Thymidine kinase and polyribosome distribution in regenerating rat liver following 5-azacytidine

PRELIMIIqAI~"NOTES

2~

BBA91225 Thymidlne kinese and polyrlbosome distribution in refluneratin 9 rat liver following 5~zocytidine Recent studies on the mechanism of substrate and hormonal induction of tryptophan pyrrolase in rat liver1 have made u~e of the inhibitory effect of 5-nzacytidinz. It is known that this antimctabolite markedly inhibits the de ~wvo synthesis of pyrimidines~ and is incorporated into various types of nucleic acidss,4. The decreased stability of nucleic acids with incorporated 5-azacytidine is believed to be the main cause of the marked biological activity of this compound ~,6. In this study we focused our attention on the effect of 5-azacytidiue on the regeneration of rat liver after partial hepatectomy. Liver rege1~eration appears to be mediated by short-lived RNA molecules, the synthesis of which starts shortly after hepatectomy and ceases at various stages of the regenerative process7. A characteristic feature of the early regeneration phases is enhanced RIgA synthesiss,t, which is followed by DI~A syntbesislo, tt. The change in the template efficiency of liver ehromatin1~is associated with a marked rise in th(: formation of liver ribosomes ts and with cellular division. We were interested in the influence of the applicatior~ of 5-azacytidine on thymidlne kinase synthesis, an enzyme which appears in the process of liver regeneration at the time when DNA synthesis is initiated ~4. At tk,e same time we tried to detect the possible changes in the distribution of polyribosomes in the postmitocbondrial liver supernatant which might have been caused by the application of 5-asacytidine. The activity of thymidine kinase was measured using the same supernatant fractious which had been analysed by the gradient centrifugatinn. As may be seen in Fig. x, the application of 5-azacytidine z h prior to hepatectomy completely prevents the normally observed rise in thymldine kinase, even when small doses of the compound are administered. We observed the same inhibitory effect caused by the application of 5-azacytidine prevailing Iz h after hepatecforay, On the other hand, large doses of 5-asa-z'-dcoxycytidine showed no effect at all This difference in effect provides evidence for the different inhibitory mechanisms of these two compoundsL A typical pattern of polyribeeomes in regenerating rat liver at the stage of maximal thymldine kinase activity is presented in Fig. 2. It is evident that 5-azacytidine entirely abolishes the heavy polyribesome region. In some cases the ribosomal pattern after ~, h of reg..~neration was the same as in normal liver. In an effort to determine to what extent these changes can be accounted for by the interference of 5-azaeyti~inc with the d~ hove synthesis of pyrimidlnesz, the effect of 6-azanridine 16, a specific inhibitor of de novo pyrimidiue synthesis, wasexamined under identical conditions. Even after the application of large doses of this compound, however, neither the polyrlbosome distribution nor thymidine kinase were affected. We may thus assume that the observed changes are due to the incorporation of 5-azacytidine into the newly synthetised RNA's, as is the case in the inhibition of tryptophan pyrrolaseL The breakdown of polyribosomes after the application of 8-azaguanine was observed recently in regenerating rat liver by W~Bs1a,17. He postulates that this Bio~kira. Btopkys. A~ta, I61 (I968) ~77-~79

278

PRELIMINARY NOTES .........

i

i

t

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"IX ................... i -4 "

o

i

5-Azacyti~[ne

~ 4 pmoles/lOOg

6

8

6

~b

~o

Fig, L Effect of 5-a~acytidioe and 5-aza-2'-deoxycytidine on thymidlne kinase in regenerating r a t liver, The compounds were administered ~umoles/xoo g} intrapexitoneally 2 h prior to 6 7 % hepateetomy to groups o[ d~) female rats weighing I35-14o g. The ~tnimals were sacrificed z4 h thereafter. The excised liver was homogenized with 3 "col o.25 M sucroBe in o.o 5 M T t i ~ H C I buffer a t p H 7.5 containing o,o~ 5 M KCI and o.oo 5 M Mg ~+. The homogenate was centrifuged (12 ooo x g, ~o rain, ~=) a n d o.x ml of the sttpernafant was added to a mixture containing o.o2 pmole [2-nC]thymidine, 2/*moles ATP, and Mba+ in 20 btmoles Trie--HCI buffer at p H 8.L The volume of the mixture was adjusted to i m t a n d incubation was allowed to proceed xo m i n at 37*. The quantity of thymidtno 5'-phosphate (m/~oles/ml) was determined niter chromatographic separation, Fig. 2. Changes in distribution of polyribosomes in regenerating r a t liver after application of 5-azacytidioe .To x ml of supernstant, which had been used tot t h e determination of thymldine kinase, deoxychal~te wa~ added (final concentration t,3 %} and the mixture was allowed to stand to rain. The separation o[ polyribosomes was effeeted TM w i t h a linear gradient of ~o-4o % sucrose in o.o 5 M Tri~-HCI buffer z t p H 7.5 containing o,o25 M KC1, ~nd o.oo 5 M Mgt+. After 3 h o f centrifugation in Spin¢o Model L 50 centrifuge (rotar SW ~4, 6 3 0 0 0 × 5 ) ~liquot~ (n) were withdrawn, diluted and their 0.bsorbanee a t 260 n m meazured with a Unlearn SP 700 spcctrophotometer, i, liver p0lyribosomes after 24 h af regeneration; z, after application of 4pmoles of 5" azaeytidtae per ioo g weight, 2 h prior to hepatectomy. d e c o m p o s i t i o n is a r e s u l t of t h e r e p l a c e m e n t of g u a n i n e b y 8 - a z a g u a n i n e i n c e r t a i n k i n d s of R N A ' s e s s e n t i a l f o r t h e i n t e g r i t y o f r i b o s o m e s .

Insl@ute of Organic Chemistry a n d Biochomis#ry, Czechoslovak Academy e/Scianees; Research Insli~ute of Pharmacy and Biochemistry, Prague (Czechoslovakia)

A. ~ I H A K H , VESELA F . ~ORM

t A, ~.]wAIL J. VEZeLq" AND F, ~OgM, Colleaion Cecile. Chem. Commun, 32 (t967) 3427. J. W s g L * , A. ~tHhg AND F..~Og~, Biochem. Phar~tacol,, i 7 (1968) 519. 3 M, JUROV~IK, K, RA~KA, Z. ~onMovh AnD F. ~OR~I, Collection Czech. Chem. Commun., 3 ° {1965) 337 °. A. I~IltA~, J, V~eEL'2 A~rVF. gO1U,I, Btoahim. Biophys. Acta, Io5 (1965) 516, 4 A . I~;lll~.g a:"D F, .~,ORM,Collection Czech. Chem. Cammun., 3o (1955) ~o91. 6 J, V ~ E L ~ , A. I~[IIAK AUD I#, ~Og~, Cancer Rea,, in the press. 7 R, B. Cavucrl al¢l~ B. J. MCCA.~TII~,J. Mol. Bio/,, 23 (1967) 459, 5 M. IruIIONA, M, KOGA A~D I. I.I~Bg~.~AIL J. Bid, Chore., 238 (1965) 34OI. 9 J..ORgws nl~l~ G, BIIAWF,~t~IAII',J. Biol. Chem., ~42 (t967) 8ox,

Biochim. Bioph~s. Aaa, x66 (x968) 277"-~79

PRELIMINARY~TOTES

279

Io L, I, ttv~uT A~I~ V. R. POXXER, Ca.¢¢r Bes., t8 (1958) x86.

at ~. L. R. Btacn~ AtqnM. N. SWAt~FtZLD,Cancer Re~,, ~4 (t964) Xr~tL I~ M, M. '~I4ALERA~ID~. A. 3/ILLEE,Pfoa. ~atl. A ~ . ~ i . O'.S.. 58 (1967) 2055. ~3 5, CnA~DBUmASVL IAE,n~ttAN,J. BioL Chem., 243 (z968) ~9. ~t4 F. J. J$OLLXtMANDV. R. PQ?XEE,Can~e~"Res., I9 (1959) 56z. 15 R. ~. HAttDSCIIU~tACttER,f. Biol. Chem., 235 (x96o) ~9x7. I6 T. E. WI~I~,Bio~him. Biophys. Aaa, 138 (1967) 307. z7 S. W. Kwxu Ar~l~T. E. W~sB.J. Biol. Chem., a4z (t967) 5542, Received May z~'nd, x968

BBA91226 I ~ of uridlne kinase octiviL7 in Escherichla coil • following "r2-phoge inft=ction or chloramphenicol treQtment The infection of Eschcrich~a cotl with 3"2 phage induces the syntbesis of several enzymes which catalyze the formation of deexyribonacleotides and thereby contribute to the precursor pool required for phage DNA synthesis .The synthesis of pyrimidine deoxyribonucleotides has been investigated thoroughly in this system l-~. The universal precursor of pyrimldine nucleotides in biologloai systems is UMP and its synthesis in T2-phage infection has not yet been studied. The inhibition of T2-phage reproduction with the blockers of synthesis of uridyiate, 6-azauracil4and 6-azauridine~ indicates the important role of this mtcicotide in Tz-pbage infectiom We studied the uridine kinase activity (ATP:uridine 5'-phospllotransferase) in E, ¢oli celts infected with Tz-pbage. This enzyme, on one hand, and orotidylate deearboxylase and UMP pyrophosphorylase on the other, catalyze the formation of UMP. E. celi was infected with phage T2 at a multiplicity of 5. After 3, 5, 7, xo, 15 and zo rain of infection aliquots were taken and quickly chilled to 4°. Uninfected bacteria, treated in a similar way, were used as a control. An enzyme extract was obtained by disrupting the cells with quartz sand in o.x M Tris-HC[ buffer (pH 7.40). Thehomogenate was centrifuged at z5 coo ×g for 20 rain (all procedures were carried out at 4°). Then the enzymatic activity of supernatant was tested. The uridine kina~e activity was checked by I~ICHARD'Smodified technique s. The newly formed ~uC]IJMPwas separated from [t4C]uridine in a borate system 7. The spots of labelled UMP were cut out and their radioactivity was determined in a Packard scintillation counter. The uridine kinase activity was expressed as mpmoles of It*ClUMP formed per rag of prorein in 3° rain. Uridine nacleotides were separated by chromatography on DEAE-8I paper in formate-ammonium buffer s. The assay for nridine phosphorylase was similar to that for nridine kinase except that orthophosphate at a final concentration of o.oz M was included and Mga÷ and ATP were omitted from the substrate mixtures. The protein was determined by the technique of LowaY et M.to. Fig. x shows the uridine ldnase activityinT2-phage-infected E. c0/i as measured by the formation of [UC]uridine nucleotides, xo rain after infection the activity inerea~d xo-x5 times as compared with uninfected cells, and decreases steeply x5--2o rain after infection, The same experiments were carried out in the presence of chlorampbenicol (~oo/*g/ml). As shown in Fig. x, addition of this potent inhibitor of Bioehim. Biophy*. Acta, x66 (I958) z79-~Sz