Searches for Exploitable Biochemical Differences between Normal and Cancer Cells IX. Anabolism and Catabolism of Purines by Hepatomas 5123 and H-35* GLYNNP. WHEELER,Jo ANN ALEXANDER,ANNS. DODSON, SUSAND. BRIGGS,ANDHAROLDP. MORRIS (Kettering-Meyer Laboratory,^ Southern Research Institute, Birmingham, Alabama; and Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland SUMMARY The extents of anabolism and catabolism in vitro of purines and purine ribonucleo- tides by rat tissues, Morris Hepatoma 5123, and Reuber H-35 hepatoma were deter mined. The results are compared with those obtained previously for Novikoff hepato ma. Hepatoma 5123 was more active catabolically than any of the host tissues exam ined, including liver; H-35 tumor was about one-half as active catabolically as the host liver; and Novikoff hepatoma was much less active catabolically than the host liver. The results show that, although a deficiency of purine catabolism may be a contribut ing factor to the uncontrolled growth of some neoplasms, deficiency of purine catab olism is not a requisite for neoplastic growth. Previous studies (17) showed that more anab 6) were very similar to those of the host liver, it olism and less catabolism of radioactive purines seemed worth while to compare the extent of occurred with minces of several neoplasms (Leu catabolism of purines and purine ribonucleotides kemia L1210, Leukemia L4946, Leukemia L5178, by Hepatoma 5123 with that of the liver and Sarcoma 180, Adenocarcinoma 755, Human Sar other tissues of the host. Also, since studies of coma No. 1, Novikoff hepatoma) than with minces carbohydrate metabolism (13,15) of deoxycytidyl- of several tissues of the hosts. Also, more catabo ate deaminase activity (4) and of glutamic oxal- lism of purine ribonucleotides occurred with total acetic transaminase activity (2) in hepatomas sonicates of neoplasms than with total sonicates indicated that certain enzyme levels of hepatomas of tissues of the hosts (18). It was suggested that could be correlated with the rates of growth of the the deficiency of enzymes that catabolize pu tumors, it was decided also to determine the capac rines might partially account for the uncontrolled ity of the slowly growing Reuber H-35 hepatoma growth of neoplasms (18), in accordance with the (12) to catabolize purines and purine ribonucleo enzyme-deletion hypothesis of neoplasia (9). Since tides. This report presents the results of these studies by others (1, 3, 7, 8, 11) indicated that the studies and also contains some data that were enzyme contents of the Morris Hepatoma 5123 (5, obtained previously for Novikoff hepatoma but * This work was supported by the Cancer Chemotherapy that are included here to facilitate comparison of National Service Center, National Cancer Institute, under the National Institutes of Health Contract No. SA-43-ph-24S3, data for the three hepatomas. and by grants from the Charles F. Kettering Foundation and the Alfred P. Sloan Foundation. EXPERIMENTAL METHODS t Affiliated with Sloan-Kettering Institute for Cancer Re Segments of Hepatoma 5123, a transplantable search, New York, N.Y. liver carcinoma that was induced by ingestion of Received for publication February 16, 1962. N-(2-fluorenyl)-phthalamic acid in an inbred Buf- 769 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1962 American Association for Cancer Research. 770 Cancer Research Vol. 22, July 1962 falo-strain rat (5), were implanted by trocar sub- been reported previously (17,18) but are included cutaneously into female Buffalo-strain rats. The here to facilitate comparison of the data for the rats were killed 8|-9 weeks later by carbon di three hepatomas. Since the radioassays of the oxide asphyxiation, and the tumors and desired compounds obtained from the Novikoff hepatoma tissues were excised quickly and placed in ice-cold were performed by a different technic and with beakers. The tumors, ranging in size from 1 to 10 the use of a different type of counter, the data for gm., and tissues from five or six rats were pooled in the total activities are not directly comparable, each experiment. but comparisons of the distributions of the activ Segments of Reuber H-35 tumor were implanted ity among the various compounds are justifiable. by trocar subcutaneously in the axillary region The data obtained with adenine-8-C14, hypo- and intramuscularly in the right hind leg of A X xanthine-8-C14, guanine-8-CH, and xanthine-8-C14 C strain rats.1 The rats were killed by carbon as substrates in experiments with minces are dioxide asphyxiation 9-12 weeks later. The sub shown in Charts 1-4. Data are given for the livers cutaneous and intramuscular tumors, weighing of the animals bearing the various hepatomas and from 2 to 9 gm., were pooled after the removal of for several other tissues of the animals bearing the the muscle tissue. Morris Hepatoma 5123. Minces were prepared by free-hand cutting with Adenine was utilized rather extensively for ana knives, and the minced tissues were added to bolic reactions by all the tissues except the brain, Krebs-Ringer phosphate buffer containing a radio as shown in Chart 1. The compounds that are con active substrate and incubated in a Dubnoff sidered here as anabolic products are adenosine, shaking incubator in an atmosphere of oxygen for adenylic acid, adenosine disphosphate (ADP), 4^ hours at 37°C. as described previously (17). adenosine triphosphate (ATP), diphosphopyri- Total sonicates were prepared by homogenizing dine nucleotide (DPN), hypoxanthine, inosine, the tissues with a Virtis homogenizer, suspending and inosinic acid. The last three of these com the homogenate in Krebs-Ringer phosphate buf pounds are included as anabolic products, because fer, and then subjecting the resulting suspension it is likely that adenine was converted to adeno to sonic vibrations by means of a Raytheon 9 KC sine or adenylic acid prior to deamination, and Magnetostriction Oscillator. After the addition of the presence of these compounds reflects a prior labeled substrates, the sonicates were incubated anabolic process. Xanthine, xanthosine, uric acid, for 60 minutes in the Dubnoff shaking incubator. allantoin, and certain unidentified minor com Alcoholic extracts of the minces and the sonicates ponents are considered to be catabolic products. were prepared and utilized for the preparation of Table 1 contains the sums of the percentages of paper chromatograms and radioautograms by de anabolic products and those of catabolic products scribed procedures (16). The radioactive areas of and also the ratios of these sums. These ratios show the chromatograms were cut out and assayed for the degrees of balance between catabolic and ana radioactivity by means of a Tri-Carb liquid scin bolic processes under the test conditions. The liver tillation spectrometer. and spleen had higher ratios of catabolism to RESULTS anabolism than the other host tissues of the animals bearing Hepatoma 5123, but the hepa The results of the radioassays are presented in toma had a higher ratio than any of the host the charts as per cent of the total radioactivity de tissues examined. The Reuber H-35 tumor and the tected on the chromatograms, in order to show the distribution of the CM among the various com host liver yielded approximately the same ratios of catabolism to anabolism, whereas the Novikoff ponents of the extracts. The total activities de hepatoma gave a much lower ratio than the host tected on the chromatograms are also indicated liver. Thus, Hepatoma 5123 and the H-35 tumor on the charts, so that the activity of any of the were very much like the host livers in the bal compounds can be calculated. Although single ances between catabolism and anabolism, but the experiments were performed with each tissue prep Novikoff hepatoma carried out more anabolism aration and each substrate, the consistency of the and less catabolism than did the host liver. results obtained with the various labeled sub The data of Chart 2 and Table 1 show that strates adds to the degree of confidence of the re there was much less anabolism of hypoxanthine- sults. The data for the Novikoff hepatoma have C14than of adenine-C14 by all the tissues except 1Dr. M. D. Reuber, who decided to stop maintaining this brain, and there was correspondingly much more hepatoma, turned over his animals bearing the H-35 hepa catabolism. Again, liver was the most active host toma to H. P. Morris in May, 1961, because Morris had an tissue catabolically, and Hepatoma 5123 and H-35 available supply of A X C strain rats in which the tumor grows. Morris is now maintaining this tumor at Bethesda. tumor were as active as the host livers, whereas Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1962 American Association for Cancer Research. Per Cent of Total Activity Total Activity Tissue 0 10 20 30 40 50 60 70 80 9.0 100 cpm IMPHxR Hx LIVER 4,076 HER5I23 Hllj«5********lllllll*:v8i 4,577 KIDNEY 6,570 LUNG 9,592 SPLEEN 6,086 HEART illlllllllllBBIBI» í^ 4,216 BRAIN I5*illlllllllimilimimi m 9,170 LIVER NOV. HER LIVER 20,714 H-35TUM. 21,433 Key to Charts. XMP, xanthylic acid; IMP, ¡nosinicacid; nucleotide. GMP, guanylic acid; adenine tides, adenylic acid + adenosine CHART1.—Anabolismand catabolism of adenine-8-C14 by diphosphate + adenosine triphosphate + diphosphopyridine various minced tissues. ALLANTOIN INOSINE IlllllllllllllllURIC ACID IMP UNKNOWNS GUANINE XANTHINE 000000000 GUANOSINE + GMP XANTHOSINE ADENINE XMP ******** ADENOSINE