VII. Anabolism and Catabolism of Purines by Minced Tissues*

Home , Adenosine monophosphate

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 tissues 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 experiments.

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). These data the exception of brain and mouse embryo. These show that "substrate quantities" rather than exceptions are not surprising, because the brain is "tracer quantities" were used. relatively inactive metabolically, and most of the Charts ~ and 3 show the extent of degradation radioactivity was present in the unaltered sub- of xanthine-8-C TM that occurred in the various tis- strate, whereas the embryo, like neoplasms, is sues. There was no evidence of anabolism of xan- metabolically and mitotically quite active. On the

other hand, if inosine, inosinic acid, adenosine, adenine-8-C 14 than with xanthine-8-C ~4 or hypo- adenosine monophosphate, adenosine diphos- xanthine-8-C TM. Less catabolism and more anabo- phate, adenosine triphosphate, and diphospho- lism occurred in the Ad-755, S-180, Ll~210, Nov. pyridine nucleotide are considered to be anabolic hep., and HS than occurred in the liver and intes- products of hypoxanthine, more anabolism oc- tines of the host animals. curred with Ad-755 and Nov. hep. than with the Chart 10 shows the results obtained with gua- host tissues of the mouse and the rat. Similar dif- nine-8-C TM as substrate. Small quantities of free ferences were observed between the results for substrate were found in the extracts of mouse in- S-180, LI~10, L4946, and L5178 and the results testine and Nov. hep., but none was found in the extracts of the other tissues. Extensive deamina- 253 78 245 329 200 285 172 145 227 tion occurred in each of the tissues, and the dis- ~~l ~ll tribution of radioactivity is very similar to that observed when xanthine-8-C TM was the substrate -Hx (Chart 2).

3 to= 43 5e toz los 141 I ~ ! !::::. - iJ.i m i - ~ -U.A.

1 4se ohsl fhypoxanthnle8 IIII , ?;for mouse liver??&: and intestine as shown:: in Chart 6 and also between the results for HS and those for L I SP K H B iLU iN hamster liver and intestine as shown in Chart 7. RAT Charts 8 and 9 show data that were obtained CHART 5.--Anabolism and catabolism of hypoxanthine-8- TM with adenine-8-C as substrate. The total activi- C 14 by rat tissues and Nov. hep. ties for the intestines are notably less than for the other tissues for all three species. Because of the DISCUSSION low quantity of radioactive material recovered for Potter (5-7) has presented and developed the the hamster intestine, it is doubtful whether the hypothesis that neoplastic growth results from data for this tissue are meaningful. Relatively loss of a normal balance between anabolism and much less degradation to xanthine, uric acid, and catabolism and that this imbalance is due chiefly allantoin occurred in all tissues with adenine-8-C TM to decreased catabolism, perhaps resulting from as substrate than occurred with xanthine-8-C TM or the deletion of catabolic enzymes. Although this hypoxanthine-8-C TM as substrate, even though ra- imbalance might exist in any or several of the dioactive free hypoxanthine was present. Con- areas of metabolism, he has considered it chiefly in versely, in all tissues larger portions of the sub- the area of biosynthesis of deoxyribonucleic acids. strate were converted to anabolic products with Rutman, Cantarow, and Paschkis (8) and Ca-

=-,,... ill J i ! --! L I A S LI L4 L5 L I A S LI L I N

MOUSE M OUSE RAT

CHA~T 6.--Anabolism and catabolism of hypoxanthine-8- CHArtT 8.--Anabolism and catabolism of adenine-8-C 14 by 0 4 by mouse tissues, Ad-755, S-180, LlO~10, L4946, and L5178. mouse tissues, rat tissues, Ad-755, S-180, LI~10, and Nov. hep.

176 46 28 93 13 128 .i -Ad -- Hx

HxR ~ IMP

m i

i I i i i --Tides i i i --Tides i

i i I i i I

m i i i i i

m I --i

i i .Hx

i I ~-U.A.i

---~-u.A. I -All. L I HS L I HS

HAMSTER HAMSTER

CHART 7.--Anabolism and catabolism of hypoxanthine-8- CHART 9.--Anabolism and catabolism of adenine-8-C 14 by C 14 by hamster tissues and HS. hamster tissues and HS.

nellakis (3, 4) have obtained data that led them to products that were formed intracellularly within a suggest that the extent of incorporation of pyriini- tissue and those that were formed in other tissues dines into nucleic acids is determined by the extent and transported by the blood to that particular of degradation of the pyrimidines by the respective tissue. In the in vitro experiments described here, tissue. Bergel, Bray, Haddow, and Lewin (2) have data were obtained for the isolated tissues. The also suggested that the extent of nucleic acid syn- free-hand mincing leaves many intact cells that thesis that occurs in a tissue is dependent upon the retain much of their metabolic activity. This extent of catabolism of building blocks, and these metabolic activity includes both anabolism and workers suggest that xanthine oxidase might be catabolism. Hence, the use of minces makes it pos- the critical enzyme in maintaining a balance. Pre- sible to obtain some information about the extent of balance between anabolism and catabolism in 79 80 37 81 112 92 80 the various tissues. The application of the chromatographic-radioautographic technic to extracts I -- Gu. of the tissues is useful for such a study, because it is possible to detect and isolate both anabolic and catabolic products from the same preparations. Both intermediary products and end products of anabolism and catabolism are detected, and there- fore information is obtained for the various steps along the metabolic path. The data show the net results of the multiple enzymic reactions occurring i within the cells, and it is possible that differences -J 3R in net results for neoplastic tissues and for host tissues may be more significant than differences in concentrations of specific enzymes. In these experiments only inorganic salts, glucose, adenosine triphosphate, and substrate were furnished to the -Unk. cells, and no effort was made to accomplish maxi- ~-U.A. mum enzyme activity by the adjustment of pH or addition of co-factors--each tissue was dependent upon its own supply of vitamins, co-factors, and amino acids. -- All. Because of the diversity of metabolic patterns of various normal tissues and a similar diversity of " l-i metabolic patterns of neoplastic tissues, it is dif- L I A S L I Ni ficult to compare the metabolism of "normal tis- MOUSE RAT sues" and "cancer tissues." For most experimental animal tumors it is difficult to obtain an analogous CHART 10.--Catabolism of guanine-8-C14 by mouse tissues, rat tissues, Ad-755, S-180, and Nov. hep. normal tissue. One possible approach in dealing with this problem is to examine a number of nor- viously reported results from this laboratory (1) mal tissues and a number of cancer tissues and have been consistent with the hypothesis that the determine if the normal tissues have any property rate of cell division might be controlled by a bal- in common and if this property is lacking in the ance between anabolic events leading to nucleic cancer tissues. Accordingly, in the present study acids and catabolic events that degrade potential several minced mouse tissues (liver, intestine, nucleic acid precursors. Numerous investigators spleen, kidney, heart, brain, lung, embryo, blood have assayed tumors and normal tissues for many cells, and blood plasma) were incubated with enzymes, but few of them have made direct com- hypoxanthine-8-C TM, and similarly a number of parisons between levels of anabolic activity and minced mouse neoplasms (Ad-755, S-180, L1~10, levels of catabolic activity. In the experiments re- L4946, and L5178) were incubated with this la- ported here the data show the relative extents of beled substrate. Since both anabolism and catabo- simultaneously occurring anabolism and catabolism occurred, it was possible to determine the de- lism of a specific substrate within a single sample of gree of balance between these two types of proc- tissue. esses in the host tissues and in the neoplastic As was pointed out in the introduction, in vivo tissues. For this purpose inosine, inosinic acid, ade- experiments did not distinguish between metabolic nine, adenosine, adenosine monophosphate, adeno-

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1961 American Association for Cancer Research. ]u AND ALEXANDER--Anabolism and Catabolism of Purines by Minced Tissues 405 sine diphosphate, adenosine triphosphate, and di- the differences between the values for the hamster phosphopyridine nucleotide were considered to be tissues and those for the HS are due to the species anabolic products, and xanthine, xanthosine, uric differences in metabolic rates.) The ratios for tu- acid, and allantoin were considered to be catabolic mors were also smaller than those for host liver products. Table ~ contains the data obtained by and intestine when adenine-8-C TM was the sub- combining the values for the various compounds strate. (Hypoxanthine, inosine, and inosine mono- in these two categories. The values of the ratio, phosphate were included as anabolic products of catabolic products/anabolic products, are also adenine-8-C TM since the purine ring of these com- given. The ratio for each mouse tumor is smaller pounds is at the same level of oxidation as ade- than the ratio for any host tissue with the excep- nine.) Thus it can be stated that in these experi- tion of brain, spleen, and embryo. As was pointed ments relatively more catabolism occurred in the out under "Results," these exceptions are not sur- host tissues than in the cancer tissues, but it is prising in view of the low metabolic activity of the possible that the ratios would have been somewhat brain and the high metabolic and mitotic activity different for other periods of incubation. of spleen and embryo. Likewise, the ratio for Nov. It is emphasized that some catabolism occurred hep. was lower than the ratios for the rat tissues, in every tissue, and that for some neoplasms the and that for HS was lower than those for hamster values of the ratio, catabolic products/anabolic liver and intestine. (It is not known to what extent products, was only slightly lower than the values

TABLE 2 ANABOLISM AND CATABOLISM OF VARIOUS MINCED TISSUES

PER CENT OF TOTAL RADIOACTIVITY

Precursor: hypoxanthine-8-CTM Precursor: adeniue-8-C 14 SPECIES Tissue

Anabolic Catabolic Anabolic Catabolic Free Free 9products products! C/A I products products C/A substrate J substrate (A)* ' (c)t (A)~ (C)?

Mouse Liver 1 I 2 97 48.5 11 48 41 0.85 Intestine 3 ! 1 96 96.0 11 61 27 0.44 Spleen 30 22 48 2.2 Kidney 13 7 80 11.4 Heart 17 8 75 9.4 Brain 75 i 20 5 0.25 Lung 3 17 80 4.7 Blood--cells 35 65 87 10 I 3 0.30 Blood--plasma 30 70 90 10 0 0 Embryo 65 ~5 10 0.4 Ad-755 i 19 26 52 2.0 2 89 9 0.10 S-180 13 29 ! 58 2.0 0 80 20 0.25 L1210 i 35 14 ' 51 3.6 5 77 18 0.22 L4946 68 21 13 0.6 3 80 17 0.21 L5178 I 26 17 57 3.4

Rat Liver 0 ~ 98 49.0 9 i 34 57 1.67 Intestine 0 1 99 99.0 0 ! 40 60 1.5 Spleen 0 12 88 7.3 I Kidney i, 8 13 79 6.1 Heart 23 4 73 18.2 Brain 80 15 5 : 0.33 Lung i 3 17 80 4.7 Nov. hep. 5 34 61 1.8 1 87 12 0.14

Hamster Liver i 4 5 91 18.2 73 26 0.36 Intestine 2 10 88 I 8.8 29 71 2.44 HS 9 37 54 I 1.4 90 9 0.10

* Inosine, inosine monophosphate, adenine, adenosine, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, and diphosphopyridine nucleotide. t Xanthine, xanthosine, uric acid, allantoin, unknowns. ~/Hypoxanthine, inosine, inosine monophosphate, adenosine, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, and diphosphopyridine nueleotide.

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1961 American Association for Cancer Research. 406 Cancer Research Vol. ~1, April 1961 for some of the host tissues. However, the cubated with labeled purines and labeled purine uniformity of the lower values for the neoplasms ribonucleotides, and the results of such experi- is perhaps significant, and it is possible that a ments are reported in the following paper of this small difference in this ratio could have a critical series. influence on the rate of growth and mitosis. ACKNOWLEDGMENTS Since no anabolic products were detected in the The authors wish to express their appreciation to Dr. experiments with guanine-8-C 14 and xanthine-8- H. E. Skipper, Dr. L. L. Bennett, Jr., and Dr. R. W. Brockman C ~4 as substrates, no ratio of catabolic products to for their contributions in discussions of this work and to the anabolic products can be calculated. Charts ~ and following people for their technical assistance: Mrs. Margie 3, however, show that more oxidative degradation Grammer, Mrs. Patricia Fisher, Mrs. Joan Harrill, Mrs. Ann Dodson, Miss Linda Simpson, Mrs. Jane Hazelrig, Miss Fran- of xanthine to uric acid and allantoin occurred in ces Newman, Miss Tommie Lou Barker, Miss Gall Yerby, the host liver and intestine than in the tumors. In and Mrs. Joyce Watkins. agreement with these results the data of Chart 10 show that, although there was extensive deamina- REFERENCES tion of guanine-8-C ~4 to xanthine by the livers, in- 1. BENNETT, L. L., JR.; SKIPPF.R, H. E.; SIMPSON, L.; WHEEL- testines, and tumors, subsequent degradation of Ea, G. P.; and WILCOX, W. S. Searches for Exploitable the xanthine to uric acid and allantoin was less Biochemical Differences between Normal and Cancer Cells. V. Cellular Conservation of Purines. Cancer Research, extensive in the tumors than in the livers and in- 20: 6~2-81, 1960. testines. These results along with those obtained ~. BEaGEL, F.; BRAY, R. C.; HADDOW, A.; and LEwIN, I. with hypoxanthine-8-C 14 and adenine-8-C 14 indi- Enzymic Control of Purines by Xanthine Oxidase. In: cate that the levels of xanthine oxidase activity G. E. W. WOLSTENHOLME and C. M. O'CoNNoa (eds.), are lower for the neoplasms than for most of the Ciba Foundation Symposium on the Chemistry and Biology of Purines, pp. ~56-66. London: J. & A. Churchill, Ltd., host tissues that were examined. Other investiga- 1957. tors have obtained similar results, but a review of 3. CANELLAKIS, E. S. Pyrimidine Metabolism. II. Enzymatic their results and a consideration of whether xan- Pathways of Uracil Anabolism. J. Biol. Chem., 227:329- thine oxidase is the limiting enzyme for degrada- 38, 1957. 4. -. --. Pyrimidine Metabolism. III. The Interaction of the tion of purines by tumors are withheld for inclu- Catabolic and Anabolic Pathways of Uracil Metabolism. sion in the following paper of this series, where Ibid., pp. 701-9. supplementary experimental evidence is presented. 5. POTTER, V. R. Biological Energy Transformations and the Although the data presented here show that less Cancer Problem. In: F. F. NORD and C. H. WEaKMAN catabolism of purines occurs in the neoplasms than (eds.), Advances in Enzymology, 4:~01-56. New York: Interscience Publishers, Inc., 1944. in the host tissues, the significance of this fact 6. . The Present Status of the Deletion Hypothesis. might be questioned, since most of the purines Univ. of Mich. M. Bull., 23:401-12, 1957. normally present in the cells are probably ribonu- 7. . The Biochemical Approach to the Cancer Problem. Fed. Proc., 17: 691-97, 1958. eleotides rather than free bases. If various minced 8. RtrrMXN, R. J.; C-~NTAROW, A.; and PAsCr[KIS, K. E. The tissues were incubated with labeled ribonucleo- Catabolism of Uracil in vivo and in vitro. J. Biol. Chem., tides, the results might be questioned because of 210: 3~1-~9, 1954. possible differences in the permeability of the cells 9. WHEELER, G. P., and ALEXANDEa, J. A. Searches for Exploitable Biochemical Differences between Normal and to ribonueleotides. To circumvent this difficulty, Cancer Cells. VI. Metabolism of Purines in vivo. Cancer suspensions of sonically ruptured cells were in- Research, 9.1: 390-98, 1961.

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1961 American Association for Cancer Research. Searches for Exploitable Biochemical Differences between Normal and Cancer Cells. VII. Anabolism and Catabolism of Purines by Minced Tissues

Glynn P. Wheeler and Jo Ann Alexander

Cancer Res 1961;21:399-406.

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