VIII. Catabolism of Purines and Purine Nucleotides by Sonicates*

Home , Xanthosine monophosphate

Searches for Exploitable Biochemical Differences between Normal and Cancer Cells VIII. Catabolism of Purines and Purine Nucleotides by Sonicates*

CJLYNN P. WHEELER AND Jo ANN ALEXANDER

(Kettering-Meyer Laboratory,t Southern Research Institute, Birmingham, Ala.)

SUMMARY Radioactive purines and purine ribonueleotides were incubated with sonicates of mouse liver, mouse intestine, Adenocarcinoma 755, Sarcoma 180, Leukemia LI~10, rat liver, rat intestine, and Novikoff hepatoma, and the residual substrates and the products of catabolism were isolated with the aid of paper chromatography and radioautography. Less catabolism oeeurred with the tumors than with the livers and intestines, and analysis of the data led to the conclusion that the deereased catabolism was primarily due to deereased xanthine oxidase activity.

In previous experiments (17) with minced neo- tected in the hepatoma, and it was concluded that plasms and minced tissues of the host animals it catabolism of purines was therefore blocked at was found that relatively less catabolism and more steps requiring this enzyme. In the present study a anabolism of purines occurred in the neoplastic chromatographic-radioautographic technic has tissues than in most of the host tissues examined. been used to study the catabolism of purines and This finding was consistent with the hypothesis purine nucleotides by sonicates of rat liver, rat in- that the uncontrolled growth of neoplasms might testine, Novikoff hepatoma, mouse liver, mouse in- be due to an imbalance between anabolic and testine, Adenocarcinoma 755, Sarcoma 180, and catabolic events (1, 18). If there is a deficiency of Leukemia LI~10. The results are in general agree- catabolic activity in neoplastic tissues, then it is ment with those of de Lamirande et al. (5), al- desirable to know at what stages of catabolism though certain differences were found, and the re- there are deficiencies and to know which step of suits for the four neoplasms are similar. the catabolism might be the rate-limiting one. De Lamirande, Allard, and Cantero have compared MATERIALS AND METHODS the activities of purine-metabolizing enzymes in Sources of tissues.--The neoplasms, host ani- normal rat liver and in Novikoff hepatoma and mals, and ages of the transplanted neoplasms em- found lower activities of 5t-nucleotidase, nucleo- ployed were as follows: Adenocarcinoma 755 (Ad- side phosphorylase, guanase, adenase, xanthine 755), C57 mice, 14 days; Sarcoma 180 (S-180), oxidase, and uricase in the hepatoma than in the Swiss mice, 7 days; Leukemia LI~10, DBA mice, normal liver (5). No xanthine oxidase was de- 7 days; Novikoff hepatoma (Nov. hep.), Wistar rats, 6 days. The Nov. hep. was grown intraperi- * This work was supported by the Cancer Chemotherapy National Service Center, National Cancer Institute, under toneally, and the other neoplasms were grown sub- the National Institutes of Health Contract No. SA-48-ph-~488, cutaneously. and by grants from the Charles F. Kettering Foundation and Radioactive compounds.--The following radio- the Alfred P. Sloan Foundation. active compounds having the indicated specific t Affiliated with Sloan-Kettering Institute for Cancer Re- activities (in mc/mmole) were used: xanthine-8- search, New York, N.Y. C 1., 3.69; guanine-8-C 14, 0.66~; hypoxanthine-8- Received for publication October 14, 1960. C 14, 4.58; adenine-8-C 14, 0.54 and 8.51; xanthosine- 407

(2' q-3')-monophosphate-8-C TM, 0.455; guanosine- nine-8-C TM, hypoxanthine-8-C TM, or adenine-8-C TM, (2' + 3')-monophosphate-8-C TM, 0.196; inosine-5'- uc. ; xanthosine-(~' + 3')-monophosphate-8-C 14, monophosphate-8-C TM, 0.182; adenosine-5'-mono- 1.09 uc.; guanosine-(~' + 3')-monophosphate-8- phosphate-8-C TM, 0.163 and 0.198. C TM, ~ uc.; inosine-5'-monophosphate-8-C 14, 0.99 Experimental ~ocedure.--The animals were uc.; adenosine-5'-monophosphate-8-C '4, 1.25 uc. killed by cervical fracture, and the livers, intes- or 1.55 uc. After incubation periods of 15, 30, 60, tines, and tumors were removed as quickly as pos- and 90 minutes ~-ml. portions of the mixture were sible and placed in ice-cold containers. Three ani- added to 4 volumes of absolute ethanol, and the mals were used in each experiment with mice, and resulting ethanolic mixtures were boiled for ap- the tissues from the three animals were pooled; proximately 5 minutes. The procedures for concen- only one rat was used in each experiment. Each trating the alcoholic extracts, for two-dimensional tissue was homogenized by means of a Virtis ho- paper chromatography and radioautography, and mogenizer without the addition of liquid, and the for elution and assaying of radioactive substances container was immersed in an ice bath during have been described (16). In these experiments, however, only 10 ul. of the final aqueous solution ~.-'-:i~i:i:i!:::-2{':i'i'::::~::::~" ALLANTOIN ii!i~ii!i!{!iiii!iiURIC ACID was used for chromatography. If there was doubt about the identification of a radioactive compo- 1 UNKNOWNS ~ XANTHINE nent of an extract, supplementary one-dimensional paper chromatography was performed to establish :~:-:;_:_;:-:;_=~:~ XANTHOSINE ~~ Xa+ XoR its identity. RESULTS The experimental results are presented in the form of charts showing the per cent distribution of ii::i!i::iiiii!ii ,,os,.E ,..,., the radioactivity among the various components of the extract. The total radioactivity, in counts per second, recovered from each chromatogram is A M P ~--- ...... GUANINE shown by the numbers at the top of the charts. The charts show the change of distribution with time. The same code for the compounds is used in :::::;:::::::::::iiiiiiiiiiiiiiiii GUANOSlNE B G M P ...... all the charts to facilitate comparisons, and the CHART 1.--Key to subsequent charts. The following ab- key to this code is given in Chart 1. breviations are used: All., allantoin; U.A., uric acid; Unk., un- The values shown in the charts for mouse liver knowns; Xa, xanthine; XaR, xanthosine; XMP, xanthosine and mouse intestine are average values obtained monophosphate; Hx, hypoxanthine; HxR, inosine; IMP, ino- from the data of the three experiments with the sine monophosphate; Ad, adenine; AMP, adenosine monophosphate; Gu, guanine; GuR, guanosine; GMP, guanosine mono- indicated substrate irrespective of the type of neo- phosphate. The numbers above each graph show the total plasm the host animal bore. Although the values quantities of radioactivity (counts/sec) recovered from the for the mouse tumors, the rat liver and intestine, respective chromatograms. and the Nov. hep. were obtained from single experiments, the smoothness of the distribution homogenization. Two and one-half grams of the curves gives some evidence of the degree of preci- homogenate was suspended in 25 ml. of Krebs- sion of the experiments. Ringer phosphate buffer containing adenosine tri- Charts ~ and 3 show that there was excess xan- phosphate (0.02 per cent) and glucose (0.1 per thine-8-C 14 present throughout the experiment for cent), and the suspension was subjected for 30 all tissues except rat liver. There was more residual minutes to sonic vibrations by means of a Ray- substrate present with the tumors than with the theon 50-watt, 9KC Magnetostriction Oscillator livers and intestines, and there was correspond- using a plate voltage of 135 volts and an input ingly less allantoin and/or uric acid present with voltage of 130 volts. To 10 ml. of the sonicate was the tumors than with the livers and intestines. added the radioactive substrate, and the resulting Traces of radioactive xanthosine were detected for mixture was incubated in a Dubnoff Metabolic all tissues, and a small quantity of what appeared Shaking Incubator at 37~ C. with an atmosphere to be guanosine was detected for the Nov. hep. of oxygen (oxygen was passed through the cham- When the substrate was guanine-8-C 14, exten- ber at the rate of 7-8 cubic feet per hour). The sive deamination occurred with all the tissues, and quantities of labeled substrate that were added no guanine-C TM remained after incubation for 30 per 10 ml. of sonicate were: xanthine-8-C x4, gua- minutes (Charts 4 and 5). Traces of labeled guano-

~o 2c <~ 0 .J 30 60 90 0 30 60 90 i- L I VER INTESTINE o I-

1.1.. 126 165 Ii0 180 478 406 401 406 2'59 185 226 187 o I00

I- I,LIz 80 r

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0 '3'0"6b'gb 6 ' 30' 6'0" ~00 30 60 90 AD 755 SA 180 L 1210

TIME IN MINUTES

CUAR~ ~.--Catabolism of xanthine-8-C TM by sonieates of mouse tissues, Ad-755, S-]80, and L]~]O

2"55 !88 248 240 219 201 255 201 169 208 179 187 >- I00 i-

-Unk. ~ 8o ~

6o, I- ~!iiiiii!ililiiiiiiiiiiiiiiiiiiiii!iiiiiiiiiii iiiiii~!iiiiiiiii!-~ ~ ~ <~ 40- N! i !i ii iiiiiiii -~176 zo. ~iiiiiiii!iiiiiiii!iiiiiiii!i!i!iiiiii!!ii!iiiiililiiiiiiiiii~!iiii!iiii iiiiiiiiiiiiiiiiiii~ii~i ~i~:~~;~i~i:iii:i:i ill iii!:', i!ii !iiiii~,ii!:i:i iiiiliii!i~iiiiiii ~) ,.=, -AlL o. 0 0 30 60 90 0 50 60 90 0 30 60 90 LIVER INTESTINE NOV. HER

TIME IN MINUTES C~AzcT 3.--Catabolism of xanthine-8-C ~4 by sonicates of rat tissues and Nov. hep.

sine, hypoxanthine, inosine, and xanthosine were quantity was formed by Nov. hep. Small quan- formed with some of the tissues. The extents of tities of xanthosine were found for all the tissues, over-all deamination were similar for the host tis- but, since these quantities were small, the com- sues and the tumors. As would be expected if ex- bined quantities of xanthosine and xanthine are tensive deamination occurred, the patterns ob- shown in these charts--much more xanthine than tained with guanine-C TM were very similar to those xanthosine was present. There is a significant ac- for xanthine-Ci4--much more degradation to uric cumulation of xanthine plus xanthosine during in- acid and allantoin occurred with the livers and cubation for each of the tumors, which indicates intestines than with the tumors. oxidation of hypoxanthine to xanthine occurred With hypoxanthine-8-C ~4 as substrate, as more rapidly than oxidation of xanthine to uric shown in Charts 6 and 7, much residual hypoxan- acid. This latter point is of interest, since the same thine-8-C 14 remained in the tumor sonicates even enzyme, xanthine oxidase, is involved in both after incubation for 90 minutes, while most of the catabolic steps. hypoxanthine-8-C 14 had been oxidized to xanthine, Very little deamination of adenine-8-C ~4 oc- uric acid, and allantoin by the livers and intes- curred with any of the tissues, but some degrada- tines. Some inosine was found in the extracts of all tion to uric acid and allantoin occurred in the soni- of the tissues except rat intestine, and the largest cates of the livers and intestines, whereas none oc-

88 120 126 159 I00 105 97 72,Hx

8 XaR 9 i-~ ." Unk. 60. i, 40 U.A.

I-- 98 66 96 s4 i041,0 ll7 136 i29 224 i47 134 ioo ~ .

.5 60

~ :~!iiii~ill~

0 50 60 90 0 30 60 90 0 30 60 90 AD 755 SA 180 LI210

TIME IN MINUTES CHART 4.--Catabolism of guanine-8-CJ4 by sonicates of mouse tissues, Ad-755, S-180, and LI~10

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LIVER INTESTINE NOV. HEP.

TIME IN MINUTES CHART 5.~Catabolism of guanine-8-C ~ by sonieates of rat tissues and Nov. hep.

564 412 532 495 655 487 635 518 I00- :%

80 i xo

60' --Unk.

40- .....~:;;::iil;ii)ii)i;i;ii;iiiiiii)i!iiii_ U.A.

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0 30 60 90 0 0 60 90 AD 755 SA 180 L 1210

TIME IN MINUTES

CHART 6.--Catabolism of hypoxanthme-8-C 9 14 by sonicates of mouse tissues, Ad-755, S-180, and LI~10

curred with the sonicates of the tumors (Charts 8 thine and xanthosine were combined; there was and 9). much more radioactive xanthine than xanthosine Charts 10 and 11 show that with the exception present. Hypoxanthine and xanthine accumulated of the experiments with mouse intestine consider- in the extracts of the tumors, and very little deg- able quantities of xanthosine-(~' ~ 3')-monophos- radation of xanthine to uric acid and allantoin phate-8-C 1. remained even after incubation for 90 occurred with the tumors. minutes, although some dephosphorylation oc- The graphs for the experiments with adenosine- curred with all the tissues. Again, less degradation 5'-monophosphate-8-C TM (Charts 16 and 17) are to uric acid and allantoin occurred in the sonicates very similar to those obtained for the experiments of the tumors than in the sonicates of the livers with inosine-5'-monophosphate. Little or no ade- and intestines. nylic acid remained in the sonicates of any of the As was the case with xanthosine-(r 3')- tissues after incubation for 80 minutes. The pres- monophosphate-8-C TM, less guanosine-(~' + 3')- ence of inosinic acid in two of the extracts indi- monophosphate-8-C TM remained in the extract of cates that some deamination probably occurred at

>- 258 291 296 248 91 199 164 139 343 328 330 317 v., I00- ~,~,~,v~ I I

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20 "

I -AII. 0 ~ ' t Ir--I i I I I II 0 30 60 90 0 30 60 9O 0 50 60 90 LIVER INTESTINE NOV. HEP.

TIME IN MINUTES

CHART 7.--Catabolism of hypoxanthine-8-CTM by sonicates of rat tissues and Nov. hep.

mouse intestine than in the extracts of the other the nucleotide level, but it is possible that some mouse tissues (Charts 1~ and 13). Dephosphoryla- deamination also occurred at the nucleoside level; tion was accompanied by extensive deamination the data obtained with adenine-8-C14 as substrate with all the tissues, and the results do not indicate indicate that probably very little deamination oc- whether the deamination occurred at the level of curred at the level of free base. Again, accumula- nucleotide, nucleoside, or free base. There were tion of hypoxanthine and xanthine with little for- significant accumulations of xanthine with the tu- mation of uric acid and allantoin was noted for mors, whereas with the livers and intestines the each of the tumors. xanthine was degraded at approximately the same rate that it was formed. Very little uric acid and DISCUSSION allantoin were formed by the tumors. In the preceding paper of this series (17) it was Extensive dephosphorylation of inosine-5'-mon- pointed out that, although there was less catabo- ophosphate and extensive formation of hypoxan- lism of purines by the minces of tumors than by thine-8-C TM occurred with all the tissues (Charts 14 the minces of livers and intestines of the host ani- and 15). Small quantities of xanthosine were pres- mals, the significance of this fact might be ques- ent in a number of the extracts, but to facilitate tioned because the preponderant portion of the preparation of the charts the quantities of xan- "soluble purines" of tissues might be present as

--Ad 60

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-., , - ..,.,o.. "; 0 50 60 90 0 30 60 90 0 30 60 90 AD 755 SA 180 L 1210

TIME IN MINUTES CHART 8.--Catabolism of adenine-8-C t4 by sonicates of mouse tissues, Ad-755, S-180, and 1,1~10 >. I- > 177 165 177 177 247 258 224 192 167 212 205 254 ~_ ~oo 9 t . . r

I- -Ad 0 ~" 6C

0 4O l- z bJ 20'

w -Unk. I%. ..:~ Hx 0 0 30 60 90 0 30 60 90 LIVER INTESTINE NOV. HER

TIME IN MINUTES CHART 9.uCatabolism of adenine-8-C 14 by sonicates of rat tissues and Nov. hep.

~ 2

0 30 60 90 0 30 60 90 LIVER INTESTINE .I- Oa" i0 71 73 72 88 104 III 97 104 I01104 I01 136 !

0" 0 30 60 90 0 :30 60 90 0 30 60 90 AD 755 SA 180 L 1210

TIME IN MINUTES

CHART 10.=-Catabolism of xanthosine-(~' q-3')-monophosphate-8-C TM by sonieates of mouse tissues, Ad-755, S-180, and LI~10.

78 112 118 109 96 134 138 97 104 93 82 I01 9>. I00 , @ 4e0 04 I-- P@ '~ @ 4. e 0~ .~ . POO40~e00 9 ~~,T.T~IiIF;%%%',~ 80. =-s 9 9

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,,9 o. 0 ~ I 30 60 90 0 30 60 90 0 30 60 90 LIVER INTESTINE NOV. HEP.

TIME IN MINUTES CrIxRT It.--Catabolism of xanthosine-(~' + 3')-monophosphate-8-C x4 by sonieates of rat tissues and Nov. hep.

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71 III 118 174 84 79 122 117 102 I10 152 147 h I00. 0

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0 30 60 90 0 30 60 90 0 :50 60 90 AD 755 SA 180 L 1210

TIME IN MINUTES Cm~RT l~.--Ca.tabolism of guanosine-(~'+3')-monophosphate-8-C x4 by sonieates of mouse tissues, Ad:755, S-180, and LI~10.

124 124 155 124 116 III 145 93 104 137 145 117 >" I00' ,oeooooooo~ k- oooooooooo 1 boeooeooeoo > oeeooeooeo k.1 o 8( .1.:.:.:.:.:.1.:.:.: ,r .:.:.:.:.:.:.:.:.:.:ooooooeooo, ooooooooo/ .J ,eoeoooeoo oooooooeo I- 60 ,oooeoooo o ' :.'.'.'.:.'" eo ;, ooo oo~ I- ,ooeooo ~176176176176176 u. 40 Ix o ~176176176 IxR ~176176 ,uR I- z :.:." I~J o 20 h-

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LIVER INTESTINE NOV. HEP.

TIME IN MINUTES CHXaT 13.--Catabolism of guanosine~(~' + 3')-monosphosphate-8-C 14 by sonieates of rat tissues and Nov. hep.

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TIME IN MINUTES CIiART l;.--Catabolism of inosine-5'-monophosphate-8-C ~ by sonicates of mouse tissues, Ad-755, S-180, and LI~10

145 156 159 125 1:57 121 99 76 114 169 126 147 I00 IMP ,r~,,~,-..,.I,.,,,,,.,I,,,,,,,,--HxR irn-n1,,,',,,,,,+,,,,,,~ a 9 9 9 9 9 9 9 9 .,...... ;,,.,,~.~ 9 - Ullll ii 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9;)"*'0"~'0" 9 ", Hx 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 I 9 9 9 9 9 9 9 9 9 9 9i.-;~,;1-o~.:..:,.-"" 9 9:.-.:K.*'" i 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 -."~.~.i":." 9 ~ InnnlUlnuUununnnnnnllnnUnnulln ,,,,,,,,,.::.+...... ~...... ~jO:,9, XoRx o lIHl!.'.'.'.','.'.'.'.','.','.','," ...... " 80 9 9 9 ~:' ~ 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 IIIIIIV.mmmmlmmm 9149 ...... ,, .< mmm .)~,,,l;~,.~t~ ..:.:::::: + 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 ~:,.,-.r ..... Hill'.., ...... ,,.,, .~m~ ,, tt:'.-.'.'.'.'.'.'.'...-~-m-.~...... "~" II II Ilm', 9 9 9 9 9 9 ,, 9 ,.,-.J~;~ 9 9IF -:.~ 9 ":':")'"-~*.~. :: .. 60 li II il,' ..... j~-"~.J.~I'O:WZO:'~iO:r mi~' ~ g, I!.:...... ~:~+m::~...+,:~.~...o, ,...... ~.....o...: ...:. IIIlt,~ + --U.A. ~...... ~.:.:"+ ...... :.-..v'..Y..":.~..'",:...;"+' " " ":'"~: "'"'":'"="'";'"" 9~1,~'o I,~ ~\\\,~,~-~\\\\'~ ======...... i+1. i~@ ...... ill I~# I I~!~1111 li~l #I I#vi 14 I~! t 40 I I HII .'.'..-':e-'-<:::o:m'~:~::o::~-o-u-( ; :!i~::::i!iiii]!ii~:ii)i!ii]::iiii]iiii!iiiiiiiii!~!ii~:ii!iiiiiiiiii]iii~i]ii~ii]~i~::: , ,. :;o:.~'o.'-~.:o:-~- .o.-~-m.x~:-o~m-~

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0 9...... IF ...... I ...... I ...... '~ 0 30 60 90 0 :50 60 90 0 30 60 90 LIVER INTESTINE NOV. HER

TIME IN MINUTES

Cravat 15.--Catabolism of inosine-5'-monophosphate-8-C TM by sonieates of rat tissues and Nov. hep.

II :'::!:~:!:i:i:.::~:i:i:!:iL.~I i I I I I i i i i I i i I I I I I I i i i i i I I I I I I i i i i i R I ...... I i I i i i ".""~2~--Xo ... 80 ra ,m 9 ~~..-.:,~. --, ,,, ' 9~:~0~' 60 ~ .,,,,.. ~:~,j~ '.,d

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r :..._..... J 0 30 60 90 0 30 60 90 I'- LIVER INTESTINE 0 I--

h 80 247 i97 271 252 190 199 164 104 125 103 73 I00 0 I I m6'~.'~'-'"'-"~-"-'-",,-~i" ...... " " ..... " .... '~ illllllllllll~'Im' '~" I~''' 9149149 I-- Z 8O bJ 0 iLIIILI]l[It!!!llNlliiiNiiiiiiie ."""-- i ...... ' ' ".__." --...... IIIIItll" I~..,,,,...... :~:...:~.:::.-.,-,':.,~,"....'" ...... ~,:..,-~:,:.,., /1/// 9,.-,:~,:.-.o:,~,-o:.~-o:.=.< ..... IIIIL fl .... :@O':':~r t~ IIIII IL'. 9~:o:.~:~,:.-~. -;-:-o->~:-r ILl 6O IIIIIIH ,, 9 9.,.>-..,,.,.,-..o:,:,:o: ilil','~-~.;~'~.~'!'~,~'~'~'~':~';:.! I IIlll ,',..<~-~s~.~,~.:~:~-.."*'~"~"""~":''~:""~:".:r IIIIIllll, .%'O':'~'O':'q.;:.v;'<.::.~;';-:< .,,, ~'-:-::,'..~':'O:-~.:-:~:-:-:~@;'O:T;:~: ,,,,,. 9.~:,~-O-~,-O:,~,:.~-o-:-.~-~;': I 9 '-~-' "-~:'" 40 84 ...... >...... ,--,-,- lll',~':?.~;'i~-~T;i~'~':~::P.:i-:i~:-~.~:m, ,:~.=~:z~:-:§

I I 111:. 9..",";{G." "0":7 -'.C;'."} ";: :.~,: P~;: :-",:: .."'::>,'.'":,<':'" "~;'.'." ".~,.):.::.Y.. :.:...: ..- ..< ...... : iil.~:~-:-~.-:~i':-:o':-:~o-:.~-:o':'o:~ /~, :o:.=,:o:.=,:o:..-,-,.)-.-,~.,-:.E,.:-:.

0 :30 60 90 0 30 60 90 0 30 ' 60 " 9'0 AD 755 SA 180 L 1210

TIME IN MINUTES CHART 16.---Catabolism of adenosine-5'-monophosphate-8-C 14 by sonicates of mouse tissues, Ad-755, S-180, and LI~10 >- I-.- 116 87 115 154 114 126 122 113 149 129 152 159 .~ I00 IIIIIIII..:.:...,...... ~,:~,u~-XoR...... ,, . . IIIIIIII~,,,,~.,,,,%,.,.,~.!_HxR _ ...... =, 9 = 9 IIIIIIIId- 9149149149149149, , , I ,/ i I ii 9 I I I I I I I l i i i m In will, t. n u n u n u n u n . .,i ,-,~-,'.";-,:~ " i I I I l I I I I l l i I ii i """" 9 9.'.'.'.":~~ """" ...... :-~"~~"~~"" .... , ..~ r,, ,, ,, 9,,. ,.;_-..:;-:ff.:--;-iNT;..i~:g:.N IllllllP:...... , .... l I l 9 I l l l 9 I l i 9il l. 8 0 Ilfl[l'!,:,',...... ' A M P- i[lllll : 9...... ,.~..~iC::::~]~"rili-~..~i~.~.-~T.:;i~.~::~:-'[~ ...... IILIIIt 9 ...... 9...... liE'"" ..<~x.,~: ,,..,,~::.~:,::-::<:,=::::~::.-.,~:-:~.,.~:..~;~ ...... ,, ,,~ ...... , ~, ~.~.:.~ ...... ;~-:;.-.~ 9 "% e , ~ ,72 ! ,~ ~,a Illill.,..,.,,.,,.,,,,,,,,,,~.,,~::~.~i 9 9 9 9 9 9 9 9i~.;l~." 9 "~ IIII1[ ~i flfllL~.~...~,;-..,.~.~-..-:.~.~...-:~,~-o..~~,,,,,,...... o.....,~..~.,,..~ ,..~.~-~...'~;~~,,~ l;,-~-~.~i~:;~i.~...~!~: .\"~ ...... -Xo iiil/, :,, ,,, ,, ,, ?..~:.~.:~.~.E;:.g...:<.~:.~i ,,::~i~r:: ~\x~~ ,'~'~'~T;!i':'~ "r 9 9".".~.~...~.:':...: :...:~:.'..:. P, ,. -. ~::,~ .~: ~!~'~'.. /1/~,".:.:. ::.O:"+j~|174 " 40 ;~::~ ~ :~: ,:~! II/,:.i~:~;i~Y~:-~::~:::.5::~~'~:'~i III, ,~:,..:.r:,..::.@.:..,'.@.:.:~:~:~.~:V .~".'.. ~z ,:~i ~ II .'~:'O:':.~EO:':.~Ee~:'

0 30 60 90 0 50 60 90 0 50 60 90 LIVER INTESTINE NOV. HER

TIME IN MINUTES

CHART 17.--Catabolism of adenosine-5-monophosphate-8-CP 14 by sonieates of rat tissues and Nov. hep.

nucleotides rather than as free bases. It was also tions. If the conversions of B to C and of C to D pointed out that a study of the metabolism of la- occur less rapidly in the tumor than in the liver, beled nucleotides by minced tissues might be com- then the relations shown in Chart 18 would exist plicated by differences in the permeability of the under conditions of a steady state. Conversely, if different types of cells to nucleotides. This latter the above relations between the sums of sequential problem was circumvented in the present study by catabolic products exist, then analysis of the rela- the use of sonically ruptured cells. In order to get tions leads to the conclusion that the tumor is information concerning the net over-all metabo- deficient in the capacity to convert C to D and B lism of purines and purine ribonucleotides by the to C. Such an analysis can be applied to the data tissues, the complete sonicates rather than cellular obtained in the present investigation. fractions were used in these experiments. Also, no Table 1 contains the data for the various sums supplement other than adenosine triphosphate, when the indicated substrates were used. The re- glucose, and inorganic salts was added to the in- sults obtained with xanthine-8-C 14 show that much cubation mixtures, and no enzyme inhibitor or more oxidation of xanthine to uric acid occurred poison was intentionally added. The labeled com- with the livers and intestines than with the tu- pounds were present in "substrate quantities" mors. The data of the experiment with guanine-8- rather than "tracer quantities," and the same C TM confirm the results obtained with xanthine-8- quantities were added for all the tissues; no effort C TM, and they also show that all the tissues deami- was made to maintain optimum concentrations of nated guanine-8-C ~4 about equally. The values for substrate or optimum pH. Although to the en- the experiment with hypoxanthine-8-C ~4 also con- zymologist this experimental system may seem to firm the results obtained with xanthine-8-C ~4, but be crude, it was chosen deliberately in an effort to in addition they show that the tumors convert make possible the comparison of the composite hypoxanthine to xanthine less extensively than the metabolic activities of the various tissues under a livers and the intestines. The data for adenine-8- given set of conditions. No claim is made for the C TM have little significance other than to show that accuracy or completeness of recovery of radioac- there was very little adenase activity in any of the tive compounds from the incubation mixtures, and tissues. The data for the experiment with xantho- the methods are not represented to be methods of sine-(~' + S')-monophosphate-8-C 14 indicate that assaying for absolute enzymic activities. However, the tumors contain less xanthine oxidase, less the precision of the procedure is believed to be suf- xanthosine phosphorylase, and less of the nucleo- ficiently good to afford a comparison of the meta- tidases that act on the e'- and 3'-isomers of bolic activities of the various tissues. By obtaining xanthosine phosphate than the livers and the in- data for various periods of incubation, it is pos- testines. A similar deficiency of nucleotidase was sible to get some idea of the kinetics of the various found for guanosine-(~' -4- 3')-monophosphate-8- reactions for each tissue. C TM, but there was no evidence of a relative de- With the sonicates, little or no anabolism of ficiency of guanosine phosphorylase in the tumors purines occurred, but catabolism did occur; where- or the host tissues; since there is no evidence of a as with minces both anabolism and catabolism oc- deficiency of xanthosine phosphorylase for any curred. Therefore, with sonicates, the balance be- tissue in this experiment, it is likely that xantho- tween anabolism and catabolism is probably not sine is not a major intermediate in the catabolism involved, and it is primarily the catabolic capacity of guanylic acid. With inosine-5'-monophosphate- that is measured. The results obtained with soni- 8-C 14 there is evidence not only of less oxidation of cates and labeled purines are in agreement with xanthine and hypoxanthine, but also of less inosine those obtained with minces, in that there was less phosphorylase activity in the tumor than in the catabolism by the tumors than by the livers and livers. With the exception of Ad-755, the tumors intestines. There was also less over-all catabolism had as much 5'-nucleotidase activity as the livers of purine ribonucleotides by the sonicates of tu- an, intestines. The data for adenosine-5'-mono- mors than by the sonicates of livers and intestines. phosphate-8-C 14 confirm the results obtained with It appears that the same catabolic steps occur with inosine-5'-monophosphate-8-C ~4 and also show the host tissues and the tumors, but that there are that deamination of the adenine moiety occurred quantitative differences in the catabolism by the as extensively with the tumors as with the livers two types of tissues. and intestines. The data for these last two precur- Suppose the catabolism by the two types of tis- sors indicate that the greatest difference between sues is represented schematically as in Chart 18. the catabolism by the host tissues and by the tu- The added radioactive substrate is represented by mors is at the step at which hypoxanthine is con- A, and the other letters represent the concentra- verted to xanthine, and the next greatest differ- tions of the products of the various catabolic rcac- ence is at the step at which xanthine is converted

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1961 American Association for Cancer Research. WtIEELER AND ALEXAN~DER--Catabolism by Sonicates 419 to uric acid. Since xanthine oxidase is involved in genase. Lewin et al. (10, 11) observed that the both of these conversions, one may conclude that breast tissue of mice bearing the "Bittner factor" the decreased catabolism in these tumors relative contained less xanthine oxidase than the breast to liver and intestine is chiefly due to decreased tissue of normal mice. xanthine oxidase activity. The facts given in the preceding paragraph and Several other investigators have previously re- the results presented in this report indicate that ported decreased degradation of xanthine by neo- low xanthine oxidase activity might be generally plasms. Greenstein, Jenrette, and White (7) re- associated with the neoplastic process. This possi- ported that a transplanted rat hepatoma had only bility is consistent with the enzyme deletion hy- one-half as much xanthine dehydrogenase activity pothesis of Potter (13) and would at least partially as normal liver, and Reid and Lewin (14) observed account for the lack of balance between anabolism that hepatomas induced by feeding azo dyes con- and catabolism in neoplastic tissues (17). The re- tained less xanthine oxidase than normal liver. As suits presented in this report for two host tissues stated above, de Lamirande, Allard, and Cantero and four types of neoplasms supplement the results (5) found no xanthine oxidase activity in Nov. of de Lamirande, Allard, and Cantero (5) and sub- hep.; this finding is in contrast to the results pre- stantiate the possibility suggested by Bergel, sented in the present report where different tech- Bray, Haddow, and Lewin (3) that xanthine oxi-

A ---~ B ---) C --~ D ---~ E ---~ F A--~B ~-~C +~ D-~ E--~ F Liver Tumor (E-f-F) >>(E+F) Liver Tumor (D-t-E-t-F) >> (D + E + F) Liver Tumol (C + D + E-I- F) > (C+D+E+F) Liver Tumor (B +C +D +E +F) (B+CA-DA-EA-F) Liver Tumor (A +B + C +D +E +F) = (A -4- B -+- C + D -t- E q- F) Liver Tumor ('HART lS nits and procedures were used. Benton (~) de- dase is the key enzyme in controlling the catabo- tected no xanthine oxidase in S-180 and Ad-755, lism of purines. It is known that tumors are de- and Feigelson, Ultmann, Harris, and Dashman ficient in a number of enzymes that are involved in (6) reported that ascites LI~10 cells contained less catabolism in other areas of metabolism, and the xanthine oxidase than normal lymphoid cells. relative importance of the various deficiencies is Lewin et al. (10, 11) found that a spontaneous not yet established. In this regard, however, it is of mammary tumor contained less xanthine oxidase interest that Haddow, de Lamirande, Bergel, than normal mammary tissue. Leslie (9) deter- Bray, and Gilbert (8) observed that the adminis- mined the xanthine oxidase activities of liver cells tration of xanthine oxidase to mice inhibited the grown in cell culture, of liver cells that became growth of spontaneous mammary tumors. "transformed" while growing in cell culture, and It is not presently known whether the lower of human epidernloid carcinoma cells (H.Ep. #1) xanthine oxidase activity of tumors is due to the grown in tissue culture and found that the "trans- presence of less enzyme, to the presence of an in- formed" cells and carcinoma cells contained less hibitor of the enzyme (4), or to the absence of an xanthine oxidase than the liver cells. Westerfeld, essential activator or auxiliary system. Further Richert, and Hilfinger (15) found that the in vivo definition of this deficiency might lead to a chemo- administration of the carcinogen p-dimethyl- therapeutic means of overcoming the deficiency. aminoazobenzene caused decreases in the xanthine oxidase activities of kidney, blood, and liver, and ACKNOWLEDGMENTS Neish (1~) found that the administration of the The authors wish to express their appreciation to Dr. carcinogen 3'-methyl-3-dimethylaminoazobenzene H. E. Skipper, Dr. L. L. Bennett, Jr., and Dr. R. W. Brockman caused a decrease in serum xanthine dehydro- for their contributions in discussions of this work and to the

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1961 American Association for Cancer Research. TABLE 1 DISTRIBUTION OF RADIOACTIVITY AMONG THE CATABOLIC PRODUCTS DERIVED FROM LABELED PURINES AND PURINE RIBONUCLEOTIDES

PER CENT OF TOTAL RADIOACTIVITY AFTER INCUBATION FOR 60 MINUTES

SUBSTRATE CATABOLIC PRODUCTS Mouse Rat

[ _. Intes- I Nov. Liver Intes- Ad-755 ~180 LI~IO tine Liver tine [ hep. I Xa_8_C 14 AII+UA 94 68 26 1~ 22 99 I 9o E ss AII+UA+Xa 98 88 97 99 92 99 ' 97 I 93 I Gu_8_C 14 All+UA 91 61 25 25 11 92 751 s6 All+ UA+Xa 95 83 93 95 86 96 91 91 All+ UA+Xa+ Gu 95 83 93 95 86 98 911 91

Hx.8-C 14 AII+UA 73 49 3 1 4 100 861 5 AII+UA+Xa 91 77 31 16 24 100 92 [ 30 All+ UA+Xa+XaR 92 80 32 16 25 100 94 i 31 All+ UA+Xa+XaR+Hx 99 92 88 97 94 100 941 78

Ad-8-O 4 AII+UA 10 6 0 0 0 5 1 I 1 All+ UA+Xa 10 8 1 2 0 5 1] 1 All+UA+Xa+XaR 10 8 1 2 0 5 1 1 All+ UA+Xa+XaR+Hx 12 11 7 7 0 All+ UA+Xa+XaR+Hx+HxR 12 13 8 9 0 5 9! 34 All+UA+Xa+XaR+Hx+HxR+Ad 99 97 100 100 99 91 100 I XMP_8-CI4(2'+3 ') AII+UA 41 25 0 2 1 74 12 1 AII+UA+Xa 45 37 3 7 6 74 16 I 6 AII+UA+Xa+XaR 7O 89 18 20 49 82 56] 37 All+ UA+Xa+XaR+XMP 96 93 84~ 84 84 97 93l 85

GMP-8-C14(2'+3 ') AII+UA 56 41 4 4 6 48 36 4 All+UA+Xa 62 74 24 23 46 48 45 23 AII+UA+Xa+XaR 64 80 24 23 47 48 45 23 All+ UA+Xa+XaR+ Gu 64 81 24 28 47 48 45 23 All+ UA+Xa+XaR+ Gu+ GuR 65 82 24i 33 49 50 47 30 All+ UA+Xa+XaR+ Gu+ GuR+ GMP 97 89 851 98 9~ 100 97 96

IMP-8-CI4(5 ') AII+UA 57 29 11 2 2 98 59 2 AII+UA+Xa 79 57 8 15 12 98 81 18 All+UA+Xa+XaR 81 59 8~ 15 12 99 83 18 All+ UA+Xa+XaR+Hx 96 83 49 86 82 99 91 64 All+ UA+Xa+XaR+Hx+HxR 100 98 79 100 100 100 95 100 All+ UA+Xa+XaR+Hx+HxR+IMP 100 98 100i 100 100 100 95 100 1 AMP-8-C14(5 ') All+ UA 63 13 4 I 1 2 97 30 4 AII+UA+Xa 77 50 27 11 12 97 65 22 All+ UA+Xa+XaR 78 51 27 11 99 65 22 All+ UA+Xa+XaR+Hx 95 80 84[ 64 79 99 92 68 All+ UA+Xa+XaR+Hx+HxR 100 99 100 98 99 10o All+ UA+Xa+XaR+Hx+HxR+Ad 100 99 100, 98 99 100 99 All+ UA+Xa+XaR+Hx+HxR+Ad+ AdR 100 99 100, 98 99 100 96 99 All+ UA+Xa+XaR+Hx+HxR+Ad+ AdR+AMP 100 99 100 98 99 100 96 99

Note: All = altantoin Hx = hypoxanthine Ad = adenine UA = uric acid HxR = inosine AdR = adenosine IMP = inosine monophosphate AMP = adenosine monophosphate X1VEP = xanthosine monophosphate Xa = xanthine Gu = guanine GMP = guanosine monophosphate XaR = xanthosine GuR = guanosine

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1961 American Association for Cancer Research. WHEELER AND ALEXANDER--Catabolism by Sonicates 4~1 following people for their technical assistance: Mrs. Margie lase, and Amylase in Normal and Cancerous Hepatic Grammer, Mrs. Patricia Fisher, Mrs. Joan Harrill, Mrs. Ann Tissues of the Rat. J. Nat. Cancer Inst., 2:17-33, 1941. Dodson, Miss Linda Simpson, Mrs. Jane Hazelrig, Miss Fran- 8. HADDOW, A.; DE LAMIRANDE, G.; BERGEL, F.; BRAY, ces Newman, Miss Tommie Lou Barker, Miss Gall Yerby, R. C.; and GILBERT, D. A. Anti-tumor and Biochemical and Mrs. Joyce Watkins. Effects of Purified Bovine Xanthine Oxidase in C3H and C Mice. Nature, 182:1144-46, 1958. REFERENCES 9. LES~E, I. Cell and Organ Culture. Nature, 184:935-37, 1. BENNETT, L. L., JR.; SKIPPER, H. E.; SIMPSON, L.; WHEEL- 1959. ER, G. P.; and WILCOX, W. S. Searches for Exploitable 10. LEWIN, I.; BERGEL, F.; BRAY, R. C.; HADDOW, A.; Biochemical Differences between Normal and Cancer Cells. and LEWIS, R. Levels of Xanthine Oxidase Activity in V. Cellular Conservation of Purines. Cancer Research, in Carcinogenesis Due to the Mammary Tumor Agent. 20:63-81, 1960. Proc. Am. Assoc. Cancer Research, 2:336, 1957. ~. BENTON, D. A. Altered Xanthine Oxidase Activity and 11. LEWIN, I.; LEWIN, R.; and BRAY, R. C. Xanthine Oxidase Tumor Growth. Proc. Am. Assoc. Cancer Research, 3: Activity during Mammary Carcinogenesis in Mice. Nature, 94, 1960. 180: 763-64, 1957. 13. NEmH, W. J. P. Serum para-Phenylenediamine Oxidase 3. BERGEL, F.; BRAY, R. C.; HADDOW, A.; and LEWIN, and Xanthine Dehydrogenase Levels during Liver Car- I. Enzymic Control of Purines by Xanthine Oxidase. cinogensis in the Rat. Experientia, 15:336-37, 1959. In: G. E. W. WOLSTENHOLMV. and C. M. O'CONNOR 13. POTTER, V. R. The Biochemical Approach to the Cancer (eds.), Ciba Foundation Symposium on the Chemistry Problem. Fed. Proc., 17:691-97, 1958. and Biology of Purines, pp. 356-66. London: J. & A. 14. REID, E., and LEWIN, I. Adenosine Deaminase, Nucleoside Churchill, Ltd., 1957. Phosphorylase, and Xanthine Oxidase in Liver Tumours. 4. BRAY, R. C. The Chemistry of Xanthine Oxidase. 6. Brit. J. Cancer, 11:494-98, 1957. Variations in Stability and the Presence of an Inhibitor 15. WESTERFELD, W. W., RICHERT, D. A.; and HILFINGER, in Certain Preparations. Biochem. J., 73: 690-94, 1959. M. F. Studies on Xanthine Oxidase during Carcinogenesis 5. DE LAMIRANDE, G.; ALLARD, C.; and CANTERO, A. Purine- by p-Dimethylaminoazobenzene. Cancer Research, 10: metabolizing Enzymes in Normal Rat Liver and Novikoff 486-94, 1950. Hepatoma. Cancer Research, 18: 953-58, 1958. 16. WHEELER, G. P., and ALEXANDER, J. A. Searches for 6. FEI(~EbSON, P.; ULTMANN, J. E.; HARRIS, S.; and DASH- Exploitable Biochemical Differences between Normal and ~AN, T. Cellular Xanthine Oxidase and Uricase Levels Cancer Cells. VI. Metabolism of Purines in vivo. Cancer in Leukemic and Normal Mouse Leukocytes. Cancer Re- Research, 21: 390-98, 1961. search, 19:1330-33, 1959. 17. ~. Searches for Exploitable Biochemical Differences 7. GREENSTEIN, J. P.; JENRETTE, W. V.; and WHITE, J. between Normal and Cancer Cells. VII. Anabolism and Ca- The Relative Activity of Xanthine Dehydrogenase, Cata- tabolism of Purines by Minced Tissues. Ibid., pp. 399-406.

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1961 American Association for Cancer Research. Searches for Exploitable Biochemical Differences between Normal and Cancer Cells. VIII. Catabolism of Purines and Purine Nucleotides by Sonicates

Glynn P. Wheeler and Jo Ann Alexander

Cancer Res 1961;21:407-421.

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