Searches for Expbitable Biochemical Differences between Normal and Cancer Cells VII. Anabolism and Catabolism of Purines by Minced Tissues* GLYNN P. WHEELER AND Jo ANN ALEXANDER (Kettering-Meyer Laboratory,t Southern Research Institute, Birmingham, Ala.) SUMMARY Minced neoplasms and minced tissues of the host animals were incubated with xanthine-8-C 14, hypoxanthine-8-C 14, adenine-8-C 14, or guanine-8-C~4; and aqueous alcoholic extracts of these mixtures were examined by means of the chromatographic- radioautographic technic. It was found that relatively less catabolism and more anabolism occurred in the neoplastic tissues than in most of the host tissues examined. The relevance of these findings to the possible roles of catabolism and anabolism in control of growth is discussed. As reported in the preceding paper of this series (S-180), Leukemia LI~10 (LI~10), and Novikoff (9), a study of the metabolism of labeled purines in hepatoma (Nov. hep.) were the same as in the pre- vivo by animals bearing tumors showed that the ceding study (9). The host animals and ages of the patterns of anabolism and catabolism appeared to other transplantable neoplasms employed in this be generally similar for the tumors and the livers study were as follows: Leukemia IA946 (IA946), and intestines of the host animals. The presence of AKR mice, 7 days; Leukemia L5178 (L5178), relatively high concentrations of the radioactive DBF 1mice, 1~ days; Human Sarcoma 1 (HS), cor- metabolic products in the blood, however, showed tisonized golden hamsters, 11 days. The Nov. hep. that the radioactive materials that were isolated was grown intraperitoneally, and all the other tu- from one tissue might actually have been formed mors were grown subcutaneously. In one experi- in another tissue and transported by the blood to ment 27 18-day-old embryos obtained from three the tissue being examined. Thus, the in vivo results mice were used. might not give a real measure of the capacities of Radioactive compounds.--The following radio- the various tissues for intracellular anabolism and active compounds having the indicated specific ac- catabolism of purines. To avoid this possibility tivities (in mc/mmole) were used: hypoxanthine- and to remove other possible systemic effects, ex- 8-C 14, 5.14; adenine-8-C 14, 1.52; adenine-8-C 14 sul- periments were performed with freshly excised and fate, 1.48 and 3.3~; guanine-8-C 14, 0.662; xanthine- minced tissues. This report describes these experi- 8-C TM, 3.69. Adenine and adenine sulfate were con- ments and presents the results. sidered to be equally satisfactory as substrates, and the availability of the compounds at the time MATERIALS AND METHODS the experiment was performed determined which Source,~ of tissues.--The host animals and ages form of adenine was used. of the Adenocarcinoma 755 (Ad-755), Sarcoma 180 Experimental procedure.--The animals were * This work was supported by the Cancer Chemotherapy killed by cervical fracture, and the desired tissues National Service Center, National Cancer Institute, under the National Institutes of Health, Contract No. SA-43-ph-~433, were removed as quickly as possible and placed in and by grants from the Charles F. Kettering Foundation ice-cold Petri dishes. After the tissues were finely and the Alfred P. Sloan Foundation. t Affiliated with Sloan-Kettering Institute for Cancer Re- Received for publication October 14, 1960. search, New York. 399 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1961 American Association for Cancer Research. 400 Cancer Research Vol. ~1, April 1961 minced with knives, 1 gm. of the minced tissue at a rate of 7-8 cubic feet per hour. Following the was suspended in 10 ml. of Krebs-Ringer phos- incubation the solid material was separated by phate solution (pH 7.4) containing glucose (0.1 per centrifugation, and the supernatant solution was cent) and adenosine triphosphate (0.0~ per cent), discarded. The sediment was suspended in 10 ml. and the radioactive substrate (1-~.5 #c.) was of water, the suspension was poured into 40 ml. of added. This mixture was incubated in a Dubnoff boiling absolute ethanol, and boiling was contin- Metabolic Shaking Incubator for 4{ hours at ued for 5 minutes. The methods for separating and 37 ~ C. in an atmosphere of oxygen. During the in- concentrating the extracts, for chromatography cubation oxygen was passed through the chamber and radioautography, and for radioassay of the separated components of the extract were the same as those described in the preceding paper (9). ALLANTOIN ~ URIC ACID ___.-- RESULTS The experimental results are presented in the form of column graphs showing the per cent dis- ~] UNKNOWNS I Xo-I-XoR tribution of radioactivity among the radioactive compounds detected on the chromatogram, and the total radioactivity (counts/see) recovered from the chromatogram is shown by the number HYPOXANTHINE HxR + IMP above the column. Thus it is possible to compare the quantities of radioactivity recovered from the chromatograms of the extracts of the various tis- sues and also to compare the distribution of the | i GUANINE m ADENINE radioactivity among the various components of m mmBB the extracts. The use of the same code (Chart 1) to II identify the compounds in all the graphs facilitates comparisons of the results for different substrates AdR 4- AMP+ ADP+ ATP+ DPN and for different tissues. The values presented for the livers and the in- I testines of the mice are average values that were CHART 1.--Key to subsequent charts. The following abbre- obtained from the experiments with the various viations are used in the charts: Xa, xanthine; XaR, xanthosine; strains of mice bearing the various types of tu- HxR, inosine; IMP, inosine monophosphate; AdR, adenosine; AMP, adenosine monophosphate; ADP, adenosine diphos- mors, since the metabolic patterns were similar phate; ATP, adenosine triphosphate; D PN, d iphosphopyridine enough to permit the use of the average value. In a nucleotide; L, liver; I, intestine; Sp, spleen; K, kidney; H, number of instances replicate experiments were heart; B, brain; Ln, lung; E, embryo; A, Adenocarcinoma 755; S, Sarcoma 180, L1, Leukemia LI~10; L4, Leukemia 1,4946; performed. More experiments were run with hy- LS, Leukemia L5178; N, Novikoff hepatoma; HS, Human Sar- poxanthine-8-C 14 as substrate than with any other coma No. 1. The numbers above the columns ill the charts show the total quantities of radioactivity (counts/see) that labeled substrate, and the data that are presented were recovered from the respective chromatogralns. for this substrate are based upon from one (in the TABLE 1 COMPARISON OF THE QUANTITIES OF RADIOACTD'EPURINES ADDED TO THE MINCES AND THE QUANTITIES OF SOLUBLE PURINES NORMALLY PRESENT IN THE TISSUES QUANTITY OF LABELED PURINE ADDED TmSUE QUANTITY OF THAT PUBINE IN POOL Substrate: Hypoxanthine-8-C 14 Adenine-8-C 14 Guanine-8-C 14 Liver 0.92 0.81; 0.36* 7.15; 7.82* Intestine 0.49 1.42; 0.64, 8.02; 8.78 S-180 1.26 1.46; 0.67 8.80; 9.6~ * Two sets of data are given for these precursors because two different lots of radioactive compound having different specific activities were used in the experi- ments. Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1961 American Association for Cancer Research. WHEELER AND ALEX_ANDEa--Anabolism and Catabolism of Purines by Minced Tissues 401 case of certain of the tumors and host tissues) to thine in any of the tissues. With all three species, seventeen (in the case of mouse liver) experiments. mouse, rat, and hamster, relatively more degrada- Although the quantities of total radioactivity tion of xanthine to uric acid and allantoin occurred recovered from the chromatograms differed con- in the host livers and intestines than in the five siderably for the various tissues, there was no evi- tumors tested. dence of preferential retention or loss of any of the Chart 4 shows the data obtained with hypoxan- components of the extracts by any of the tissues, thine-8-C 14 for several mouse tissues and Ad-755, and hence comparisons of the distributions of radio- and Chart 5 shows similar data for rat tissues and activity are justifiable. The quantities of radioactive compounds that 234 133 138 were added to the minces were relatively large compared with the pools of the acid-soluble pu- 7 rines. Table 1 shows the ratios of the quantity of added labeled compound to the soluble pool of Xo 157 89 7:5 78 78 156 108 82 XoR I i ~ / i - HAMS TE-R CUART 3.--Catabolism of xanthine-8-C ~4 by hamster tissues IIII and HS. L I A S Li L I N Nov. hep. Residual substrate was present in most MOUSE R AT tissues at the time the experiment was terminated, and for all tissues except rat liver and rat intestine CHART ~.--Catabolism of xanthine-8-C t4 by mouse tissues, there was evidence of both anabolism and catabo- rat tissues, Ad-755, S-180, LI~10, and Nov. hep. lism. More extensive degradation to uric acid and allantoin occurred in the liver and intestine than in that respective purine. The pool is defined as the the other tissues. If xanthine, uric acid, and al- quantity of purine isolated following the acid lantoin are considered to be the catabolic products hydrolysis of a trichloroacetic acid extract of a unit derived from hypoxanthine, these charts show weight of wet tissue, and the values for pool sizes that less catabolism occurred with Ad-755 and that were used in calculating the ratios of Table 1 Nov. hep. than with any of the host tissues, with have been previously reported (1).