Â±645 +27 Â±239 Â±24 Â±417 Â±9Â±8010203010584130.4 (60)Thymidine Â±1Thymidine 27670

Table 2 Behavior of opposing pathways and key enzymes of synthetic and degradative utilization of thymidine in differentiating rat liver Results are expressed as percentages of values observed in liver of adult rats. Data for incorporation of thymidine into DNA and to CO2 were computed from Table 1. Thymidine kinase activities were calculated from results of Klemperer and Haynes (4), DNA polymerase activity was calculated from data of Ove et al. (11) and dTMP kinase, dCMP deaminase, and dTMP synthetase activities were determined from results of Sneider and Potter (15). Thymidine and undine phosphorylase activities were calculated from results of Stevens and Stocken (17), and dihydrouracil dehydrogenase activity was determined from results of Queener et al. (14).

Synthetic pathway Degradative pathway

Ages dCMP dTMP Thymidine Dihydrouracil (days)12346714182224253060ThymidineintoDNA3,0002,2601,8001,6001,200500410300260230100Thymidinekinase2,8442,5441,522600200100dTMPsynthetase13,100kinase deaminase polymerase1,4571,157900557314100Thymidineto CO2phosphorylase45 phosphorylasedehydrogenase9

4,00013,600 3,867 256060 576630

50646965918589899(191100 72100 3,1006,300 3,667

2,1002005,5003,400

900100 100 100DNA 100Uridine 100 values of the fully differentiated rat liver. The incorporation of of some of the key enzymes involved in the catabolism of thymidine into DNA (Table 1) and the activities of thymidine pyrimidine nucleosides. Thus, the degradation of thymidine to kinase, dCMP deaminase, dTMP synthetase, and DNA CO2 (Table 1), the activities of thymidine phosphorylase, polymerase in the newborn were approximately 30-, 28-, 38-, uridine phosphorylase (17) and dihydrouracil dehydrogenase 40-, and 14-fold of those in adult rat liver (4, 11, 15). The (14) increased, gradually reaching in the adult liver activity of dTMP kinase was an order of magnitude higher, approximately 2-, 4-, 11-, and 1.7-fold of those observed in the 131-fold of the values observed in adult rat liver (15). The liver of newborn rats. activities of the overall pathway and of the synthetic enzymes The results shown in Tables 1 and 2 indicate a sequential then decreased to the low levels observed in the adult rat. derepression of the genes governing the enzymes of the The right side of Table 2 shows that during differentiation, catabolic pathway and a gradual repression of the enzymes of concomitant with the decrease in the synthetic pathway, there the biosynthetic pathway (24). These data also suggest the was an increase in the degradative utilization of thymidine and operation of a coordinated behavioral pattern manifesting

552 CANCER RESEARCH VOL.31

opposing changes in the activity of antagonistic pathways and overwhelming predominance of the activity of the catabolic enzymes during differentiation. pathway over the synthetic utilization of thymidine. From this Effect of Actinomycin on the Development of the metabolic relationship, it might appear that the gene Degradative Pathway of Thymidine in Rat Liver. Table 3 expression of these opposing pathways is now completed in an shows a segment of the behavior of the degradative pathway of unalterable balance in the fully differentiated liver. However, thymidine to C02 in rat liver during the first 5 postpartum Table 4 provides experimental evidence that the potential for days. In this period, the degradation rises by 69%. Daily altering the steady state of the activity of the synthetic and administration of 2 injections of actinomycin (50 /zg/kg) catabolic pathways is retained in adult liver and can be completely prevented the developmental rise of the unleashed by the physiological signal system generated by degradative pathway. These results support the suggestion that partial hepatectomy. In the rat, partial hepatectomy leads to a the increase in the activity of the catabolic pathway during rise in hepatic cell replicative function involving mitosis, development entails an increase in the biosynthesis of certain causing an increase in the number of cells and in liver weight key enzymes and that this process requires the production of until the preoperative liver weight, liverbody ratio, and the new RNA. Addition of actinomycin in vitro to the slice system total hepatic cellularity are reestablished (22). The results of (25 /ug/50 mg of tissue in 5 ml of medium) had no effect on investigation into the behavior of the activity of opposing the incorporation of thymidine into DNA or the degradation pathways of thymidine utilization and breakdown at various of thymidine to CO2. intervals after partial hepatectomy are presented in Table 4. Behavior of the Activity of Opposing Pathways of Synthetic After operation, no significant change is observed in the and Catabolic Utilization of Thymidine in Regenerating Liver. activity of the pathways for approximately 15 hr, which agrees In the adult rat liver, the ratio of the activity of the with earlier results obtained with the use of in vivo injection of biosynthetic :catabolic pathway is very low (Table 1), thus thymidine by Bucher et al. (1). At a period 18 hr favoring degradation of thymidine to CO2. The resting state of postoperation, there was a 5-fold rise in the activity of the DNA metabolism in the adult rat liver is an expression of the synthetic pathway and an 18% decrease in the activity in the catabolic one, resulting in a 6-fold increase in the ratio (Table Table 3 4). This trend continued, showing a decrease in C02 Effect of actinomycin D on thymidine degradation to CO, in production from thymidine and an increase in the developing rat liver incorporation of thymidine into DNA, reaching a maximum The mean values and S.E. from 4 or more animals are given in each imbalance about 24 hr after operation. Consequently, the group. Actinomycin D was injected i.p. twice daily (8 a.m. and 8 p.m.) ratio of thymidine into DNA:thymidine to C02 reached a at a dose of 50 jug/kg; controls were given injections of distilled water. Percentages of 1-day control values are given in parentheses. peak at 24 hr, with a 15-fold increase. The activities of the opposing pathways of thymidine utilization subsequently IO3)Age Activity (dpm/g/hr X returned to nearly normal range, approaching the previous adult levels at about 96 hr. D- of rats These results support the concept that it is possible to (days)135Control rats824 treatedrats600Â±92(73)6 unleash the genome potential in adult, differentiated rat liver, Â±65(100) resulting in a repression of the derepressed pathway of 1211 Â±74(147)Â° thymidine catabolism and a derepression of the repressed 1389 Â±43(169)aActinomycin870 Â±113(105)b pathway of utilization of thymidine for biosynthesis of DNA. " Statistically significant difference as compared with values of This interpretation is in agreement with that of Labow et al. 1-day-old rats (p < 0.05). (7), who suggested on the basis of their investigation of b Statistically significant difference as compared with values of thymidine kinase, dTMP kinase, dTMP synthetase, DNA untreated rats of the same age groups (p <0.05). polymerase, and dCMP deaminase, that "it may be considered

Table 4 Thymidine metabolism in regenerating rat liver Means and S.E. are given with the percentages of control values in parentheses. Three or more rats were used for each time period. All values are to be multiplied by the exponential indicated. Assay methods for slice experiments are the same as described in Table 1.

Time after partial hepatectomy Thymidine into DNA Thymidine to CO2 Thymidine into DNA: (hr) (dpm/g/hr X IO3) (dpm/g/hr X IO3) thymidine to CO, X 10"

0(control)15182124364896171(100)19 27(100)1798 Â±0.4(100)9.8 2(112)84 96(97)1526 Â±0.8(107)56.0 5(500)128 (82)1278107 (610)106.0+5.0Â±7.0 6(750)155 74(69)1197 (1150)133.0 12(910)132 49(64)1346 Â±10.0(1450)99.0+ Â±4(780)76 112(72)1809 7.0(1080)44.0 Â±3(450)50 77(97)1843 Â±2.0(480)28.0 Â±3(290)1859 + 54 (99)9.2 Â±2.0(300)

MAY 1971 553

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1971 American Association for Cancer Research. J. A. Ferdinandus, H. P. Morris, and G. Weber that the rise in enzyme synthesis following partial Correlation of the Behavior of the Activity of Opposing hepatectomy results from either a derepression or an induction Pathways of Synthetic and Degradative Utilization of of closely linked genes." The yet unknown forces that operate Thymidine with Hepatoma Growth Rate. Table 5 lists in a reciprocal fashion in controlling the opposing pathways of hepatomas in order of increasing growth rate and presents data thymidine utilization are capable of restoring the balance of on the incorporation of thymidine into DNA, the degradation the adult liver steady state after the wave of hepatic to C02, and the ratio of the activities of the 2 pathways. It is regeneration reaches completion, as is shown in a graphical important that, even in the slowest-growing neoplasms, the fashion in Chart 1. incorporation of thymidine into DNA increased nearly 3-fold and, concurrently, the degradation of thymidine to CO2 decreased 49%; the ratio increased 6-fold over the value 600- observed in the liver of control rats. Parallel with the increase 200- in hepatoma growth rate, there is a rise in the activity of the synthetic pathway and a decrease in the activity of the catabolic pathway of thymidine. In the most rapidly growing 150- tumor in this series, 9618-A2, the incorporation into DNA increased 31-fold, whereas the degradative pathway decreased to 0.041% of the values of normal control rat liver. In consequence, the ratio increased nearly 140,000-fold. Z a The gradually widening gap between the synthetic and

50- degradative utilization of thymidine, the resultant predominance of the synthetic over the catabolic pathway, and the relationship to hepatoma growth rate of the ratios of thymidine into DNA:thymidine to CO2 are summarized in \ rn i i r Chart 2. O 20 40 ! 24 36 48 96 GROWTH PERIOD Partial HOURS OF An Integrated Picture of the Behavior of Metabolic ( Days ) Hepatectomy REGENERATION Imbalance and Key Enzyme Activities of the Synthetic and Chart 1. Sequence of behavior of the activity of synthetic and Degradative Pathways of Nucleic Acid Metabolism: degradative pathways of thymidine (Tdr) in differentiation (growth) Correlation with Hepatoma Growth Rate. In assembling the and in induced proliferation (regeneration). Three or more animals were evidence for the behavioral pattern of the activities of the killed in each age group for the liver development study, and 3 or more animals were killed for each time period during the partial hepatectomy opposing pathways in DNA metabolism, we noted the close study. Assay conditions for thymidine incorporation into DNA and linking of hepatoma growth rate and the ratio of thymidine degradation to CO2 are described in "Materials and Methods." into DNA:thymidine to CO2 (Table 5). These events indicate a

Table 5 Correlation of activity of synthetic and degradative pathways of thymidine with hepatoma growth rate Data are expressed as percentages of normal liver values." The growth rate is the mean transplantation time given in months between inoculation and growth to a size of approximately 1.5 cm in diameter.0 Assay conditions for slice experiments are the same as described in Table 1.

DNA:thymidinetoCO21006158105,75013,5002,210,0005,620,0008,700,00011,500,00013,900,000

TissuesLiverHepatomas96 (mo.)S.84.53.02.51.00.80.70.50.4ThymidineintoDNA1002802923707061,8902,3604,5203,9003,180ThymidinetoCO210049.036.06.96.50.0940.0500.0640.0450.041Thymidineinto

18-A9618-B78005123-D3924-A7288-C77773683-F9618-A2Transplantgeneration6649812741017027215Growthrate

"Normal liver values: thymidine into DNA = 11,300 Â±250;thymidine to CO., = 1,065,000 Â±17,000 in dpm/g/hr. b Spearman's rank correlation coefficients, r' = 1 -[6 Â£d* /n (n1 -1)|, were calculated from the difference, d, between rankings, with n = 9 for the orders of the 9 tumors in increasing growth rate and the activities of the pathways for ranking of growth rate against thymidine to DNA activity, r' = 0.933; for thymidine to CO, activity, r' = 0.983; and for thymidine into DNA:thymidine to CO2 activity, r' = 1.0. All these values were significant (p < 0.05).

554 CANCER RESEARCH VOL.31

observation) and for the activity of dihydrouracil dehydrogenase (14). With consideration of all these correlations (2, 10, 12, 14, 16, 19) and those summarized elsewhere (21, 25), the increase in the ratio of thymidine to DNA:thymidine to C02 in the rapidly proliferating tumors is the most striking for the extent of alteration of any biochemical parameter studied so far in the hepatoma spectrum. The ratio correlates with hepatoma growth rate over a 100,000-fold range; therefore, this ratio should have considerable interest as an indicator of tumor malignancy and growth rate. The progressive imbalance that is linked to the increase in proliferation rate in the hepatomas is much more pronounced (Table 5) than that observed in regenerating liver (Table 4). Since the regenerating liver grows at a rate comparable to that of the rapidly growing tumors, the alterations in the hepatomas appear to be specific to the tumor system, inasmuch as there is no similar extent of change in regenerating liver. A comparison with the rapidly growing, newborn rat liver also indicates a difference, as in the liver of newborn and young rats in the presence of a high synthetic > OÃ¯ câ€” 1/5 e- câ€” co co utilization of thymidine there is also a strong degradative HE PATOMAS HE PATOMAS Increasing Growth Rate Increasing Growth Rate pathway operating. Thus, the very marked imbalance that Chart 2. Behavior of the activity of opposing pathways of thymidine emerges in thymidine metabolism in the hepatomas, which is (Tdr) utilization: correlation with hepatoma growth rate. The growth linked with tumor growth rate, is not simply a manifestation rates for the hepatomas are given in Table 5, and the assay conditions of rapid cell growth, but seems specific to the neoplastic for thymidine incorporation into DNA and degradation to CO2 are alteration in gene expression. These observations further described in "Materials and Methods." support the Molecular Correlation Concept. The close linking of the biochemical imbalance to growth rate now permits the integration of the biochemical changes in close linkage in the expression of the replicative potential of cancer cells into a meaningful pattern. Since the correlation of the genome with the extent of the progressive imbalance in biochemical alterations and growth rate has also been shown DNA metabolism and, as reported earlier, in carbohydrate to be applicable to kidney tumors (8), mammary cancer (6), metabolism (18, 20, 21, 23, 25). The results underline the and other neoplasms of different growth rates (5), it appears importance of ratios of activities of opposing enzymes and the the approaches of the Molecular Correlation Concept are pathways as indicators of the link between replicative, valid for various types of neoplastic cells. translative, and transcriptive expression of the altered genome in neoplasia. REFERENCES In view of the interpretation that these alterations in the activities of overall pathways might involve altered gene 1. Bucher, N. L. R., Swaffield, M. N., and Ditroia, J. F. The Influence expression, it is relevant that there is considerable evidence of Age upon the Incorporation of Thymidine-2-C'4 into the DNA showing a pattern of correlation with hepatoma proliferation of Regenerating Rat Liver. Cancer Res., 24: 509-512, 1964. rate for a number of key enzymes of nucleic acid metabolism. 2 Elford, H. L., Freese, M., Passamani, E., and Morris, H. P. Elford et al. (2) discovered the correlation of ribonucleotide Ribonucleotide ReducÃaseand Cell Proliferation. I. Variations of Ribonucleotide ReducÃaseActivity with Tumor Growth Rate in a reducÃase activity with hepatoma growth rate, and Ove et al. Series of Rat Hepatomas. J. Biol. Chem., 245: 5228-5233, 1970. (12) recognized a similar positive correlation for DNA 3. Higgins, G. M., and Anderson, R. M. Experimental Pathology of porymerase activity. A positive correlation for an additional the Liver: Restoration of the Liver of the White Rat following number of DNA biosynthetic enzymes was also reported by Partial Surgical Removal. Arch. Pathol., 12: 186-202, 1931. Elford et al. (2), and there are also positive correlations in the 4. Klemperer, H. G., and Haynes, G. R. Thymidine Kinase in Rat results of Sneider et al. (16). These data show that the Liver during Development. Biochem. J., 108: 541-546, 1968. activities of thymidine kinase, dTMP kinase, dTMP synthetase, 5. Knox, W. E., Horowitz, M. L., and Friede!!, G. H. The and dCMP deaminase correlate positively with hepatoma Proportionality of Glutaminase Content to Growth Rate and growth rate. For the enzymes involved in the synthetic Morphology of Rat Neoplasms. Cancer Res., 29: 669-680, 1969. pathway of pyrimidines, Sweeney et al. (19) reported the 6. Knox, W. E., Linder, M., and Friedeil, G. H. A Series of Transplantable Rat Mammary Tumors with Graded Differentiation, correlation of the activity of aspartate transcarbamylase and Growth Rate, and Glutaminase Content. Cancer Res., 30: dihydroorotase with hepatoma growth rate. In contrast, there 283-287,1970. was a negative correlation with the increase in growth rate for 7. Labow, R., Maley, G. F., and Maley, F. The Effect of Methotrexate the degradation of thymidine (Table 5), thymine, and uracil on Enzymes Induced following Partial Hepatectomy. Cancer Res., (Ref. 10; J. A. Ferdinandus and G. Weber, unpublished 29. 366-372, 1969.

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8. Lea, M. A., Morris, H. P., and Weber, G. DNA Metabolism in Liver Nucleic Acid Metabolism in Foetal, Young and Regenerating Rat and Kidney Tumors of Different Growth Rates. Cancer Res., 28: Liver. Biochem. Â¡.,87: 12-15, 1963. 71-74, 1968. 18. Sweeney, M. J., Ashmore, J., Morris, H. P., and Weber, G. 9. Morris, H. P. Studies on the Development, Biochemistry, and Comparative Biochemistry of Hepatomas. IV. Isotope Studies of Biology of Experimental Hepatomas. Advan. Cancer Res., 9: Glucose and Fructose Metabolism in Liver Tumors of Different 227-302, 1965. Growth Rates. Cancer Res., 23: 995-1002, 1963. 10. Ono, T., Blair, D. G. R., Potter, V. R., and Morris, H. P. The 19. Sweeney, M. J., Hoffman, D. H., and Poore, G. A. Enzymes in Comparative Enzymology and Cell Origin of Rat Hepatomas. IV. Pyrimidine Biosysthesis in Morris Hepatomas. Proc. Am. Assoc. Pyrimidine Metabolism in Minimal-Deviation Tumors. Cancer Res., Cancer Res., 8: 66, 1967. 23: 240-249, 1963. 20. Weber, G. Behavior of Liver Enzymes during Hepatocarcinogenesis. 11. Ove, P., Jenkins, M. D., and Laszlo, J. DNA Polymerase Patterns in Advan. Cancer Res., 6: 403-494, 1961. Developing Rat Liver. Cancer Res., 30: 535-539, 1970. 21. Weber, G. Carbohydrate Metabolism in Cancer Cells and the 12. Ove, P., Laszlo, J., Jenkins, M. D., and Morris, H. P. Increased DNA Molecular Correlation Concept. Naturwissenschaften, 55: Polymerase Activity in a Series of Rat Hepatomas. Cancer Res., 29: 418-429, 1968. 1557-1561,1969. 22. Weber, G., and Cantero, A. Glucose-6-phosphatase Activity in 13. Perg, C. T. Quenching Correction in Liquid Scintillation Counting. Regenerating, Embryonic, and Newborn Rat Liver. Cancer Res., Advan. Tracer Methodol., 3: 81 -94, 1966. 15: 679-684, 1955. 14. Queener, S. F., Morris, H. P., and Weber, G. Dihydrouracil 23. Weber, G., and Cantero, A. Fructose-1,6-diphosphatase and Lactic Dehydrogenase Activity in Normal, Differentiating and Regenerat Dehydrogenase Activity in Hepatoma and in Control Human and ing Liver and in Hepatomas. Cancer Res., 31: in press, 1971. Animal Tissues. Cancer Res., 19: 763-768, 1959. 15. Sneider, T. W., and Potter, V. R. Deoxycytidylate Deaminase and 24. Weber, G., Ferdinandus, J. A., and Queener, S. F. Role of Related Enzymes of Thyriidine Triphosphate Metabolism in Metabolic Imbalance in Neoplasia. Proceedings of the Tenth Hepatomas and Precancerous Rat Liver. Advan. Enzyme International Cancer Congress, Houston, Texas, May 22-29, 1970, Regulation, 7: 375-394, 1969. in press, 1971. 16. Sneider, T. W., Potter, V. R., and Morris, H. P. Enzymes of 25. Weber, G., and Lea, M. A. The Molecular Correlation Concept: An Thymidine Triphosphate Synthesis in Selected Morris Hepatomas. Experimental and Conceptual Method in Cancer Research, in: H. Cancer Res., 29: 40-54, 1969. Busch (ed.), Methods in Cancer Research, Vol. 2, pp. 523-578. 17. Stevens, L., and Stocken, L. A. Studies on Enzymes Involved in New York: Academic Press, Inc., 1967.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1971 American Association for Cancer Research. Behavior of Opposing Pathways of Thymidine Utilization in Differentiating, Regenerating, and Neoplastic Liver

John A. Ferdinandus, Harold P. Morris and George Weber

Cancer Res 1971;31:550-556.

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