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1982 The influences of cytotoxic drugs and L1210 suspension culture density on de novo purine synthesis Joyce Ann O'Shaughnessy Yale University
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Recommended Citation O'Shaughnessy, Joyce Ann, "The influences of cytotoxic drugs and L1210 suspension culture density on de novo purine synthesis" (1982). Yale Medicine Thesis Digital Library. 2999. http://elischolar.library.yale.edu/ymtdl/2999
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The Influences of Cytotoxic Drugs
and L1210 Suspension Culture Density on
de novo Purine Synthesis
A thesis Submitted to the Yale University School of Medicine in
Partial Fulfillment of the Requirements for the degree of
Doctor of Medicine to be awarded in May, 1982.
Joyce Ann 0*Shaughnessy Hed L/b-
<113 Yi^ 3079 Acknowledgements:
I wish to thank Ed Cadman and Chris Benz for four years of guidance and support and for the opportunity to work with them, and
Robert Heimer for his assistance and advice.
I am grateful for the funding provided by
Ed Cadman and the Yale School of Medicine
Summer Student Fellowship Program which supported this work. 3 ti ‘3n; : iqa i won'-
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ABSTRACT
Pretreatment of L1210 cells with methotrexate has been shown by E, Cadman et al to enhance intracellular accumulation of 5-fluorouracil and the formation of metabolically active fluoropyrimidines, Sequential treatment with methotrexate and
5-fluorouracil resulted in synergistic cytotoxicity. These effects of methotrexate were correlated with increased intra¬ cellular levels of PRPP(5-phosphoribosyl-l-pyrophosphate), the cofactor required for the conversion of 5-^luorouracil to
5-fluorouridine-5*-monophosphate(FUMP), Methotrexate is a potent inhibitor of de novo purine synthesis and it is likely that methotrexate’s anti-purine effect resulted in the increased
PRPP levels and therefore in synergism with 5-^luorouracil,
The anti-purine effects of 24 cytotoxic agents were measured l4 by quantitating incorporation of 1- C-glycine into purine bases. Possible synergism between each of these agents and
5-fluorouracil is discussed based on the agents’ mechanisms of de novo purine synthesis inhibition.
It was found that de novo purine synthesis progressively decreased from peak values in early log phase grow’th to/N-/ of the maximal level during late log and plateau phase growth of L1210 cultures. Increasing concentrations of exogenous hypoxanthine, a substrate in the salvage pathway of purine biosynthesis, were shown to progressively decrease de novo purine synthesis, making increased utilization of salvage purine synthesis along the growth curve one possible explan¬ ation for the observed decline in de novo purine synthesis. J
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'ri- ;.jixr,YXg-i to :ioi^? .•oq'toon!: i*nitst'irna*/p :;LS ?-.‘r..,^: £.5G:1- to nc-32 riijfyrfotf tnci.g'ioiry.a alotsao*? .avacd £jjai'is1wG(n 'stfiG^js •='ilj rto b'g^^scf Dstaauoaib llorxwoioi/It-2. .rroi£ sxesifvnv.a affltyq ovon »Jb to ^nl'iuq cvon eo Tfidjf Onwot aaw &X y. - ■'■ t>tc .'*-^0v- nrwo*:^ aas.Aq 30! rr: sat/Xxiv jf#»q motl b®8a«'Xo#b ^ rwo'X]i uiTsts.'t -l i* ^oI oJaX ifft^ b IdvaX XjmbIj^ibo td^‘ to 5L£Ojrt£sc>X£' to anoid'siJrraortoo igiiiaiotonl iSa’tt/iT'iiJO OXSXwt to an^’iL-q ''oswntsq a^iovlss ©rf^ ni eJ*n;?ecii/a 9 ,i«tIfltrt«xoqX'^ cvon ob ©aetj'TQsh vIovlaae'i^O'Tq of rtvrarra •aia^d^q^sold s^fivlsa to rtold'^fiiXir/o‘ b3a«sior5-. srjiivjfitt , »i8'dri3'rr'^8 »niiuq -aoXqxe ^loiaaoq sm svzyo ^nolB • tfni'itrq .8/5y»d;riT^s aaiiyq ovon ®b ni oaiXo^Jb fesv-r^ao'-? lot nolfM 4 A reduced rate of de novo purine synthesis secondary to length ening of the cell cycle from early to late log phase growth is discussed as a second explanation. Finally, a progressive decrease in cell volume with increasing culture density re¬ sulting in a need for reduced overall purine synthesis to main tain the required intracellular purine nucleotide concentra¬ tions is discussed as a third possible explanation for the decrease in de novo purine synthesis. The anti-purine effects of cytotoxic agents must be interpreted in the light of this natural progressive decline in de novo purine synthesis during exponential cell growth. J ■ j - ■ Ci3i:'i:0‘-sz ovoi\ Iter #i-\a'T ■sr;3X O' ;roit -.■.,■ I£9'. M-j' -•'lo'i'c ; ».J,; U;. •' . io-. o'•riOisXtjX'i b«a3e3«i:b « .Is. i-jtTii T r*.^4 iJ .'-VTOiii fitfiW S;..':jXov XX^^j rti i ■ :■’ .•.;.. i./- ..i:5'iov‘3 'roX beii»i e jrri^’Xy •h' JiA' - . £>-rto'’i :L > .''o.. • -^zi:jLl^oe't. iU nie .v.>: .;.u-:.i.:;.. c ■ _' . ie-.o- ;:'3iriX s Gfs bepsyi-?.ib eX fcitoi tiT -y 3 . r:-j-M s-riX sTj* >■• .rt r^s dni^i/c vfon ■*£) cU eta^'xa# - --* ”• - .2 ;.v -*■ ■ •.-,! ed .T?jj7} jpjfifcais qixo^o^o * oce .vn. lv*-. rri *t;xIo9& »vjtast'i^cj'xq IsTuXs .1X^45 X^iJ’n^aoqx® anl*ta/ 1' 5 INTRODUCTION In treating cancer with cytotoxic drugs, clinical trials have shown that combinations of drugs generally result in better tumor response and patient survival than does single agent chemotherapy. Until recently these drug combinations were most often chosen on the basis of different proposed sites of drug action, different toxicity or simply empirically. Rarely has sequential drug therapy been designed from known biochemical parameters that would facilitate effective drug activation or other advantageous interactions between drugs or between natural metabolites and drugs. Certain sequential drug combinations have demonstrated synergism in inhibiting the growth of animal tumors(l), and the biochemically rational design of sequential drug therapy has become of interest to many investigators. Methotrexate and 5-f’luorouracil have been used success¬ fully both singly and in combination in treating many human neoplasms including breast and colon cancer(2), Ed Cadman et al have shown that methotrexate pretreatment of L1210 murine leukemia cells in concentrations which produced near maximal inhibition of dihydrofolate reductase, enhanced intra¬ cellular accumulation of 5“f‘luorouracil(5-fold) and increased 5-fold the formation of the 5-fluorouracil nucleotides, FUMP, FUDP, FUTP and FdUMP, These alterations correlated with synergistic cytotoxicity. In cells pretreated with methotrex¬ ate in concentrations between 0,lpiM and lO^M, intracellular 5-phosphoribosyl-l-pyrophosphate(PRPP) pools were increased between 2 to 8 times over control values. The increases in :(ti-<'r. s?ix.''>.To^"Yo :isi%s -lACimiD :^hltS9Vt''ii^ ^. ■• .'J :x.;'r-sr;'is 3'ifr.o.tn^riTw'^:rt‘i »va . !<■■••.•■'■ ■’ ^ h n:'::-:'7 ...i'.'-L"/7Ui'» ofTS #iirr^ '! ■ '•••. .:r-!.':(rc;: lirn^ . ■',■ Mrotii'o'vq .rnr^^ri^r i.i''^ '-o *^i'T c,'- ^.fOJ ;> .,.*-„^,i. 'irt) j.Lto-'^' ^r-'s^c'fa ,noi.Toa S*''**^’ ..;; :- . .>r ,■'. i,b, .?nc*i/p«a B« . 'ri: ■"% 1 z ' •!- e **-/ 'i;;." .ii/iiw' ^ .'■'tf-r•,wjn!,i»'^ t.«0:^'iSllrtOOll ■ ■' --. rf- i' 'SnoiT. '^!»7^*‘fc '■ ai,7iJ5tiV.-vV/^,fe .-jfj ««i’tjVjf I'd) • '■■■■ ■ '!■ .r;p,)''!t.' b:v» ^■■'3,riX^;,-.,j :^,^5 i's'iwtar!' rt( yir.' j.i. _ r . :^?i,,. ;^ rr--*- ,ri L/ff isr^d J,i3 a{TOit^J3WT.f'rfW^bts jp/lj -r . .• .rr v. ... “r'i''’ai.Tc-i:'■ ••.-^.x , ■ I ^ -Df,..'-X X.jtTjIft* to. rtTwcs^ ®fl^ *' ' ■ T'"V,;.,': ■, - i.:. -,■ :_v-jb p©« »tails'99Vni ''•-' - '-■^^- ■ - ■• Ll^yr \. r-'^cu i.^ has 9-7‘iixiis*i#D4tfrii? -. ‘ .X-. - ,Tj .'•vj , j r,i fee:* V-^.inia (t>^od ^IXa"! .. .. ;•v,^■^^^r/:■ i .L.:i- i^Ai; 3'aA«*'xcf ifJXbwXijni 8wraj!Xqfo« 7 f'"£y.bar.?5j.x .xa.rtb rwOa1« #7jBf( Xa ti T X ■'".(•'lb ^3'- A mr ^bs-■>.oo n,i iXIeo alxintijsi stiliua r: • M.'Te": 5 la XssJiXJtf : ft ^3--i.c;.T-i: ■ ;. „. ' !:'4.n'].-\:; ) I, ;.;-*x:s;.:''^'.vi/i.--c_ lo aQl^;i[ijmjao3 'tttXi/XJ®?; .-'! ,59.6.11 ,:■ _,i o.wn li:->!■».•/ *0 fToXtatnio'^ *4? ,"{-r,cv; .■-:*• '10a aaoitma HT4H t^QV'^ v-^ibobJ-Si-n r!:iy/ ca:f^eTj^jq nIC&X) al »XtaXa’^#nt« hci.» ^4j^x .0 Aj»’9.vf-'saoJt f M^i'asa-aoo^ iti bsaASioaX Slew uXO'dq' ai 39B&9ionLt #r. r Ttavo 6 , oi' S nsav/t^d 6 PRPP levels and in 5-fluorouracil accumulation could be pre¬ vented by the addition of leukovorin(N^-formyltetrahydrofolate) in concentrations which rescued cells from the inhibitory effects of methotrexate. If the PRPP pools were reduced fol¬ lowing methotrexate pretreatment by adding hypoxanthine which uses PRPP in its conversion to inosone monophosphate(IMP), the intracellular accumulation of 5-fluorouracil was not enhanced. These observations indicate that the enhanced 5-Tluorouracil accumulation within L1210 cells following methotrexate pre¬ treatment resulted from methotrexate's anti-purine effect which led to increased PRPP levels. The augmented PRPP levels produced by methotrexate were most likely the result of de¬ creased PRPP utilization in de novo purine synthesis and de¬ creased endproduct inhibition of PRPP synthetase by the re¬ duction in the purine nucleotides. The augmented PRPP pools were then able to be utilized in the conversion of 5-f’li^oro- uracil to 5“f‘luorouridylate(5FUMP) by orotate phosphoribosyl- transferase, the rate-limiting step in 5-fluorouracil intra¬ cellular metabolism(4). This is the primary reaction regula¬ ting the rate of intracellular 5-f'luorouracil accumulation. Methotrexate pretreatment of L1210 cells then incubated with 5--fluorouracil resulted in a 5-i'old increased incorporation of 5-fluorouracil into RNA and in synergistic cell death as determined by soft agar cloning. Methotrexate pretreat¬ ment not only inhibits de novo purine synthesis leading to increased PRPP levels, but it also inhibits thym.idylate(dTMP) synthesis. 5-Tluorouracil, in one of its metabolically active forms, 5-fluoro-2'-deoxyuridine-5 *-monophosphate(5-FdUMP), ... ' ' I - cl:-'-, nil.03':- ni ,l'*n-3 ^X;. " * ' ^'i''"'* T •• ,iL*-i ; I v:-r *'■'' ’''Itr-i'rox ■. :- "lo .iOXS’lI'^fca S';!' v;xf •xiXi.'l'.i ®,’ir ''‘IJ .'-' ti9uos.9'i Bihviv enSn»ocioo r - • . -' jICGC '^s.^S tu 1 - •^..'-'xyf-TXon:. .>;n >0 atom'll i'- ,e,r- .:v,3 Jt. :vjj xvi; a-^'.T ^f^x&'UoutM i < ■!’’.I) ?X]3''.i'r--onqc. o:« n'aoV'tJ; -X * .;a; aJi r;i g®i v:.rtF.n.-!5 i- i ■ - j i ^ ^ ‘ -Oj? •'rAlu'X.lacjSElfrf'^ : '■ r.ix Xa.X" • W.i " oi •«<* -s .' '. ‘jiJ7.-tT, -O! -*.«!3 ' '.I .iu alfl'Jlif /lOitaXi/IQLtOfl U S' - •(;■ ^;it .i'ii-'T' ■ .-’^ t;#r>'.;aLX . . ■2i»t''';o:'vi c-t Ui-X ffolc -vi- ' V tizi i ■■-'• j ;':....I' “Xiw f»Xj3iXe'i-to.'.: -' 'l‘•.^ ai3to:.-i7" f’? TJi'.i ■-'i .-,tx ■•; : , X:'’’ f-:oxT.crJii'tnI TC'<'bC’Tqljna J>a4j|an eToo-r ’ £»v+'1p y."'.’' ,' . j ? ['j'.anxic cr ‘*i{X iii ftoii'Oi. -•''roj.Ii-? *^0 -^4 ‘ s-*‘i-’■ .To. b-::Xiro •id qr >9ldA fisrtJ m - -'sad xionqt..!- .rr.jc _ .• 'ot llPfiz -ji-i r i Xir:'i5'ijy--C'."1 ! 11--^') jji diir 495:jri»lear5 - .'rr ••r.' :2i si;, i .< r‘.laXuIIl ,.ac'i'aXyflixDOi* 1 vXlac s*! ta^ to ai’S'i adJ’ .in'iw tosX'Sdt'oriX n^dr >;/: ro " ra tr^mXsa^Xinq a7£x»'n^oftti .roxTsrzoqrcoorrl ;u dXci-^, i .nx -LlojrxjO"¥OMX’5> dtest XXhc osj'ai,'?''. xf-fv? rU Pcis o?A- sio^iif^Ttu/Xt-i?, 1 sxa'x7on^^^ ,:^ninclo Tton v:cf b«rixa5'i9^«l> 1 c& ^albs«£ eisa/f^-fiYs swiTiyA ovon 9b Sopi ^fr.t {^4il'rb}e>Ti5Xx^^i3!Y;i.j' 0«Xcfidni oeX^a tX ?yd ♦.sXavaX b«8«®'ror •ivi^ofi ^tXijeoiXoJ^j-sjTi sc-x to » , (‘tMUf3'^-c)«'ifaAqE0«qoAOffl[-*^~«Ai&iix'V7Jfoafc-'*S-o'ioyXt*^ .afUA? 7 also inhibits thymidylate synthesis by inhibiting the enzyme, thymidylate synthetase. Since the increased formation of 5-FdUMP is unlikely to substantially increase the inhibition of thymidylate synthesis induced by methotrexate pretreatment, the likely mechanism of methotrexate - 5**flT-iorouracil syner¬ gistic cytotoxicity is the enhanced incorporation of the metabolite fluorouridine triphosphate(FUTP) into RNA(3). This proposed mechanism of methotrexate - 5-TluoJ^ouracil syn¬ ergism was tested in monolayer, cultures of human colorectal adenocarcinoma, HCT-8 cells. Sequential treatment with metho¬ trexate and 5“Tluorouracil resulted in a 3-fold increase in total intracellular 5-Tluorouracil accumulation, increased PRPP pools associated with enhanced 5-Tluorouracil metabolism, and synergistic inhibition of human colorectal tumor cells' clonal growh:h( 5* 6). The hypothesis of Cadman et al is that methotrexate enhances the metabolism of 5-i’l'uorouracil to fluoronucleo- tides which are incorporated into RNA, by inhibiting de novo purine synthesis. Other inhibitors of de novo purine synthe¬ sis, therefore, might prove to be similarly synergistic with 5-fluorouracil. The purpose of this work was to quantitate the degree of de novo purine synthesis inhibition by several purine and pyrimidine analogs and antagonists, nucleosides and other cytotoxic agents by measuring the amount of 1- C- glycine incorporated into adenine and guanine. Additional work performed by Cadman et al investigated 5-fluorouracil intracellular accumulation, PRPP pools, fluoronucleotide levels and cytotoxicity resulting from sequential treatment ' .■ •’i " V'’V.'; •:J o®i :r':r h:\ ' , y^aUffnd^dX'^ '.- i i’?'-:r.' V' . ^ - ■ ."uj,2j':iV5 o* ' -vii^ .;,-' (IT'^ .3i;39.rt3'nYS fi 1 ■ >i r_ ■-. t>r:r 5^’ <:7xolxcJoty:^> ^itEi ■ .'.no*. : '■ - - ■•,r i.-M. ^^rr; ^ 3a^ui lfc‘3P070'iq alii .. -^uv'-T T„u) Bjfw fwaijn ■“"-'’i ■ .-<1 • ■■-•'‘^ , L-:-., „■-! . •■:! ’ 0 ■ "’OH ♦awoiTjts'iAiOonat *■'■' ■ ■ , ... •■ • f -'joroL' i'l-? bns^ 9fax9i ‘ •'. ■ 03:.' ' ■-; Tai^''XyaaTEtiii lato -'■''' ' '■ ' ■c.--3»A : .'^ aXooqr ‘uxi.* 'ji'*.'r i,., -••*:.•( ' ^ -.:>xXi^’i.'‘i; I' *5.1 •^3Xa^«^v^^ tm # ().lJ“v»ois ij*noX - '■■■"^'‘i i .'•'1..*!} 'to >iX8Sfttoqx^ ~ ''"'• . . ■“ "■ 'io jTraH,od«i‘9*'3 Baonjtrfn 0'3:--rToqTOon,f auM (ioldw a^bi I ■■’.-.'L ■ j -'io.'. :3.'talaeiTitys aaiiu . •■■* . , sYola'iart;)- ,aJt ■■ - ''■■■'■ * - ■»/ ;■■ *:o «.if .liojrxKoiQwit- '-x-riX^'u* - :2i 3**-’:.: .^5 .-l.t tb lo 9e^^»b trt 3.- ?:■■:> ,a?c;uv 3?"s aijs 'Tvyjlfi’iz <»u :btmir[X^ bx,:j| tftlaui '.' 1.3 -‘■jowOjns 3riJ olKo^toT'^d Tiito fcn, i Dc.“. ,9n.t^ix;.3 orti.ri'oioi otni: X)*J‘iin[oq-iooni 9Ctloxl\ I iO'67/.'O'lOl’- 1-c 057£^iiJatt*.''rT,t £b j*® ii:SiTIO*0 taiTXQl*l0Cj jIioi t.r51^c«Idii'rTO'YO)u.n . ♦nO'i^’jaXwwwooB 7aii.fIi«Q4nJ'n dnttn*?-£©’?'t XsxXf?-£» of L1210 cultures with several anti-purine agents and S-fluor- ouracilc The combined investigations were performed to determine whether cytotoxic synergism of 5-Tluorouracil and another anti-tumor agent was related to the agent's ability to inhibit de novo purine synthesis. In addition, this study investigates the relationship between the growth density of L1210 suspension cultures and the rate of de novo purine synthesis, examining the purine salvage pathway as a possible alternative method of purine synthesis at high culture den¬ sities, Cell cycle length and cell volume are also dis¬ cussed as factors influencing the rate of de novo purine synthesis. ‘ jfjq-j'/T® Jgn-?V‘5.'{ .'.tiw (59TU/tl0O 6i%II 1 ‘--■r ,"*. 0' ^9^ivdWoo ^.-IT .liojrxx ■ •'0 3 u ■•T'’-' ; ■ . ‘Ti’-jiB,"! oix : •'v " co -larfcTsriw li'.'a oT oo’' icntuc-*l9(itOX zl.ir ..ivi. :Dvon o6 iic^frirti < ■• Itv^n-T^ 9rf7 Ti-; .v5?it arj ?-..j-.jsjvrri OL-x Cv'oit ,*. v.->v u^*£;a ?^£.--X«-■ 9ni;“:i;7 f.-'u 'arJj.ii-yi^x«» .3;i89i ~hS^ '7-2,*.-a ■- ■’. ^’Mulzv It': iili£- '-'iOV^ XC«»0 ,3#i^J !sr7.’-^q ovDi' ': a ‘snd' aa ^aaai alaBiitns 9 MATERIALS AND METHODS Cells L1210 murine leukemia cells with a doubling time of 10 to 12 hours were maintained as stationary suspension cul¬ tures in Fischer's glycine-free medium plus 10^ horse serum and were grown at 37®C in a 5% CO2 atmosphere. The stocks were diluted twice weekly and monthly cultures for myco¬ plasma contamination were negative. For each experiment testing a proposed anti-purine agent, cells were inoculated at 1-2 X lO^cells/cc and were harvested in the logarithmic phase of growth, generally at 2-5 X lO^cells/cc. Staining with trypan blue demonstrated^ 99% viability until cultures entered plateau phase. After 120 hours in culture, viability was reduced to < 70%(7). Cells were counted with a model ZBI Coulter Counter(Coulter Electronics, Inc,, Hialeah, FL). Drugs 1-^^C-glycine(20mCi/mmole) which was used to evalu¬ ate de novo purine synthesis was obtained from the New Eng- 1U- land Nuclear Corporation(Boston, MA), 8- C-hypoxanthine (56mCi/mmole) which was used in assessing the purine sal¬ vage pathway of L1210 cells was also obtained from the New England Nuclear Corporation(Boston,MA), Methotrexate, N- (phosphonacetyl)-L-aspartate(PALA), vincristine, 5~azacyti- dine and 3-deazauridine were provided by the Drug Development Branch of the National Cancer Institute, Bethesda, MD. Pyrazofurin was provided by Lilly Co.(Indianapolis,IN). All other compounds were purchased from Sigma(St, Louis,MO), saoMSsy. .,?A.aiA'.-3TA ails lo il-T,■'■'•' iixiTrgttL'si OiStI -lif ' rtcI-in'XTQl/? 'x.T:ri^O.C.+ Bv S SB "> '.iTL-Oli SI OS' 0 .rtb'tya ^eiOi"! -0. wwXq .Msttjrx 35-*:l-,9ni'-•/!’1 ' ’'iii'.':;* s-al ••tfci - ■-■ouS'-? 5."iT . c'f•3;'^q3cm.r^'3 ,^0^ Ilf'f ’ /ti "'''Vf /-• '''./OTr^ d'lww bn -oo'^ni loi stt-rvvlL.D ^ naSwIib /Bia J‘fT.?*i,’Ti'l 1-^.'S[cf^oixi 3ri^'vq-1 f^aoqoiq s 4J1 0.HCi* nl dbj-a ^.-t® v : cIj-^ 01 X S-f S : - T ■<■-•■ i '•:-CX£'i jXia* iP.r^.'OTt® to !r* xj IX*ny * ^ t-1»oiti iiii.q'iCTS' dti Y.ti/.i'fjsiv , ii'Xi/Sljj*’ rj 0:'-. »'■ ' ^ .^csi'-iq .-jaaS'^Xq baiaS^r I^&oir, ^ '-(tiw baJTiJOO :y"!0W OS boOWboT 9t .(J'l iffeoLsiH „.c/tI * '.'O'-xSouX^ t:9' 'r-^TiT^joD z^tLuoO II ' ill -uIiitY9 oj basw a^w rfcX <•:..' ( yloiron'v.: 0«02 ) -I / ws!X 9ilt f>or.!r.j'-Xo' pfp.arJS'axfe ovoft ab #S d l; dnIrfJ’nA7Cf>qf//f-C -8 . ( Af/I -laa sniTwq ^niaaaaas ni toeu 3bw (al t.'9M flflS m'dit b^rTiSS^cfo 03X3 aaw aXxeo OlSIJ >0 •lJ> -*»( , ataxaTtoriSsi-'T . ,nocr2oii)rcoixi5rroq’ioX isaXoi/K bajaXyi ,&rtiSaiqonlv , (AlA‘i)eTBSiBqs3-iI-(I'^taojflno/ /nte«aqoiavaQ O’rt?’ \;d babi'voitq stsw ^rri fel^tyfl^eob^X »nJ iSbaadS®® qeoiiitO X«fioiSiRM ari-t lo rlonsn , (HI .eiXoq&njsilxrrl) ,o0 'iXIiX bebXvo'iq «;«w n2"!«to5ai\ .(0M,8j:t;oJ .SS)4!aTai^ fftori’i ■D®a3dq'it;q «rfaw gbrofoqifloo lartto IX 10 Growth Curve Logarithmically growing L1210 cells were inoculated in¬ to Fischer's medium plus 10% horse serum to achieve a concen¬ tration of 1 X lO^cells/cc. Aliquots of lOcc were trans¬ ferred to sterile culture tubes(3 tubes/time point). At 24- hour intervals, the tubes were carefully agitated and 1-cc aliquots were each diluted with 9cc of 0,9% NaCl, The cells were dispersed with gentle pipetting and cell numbers were determined with the ZBI Coulter Counter, De Novo Purine Synthesis Determination 14 The incorporation of 1- C-glycine into the purine bases of nucleic acid was used as a measure of de novo pur¬ ine synthesis. By a modification of Henderson's technique (8), lOOcc aliquots of L1210 cells at various phases of sus- 14 pension growth were incubated with 1- C-glycine for 2 hours. In some experiments, cell cultures of 2-5 X 10-^cells per cc were incubated with anti-tumor agents for 1-2 hours 14 before the 1- C-glycine was added. The cells were centri¬ fuged at 1000 X g for 5 mins, and were washed in cold phos¬ phate buffered solution(PBS) with ImM glycine(unlabelled) three times. The supernatant was discarded and the cell pel¬ let was incubated with lo5cc of IMHCIO^^ at lOO'^C for 1 hour to depurinate the nucleic acids. Following centrifugation at 1000 X g for 10 mins,, the acid soluble supernatant was treated with 4N KOH to precipitate out the perchlorate salt. The supernatant, containing free purine bases, was neutral¬ ized with HCl and was analyzed by high pressure liquid oo I t-'i..''v •-1. ’’I 5;-*^:'cij / .,-j'<:rnr3i-rp^A4 o. N ■.■•■'-' -- •,<'"! 'ii-lc *i’ iartosi'^ c - ■ . '.v ?r;0: . -;7': . ■..' f***. Zr-",!'’-3.' X : lo rtoii'ffi .. ■ •.. ^-r . _ .•- ot Cxdne' .IV ' -I'■ 7::' , i. ov^s'ni lUO * -• *• • ‘ I* . 1:. ’C vf“>'v yr-yL-piX ■ ' .-- ,ir^Iw itjo '•!0: ~ ■ rcl.t '^i'V ■■ ' ^.:-r: ' - i': roo'I ovoJl e » .7.- mT' . '-w fc;_: '-i-Ioun Ic 8^z£ - k ^ - r, ^ ■ .4 ^ \ j Z ■■'‘ O'.M ,- ’1’: .af Esjljrfryri ^rr • to -"otifrJ'iS ooOCi <(0 : .. " i’ .V ■■• -•’ 9w rt;wo*x,^ froXart9 i r . •■_ ■ ^ : -•.» Xi : B(rc.~: ft! ,artiJO ; . ■'. ■ •'O'-.- ■ '• • V ■ ■ 5, xx^cL-ortr ftiaw 0*5 la • - -> '■. If'Ub 50,' -9**"-1 artJ a-xol* .. .. -■ 'll 7 J ' .' L-; I'T •. ' i 3 X. 000! ra ba^if • : ^ ir...;': 'r.' ■ _. : ^ J , •■, ioa atari ; . ,. - 'T : w , , 1oi 5^'. 0 j liT . a i-n :• T aertrt i;ni.i - :rf :"0C: ‘5 'ta . , : 'i *v/ ;‘5X6CSWcrri a.8«r ta .7'7r.v^. :l'i.'^rr?5o ^ftx’.volXo’^ .. - o '^Ioyrt 9Jsrti'ii;q9fc 0 Tn.£.T.7,'nCr-CJ/'S iiiXi.'.'OS Jbx 05, *s n ,.3rtiin 0! 'lol ) X 0001 J l£fi S.T •Oi-IO'I'5'T i-d^ tlfO •SX'J.i'i.q xo97q oj H0?i W'i* df^yt feataa'i i'isw ,:iBe3<< oa'il. ^ai'ii^.ia^rjoa *^.t53enrx^qs/® ad sxt-nxi 9-1:. >a4'io ftairf vcf t^sx-sct£. '.r-'f ba^ lOK ciSlw ooa 11 chromatography(HPLC) on a Partisil ODS-2 column(Whatman, Inc,, Clifton, NJ) eluting with a O.IM sodium acetate buffer(pH 5»5) and acetonitrile as a linear gradient from 0 to 7.5^ over 30 mins, Nonradiolabelled adenine and guanine were used as markers to determime column retention times: Void volume containing glycine that was not incorporated into purine bases, ^ 4—7 mins,. Guanine ^ 12 mins, and Adenine ^ 38 mins. Absorbance was recorded at 254 and 280nm, 0o5nil fractions were collected and the radioactivity was quantita¬ ted, The amount of radiolabel incorporated into adenine and guanine was calculated as pmol/lO^cells/hour, was expressed as a percentage of control and reflected the de novo purine synthesis rate for each experimental condition. The control cultures for each study were harvested for testing at the same culture density and v/ith the same nutritional status as the experimental cultures. r.r: V Tit'l >. M':i 'P'. :t'":- Mt >.0^ B C'VH •Ci'V'i. vr./:: o:t «,.' ,:io’^:t T s aw. eXi'id'ifioteofi bit i,-.‘-'.: ':.yu 4tu:7r>;^f »ei^X ■ '•r:;9('fli'^ f-f ;i -tx-X” . o.* ^T^'T.w'..r:. ■-;;“n zeav s>ni,a^*X§ ■_ •. .■;. „ ,oi i'l ’,': '^TX^UU , ■ virtijj '[•-' . '.•■■i>i: ,■ c.-:.E 'r""X ■' ■ ssw 'i?oai”Ci’ToaCfA »)(inX ;;.j;'::-^ \-.3" ^TX^-OjEO tbS'' v.ir DfTfi foatt‘i'XXOO ancIXOBXl •'ni-i' , '-: ■- ‘ i "ST:";'~:ou;“o:,r.„^ '.'-j-.i sa ;![:a IxniofliiJS #fee . ,■'^o-i ... X; ' ycIfw «*w aninaw :7 . -vr;.: ■ X =•.-.■ "jX I'l!9^1 f,..i£‘. . j'ld’npO '*‘ 0 a^^Xi'-feoTS^ « a , - ' ‘-.iX *, 1* ^:,, :'-r? IeX:-rUtr. _£;qK9 TLO'i aTii'i, alBdX.STitTC ^rn:" , £; .xirs':' toX ■5'iaw '/bLuts .nai?!'« ’toJ. -1," •-' * ^ ~ z -1' j ' L 1,!,-■*'■ iii'2 isXn©«aX‘3:» RESULTS Effects of Anti-tumor Agents on de novo Purine Synthesis The effects of 24 anti-tumor agents on the incorpor- 14 ation of 1- C-glycine into purine bases are shown in Table 1 and in Figure A, In general, the purine analogs and antagon¬ ists such as hypoxanthine, inosine, adenosine, tubercidin and 6-thioguanine were found to be more potent inhibitors of de novo purine synthesis than were the pyrimidine analogs and antagonists such as 3-deazauridine, pyrazofurin, PALA, Ara-C, thymidine and 5-azacytidine. The RNA and protein syn¬ thesis inhibitors, actinomycin and cycloheximide respectively, were also found to significantly reduce de novo purine syn¬ thesis. Effects of Culture Density on de novo Purine Synthesis It was observed that the ability of an anti-tumor agent to inhibit de novo purine synthesis was dependent on the density of the culture that the experimental cells were harves¬ ted from. Table 2 illustrates that, in the case of 5--i*luoro- uracil, the anti-tumor agent could inhibit de novo purine synthesis to a nearly identical percentage of control given the same experimental culture concentration. Seen in the case of methotrexate, however, is an inverse relationship between culture density and the degree to which this potent anti-purine agent can inhibit de novo purine synthesis. The 14 control value for the amount of 1- C-glycine incorporated into adenine and guanine in the methotrexate study per¬ formed at a density of 1.2 X lO-^cells/cc was 25.5pinoles of -?eq'!':joai ^lu* ac £'lauf: -.-l.- 1a : '.lisT ni -vori^ t o.'nx lo noi^ -rr'.;,3B.-fts '-r-ji r.3.'!i -... •'.-o' r-'J , rl .A ft,t Jw ~ii.i c'i Ai-i? rja doija Ri^i !.:.inj TAyr,-?*: :~;-v.-n 3." b.'^-aZ fcfi b^oL&i.'.£ 'erib:,:il-z',_-i ■ at-w Aaii.* c;i =^^aJ.Tx;s 'vvpn aft ^ L - . •'i - VV-. V , t . _ i XJ I .vt; ■ BE£Ab-C .':c-;;^ bn i :aJO'l,..T Li'~ -noM ^^riT ,9iTrb£?-'csj:B--i. br.'s “f:'! 1; , •■ fev*,"'vt'q-ie ?: &bi ■^n.l’i.uQ j ’ ■-'•^ -"’ aoilrrrix^ oX ci.'u/o'V oaXfi aia .siHaii : --. ,-tn :,■. O >q Sfaull %p -Jl.: cew vfi: ro -neorr^-.-' . ahI't-jc -vor . r• :i^Lrinx ot Sr,9-g S#v srf i; -?• U. .-. ■:':. :--:^r9 3^7 ,: .s/< • •.--^ s-1t l:o -r.-TC-. J ~l , 3^X0 s.'- ;■ ; . ^ ..\7 39TjB'lJ2i. liX H'Xb To dl ♦fflO'll be. ■■'.ii'Ti.q QV7^ xxJxncJ L-Jjuco crxfdgx -toi'u. ;r-. ^tX3 triJ- ^IIos% .lavi-a lo'i-r'..: 'io *X9C- idrir-db. .:-‘sdfi 3 ;>j- Ei ?rlt nx ustiS ii'r.Tfrac'.'TCc -.cc v-si"*«4I8 ad; crldsnox*i>istT t-3 'ij. ji^^vx'wofi , sscaTjod^ar,? lo aasi tasfc^q iiidT noLciw ot vsTsab 9cif ijr_s ^tf’l^ndb am/^Xyo drfi VsiaerlJTT^E 9'ixn:" ovorr ab ft^ilairtl aso *fj»^ji srJiTtvq-x^fL •* t- w»i'B>^o<5pEOOfrl BftXovXib-O ~'- a lo txufOfns arii# toIc ai/Xav Xot^rto: -Taq '^bud’8 Bitzf itX tfiX/iA'-:^ bfta ♦nirabA qcfn, lo a»Xo;mic.es aavf oa\2XX»p^0i X S,r lo x^iansb b t£ bwoVo: 13 14 TABLE 1 1- ,C-glycine Incorporation into Purine Bases 1U Zxnosure Time Concentration 1- C-glycine into Adenine and (hrs.) ^M) Guanine/lO°cells/hr (% of control + 2,1%) Purine Analoe:s Hypoxanthine 3 10 13.0 Inosine 3 10 9.4 Adenosine 3 10 20.7 6-mercaptopurine 3 5 58.7 6-thioguanine 3 5 15.9 Virazole 3 10 39.1 Tubercidin 1.5 10 17.4 MMPR 3 2 61.0 Pyrimidine AnalOi2:s Ara-C 3 1 84.8 Thymidine 3 100 72c5 Pyrazofurin 3 5 93.8 5-azacytidine 3 30 62.6 5-f’luorouracil 3 100 20.7 5-fluorouridine 3 100 26. 1 5-i‘luorodeoxyuridine 3 100 53.3 Others Hydroxyurea 3 100 90.0 Methotrexate 3 10 11.3 Leukovorin 3 1000 108.2 Vincristine 3 10 15.2 Actinomycin D 3 10 g/ml 8.7 Cycloheximide 3 10 10.9 Azaserine 3 10 14.1 L-alanosine 3 2 79.0 PALA 84.8 * * n ■ ^ ■.r::': i. utfii ;T'Ti::; 301.00^1 -f ■ JUlgjy • i • -o * 'f''r £r.". '.V mi ‘ .> o (. ai.i) -- • , dt stjbx6t:pi gn!’ wor •■' t ••i r r 94TX aorngi giii : 1C ^nlrusL lOirfT- . , eIdJrxJ nl bXa'igdi f , ..:.[mA atrLtMllj i: - . ■ li'XJ.'OLOfCT' , :.:d r r ♦ajruro^ot'Il- .OS ,'' - 9/ti ^si’^t/o^ou^^ i .ds 1 ^ 1 >'l’! r bl'Tuy xoe tWTICuX1> " . r ' V’v'J JS3^ 0.0^ 00 X • ♦#jt3trT^ortt e.n o: f s.ao! ooo t nliovajii.' 0! «: C .tia'cworri^^ ^.3 l.Ai> Of c or n • f)l«lx*((oXo 01 c «nX‘X«s£ % 14 Table 1 (continued) Abbreviations: MMPR, 6-methyl-inercaptopurine ribonucleoside; Ara-C, 1-^-D-arabinofuranosylcytosine; PALA, N-phosphonacetyl- L-aspartate. 14 The control value of moles of 1- C-glycine incorporated into adenine and guanine/lO^cells/hr varied with the culture den¬ sity and position of the control cells on the growth curve. For middle log phase cells at 3 X 10-^cells/cc, the control . 14 value was 9.7o ± 2.45 pmoles 1- C-glycine incorporated into adenine and guanine/lO^cells/hr, The of control" values for the anti-tumor agents represent the amount of de novo purine synthesis occurring in the presence of the anti-tumor agents relative to the control cultures. These "% of con¬ trol" values were reproducible within + 2,1^ when the agents were tested in duplicate. Table 2 14 Drug Concentration Cell Culture 1- C-elvcine into Concentration Adenine and Guanine UM) / % of Control + 2.1% (XlO-^cells/cc) 5-fluorouracil 100 1.2 15.9 5-fluorouracil 100 1.3 20.7 Methotrexate 10 1.2 16.5 Methotrexate 10 6.0 50.0 14 As in Table 1, the control value for the amount of 1- C-glycine incorporated into purine bases varied with the culture density. For mid-log cells(3 X lO^cells/cc) the control value was 9.76 21 2.45pmoles 1- C-glycine into adenine and guanine/lO°cells/hr, * ? c- i,?}ooXotf£iod£'i ‘^('i ^ tUC Oif” t 3ftOX^.flXr^9^dC}i -T^acq-oon/ Xo'tJtfton nA ■ rr,;.^ ^i'-:L\rI,.'; i n<^t-..c' -ftNa: f 5-''Ot'^'.^vninsys. »rtin«b . .vV":*ij *->*’*' -rifY nro “io rtoij'iiacq btiM io'ijnco t}/1o ,.“r,‘i-cXXtS!!}'^ tL sXiso sssrlq ^oX #Ibl j.trtr i’'£r:r •■■:: ■ =r;"oo;Ti ssaitigoionq c.'^-^ Z. ^wXfi V 'lo-j.ior ^'’ ' ' •id^',a,rx=7o'^0l\«nirijst/a tMts fiirtifldt ••jYorr ab ^iTi.fcfn£. 3d3r .'-‘iomu:'--i^fis #ft^' tic i:,,;v'o»-l:\-i^ ’ •’' '^o 5ori^i3''*q -^n*' -’i anixsuooc aXa^tti'fiX® a4'ti'iA ■■r‘'o "M* j?" •js-'T .:.' 31” t*.Xi^^d .o'lZ'Cico ajXT c-T avi.i'iiXa'i ao'-negw rerfv; vrX .r. “ r:Xd7J.‘ eXcioubo^qm ••ulaT “Xck; ,f.XXqyi iX •"Xi - -. I-.- n'*^' £i2iis2iasaa ^,i .s l::iig:s£v...>$-x rj 'S . sX 13 r 01X > oox t.t 001 Xiojuvotoyr^ l,dL X.X oi o4^ o.oe 0.3 ox '^tJEicn^orid’ •nloYX|i-’0*^^-X "to .tawoiTijs srfq 'lo'l 9i/Xr,y Xo-iX^toiS ,a tXdaT nl ,Y?i»n#b -a-rt-'^XL'tJ adS*' rtXlw 'lbaiq:,sv S:a^d ■aal'xwq, ba^’ffioq'xooi bY.«? qq./ abXfiv Xa-r^^rtoo afl^ •{•oo\?.XXao?Oi X cJaXLXto jpX-bin 1 . ' ■ Si .u Xv , ?.nXrtsbi .&q3, anix^aba '--C «aXocq^*^.S L&'' 1 . ■■'■,.,,#• 15 Figure A Inhibition of de novo Purine Synthesis by Cytotoxic Agents - MMPR (2;aIVl) 6-mercaptopurine Virazole (10;uM) — Adenosine (10;uM) - Tubercidin (lOuM) Purine Analogs 6-thioguanine {5)M) Hypoxanthine (10;^M) Inosine % of Control 4—I-1—I—^ 0 10 20 30 40 50 60 70 80 90 100 _ 5“fluorouracil (lOOuM) 5-fluorouridine (lOO^.M) Pyrimidine Analogs - 5-l'lT-ioro-2 *deoxyuridine (100;uIV!) 5-azacyxidine (30;uM) _ Thymidine (100;liM) _ Ara-C {l)in) Pyrazofurin (5mM) _ % of Control II I ■( 10 20 30 40 50 60 70 80 90 100 _ Actinomycin D (10;ug/mi) Cycloheximide (10;uM) Methotrexate (10;liM) Others Azaserine (10;uM) Vincristine (10;uM) L-alanosine (2;uM) PALA (1>jM) _ Hydroxyurea (lOO/jiM) _ Leukovorin (1000;uM) Mean (100^) Control Rate = 9.7^ ± 2.45 pmoles 1-^^C-glycine into Adenine t Guanine/lO°cells/hr. M, lj ^M;^'v) KCM ' 'I ■ frtnO ^ ) »Xi*5iiSTiV *- (WuOi) s^n!i.aox^sbA (M/uyOi) nibiaiscJyT -- } 3aXn3iv:^oi.f{t-3 —^ (f'Z'.OrJ Br.ldfasxaqxH ——— ' MuO f) fttii a on I — Ji/r;0-i >0 ? , -r -1-1-f—r-|-1--I-f-f '■; ?/'. C'P GB 0^ Oo 0^ 0^ cc OS 01 ( iioaii/otoi/Xl-S. _ ' aaoA t (M^OO!: ) SiUXx'X.W'cX'^^ai* S-r.-ioi/Xt-^ - "■ ....— ( M£a,0 C ) £■ r: L b 1V t S jS v" .3i?"-,y _ (SfsxO 01^ sn ; t': r, C ___ _ ; Mul) _ _._,,,_«*.,,,ii^ ; ' i'i 'lij'!: o a s'r/’^ _ i.j'IjnOC ’X I I ! t 1 1 i I I-j.-,|-j-j-j,-h- 4-h-—h— 00 i 0^-- 03 07 ^ Oc Oi Q4 OC OS 01 ' '■ X.4\'V ,p : ) XT nX D'^dOivl ^oA ^ _ (MK0i:> afeX^UxaitoIaY^J __ ST jjlTO iM.uOl) s3‘s.»«'T3f‘d4 (MiiOX) •^nx'iSBja.aiA (MuO:) ■jftli'diicniv ||jj] (Mt;S) sni 3ocislE- J ______ (Miv^) AZA^__ li'fiiu.OOI) 34i‘li/TCX0'li)\Jtir ■___ ■ . i ', i ' ■ ——— „....«,.„..,....,_..,.„w. « »;rsH loi^noO (*^00:) ns»l' ,’X^\,gIX9o'-Oi\aiain;?uC -r dnlftabA olni 16 l4 / 6 / 1- C-glycine/10 cells/hr. The control for the methotrexate study at a density of 6 X 10-^cells/cc was 3.^pnioles incor¬ porated into adenine and guanine/lO^cells/hr, It therefore appeared that the amount of de novo purine synthesis de¬ creased as the density of L1210 cultures increased and that this decline in de novo purine synthesis influenced the ap¬ parent anti-purine potency of methotrexate. Figure B depicts the growth curve of L1210 cells over a 120 hour period. The cells grow exponentially to a culture concentration of approximately 5.5 X 10-^cells/cc at which the plateau phase of growth begins. Table 3 and Figure C show the relationship between culture density and the rate lil of de novo purine synthesis defined as the amount of 1- C- glycine incorporated into adenine and guanine in a 2 hour period. It was found that the rate of de novo purine syn¬ thesis decreases steadily as the culture density of L1210 cells increases. As seen in Figure C, the amount of de novo purine synthesis progressively decreased to15^ of the early log phase level(100%), even in the face of exponential cell growth. During logarithmic cell growth, DNA and RNA synthesis precede actively. Since the rate of de novo pur¬ ine synthesis clearly diminishes along the upward swing of the growth curve, it seemed possible that the salvage path¬ way of purine biosynthesis might provide a portion of the purine nucleotide pool required for the actively dividing cells. To examine whether s: Iv^^ge purine synthesis could account for the decline in de novo purine synthesis along the growth curve, increasing concentrations of exogenous T*ao^ .:£rt\pXit5 OiXtniDxI^-:) -X 3 .r !. c. A.i.; Lso^OX X to \:tiart»tj a t» . :»i,*^0Z'' ^ni:;;iJ0^ tiu?. ©nlfUfcj* otni batartoq % ‘ J'r." ■' ?.’'''t' '4 oven 3fc I -' vi'ii(jS 'iilX tAfit CA£. V ' ' ‘ ^ S. ' -•-3 CZS.Xi *> a-fi fi.-,n •-. ' ’f s/'.'•*’ fT's or.inij^ . v*!’: •>!) ni <*nXXo#t> siifS .,13X0'2 CIT-Jin 1 -j;’-057 :.ni-..'--itn^a tnrxiiq ,.,r. : •• ■:'V£ o/'x ':7 e eiL3i^ ., .-, ■' ^ o ^. T ,tr‘7i’X*q Ti/ort OSi a ; !>;■ ^ •':r©./if£nx\>-iX'!-f6 '^0 fTotfMii’aawxoi} ■ :. i:~z ^ fl’vvo'X^ ‘»aU'{tq tfss^fllq •rft . -. il^ = r.JBd qi ft ./W9ft8 L^. .. ..^ - , ,. 3ir-'!r:-f>nvj=: ^nirujq evon at to ,vt % - A ■.•• .i-L..-; •j.'-Z vh- /.v:0410arcJ •5ini:o\^X3 . .,.y.^., .0 : _* ■■ !L12 i- ^ ■^LJiAS'.ra ae^C'itcosb »lemd^‘ i •• t., ni .ass.-a-xanl 3L£«o w*1*T I j ■■’"•■- ' ' 7c?5 3v-.Mi.' ^ ivi aa'S'i'^o^o; aia'«itiJ'nY8 •fltl'Xi/Q lEi jnanQctx^’ to *»*.*'/'*■ vi'. ::i no'/f? *vdX uaart'j ^pX >£l'i48 AV'H f- -r Av;c , ■>jf>i7'27j : jo Gx-nri-rins-^oi initvG .iitw€rr^ XX#o -•114 vV'<:rr r,t: to ■••cT^'T '3'^niS .\;X^vX)o« atuioa^u, i^taaiUtcvca t-'. ^«tX'V3 -Mid pirtoX^ 3©^(?..rniinib oi^adtetxa aai ~‘tifaq 9^av£j^:< •^.•■’‘ -tAv* ':’ifJ233'0q o»tri»ea Xi ,ovn[i^a alwo'iia •rtJ' d/(^ to noit^oq s ab^voreq Tn^..:r to yrifciv5t v:idvicr»£> nol bo'iXucd.n Xooq ^ftijo^Xoan .aXXoo « _ • blijoo lioaricXirtd anX'lwq *» ':*i't3’«;'w iri-njac* OT ^59X6 sXA'{3 ov<5 Figure B Grovrth Curve of L1210 Cell Cultures Time(hours) Ij’. jin:' ClSli to a...rK»ls 18 ■Pin-n-rp n L1210 Culture Concentration vs. Rate of de novo Purine —■ -— -Synthesis 36 ■ 32 24 20 he in ') 12 4 + 0 1 2 3’ ^ 5 # Cells/cc (X 10-^) 0 19 Table 3 L1210 Culture Density vs. Rate of de novo Purine Synthesis Early Log Mid Log Late Log Plateau )ells/cc X 10^ (0.3 - 1.2) (1.3 - 3.5) (3.6 - 5.6) ( 5.7) m Rate of novo Purine ithesisK 31.0 20.4 9.8 5.6 noles ^C- /■cine into }/hour/lO°cells) hypoxanthine were incubated with L1210 cell cultures. Hypo- xanthine is an intermediate of purine catabolism and can be utilized in salvage purine synthesis. Figure D shows that as the availability of hypoxanthine in the culture media increased, de novo purine synthesis was reduced to, at 10“-^M hypoxanthine, 17% of control. The control value for de novo purine syn- . 14 thesis was 10.2 pmoles 1- C-glycine incorporated into adenine and guanine/ lO^cells/hr without any exogenous hypo¬ xanthine in the media. The experiment was performed with L1210 cultures in log phase growth at a density of 4.0 X 10^ cells/cc. This relationship between the progressive decrease in de novo purine synthesis and increasing concentrations of exogenous hypoxanthine suggests that utilization of the sal¬ vage pathway of purine synthesis leads to reduced purine syn¬ thesis via the de novo pathway. The effects of increasing concentrations of exogenous 14 C- hypoxanthine on salvage purine biosynthesis was tested to demonstrate the existence of the enzymatic machinery needed for salvage purine synthesis in L1210 cells. Figure E 14 shows that a significant amount of C-hypoxanthine was in¬ corporated into adenine and guanine when the exogenous j ■jr\ j2iL_ifaai • I _■; ' -'.c - :.■■ - 'S.i - t,o; 'oi X so\i •rf'ftt ii ' «xxto^OATt iV. -:s:^rc/'i^vs j.'ti.MTrusxoqx/l *■•1 r •. ■ -j irn -n■* I ■• ‘; u ^ r -• - r_. _; fc".*':-d.'iTi •.£■ bI ^ixJ.fit'nMX 8: /■‘irit '‘>'0:18 a e :i ii.q 5-SV ■'.£ /li fc-ffsiXity !■! I,i >£iXb^»Di t« rtrvt iyo ■^aj 'irT-.a:“,T!^:ro'j y; " 't' \; *il IdJiXj! s-va 9fif 4 *■ 'iitrti C9rn"''j 'I'AIOA,’- Ulij-•■/ ■ -_.-r' ■ylG,rrr i,0^ -jiW f'fi Jf/O'itx^rv IH .bi'Isg'''; ^t^,r.rr£i75 tfricheba 11^0 =>ftrriC'l-liiq itiiW 7iTv-ai'raiq:ci' voT .G.'r;&i'’ .,11 7Jl ^niAUnJUC '01 X 'to ;£ T(9 axortq id eawtiyo *.it rrocvj^l qirteiioijg'jiT ,oa\nII»o io artoi^.3^;gr i,33r'a-;‘Ti 5i48l^*~'a ovoii ni 9^.t '^a -Cl rsr/aryo/T»504t -.r;a anriyq . -nytai oj ab^vS lijiaddlaYs tirJ-iyq '!.> Ajav »v-£wriJ3q o'/-..T * b ^.f(S mir otiBwiii" 3i;oj[t*5^oxe "io anoij's^^ffravioo :§nx'as»'ian[i lo ®4,T ijsj'ss* suw exBflrid'ftYsoicf erfi^yq ito ml4ffiXmX’0q'Zii YTyuHaism oi S .sXXsG OXStX ni E.'.c-ct/q #^4v£ja[a b&b^^ti -ril s itMti* awoiia s^0fi«^X6 aril- itB/fw 8<^’|nv3C^ o;^at b»^«ioq^o 20 Figure D Exogenous Hypoxanthine vs, the Rate of de novo Purine Synthesis The control value for the rate of de novo purine synthesis with 14 no exogenous hypoxanthine in the media was 10,2 pmoles C-glycine into A+G/lO^ cells/hr. I H\ ..'VCn* -.I *ru r i d~ ('i» ox V •V*r- ■- '6t (M) w/Tiii.taai,-:3^47i ayo/t'^:^03^.'"!:o «Qi4‘Ai'*x^n’9‘0fP?3 JkV “ a|^'',aa^ nl" &ftx4^^\^oc(’|rt •?j.oiiiiao3c» a .'/‘.h;-:; ' ■'■*i;x{>\ii:JL,L9^ ^UiXO^A otf’rt . ■< ’',“4^ 4^ t‘ V ',‘jfl ■ f^. I ',“ V 4u - -.1 ';i'',"'’r(i?V(L' . 21 14 E Exogenous C-Hyooxanthine vs. Rate of Salvage Purine S\TLthesis ?Y... sr-1il?a^ 8KBfl«^oxa a nu^n concentration. of C-hypoxanthine was l;al\i or greater. This experiment was performed on log phase cells at a density of 4,3 X lO^cells/cc, Figure E demonstrates that when the con¬ centration of exogenous hypoxanthine is in the range of the L1210 cells will utilize this substrate in salvage purine synthesis. Figures D and E together demonstrate that de novo purine synthesis progressively decreases as the salvage purine synthesis pathway is increasingly utilized which it will be if a suitable substrate such as hypoxanthine is present in suf¬ ficient concentrations. Therefore, one possible explanation for the observed decrease in de novo purine synthesis throughout the logarithmic and plateau phases of L1210 culture growth is increasing utilization of the salvage pathway of purine synthesis occurring as cell death and purine catabolism provide the necessary substrates for salvage purine synthesis, 14 Intracellular pools of 1- C-glycine in early and mid¬ dle log phase cultures were measured after cell cultures were l4 incubated with 1- C-glycine for 24 hours. The amount of i / 6 • intracellular 1- C-glycine/10 cells within cultures harves¬ ted at 9,0 X lO^cells/cc and others at 3*8 X lO^cells/cc was not significantly different after 24 hours. Therefore, growth phase specific variations in intracellular glycine pools could not account for the decrease in de novo purine synthesis observed with increasing culture density. .) ,yi A p ' ‘ -:o A(:: a*?-'/ to I'foitjrsi'ntortdo -. ,tfaq ;goI rro .■-■fc"iot’2«;tq sjxw yniHai-teqxe ‘j .ii:'’- :;w :'.f-;r,.r.raarioin€'fc S .oc\aXI«o^0x X ,••.1 'T ^‘.x .'?, 1 '■'^.'vTnj'^xvqvn Bi/ont^cxa to ctolTantMo * ».■•.; i'-i'. i.s> r:i : •■•a/B. 2i.1T lllri allao OtSiJ' rdd' •. »b .r-v^ b ", i i ■.■.'.j. :i 2'tiu3i''i .BiteliJ’nxa K-'-!,uq ail" I’.s 'i:: '- Bta9dt(T^3 tail'iSJXi “. ■' iia*/ “ ■ cii \*nv(tlt»q aiaefsl^rncs - ■-- iTj, ''•: :'-*-c.7 si j/ti. T ■,:-;.x.:'7.^O.up, J’£''Tac-w5“ B •' .-..nr-^T :-i''':•'=g'‘ .SvioiTr.#Xoil ’ i. .■ ' sqLquq OVOii jb .2x 2 9D L'^'^ZB^dO Blif lOt . , C o. *. g.L.iul.rii‘^Tc i U'raiJfJJQZJi^ •■ . ••rlj* :• ■ r ggi/'3n:. 8:i ,1twoT3 r2x-oJ5 ?-> rns fb Tr sianiTuq '/r, '•., -i > rx-^reclL'a , i-seM'.an *ftivo*iq • '.• f:. ^ rJ =*ai-?7U a-'tc alo-., 7 ;XcI_*‘OinTnI T‘- .-.JO Tiyc T’.y;Xt.:; •.*•.- xaittlufj aa^riq 9Sb .* .7- .’.g y,7? .amirnri v'i a*?i'jvig-0 * -1 rttiw 5»^j|nfaoni niiiTiw z»luX£8SMZtnl iiis!\y iO .71 i.-3C'''0J: X 8,^ Ti5 gnsJ-Tj Vfi:s X 0,9 tM 6«1 , v7C 1 . a'WDd T-3 rtj8 T'^a-r*: * i Is ^Xv^-on , f,’? ■• -i: :'iO£7Tni ni iniii.i . 'i.'n -u- >il g’ijet^'toob » BVJtXvo srriBattorti oavtittfo tla^rtv-fPCa 23 DISCUSSION Effects of de novo Purine Synthesis Inhibitors on 5-Fluorouracil Metabolism and Cytotoxicity In conjunction with the above work which quantitated the degree to which anti-tumor agents inhibited de novo pur¬ ine synthesis, Cadman et al. examined the effects of five of these anti-purine agents on 5-fluorouracil metabolism and cytotoxicity. The results of the influence of methotrexate, tubercidin, 6-methylmercaptopurine ribonucleoside (MIllPR), azaserine and L-alanosine on the intracellular accumulation of 5-^luorouracil and on PRPP levels are summarized in Table 4 (9). As noted previously, the effect of the folate analog, methotrexate, in increasing the intracellular accumulation of 5-fluorouracil is thought to result from the inhibition of de novo purine synthesis by methotrexate. Methotrexate is a tight-binding inhibitor of dihydrofolate reductase. It was found to be a potent inhibitor of de novo purine synthesis, reducing it to 11.3?^ of control. Methotrexate prevents the conversion of dihydrofolate to 5*10-methylenetetrahydrofolate (CH2THF), thereby depleting the CH2THF pools.(See Figure F) CH2THF is required for the conversion of deoxyuridylic acid (dUMP) to thymidylic acid(dTMP) and methotrexate therefore inhibits thymidylic acid synthesis in the de novo pyrim.idine pathway. The CH2THF pools are also necessary for methyla- tion at two steps in de novo purine synthesis and therefore purine synthesis is significantly reduced with methotrexate. s!lssd_lXai.,.®Oi3_2i24ili bas nisiloaMfm •3'- iDM'^icfi^w.' 'jor.t'-'!‘«lTa4 .^oxrfw 0^ &cif iz:-iOo '.-N :ti' s:3-ft«3i: nirLi'^t/^-i^rris seerf# 'oi!'•.)': V. ■■".’tj;, "Vo ^;;M.xfRs“r . iSlolxototxo 'i >'i-Jj.ioe ^:n- 'Tc-^i-: + ~ '.:.v -:vi-.. •■* j-•. Ii-;:.: rro -aaliroitBX^-.l OiTS fial*i*iiBas . ^■-: ■ 1 iTO toa ^ia/nL/OTOuXI-?. lo .(9) 4i 0X i'-iTo'i, :•" I'isllf. ^j-fc , oafocx sA i,e£/.0 L ■•■Qo'ij'nX ?»iil7 i'.ii .ijiav-iui’r t ii2 ■'■ .^u:: ..'iL'-ue^ of :f'd,|i/or] a.». Iloonyirioi^X^-^ \ ■3 'i Q." '3^4 , ■■^.^ i^-srcsr ‘^cf sia^jdjr;\,3 Bnlru'q 3V6« afj r T ^ c, be-' 9: .iXo'tcT bx-r^f b I: lo3*fblf(!:i t < 3.3ii,rr'iii erfiTLfc -f «b 'to lOTidi/fnl 2*/(9j*oq a ad oj- bnuol 9rijr3'jro-:'aM ^io'ts’nc-^ \o .t'X ^i30b«i Oj vualo’lc'sb'cdXb lo rToi»*;»vaoo (■■? 5'w-i,:l :36»I; ..34.000; 'iHT^HO 9ff7 ' biO« ci.i'4[:’i''iijv.xo$b lo rtoi^’savrtoo arft *iol li ^HT^HO s-rolsTarft »v fjRs ail'^bXfnxrl;^ ot ( «.te£x;.TX'^‘4q ovon sb srfy xi| ale^ddmds 5io« olI'^blBr^riX' atidXitnl tol oaXjj «!« aXooq IKfjjHO ®ri1? ,Y,|r*r(d«q e'laia-ia-dd bite eb rti te iroid xi:2*Xw. \;X:|na6oilXn^ 8lB«rfd.T Figure F Effect of Methotrexste on Thyr.idylate S^mthesis de novo pyrimidine synthesis CH^THF » 5*10 methylenetetrahydrofolate DKF - dihydrofolate DHFR “ dihydrofolate reductese ^iTl] - Methotrexate -ovon •• J^’/?*’::i-:.tx*iye5-»r:WH 0£ ,^ - bjj.LcJ 4*.*; £>X/fib - '? j? i; - tJrXolot6>crtib - fl'^ Ar iL'. . E .. *•! . t-i I 'h ^.X *!■ 4 ) v. I. W' r:? o- ■ ■ •i V ^■'*v /il.' . ■ ...■ «■ , jfV Vi ' k -■ ■ enow \n^jK i; .TiAeru W lo l !S V " W' S^-\ !■:.. ttViilvjfiLt liiC’ ,i.4-;' ' ,;M'; ," ‘" '■> ;■ ■»,., • *■ t ■f ;■ la- 25 Table 4 Effects of Purine Synthesis Inhibitors on 5-Fluoro- uracil Accumulation and PRPP Levels (9) Drue: Concentration 5-FU Accumulation PRPP (pM) (Treated/Control) Methotrexate 10 4.32 8 MMPR 10 4.40 15.7 Azaserine 10 3.00 15.0 00 o L-Alanosine 10 o 25.0 Tubercidin 10 Undetected 0.1 In control cells, 5-fluorouracil accumulation was 0.025pmoles/min»lO^cells and PRPP was 7± 1.2ng/l0^cells, Cell cultures were exposed to the drugs for 3 hours before evaluating 5FU accumulation and PRPP levels. MMPR - 6-methyl-mercaptopurine ribonucleoside. J l-r la Stattts » ,Itfsi ' -' g^gaj S'inri i;a4 CTofc aJ^fimoaA ilos-ui '9'iT ') ', i ' ■Jj. ■* n■ • r*• ■ 0nt^»a«sA . r &ni«onxXA-iI L jt-" '^.‘'tvon'.! or ni&ioaacful! 29W : i I,:0«'1|.'0'1: i. i'r - i Xotmoo nl 'v-rtX.: _, L\*ts alleo^OI »aint\3»X'^^4l^S0 ■■t’HC p.'LLor'. •* -icT -?iri7 ijsr.soirxs stow s^yuftSjjo IL9O 'v^.T-: .TS 4Oi-j3X'v'fllLC0i)a S/ti^JMJXflVf . •■•b.':!<^^XjL’i:ocf:'r rqi33T9(K-X^;rf:»'iHi!-d - H*iMM 26 (See Figure G), This two point inhibition of de novo purine synthesis leads to a build-up of PRPP due to decreased util¬ ization and to reduced feedback inhibition of the de novo pathway by purine nucleotide endproducts. Since PRPP syn¬ thetase is a regulatory enzyme inhibited by the purine end- products ( 10 ) , decreased purine synthesis results in in¬ creased PRPP synthesis and therefore in elevated intracell¬ ular PRPP levels. This PRPP is then available to enhance the conversion of S-i’luorouracil to leading to in¬ creased intracellular accumulation of 5-fluorouracil, Hypo- xanthine completely prevents the purineless state induced by methotrexate, a state which contributes to the lethal effects of methotrexate(11), However, both thymidine and a purine source are required by all cell lines examined to completely eliminate the cytotoxic effects of methotrexate(12). Tubercidin(7-deazadenosine) is phosphorylated to its active metabolite by adenosine kinase. In its active form, it inhibits PRPP synthetase(13) and therefore does not re¬ sult in increased PRPP levels nor in enhanced intracellular FUra accumulation. When L1210 cells, pretreated with metho¬ trexate, were incubated with tubercidin, the PRPP elevation and increased FUra accumulation seen with methotrexate were prevented(3). This suggests that de novo purine synthesis inhibition resulting in increased PRPP levels is the key effect of methotrexate in augmenting FUra accumulation. Another purine nucleoside analog, MMPR(6-methyl- mercaptopurine ribonucleoside), is also phosphorylated by ovorr ar; la eiHT .(*5 »»2) ‘-Sx fu t-r^i-'e'xoen Oy TCS' ^i/''-'.lS5''.ri,jo'''a aia^rttrr^B v“oa ijb S’-'j" 10 :{ -;;'.^T ^otrJZ ' ,oiTv/oo'rjjbn© gfSi^oeXot/n ^aJt’Hsq \:-3wrf^aq -brfs eniiioq srfO* v^d” jbcticffiirTX ®ar'.'a?rT» » al -rri ni -3tX-x'50-:i aniii/q t^jsBa'Xoel) , (OX )atowJ&oiq -Xi.yo/ZTj'inrj. Dst^vaXa rri a'io'fft'iadx SrijR 3ia©d3“fni^a fctaA^'io or &£0BliBrs rr^.io si ainT .al«v®l SlEifE uili/ -.Tx 07 •'Ii'in''!-'? o»t lios'SOOTOfc/X'i-^ lo ftoinavnoo 0(i& 'XiiX-T: lo r.olJ^Lirmuoo& iiiIuXI»ofi7L^,fiX beajBaio ’i'j fcaoi'bni aeeXiini'tvq ai.t sxrrfdva-iq ■'T^IaJalqoioo »niril‘njix 'ooi'la X7.iJai 9f!0 ot CB^ufi't'tcioo dzldw 9fM7a s i9tffx®'i;fori^®flT B buH gnibiiT^obt p(';?Qd Alt} ’.;Xatf^Xq'flioo o7 heniiioxa sartiX XXao IIs ©'tj to'xvoa ,(SX) ’»ot;irlX©ri 'io axasn? 9tmlttlL9 oJ" bstslyo'tv/'^qeoftci; zi .X‘itXeon&fc®a.se'6-?)nibi£n:^®dy'T ..Tiiol: 9Vi;*-o.9 sJT aT »-?.i»rriT( srTiacnaJtJF. e^iXodfiJaet ®vl^t04B -ei 7or S9O0 fans 9( ■.sXuXXsosT^nc bsofTiS'.rfna ni ’lo.ct bX®’/®! rfl tXua -o;!.79::: ii3-i>v faa7fi9T;teiq ,aXX»3 OX$Xa ffortW .noi^^XjjftwooB jrtUE noiO^v-^I;? ad."^ ^cilolonedui ti^ly b«fsfdfuoni a'xo/v a74x-^'3:^o^we 3i3ed:rnn^2 sni’iuq ovon afa .ixdt .(C)b9J'aav»iq Y,^ii adt el bX»v»X faBa-saT^mi rri ntoXd^Xtflfini .nQij-«j:A/ -Xyrttf’scr-dj^tEIOI (j^olsns afeiaoaio'*#,! aniiwq tad^onA lid fa9v'sX\;*so4(caodq 9rr2'wrj[o^q[MOZ0fO 27 Figure G Pathway of de novo Purine S:\nithesis Phosphorihosyl Pyrophosphate(PRPP) + Ribosylamine Glutamine Phosphate Glycine • ATP 5*10-methylene i Phosphoribosyl ^etrahydrof Plate Phosphoribosyl f ormylglycineamid e Glycineamine 4 Glutamine + ATP Phosphoribosyl ATP Phosphoribosyl f ormylglycineamidine 5 aminoimidazole HCO. ATP 1 Phosphoribosyl Phosphoribosyl Aspartate aminoimidazole ^- — amino im.idaz ole 7 s uc cinoIOC carb oxamid e carboxylate 8 I Phosphoribosyl 10-formyl- Phosphoribosyl tetrahydrofolate aminoimidazole formamido- carboxamide aminoimidazole carboxamide Adenylosuccinic Acid Xanthylic Acid i 12 14 I Adenylic Acid(AMP) Guanylic Acid(GMP) ENZYMES 1, Amidophosphoribosyl tranferase 2, Phosphoribosylglycineamide synthetase 3, Phosphoribosylglycineamide formyltransferase McariJfiYi.,e.t> .to' Y.£yfiiie1 .•XiSI X'te^&dl-xodqaod^ f ) w-fMfiq^oA<^o"rx^ + ■ ' e f'3f(qe.oriT. - 'aoni TC «j- p:Xbi.a%brfrfAncr'‘9? lodqsod*^ .—'—•»»>'.'...fa- ..•i:.b),a jnxoy,!?^ C ebluTirdr. 39‘ -.00^ ! q^T.!* *•'■', •* 2'JjA X is^adiiodqaod^ -> aloSlMtlf^lOClliBi ^ 6 X iLtx o #io*£bi v*!irD)6ioA oiX\iTjaaO l‘tMA)5lQA oil’Crtfb Xxf«diiod’3:aoirtqobX'sA ,1 a-axi^efftrivs ^bMt>9frioxX5X\»oj/X'SQ/tqioriS[ ,2 j a ST ©t Bi'Ljni -C^Tol Xifyiafti »odi To4q»o rfJ , *^OT«‘'vr,', , 28 Figure G (continued) ENZYMES 4. Phosphoribosylformylglycineamide synthetase 5. Phosphorihosylaininoimidazole synthetase 6. Phosphoribosylaminoimidazole carboxylase 7. Phosphoribosylaminoimidazole-succinocarboxamide synthetase 8. Adenylosuccinate lyase 9. Phosphoribosylaminoimidazole-carboxamide formyltransferase 10. IMP cyclohydrolase 11. Adenylosuccinate synthetase 12. Adenylosuccinate lyase 13. IMP dehydrogenase 14. GMP synthetase adenosine kinase and inhibits the rate-limiting enzyme of de novo purine synthesis, amidophosphoribosyl transferase(14). MMPR did elevate PRPP levels and enhanced FUra accumulation. Azaserine also increased intracellular PRPP levels and FUra accumulation. This anti-purine agent is a glutamine an¬ tagonist which inhibits de novo synthesis at the two steps requiring glutamine for amide transfer(15).(See Figure G) Inhibition of the first amide transfer catalyzed by amido¬ phosphoribosyl transferase probably leads to the increased PRPP levels and subsequent enhanced FUra accumulation. L-alanosine, an L-aspartate analog, competitively in¬ hibits the intracellular transport of L-aspartic acid and inhibits several enzymes involved in de novo purine synthesis as; s -:»('!. n'v.a '^lo:i.eMsi:ic^aw:Ml%m<3Hoiii^ms^' , ••' , ■jj"/ ^:IQSi?;&lflS^'xc,nI;st■«It'«oclJ^:'»Io4)^'»^ ., \»i ' '. • ' ■ ,-i “i■.•!i;.;>: ''.'iecciii■: “ua**' ■-' - •- -. - jvr-’' i? ^TO, *'0i *,IJ ssoyX Si aMI »CJ ■ aD!<|5 *4J 'I'o ->wy^T;-9 ;^, ■'?..j LT'^. --f: 5.- bills Qisnl^^ m^i&oa^bi .{'-:/:^Gs:i6tanij'T." I ^./?c : i-ir.j'fqaiv,-^qo'ti<:! ® , .;^3srfJ'Cr^s wqh sJ .no/.^’sXiiTffboos a't’fil br^-^i^srtne brts ai'jv&j: sa'svsi^ Mb ? alsvaX "'Li'a! tIs>ajaTtv*ni: bses^TT'in;! eni'i'a^^saA - 'H i)n2ins4‘wXjj a ffli trti?-^;3 9r:xiiiq-,r;}TtB alriT '>*aoj.tS-U>miS'Oiin er%^ eqtf:’-F; owJ- arft ifx ovon sb, 3J'ld‘i':tnii ifiifto;|sJ ^.0 9n;-si'i «<:’??). jrisla.'rsij ^^bxfns "lol sni{a«;J'.aXs!l 3|rt.Hii;p«': ^obMa x4 b»atX'B:^s3 'lotment sblm& 9‘44 'io adltKlltUil b9e£9'%i>nl seiT of ^b/?3l zldjsd^jnq •a^.^xnatacraiX X"‘,';?Mb »r).0'.f;i7iai;Winr^^a.? 'bsun^ilfj© TiTaw.peMvfi .fkiS" piavsX . > -><.t xIaviXi *sa;Ttoo ^i-,sTi?a?S'SS,~>I ct£ ,«^4U'30frars-'vI,, ' 5fT.8 bXaa l^o T£.Liili9ii>4rx^rS 9/if «4‘i(fljfi fJa»4twi£» anlwq Oimn SiX . ’^yXo vni Xs^av#!! stldldrtl 29 including adenylosuccinate synthetase, phosphoribosylamino- imidazole-succinocarboxamide synthetase, and L-glutamine synthetase(9)t Inhibiting de novo purine synthesis at the steps catalyzed by the above enzymes led to increased PRPP levels and to augmented FUra accumulation in the L1210 cultures treated with L-alanosine. The inhibitory effects of these 5 agents on de novo purine synthesis is summarized from Table 1 in Table 5» Table 5 Inhibition of de novo Purine Synthesis by Cytotoxic Agents Drug Exposure Concentration 1- C-glvcine into (hrs.) (piM) A+C/lO^cells/hr. of control + 2, Methotrexate 3 10 11.3 ^'IMPR 3 2 6l.O Azaserine 3 10 14.1 Tubercidin 1.5 10 17.4 L-alanosine 3 2 79.0 Each drug had a significant inhibitory effect on de novo purine synthesis and each agent, except L-alanosine, inhib¬ ited both adenine and guanine synthesis equally. L-alano¬ sine inhibits adenylosuccinate synthetase which converts IMP to AMP, Thus, adenine synthesis is inhibited while the pathway from IMP to GMP is unaltered. The reduction in purine synthesis seen with L-alanosine was due entirely to decreased adenine synthesis. -o.nxinfilvaQ-'/i'iivSa&iodcj ♦ arrl&wXorri ^airrjst'ji3-*J bnis «93£j'?dtrT'<(;9 *jLlOTsa'.icf'aisoonio«i/a'>*#Xoi:j8'&ii!iii p;(c t/i -^niurf? ovou -ofc ,(?)«o«t*fiyrr^8 l^ft-Stf-ji’yor-: o.t hn,. amr/^ssn) 5»voo*£ s?iit br?«’^I«^ao aqa^^a ‘•jx.'l*li/fj ri_::f ^riC’ lU r:oi^si'i.>'irr!ii2C« aifn &s^)nr« .eni&Q^Tata-j; rttiw baS-aa-i# '■von m5 .1-^ 3,rr.ygs e aaoi'i.i ',•■ ;3sfoc^'.Uy loldal adT ' ^ Z’ c-Ld'-? nl _ I 'iTosT .'Tio'^: i b^&l'xxriimjia i\l ♦ni'iuq sajyiss^ ;Maa -i.X^'-^“ai\D-A (.sT.'r' ■ c 'lO ; _ • r ' ' . C ors^n-toriteM flSCMM '' • C »it4t#*ifcSA fiXblo'itcfi/T #CtX- 'rLBlil-nl r #n'**t*> • ^ r\ v:'iotlQ'Xddi ^itsoX'iii^^ia ‘* ^jui m/zb tiosS -c/i.jnl tanieoa^Xr.-^ J'qooxa ^Tne^a rfoji.® bn^ •al'iuq -ofuil*-! .' '-.MI acr'T.-vnoa dri.Mw 92i;j0ilj‘fniia eJ-JsrtiOipyaoX-XfWit'jS ati^Xitcii @Xid\v ba:-Mirtcr| ai ai^a3i1% fTX noiJ-oi/h^z edT .D93a^X^iiti/ 3i oT ‘TMI ao-tl ^/swti^sq o^ \;£®'54^rta PArip axiw rf-rteea siaaiUrryia §friTx/q .8ia»rf^iT\ca »/3ixroba baaiearioab 30 To examine potential cytotoxic synergism between these 5 anti-purine agents 5-Tluorouracil, L1210 cultures were treated sequentially with an anti-purine drug and FUra and cytotoxicity was determined by soft agar cloning of the cells. The results are shown in Table 6 (9). Table 6 Viabil ity of L1210 Cells (9) DRUG 1 DRUG 2 uM(l) uM(2) hr(l) hr(2) ^Viability Control 0 0 0 0 0 100 Methotrexate 0 10 0 4 0 60 0 FUra 0 10 0 1 100 Methotrexate FUra 10 10 3 — —> 1 10 MMPR 0 1 0 4 0 100 MMPR FUra 1 10 3 — —> 1 6 Azaserine 0 6 0 4 0 77 Azaserine FUra 6 10 3 — —> 1 10 L-alanosine 0 10 0 4 0 106 L-alanosine FUra 10 3 3 — —> 1 41 The arrow(->) indicates that Drug 1 was given for the indicated time before adding Drug 2, Total drug exposure was 4 hours before cloning the cells in soft agar. Synergistic cytotoxicity was seen with the anti-purine agents which increased intracellular FUra accumulation. It therefore appears that pretreatment with anti-purine agents which results in increased PRPP levels, allows for increased activation of FUra to FUMP and for increased FUra accumulation, resulting, therefore, in enhanced cell killing. ,1. ■ ■ ! \ ''.11:^7 /s jfX'37'SrY’o oT =’^'11/;? C>jc --•il'V t>n.s gj.f'To titf* 70-i? fTS i'^ri'A* irvsjr»»!:}• c BrlnoiO *to:i ?3'?n£^i>T9h T^jfi;,ojtxo^o>fXd bnx ? 'oiS^r .-li -Yv*ro7;‘» ' t.tXi/»sT .ailed ?) ixi^Aiv tXd,'5r . (I} irf ■ : LJ^ : Dufta 0 - 0 Xo-fXrtoO 0 0.' 0 0 or 0 ..7v" 0 I ■'ir-:* 0: n f * K •■) * C r r £>■10''. HWI t • u j*i r. ♦ni-iietxA cr o ,.",1.11 ani*s®8aaA 0 <'L •ai toruil j-rvi r or ■>,,.♦'57 anIaorTjiIt-4 •'*j; as-v I ;3i;*3tl X6ri7 Sj?'jEoi,f>r;i *"'"-)»voTTa difT XiX3^ ,£ gui^l Ddsy avibni eriJ" • 'TE3£ 3^o?t fti 3lX‘,»p 9(i& '%0'taolo e-jo^td aiiworf ^ taw i; ’£iis»>a arfii :7-^xrtis difiX dTxw rts^as gii?iy IdXxdfojhp? oii^tijitnxS '■. 7i %iXel*jaSiii.iif06& ijgluXXeajrtv0-i itaaatiiijrti rtoi^lw XiL'ij'fn tOittw sXrrs^ii* Br:i*ii;ci’-’tfci£ 4^- ‘ ‘ni>.7'X£i#'xC'ti ■lo r'.^lsari:io£; h^ikamcal Tbt fo«8t«T:o£Ti ni a'iX'i -xol tna 'IMU’i dlft. .^kni ^XItI Ll'riim hAnnmrtrf mt a-ya'^ 31 This proposed mechanism of cytotoxic synergism is con¬ sistent with the findings of other investigators. MMPR in¬ creases intracellular PRPP levels and has been shown to en¬ hance conversion of 6-thioguanine(6-TG) and 6-mercaptopurine (6-MP) to their active inhibitory metabolites, 6-thioguanylic acid and thioinosinic acid, respectively(16,1?)« Guanine-Hypoxanthine 6-MP + PRPP -rr-r-rr-—---> thio-IMP f PPi (18) Phosphoribosyl transferase Guanine-Hypoxanthine 6-TG + PRPP -rr-r—rr--r-> thio-GMP + ppi (19) Phosphoribosyl transferase Cadman et al, also examined the intracellular metabolites of FUra in L1210 cells pretreated with methotrexate(3»20) and MMPR(unpublished results). FUMP, FUDP, FUTP and FdUMP were all elevated above control values in proportion to the increase in total intracellular FUra accumulation. The increase in these metabolites probably occurred as a consequence of increased PRPP levels since the primary metabolic pathway for FUra is conversion to FUMP by orotate phosphoribosyltransferase, using PRPP as a co-factor. orotate FUra + PRPP -T-T-rr-77-1^ FUMP + PPi (4,21) phosphoribosyltransferase ’ The enhanced cytotoxicity observed following sequential treatment with an anti-purine agent and FUra may be the result of increased intracellular FdUMP and FUTP levels. FdUMP in¬ hibits thymidylate synthetase and FUTP is incorporated into RNA. Complete protection of mouse L cells from FUra cytotox¬ icity requires the presence of both thymidine and uridine in the media indicating that FdUMP and FUTP act together in ? ■•tor ; uirai-a.;^vo io eiilT --■£ .c- ■’■7.^3x3:a§7r:i 'i^)ri;ro "'io -.■ ■ -■ :i£r'tn. fit '.' ( O'!.-- ) tfti,';i5i;.;iOX;f;?-d to ilOi«'*aVrtOQ BonMii V, *.. . ^ ‘ •^^J‘i..oo£cr-3iq v.*ioti'd'i-fXt-i. ot r .-^:' :X»vij-ot?qe!3T ,xj£C£ t/rrisOiticiriJ' j&it* -e.rf■ ------~ ' • ^ • t' ':;8'tcft.'jc'Xqao.{^ T ^ ' ■ ' -i ‘.-dw b^ni.TT.dX? oBio ,Xs 0g n^jNrO -s','.-‘jii d.ti'A X'srGy'tjsiq alX^o Ot?Xvi nl inU ■■■ ' " '■■• .-iMUl .(aJli/sei xtid«iIcXiiqf(itv)H -7; :i :.,.7 o? riii.t'ioqo'rq rri asulfiv rp-tfitoo #7ocfii - -^-^r itt .. v" .£tri-B.a;uyc.o£ i^.C;jXiBtije'TMi XjBS'Ot- v-- r: --- .-x *»■ ■. «JitD r a? r>£“rji;o3i5 yXc/L'r.to'iq X XotXatsOT ;-> . V'S’V 'jg.; o l Xocfsi"tfcn V^T-’nTl’lC 9dl OcnX-?. »i .---■- . , .0/Xr-xoiiq3odq 9.r.a*0'i3 %:d'<5^ ot ro?'3^i«vnoo .'Toxoj&lL-oo »a e tpt-ri.o •■ - mit ^ ■^Qs^s&zzrts^ tl. ^:afl^dt^’ortqao'T^q =1^^ T • t'* I . i SniwoIXoj: ba'ne^o'o j &aa£t'.^rirts aillf r j :u3^t sd .-At, £fs fnmitnwit -ai to -iSrwXXsoa’as^fri lo i--'B-r.3T:ccr iftsiti aX <*:Ty'i tjaa ^fslxtiis^nj Qti -xo3-odyo attUTI moTtl eXi^o X Ic troi.tcj^To-rq •^'•rqntoO frX bnj oiTXtxiifYad cfi-ocT- 1:o arft sa^iypai '^^ioi »Ti. ;;ojj ^TtH i;n^'■?}«i‘j;y j^rjidjsdiXiftX aiftsoi 32 killing cells(22), This study illustrates how one category of anti-tumor agents, the de novo purine synthesis inhiibitors, can enhance the metabolic activation of another group of agents, in this case, the fluoropyrimidines. It also demonstrates one approach to designing biochemically rational sequential drug treatments in the search for synergistic tumor cytotoxicity. Effect of Cytotoxic Agents on de novo Purine Synthesis Ed Cadman et al. demonstrated that the anti-purine agents which enhanced the effects of FUra were those which led to increased intracellular PRPP levels. In addition to the 5 agents discussed above, several other anti-tumor drugs with various mechanisms of action were found to inhibit de novo purine synthesis. Pyrimidines and Pyrimidine Analogs and Antagonists Ara-C(1-B-D-Arabinofuranosylcytosine) is a deoxycy- tidine analog, the active metabolite of which is the triphos¬ phate form, Ara-CTP. Ara-C exerts its cytotoxic effect through the inhibition of DNA polymerase, although incorpora¬ tion of Ara-C into DNA may be an important contribution to its anti-tumor action(23). In the presence of Ara-C, de novo purine synthesis was found to be 85^ of control. It is possible that inhibition of DNA polymerase with a concomitant decrease in DNA synthesis leads to a build-up of adenine and guanine nucleotides which then inhibit de novo purine synthesis. Increased intracellular concentrations of purine ribonucleo¬ tides have been found to inhibit the first committed step in de novo purine synthesis which is catalyzed by the enzyme amidophosphoribosyl transferase(24, 50), The increased purine •’ V (Si} aXX to S(ti III 3{ '"(rr j- .'-rv/v to MO wort a^vTisitaijIfi vfn^^'a ai^'flj BlB^^dsopc^ ml*usq orcn 9h * - h i ^ Ifi- qi:or^^ iLQdtortm to 3iXo •I 08X.> j-i adt ♦«iaac » .7'jfn?‘ a<- ilx'.'tot’/c '.'OS^hv lot rtoT:.«at arft ni^ B'dP s it T ■: r^:jictQmQf> 1*9 njafnbsO tfS v i-o.’j to &Jos>>@ bf^onscinQ rioirtw atne^e i7*. . .4tL£ .??.Xav^j: 'UBloXisoaitni taaaaianX o^* '7-uiv'-i?c£5 Tarrto X£^!)V6a ,«vq.Xs baaat/oaifc '^•i -t pt bfii/ot 915W fToitp* to ittnainadosw Buoitjav ♦ aXe&rtXrnije ani'u/q -.Vi' 'r,^v.X0x-»c £ 3: (3ftisiP t^^.o.LV3or,s'Si/tonM^A-a*a-X)0-B*iA ■• i -i.-fi 2i floijtvv to ^rtlipffa7«a dvxjpjs .jolafra anibit olxQtct^i-. 2li ax'iaxa O-j-A ,'J'ri5-.a*iA ,itnot ttiuiq -wcic-ni ;^r;ortjXa .aa^^gor'^io^r .A55a to fioltJtdicUrl adt rtgi/o'irf^f aol ij^f'ifnoD r.^sT-ioqmt rta art Vjani AHXI o#^nX O-frxA lo noit von Jb ,0-*-A to aon&esTxi artt nl . (CS )rioXrt5>£ •iojw/;f.iJ-no art! rtl .Xoixn:,? to ort o:f terol aaur aiaarfrt-n^a tnizuq ': t.i«rti£nor-no? a daiv. BaarrertixXcq A«<: to rroitlrtlAii t^df aXrtXaaoq iiTTfl eninabfi to qu^Utud s 0^ «^«X aisitdtnxt AiTC ni aartaioab aieontn^^e ovob ab rlrtidnX aarto- rfoirtv/ ptbifotioua ■‘O&Iok/noali to sfroi^^'itftaPiTOO 'ir.Xi/lltos^tax basaaioftl J battiaimao Xa^it ane rtXrtlrtnl ot fc-iiot naart trjid saWt jr / arf.r ai rtpInV sX«artr^r:8 awi^q ovon ab oi 33 levels also inhibit phosphoribosyl pyrophosphate(PRPP) synthe¬ tase, the enzyme which supplies PRPP, an essential cofactor for amidophosphoribosyl transferase(10,51). Given this pro¬ posed mechanism for the inhibition of de novo purine synthesis by Ara-C, this agent would not be expected to enhance the cyto¬ toxic effects of FUra since no significant increase in PRPP levels would occur with the elevated purine nucleotide levels inhibiting PRPP synthetase. Thymidine(dThd) is a pyrimidine nucleoside which in¬ hibits ribonucleotide reductase in its triphosphate form(dTTP). Inhibition of this enzyme is thought to result from feedback inhibition by expanded dTTP pools and has been found to cause reduced dCTP levels and therefore decreased DNA synthesis(25). With decreased dCTP levels limiting DNA synthesis, an initial increase in purine ribonucleotide pools due to decreased util¬ ization may result. The observed reduction in de novo purine synthesis by 27.5% niay have been the result of unused end- products of purine synthesis feeding back and inhibiting amidophosphoribosyl transferase and PRPP synthetase. Thymi¬ dine has been shown to increase incorporation of FUra into RNA and to increase the cytotoxic effects of FUra, though the mechanism for this has not been well-defined(26), This drug sequence has been found to enhance the interference with ribosomal RNA processing that occurs when FUra is used alone(27), Pyrazofurin(4-hydroxy-5-B-D-ribofuranosylpyrazole-3“ carboximide) is a nucleoside analog which when converted to its monophosphate form by adenosine kinase, inhibits orotidy- late decarboxylase, the enzyme which converts orotidylate(OMP) ;• - .1 hdlrir^I 8X«v»i '."i-.*-jaaa aeiisjcrf^ii rtr?irfw sm'cs.i'i*' fOeaf -o'Vr --• - .-.<>■ 'C , {i’: , ;.VJ-?.oHiiarfq«ortqofaljBj8 lol 1*^1* ..... V.*, ii^iij ova;' 5fa *0 sii^ toI nefaft8/fa®fli ' o+ E;v,j*09r|x.-« tort MLOw ta-:^^ aitif ‘ -c'■ t-i'::f,: ■ '-•n aortia *110 sJotll# olxo^ el-, r ani'ir-q fa-i^^ysjCj 1,7 tuo^io faXwow BXtvai .3<;.^tartf-yc .tiniiiX.Mni «I#ytX •:<•; .10 ; .j*.': ;t^ -/a.: J’.'bfi**! Qfai^.od.i.di/rtocfl'i B^“Zcflri :^.;?.S'Z 7- ■^■.^iroriT i-jX iii£t.'^,;.,'tj? aiu.T ‘lo noitMfailrfil . £3ri tn3 Uooq J>'-afan.iS(DC9 yo Aoitlcfiririi i - fav''i.qsioa u j’„<* a *jid<'(r :,'ii *^..015 fallow 6aTf ■ - ■- .-J-rV.'.,- AVfC s^ivrixr^^r '^T0^ in-t. v»vaiifa rfJiW •-•--.i.c • ’ 0' Ml alo-jq afaX-'a^Iour'a 7a.i sajeaiofli f'.' -♦ . T .’toiJ’JBii ’'-•ri,.-,; ^ ‘wA^aei v.; *'rs v«l iXearirmtc ‘. -“ / :v:3 .■^.C'ljd'' ^ru C‘aa''l rr^'H to s^fox/boK^t :-’' .‘^'‘•a.7i^. • r/2 tax; *'7 =;7-.Vl:xi-.oV'' ^Xv.li'2-*1iiortq8oriqo6i<>L8 :.‘ rr'.'1 ‘- ::i■• 37 7.^;7oafTx aigy.a'ixrti . ynu:; nso ?r .yxOfi *10 stomOIv aavinarti f?7 br*8 ,AHfl -1Vi;; Lenxtifa-XXsiw nsed *oct 'loA wiiXrffixfasm l: •.a-oruBiXtie oj ad« - sr.o,:.£ ai artl'X a*i«ooa 'i’Gsi'^ 8^ia34a7^a*K7 AilH XATioaotfi*! -C- aXo-ae'rr^q . • .r^.n loba'r^*! oa' ba’^idvnca »i;xso*->Iotjyf ^ -ti \ isbimixocfiflo -yfafafo'xo aTia'iiii-rx !nrt,v9Pj^al3» xo '^J’adqaorfqonofli ati AX'tfaiTTO'IO dol^fw 3lYf^S7<'^ :-.ti- , a€AXyXO(f7fiO«b 9^*X 34 to uridylate(UMP) (28) in the de novo pyrimidine synthesis path¬ way. This results in reduced pyrimidine ribonucleotide and deoxyribonucleotide pools and in increased orotate levels as decreased UMP levels stimulate de novo pyrimidine synthesis(29). Pyrazofurin inhibited de novo purine synthesis only minimally, (93.8^ of control) but this slight decrease may have been sec¬ ondary to an increased utilization of PRPP in converting the elevated orotate pools to orotidylate. This effect of pyrazo¬ furin in increasing consumption of PRPP by orotate phospho- ribosyltransferase, will not only decrease de novo purine syn¬ thesis, but will antagonize the actions of FUra by decreasing FUra*s conversion to FUMP, the first step in the metabolic activation of FUra, Studies of the enzyme kinetics have re¬ vealed that the conversion of orotate to OMP is energetically more favorable than the conversion of FUra to FUMP(4), Cad- man et al, have examined the values for the conversion of FUra and orotate to their 5'phosphate nucleotides by oro¬ tate phosphoribosyltransferase in L1210 cells and found them to be 520piM and 12pM, respectively(3)o A.S. Bagnera, using Ehrlich ascites tumor cells, has shown that increased utiliza¬ tion of PRPP by phosphoribosyltransferases is a potent mechan¬ ism of de novo purine synthesis inhibition because the Kjyj for the phosphoribosyltransferases including orotate phosphoribo¬ syltransferase, is in the range of 5“40piM, while the Kj^for the amidophosphoribosyl transferase of de novo purine synthesis is ImM(lO). 5-azacytidine is a cytidine analog which is incorpor¬ ated into RNA in the triphosphate form, 5-aza-CTP(31). 'M -.ncjv/rt;.-: ^ ;ifci.Tiir»:q over fi) edt rtl (8S) (ot ei-?i«i a.tXfre^ s'irtT .^sw j,. .,., o j l.•‘„l'a.fi^r^i;^ t fii fcdjje •6^^^^#Xs4/^!0‘i^x^^2X09b :?^in;:"':c ovor. >b eX^v©! ^ b^afid^bsb f f ’ - t' -r-„ avon fc©tidX:lni ni'iylosi5’i>5 3V-1 Ji-i 33i5sto.^^|) s-rtgXXa sicit tuc’ (Tcrt/ioo lo 3^5.CQ’) . ' ■ ;.'*>t;o ri '!;o iliyjj beB£f»‘i''.r;‘i '^Tsaferto "' ' ' z - r r?i.iT ,-d7~I.xbi7Qn^ c& aXooq o.TeTO'to bat*v#i» '0 1i\ »rof r^qmi.'£^-30 ^'ricfiSTOfti ni niTjL/l w.*,:- •-ni-.- 'v:,'! 90 9c-^e*rr-3f) -ino ^on IHy' .Bs^'istmas^^x^odi^ »L : f-i’-j -.s tv4;;- Xi:w jud ,aia9tit oixol£-.-.:n 3ax .tI' 7 snt ,^iym o.t ro;e'x*V!!Oo a'rsUt --•1 57SQi.-=»nrcv er;; >o ae^hvSa Xo nol^gyitdg , ; i '..:o 07 ?;;.:-o ro >xt rroi.n-r^vnoo ©rt/ TsTft Da/^iav --.•i:. /riU i foxerwvr:-;- 'iidt' »Idm>ys^ rxo« ■ vr.‘ X' S7i...cv , y. ‘.»dJ iBr'j xT9 trV£d ,1m tf ^UKI * -*I0 v;'! a-^r: ‘o f» .', .a I 7.t. :CrtC.*iq*? ilvaj C'i -jjz^joto t«ji nU^ 'lo • ‘ ^nanj- fcirKJ.>'>: /^nj «iXo.o orS'Kl ri Xx;«o'J,t*Torfq*ortq •4'a:r .3.A -(t )\fX9vti7-7^33i*x ,,?8i^SX fru atf ' "■ ■ ‘-'5J '.it .«ye^f8 aart ,3j.Zvd aatioaa rtoiXTCrtH -.*:;'J^ - : ro'-vq a ai -mi^-.slefts'tcrlxfiocfXT’aTJdaoKq q TO. it, 3Si,*oa griiit'XofiX »#'iE’3a^.r^ne*i?£x;:ao.ifi’o 5(it ^47 -i!i!w to omjKi fii «1 ,tajn3tan^X’ axM9f(f(r/P unt^taAf to oasTatnnsTf r^^ociTortqeortqofaiiiLe .(OX)MmX al -toirsoonx ai rfoiflw ggjgrs errIiijLTxa £ al vaibitT^ossa-^ ' . i U ,TTnot ni AWg oStei 35 5-aza-CTP distorts the secondary structure of PwNA and the in¬ effective, fragmented nucleic acid is rapidly degraded into its component nucleotides(32), Taylor et al. demonstrated in¬ creased total intracellular nucleotide pools in Chinese Hamster cells after a 2 hour treatment with 5-azacytidine. They also found a 50% reduction in PRPP levels presumed secondary to feedback inhibition of PRPP synthetase by the increased purine nucleotide pools(32). In this study 5-azacytidine was found to reduce de novo purine synthesis to 62,6^ of control probably due to the degradation of faulty RNA molecules with the subse¬ quent liberation of ATP and GTP. Pretreatment of cultures with 5-azacytidine would antagonize the cytotoxic effects of FUra because the decreased PRPP levels caused by 5-azacytidine would retard the conversion of FUra to its active metabolites. 5-fluorouracil(FUra) is a pyrimidine analog, the active metabolites of which are 5-FdUMP and FUTP.(See Figure H) 5-FdUMP inhibits thymidylate synthetase, the enzyme which converts dUMP to dTMP, thus, treatment with FUra results in dTTP deprivation. The *'thymine-less death" is thought to occur as inadequate dTTP pools inhibit DNA synthesis leading to degradation of the fragmented pieces of DNA(33)» FUTP is incorporated into RNA and there inhibits the processing and maturation of RNA precursor molecules which are subsequently degraded. FUTP also causes an inhibition of ribosome formation, miscoding during mRNA translation and mismatching of tRNA*s and the appropriate amino acids(3^»35). The reduction to 20.7% of control for de novo purine synthesis found after treating L1210 cells with FUra alone is probably the result of both the brrs •'■;.... -.^i r .' -i-:-* ni him ol^loua 6*9tn4WR':5Jt'Xl - ciiC’Hsb , , tj ■'loX'i^T I. {2C) 8#fei^'oeXox/n atl '•rr, -E.-: -.2sr-:. ■;- :i -sfeitcalayn! la.+o?- fcesjs^xes - J-nlibitriJlw jTTQffirrgif 'SLoti S r 't&7la 9II90 .T- ■: be.r.ws^-q sX^/^X q e--.i^:--,Ti 3.1- MX -c-iiidxrtni ipj&dfcaat 0: -vfitw ^ ci:^T nl .'C^^cXwoq ati^oaloun • :j ii". .100 to ^ -"i^^a/itrrva aiTt-ii-q cvon tf-tX :':r-- -y^.. 77 a;:;1 v^jlqa": to acr b :. -^01.7i., .'/.TC KqjB lo q9i7.n* i3_i^q .0 oiao.t-jvo -9n7 sai:io^^7pt£ bix/pw anibX;>^3jj:tja->^ - ;.:3 iita3CjiO f^X-fV^X beasaicj^?f> enr eaif*Oi>d • S JTrXo gvi c'tU'? 1q fT0i3r?vfro3 »<1r»’ ln*^#T « * ^ r TV I r - K f T' * j w , ^0 i- T-"■ ♦ r -.: i i? I’fYo 'i X i ;^-t U '•!) £ I aim.' o n oj/Xl -c Db"r-= M -7r(.i sas coxdw lio 35?X'iIod.!i3'©in 11 4 3 ?!} ,icrx./ ^ nvii:y . rf:f .•-:;ct9r{Xrr\;a atldiitni r.^t-3Vi-;~.b 'HTTb ni .-' ’ rfJrw .rom;}'»9'iJ- ,^MTb i'x^.yw^CT. a/i uifo jO ':.r if z^.u-f}'17 ’*at-4sfc saeX-er.fariT ':•? 0 gniDaaX 3i;rsnm>;a »!C fXooq ^TTb o^ni t39~ EX -ixUa J zF^AilC lo EsoaXq l?»^ra A/n >0 r.t,r^'74iin fanx ■irrlBc^ioo'rq g/tq sqirfXjifu bna AHS iiw , .bab^igs-o •!j.:.7gi;p«>,/ jrXb-TPixin ,noMiiT-20l ^iio^dx-r "io noitXdirfitl etisij-«o oeX^s 0i.j Xna 3 "be ^i^XsrfOiJj^sfXia cue .'tc lSibX3Xt,sqr? t^Hci ^*:iTt/b 'o JifT.Oa oi noiy-oi/be-i erfT ♦ oniara acfjiiTrqo'iqqa Six yrXqjie'xt [crircl eXss.iq/r^a sni'iuti c/ijn ^cl lyitrtoo •rfy ritad Iq d-Xx*-®*-! ^^Icfacfo^ si sqoXa «tU’^ rttivr eXX®9 i4u 'k 36 Figure H Metabolic Conversion of 5-Fluorouracil v43) FUra — » FUMP- FUDP- >FUTP-> RNA 1 1 1 V FdUrd- ■> FdUMP <-— FdUDP 5 y1 dUMP -1-> dTMP 7 Enzymes 1. Orotate phosphoribosyltransferase 2. Thymidine phosphorylase 3. Uridine phosphorylase 4. Uridine-cytidine kinase 5. Thymidine kinase 6. Ribonucleotide reductase 7. Thymidylate synthetase "thymine-less" state and the production of faulty RNA molecules. Thymine deprivation leads to decreased DNA synthesis producing a build-up of purine nucleotides. The degradation of defect¬ ive RNA molecules contributes to elevating the levels of pur¬ ine nucleotides which inhibit de novo purine synthesis by feedback inhibition. 5“fluorouridine(FUrd), also a fluorinated pyrimidine, is a precursor of both the active metabolites of FUra, FdUMP and FUTP, (See Figure H) Therefore both degradation of faulty RNA molecules and inhibition of thymidylate synthetase with ^ i't - w^rA^ftrsO ,| . »t^I Vtiii t, (..:; icU ft T , S >RJ:^^?^ arrifl'i,,4 9>!rrlhbr^Hf . ^sjb. j tik/bo : «* aliJ'of ifix/fjodifl ’ -I =: j f**oi;>;>vi>n ;q arts ^4•id 0 »•! *• trrla\;/< ji . ri-j a 3or: .<^11 ussadi^'.^o 0^- frol^-irviTjj^t frtiflrs/rt J ' ■ J : >-• re r3XtsJb/‘^3«t^ $f(T .Tto '.s ^ • :.\.*.T • o aievyj e/t^' ot zorJ issXi/otlofr AWK dvi * •O’' ?ir»33.irr;'^3 w{7ciU fij^rranlThuXl a oaX^ *■(o*sU^ .iTfin ^0 9drriIoa-a^»r dfod U torr^^oy^q i'ei VitliJdl. t'. tTsoiJ-^ket^a: rirtatf ;h evZ: ,<17^1 ttfu #aat3.4;rAv;i* 'o noi.^loi.4ytI Ltit amo 37 the subsequent production of fragmented DNA molecules would result from treatment with FUrd, De novo purine synthesis in the presence of FUrd was only 26,1^ of control probably be¬ cause, as with FUra, the purine nucleotides liberated from the nucleic acid breakdown inhibited de novo purine synthesis, 5-fluoro-2'-deoxyuridine(FdUrd) is phosphorylated to FdUMP by thymidine kinase(36) and is cleaved to FUra by both thymidine and deoxyuridine phosphorylases(37). The primary metabolite of FdUrd is FdUMP which inhibits DNA synthesis by creating a thymine-deficient state, De novo purine synthesis was reduced to 53*3% of control with FdUrd probably as a re¬ sult of the build-up of purine nucleotides following decreased DNA synthesis and from degradation of DNA fragments. Some conversion of FdUrd to FUra may also have contributed to FdUrd*s anti-purine effect. Purine Analogs and Antagonists Hypoxanthine is a purine base which is converted to inosinic acid(IMP) by the purine salvage enzyme, hypoxanthine- guanine phosphoribosyltransferase. PRPP is the co-substrate in this conversion donating a phosphoribosyl moiety to hypo¬ xanthine, IMP is then converted to AMP and GMP which feedback and inhibit de novo purine synthesis. Treatment of L1210 cultures with hypoxanthine decreased de novo purine synthesis to 13.0% of control by providing a substrate for the salvage pathway of purine biosynthesis. Because the salvage metabolism of hypoxanthine utilizes PRPP, hypoxanthine would be expected to antagonize the activation and therefore cytotoxic effects of i.' iw 0^. 1,0 tn*jjp%e •••xxi. ovon ag rtcfxv/ fctm&Mfriir mo't'l flu --f ,i * o ' . £g'iX''.oc Ic «x;l ,dS xIs^q to ’. :. -.aXint-s^^tiXoviXciun srriiij't -r^T dtiw sji ,«et/ao 7>c'.\’-u:kX Qvc:t yf' 50^x3“/.f * ri, '■-••I 1 -f r EX ' fyiU ) diTi' S-o'touX'i*^ xiVT ). 5 , IT-;.,.G si ..b/T^ (•'C^)98HnxX <»nlbbcr^{it - :.''fsIv'iorl.73Ciiq dai Jana $nl tlm^^ .; ■ i,' a>^3 eXx'I.'.u:: .ioiriw si b'iUb''T To stilctfjstaiK j*.'T*r.. ? ;>YC.1 s,^icfa«*io •: i-. 3.e vixEG TJ ‘tTiw Xo-x-fncb to o.t oeowbei esw ‘- '. I C *i.'I .V«bxj-09X0U;-T 'JHX'SUq IfO qD'bXii/ . ■ Al'i'i 1 j j{0x.^ b nTo*!! 8iaoflt£r(a AKQ L -iiViJji i?xx '^sm GTi'- at S»iyr.'? to noi8*x»vaoo . .-J'j rit» wriTarq-i^-ns a’bTcObH iirjs EiO|iCa *.'•-r-,--’vr:oo ex ;tDir-'w eni'iuq >i si djf!itt.7;‘!jBxoqYii 555^/las 3ni-iL'q -ad^-.oinisoni .•«Tx-i ’ V 1^-3i .sss'i&tarrp'is+Cv.eofi^i^oftqBodq tninsi/a • oc '.d f: ’r-iio:' IxsvrSlzQiiqaoiiq s j^ni^tAttob (toxBa^vtrao ai/ld^ ni X.* : .'i3xdw Vj4{} ixt^ '^AA ot barTev/joo n:fe4^ si .aaid^nax . .i ro jn-902rj>«iT .siesdc^nYS afiitinj ovon »b tididni bna TiEarii'jtya vi’t'xfc'q ovon ob bsBsaoosb 9aid'a:n:a»pq\:d it^iw ao-«/:>’Iuo “^£v,Ix;p. -adr o.i 5*X;?o-3'acfLa s ;|^^ibivo^q lo'itnoo to ot ^eiXodste/ri agavlx/.i. ‘>rft .3ia«d^ir^:njoid oni’xi/? to f>!9tc-#qx8 ad oXwe^w aajiilitr;' onidtn£XC ■ f'i' „•.*'; ' 38 FUra, Cadman et al, found that adding hypoxanthine to L1210 cultures pretreated with methotrexate prevented the methotrex¬ ate-induced elevation of PRPP levels and enhanced intracellular accumulation of FUra(3). Inosine is a purine nucleoside which inhibited de novo purine synthesis to 9.3^ of controlo Inosine can be hydrolyzed to hypoxanthine and the free ribose moiety by purine nucleosidase. Inosine may also be converted by purine nucleo¬ side phosphorylase to hypoxanthine and ribose-l-phosphate. Nucleoside Phosphorylase Inosine -Hypoxanthine + Ribose-l-Phosphate Pi H2O Inosine —-Hypoxanthine + Ribose (38) Purine Nucleosidase This hypoxanthine can then be converted to IMP by hypoxanthine- guanine phosphoribosyltransferase with PRPP acting as the phosphoribosyl donor, De novo purine synthesis is probably inhibited as the AMP and GMP formed from IMP feedback and in¬ hibit amidophosphoribosyl transferase and PRPP synthetase. Utilization of PRPP in salvaging hypoxanthine may also contrib¬ ute to the observed decrease in de novo purine synthesis(10). It has been shown that in cells such as L1210 cells which possess the enzyme purine nucleoside phosphorylase, inosine promotes the incorporation of FUra into RNA by providing a ribose-l-phosphate moiety v;hich converts FUra to FUrd(39,^0). FUra + Ribose-l-Phosphate -> 5-Fluorouridine Uridine Phosphorylase From FUrd, the activation of FUra proceeds to both FdUMP and vi-CiiJ-^.aoqYri :^afi3ifa J-fnif- htii/at .ijs tt ,5^^, X'-' ' " •I.' 4,1" ..o, ©?ax'3i‘^o •• D /S't; w .tr b^^onsfiiis . 3S3v-3l 4‘im >0 fiol^rbv^j:* b«unjkjtl^ets ,.(CV^'^U‘'5 to nol3^Mlimi/doa ~.‘i7 rt'.:ir V '^r.ii=i'.r.„ ,. la-xtriGo h) 5^C*9 cj*wiix/q ovorr g^'il Liijs srtiriiTrc&xoqxff of bajtxlO'Sb'S^' < omo'-:vvnc' d 02X4; mlsonl .•aabiaoeloun '-■ Iq-r-sf.oci-: biTL-: -snirtJq^xoqY^f o;^- daal'oortqao/fq ebia aX^isoax-v’-S/’t! r -2q£;iYTO,-1q‘5orf«I sni.'f.trr <••' I--— fMiiaonl ■5r} c--< 4 -..rrrn-rr.4.soq-,':i: <-^— -aftiaoftX aesbr sOfiIoi/?1 uaiw't Tv.n - •-’ b3J*j9‘.iioo r'drft rtjsa oni-’^^nsxoq'^rf iirtT •'--’^ bJiw (»rrinaiu^ 'r 2is9d:faY? ?n£*iuq ovort aH .Toneb •'XBOi^l'xortqeoriq ‘i 'WI n)n*rt b®rtT":bl bns orit as bsfidltinl > a^^^..Ya at.3 392*1$; lYabb'iqdbqaortqohiiRfi Xibirt :-n-c x£ arf:,f.7naxcqYrf-§oiS-r'/£.'}» iri Yo notfaflXiffl 0. ^.•./« >a£Ujq ovo^'T '-5 fli bav'^asoo Oiif of sfu .‘.:>i:’.v aiJj- iirrrj 2® ifcce sIi?o nl ferif iitfo/(ii nsa(f as/i fl ?:.£soai ,^•'jxlV/ioriq2 3;(q &bi.3b3Xni.in art^'ijjq -au.pxjflr® atif Rse-asoq a anifeh-o-rq v,cf AWH • o:f arfJl 2v -rav,*:oo .1oi.*iW yX^Xoc •^^/tqaortq-f ^.^aocfii or-lt f luoio^rX'^' ^ -»■ **ii;i ■ 3ni'.’iTO iasXYaoi.qaoin ., ’-•^■is /ij-ocf ot eb^soo'xq ^iV'i lo noX^Bvi^ojs *5iin mo*rt 39 FUTP(See Figure H). Inosine, then, would be expected to en¬ hance the cytotoxic effects of FUra by providing not PRPP, but ribose-l-phosphate to activate FUra, Adenosine is a purine nucleoside which inhibited de novo purine synthesis to 20,7% of control. Adenosine can be converted to inosine by adenosine deaminase which, after met¬ abolism to hypoxanthine, can be used in salvage purine bio¬ synthesis, Adenosine can also be phosphorylated by adenosine r-' kinase to AMP which will feedback and inhibit de novo purine synthesis. As with inosine, adenosine can be catabolized to its respective purine base, adenine, by nucleoside phosphor- ylase producing ribose-l-phosphate which can convert FUra to FUrd, Adenosine suppresses de novo purine synthesis by con¬ version to both AMP and hypoxanthine, the latter which is sal¬ vaged to IMP, Adenosine has been shown to markedly stimulate the uptake and incorporation of FUra into the RNA of Novikoff hepatoma cells by providing the ribose-l-phosphate for activa¬ tion of FUra(4l), 6-mercaptopurine(6-MP) is a hypoxanthine analog which inhibited de novo purine synthesis to 58.7% of control, 6-MP utilizes PRPP in its conversion to thio-IMP by hypoxanthine- guanine phosphoribosyltransferase(42), Thio-IMP is then con¬ verted to deoxy-thio-ITP and in this form may be incorporated into DNA, Thio-IMP prevents the conversion of IMP to AMP and GMP by inhibiting adenylosuccinate synthetase and IMP dehydro¬ genase, It also inhibits amidophosphoribosyl transferase by mimicking the effect of the natural purine nucleotides as negative feedback inhibitors(14 ), Thio-IMP's role as a pseudo- ■ : ,:i >0 sixoTot’^d »o«Bri ■ ■- ■ 'i,biaosl‘'V\ saiauq^fi al mtuoiivlA : ■ ’9h'.A .,.Lc’;-^lo'c ''r* -^'■, . OS 0* anlTUxj ovon - . ‘’£_''^’*' '^--•''i:f!j:e r> X-- 07 StmTH’^YfiOO ^ftsTj/n i.-?:.''. j-.v; :. w»o<6.i -mj cfso ,»ni^7{texocpi,fi crt mBliodM' • >•• ■ ' '■ T 'Oitc ad oalA rtfio anlBOi^abA ^ai»oi{7ity£a [j[2Vf d'jtr.w <^A ot «Qptxiil lo-if.. 1 - j ,^.3- . 1. ,i)rUsOiti ffJ-N zh ,&i99iittvi9 ■I -*' jjiai'r. 'id ,3i'iir{isbQ ,«3eis ’ r.j*:: rtorrivA f •#«odJ:'5 ‘^ixloubcnq daaX^ •-.iait.'.-. i:';.5 i*7i^uq r.vr.n &ib 960!a**iq<|t;^ eniaanBbA ^tnU^ '-’■ 'u.*;jjI . •?.ii •.^r;5xoq'trf rif, -1 ::f -A-iiic: ot ,T,.c.i‘>: .-lifet BC'JaoasbA • .'?MI ot bajiBV A 1 • • .v,tp. s.iJ- siU’-I '>0 noti-:;ao^r^^6fTi boa t-Hatqy »rtt ' e -• - '.1C-^-r-po "f.rr -iitt ^nicivocq 'Cd bIXoo mo7.:qoii ,it4)jsrLm lo nolt •'J. ' L^rc gni ttu-cyocvn i- ni )^nJ'^A'xyo>q«o't3m-i .i:' u*r.'oo oJ“ 0i?!i,*r, rn-^e erri-it-'q’^'bvcn »& 6#ti -vf. xcqvr' qo 7Wl-.<)xdi ot noia-t&vfioo atl ni aoalLldu ij >2i ‘iMI-oi/iT • (S4i)-«saPTt#i{*,ff •j7:rT'-q-rrcni orto^- ax/^* ni brjs ot Jbet^ev Ld& qwr io ,:OTU'reVi''00 :>.tlt^V'»'Tq *aMI-oi:iT .Alia otni - -.y,i orr^ sBflferftnve 'id S3J0 •:d 9 3BT3'l£nTi. .f’^odi'ioffqa • atxdtrixti ciXja tl .Baarsaa a* aofti.;o.s.xoxfc X^rr^n ant lo ro#!;!# tilt >ofM;,4aq 4 34 bXot •♦-i-il-otrt’? . (: ii^inoticfXrtnX jioadbftl tvlta^an 40 feedback inhibitor is thought to be the major site of inhib¬ ition for 6-MP because the fro thio-IMP in inhibiting amido- phosphoribosyl transferase is 4o4 X a concentration generally achieved in cell cultures and therapeutic regimens(42), 6-IVIP Would be expected to compete with FUra for PRPP, the sub¬ strate both agents utilize for metabolic activation. 6-thioguanine(6-TG) is a guanine analog which inhib¬ ited de novo purine synthesis to 15o9% of control. Like 6-MP, 6-TG is converted to its monophosphate form, thio-GMP, by hypoxanthine-guanine phosphoribosyltransferase, utilizing PRPP as the phosphoribosyl donor. Thio-GMP acts as an allo¬ steric pseudofeedback inhibitor of amidophosphoribosyl trans¬ ferase, thus depressing de novo purine synthesis, Thio-GMP inhibits IMP dehydrogenase which converts inosinic acid(IMP) to xanthylic acid(XMP), Thio-GMP also inhibits the conversion of GMP to GDP by nucleot'ide monophosphokinase (42, l4), thus allowing the relative accumulation of GMP which will inhibit de novo purine synthesis by endproduct inhibition. 6-TG would be expected to antagonize the cytotoxic effects of FUra since both agents compete for metabolic activation by PRPP. Virazole(1-^-D-ribofuranosyl-l,2,4-triazole-3-carbox- amide) is a nucleoside analog which inhibits inosinate de¬ hydrogenase, the enzyme which converts inosinic acid to xan- thyic acid, the precursor of guanylic acid(GMP)(44). Vira¬ zole has been found to be structurally similar to guanosine in its crystalline state. Its active metabolite is virazole- 5‘-phosphate and it is thought to be a substrate for nucleo¬ side kinase(44). Virazole decreased de novo purine synthesis _.f;, v,- 3». -TfoililAttJ. i^6 'I : ^ j ,'»r“0:- X T^4i 0ii aseis-la.-tirt^ r^eoif ^'xoiqaodq • ,■-.’c-if.:i'- '-;::..-iw,i,t iXt3g&-qi - 4 •- .-f.r ..^ TO*" i-ftiy^ .sjaq.'no-^ o" ’:e.3o»^*« #(f tU uo^ ■* ^\ ■ Oi 5'iXoij:;! j": .i'V 8.; 'j&ac*t:i'Q ^.i: 2 :-': 'v.'-i ,0 ^r'/cX o.}” tjii‘tsjq ovofj •{} .;,no'l 9:t3i'iqi^0/?qo.'t(?.r 3i-i ot ^*?f*tiV^OO ai OT-d ^•■^' i-. ■'-3^i-3'^'^s^Tivr>ccii'i6riqanrfq •ninjsvs-s/iirfSrtJBXoqtr< ' .:, '\iOi:-oi.i: .Tonob Iy0d-.liro(iq9-.;'fq oa •' •'- ---‘•'"'U^ri^iqol: -.? “to ipvldiru''i 3Co8Jij»*')r'i’ii*/9aq oints^a ■ i •34fi^jjq ovon ifi) .esai^l '’.. j^.'i^cnr stiavapo rfoinv qwi a^Jtoirffii ’■• .- .■ ^ ... oais .^'^^tX)^*''^A oJU^Cfi^fXMx os .'iif . :-^)d>jai7i:.0 4H ::o,r -.T^r:. . w .IpIK-.v ■'u.ir, 'Vo n0iJ'5lt>.‘nuoo-8 'r^iaale-i '^U ^i^oXlB •^X- ’ . ai/©wboaqbn*) cd aia#/!?nv;e pvoft 8i) !!:*/•' '^ . a^o-Dl's 0ixc.t0'"v,’J ailT «xXao.r- ^■ •.* t vd rro:?Bvi^tc ■ 'ict ric.od oonio •#o •!»/af:XBoriX sJidfcri ,s.oX£rtiB e.^«o«X8iuifl 8 ai (s^fnia - \»3X or jjloa oinibonx 3l*T:svr;oo i(Mian8;jo*ibic-ri - «*ix / #'. 1^0 (''^'■ Tf * w * to „'*!oe^w»e>i<'; 8 ^vAnoami^ os a x.i^a^uSc/U^ysi ^,4 oS toot ftoW «j4 aXo* 9i 9Jliocfc:*^v\ otjcitbt 8J-I .aX-.'o:?-* 8^1 (li ' - 'j-j.Cotrn ‘tot ftr^zr^^L'z s ad orr ax fl to ai^fflqtorfq-'^ j 'i; s'a.-iv'aAfi i sjn-.i'iyq ovon bd'dJE#ioec s^losxrri^ * («Ui)t&aaiji «2)i8 j -'''‘!'ii J 41 to 39.1?^ of control. Inhibition of guanine nucleotide synthesis was twice as great as irihibition of adenine synthesis. In add¬ ition to reducing the guanine pools directly, virazole may also have decreased AMP synthesis because GTP is a necessary cofactor in the conversion of IMP to adenylosuccinic acid(SAMP), the precursor of AMP. With both AMP and GMP synthesis decreased by virazole, the precursor IMP builds up and further decreases de novo purine synthesis by feedback inhibition of amidophos- phoribosyl transferase(24) and PRPP synthetase(45). This in¬ hibition of PRPP synthetase would result in decreased FUra activation making virazole and FUra an antagonistic sequential combination of agents. Other Anti-Tumor Agents PALA(N-phosphonacetyl-L-aspartate) is a transition state analog inhibitor of L-aspartate transcarbamoylase, the enzyme catalyzing the first committed step in de novo pyrimi¬ dine synthesis. Treatment with PALA leads to decreased pyrimi¬ dine ribonucleotide and deoxyribonucleotide pools and to a marked decrease in orotate synthesis(30)• The reduced pyrimi¬ dine nucleotide levels limit DNA and RNA synthesis and purine nucleotides accumulate due to decreased utilization. PALA may interfere with de novo purine synthesis(85^ of control) indirectly as the elevated purine levels reduce de novo purine synthesis by feedback inhibition. The reduced orotate levels would enhance the activation of FUra to FUMP because FUra competes with orotate which is the preferred substrate for orotate phosphoribosyltransferase(9)= ia-aj-ircr-rg arfin^i-^ to mtticfiftnl .i^TJ-rro© to o ■■' ■' . 6* ) j,-.V - ,,a ‘^ni.Tfft:^ fsoitficfliittl «b T49^ ejs «clwt -a'Jsw .>'.-t-> ’^r-m 9-':-”T. ®Xo«x? ©rft ytiov;b«*J rtoitJt •‘.OC ■/-’^33 3'j:-:v :5 »I-aortt£TMa fe0«s#'^roe4J 9V«fl •■■■•• oiaicoui>dX!4«^.i»3 ci.t m to rtoie^tvnoo oiicf ni' : c,MA nf^d riJ-tW .ajTA to Tonwodiq ii-EjSi: ro^b 'JauT'urJ tr:z ibXxucf ’'ii'II tosnifoenq ffij* .^XosBiiv ^cf w^.lqobi..t zo r^otf tdiiZrti. :iiosdb -i.v -,A r 4 a.3£j .9nTny:a bets {^^)»9il~z^\t2nsnf Lx^odl*iCiiiq ^- ’’’' bd3i.?iOdD ni ;£i.-‘p9'3 BXifow to ftoltJtcfXri IC-'L cif j; ^.aogi^n rr,s rrs qt^T'T ijns oXosisniv rtdlttmrlS'OB, lo ItOitjflitflBOO ■at ..^ • ■ B ti (9-^-5;f*:-‘?q'3^i-J-Iv^»'5ATOrtqo<>rtq-K)AJ[A<1 * I :'3^XY'>.Ti-o - . !iir’ Iriiivq oo-^ne-zo^h oJ* ?.:>or -VlAt ft?r.>-w eaib ^ or tr.s aiocci -jbi’Oy£>o:^ncriTy:xo©fe Xo-ji' -%i-iTYq b»oi/fi<^T . Jt*)«rao/^rit\;a a^jitroiio ni Qrmtnoi^t baaiTteffl on.i aies,i-»fr^a AW« brta MIC riariX ^lavaX abirotloim 4nlb A^A'": .ftoirajiliTw baaBaroab ov wo atoiamwoo* •abiroaloun (loTrrfoo to ^SJeisBiifTiXB arfiiittq ovoit ab clttw arjit-iaj-rti Yjjfu 9ri-ufq ovoa 9t dObt)o?t aX -r^to'jo booebo-t 4.fif t.-rditM'ioal jfojKfbi^at y aayaoe^f ot Aiivl to rtoiraviroa #4^ aoitjsftrT# bXuow tot ortt «i rfoiAw ©ratoto rtr-iw teifeqaoo ’’ ' . (^y^awtfttaftattXYsotfitodqaorlq trar-oto I 42 Hydroxyurea inhibits ribonucleoside diphosphate re¬ ductase leading to reduced' deoxynucleotide triphosphate pools and therefore to reduced DNA synthesis(46), Hydroxyurea produces increased levels of ADP and GDP which feedback and inhibit amidophosphoribosyl transferase(24,45)i thus decreas¬ ing de novo purine synthesis. In this study hydroxyurea was found to decrease de novo purine synthesis only to 90fo of control. Hydroxyurea would be expected to partially antag¬ onize the effects of FUra by blocking the conversion of FUDP to FdUDP and therefore to the active metabolite, FdUMP, a re¬ action catalyzed by ribonucleotide diphosphate reductase. (See Figure H) FUDP, however, would still be converted to FUTP and incorporated into RNA in the presence of hydroxyurea. It has been reported that hydroxyurea does partially block the activa¬ tion of FUra in Novikokk hepatoma cells (41 )<, Leukovorin(5“formyl-tetrahydrofolate) can be converted to 5fl0 methylene tetrahydrofolate within cells and therefore can rescue L1210 cells from the anti-purine and anti-thymi¬ dine effects of methotrexate(4?). As expected, leukovorin did not inhibit de novo purine synthesis, but rather, in¬ creased it slightly to 108,2^ of control, Leukovorin would not be expected to be synergistic with FUra as no increase in intracellular PRPP would result from pretreatment with leukovorin. Vincristine, a vinca alkaloid, binds with tubulin, the structural protein precursors of microtubules, preventing their assembly and inhibiting the mitotic spindle. Vincristine arrests mitosis in metaphase and the bound tubulin molecules /S' , (^.4^5i.S'>d.ra‘4a A^a M^xolmadt tan o.ri dnfat:o9<'j 'l.^ jUf:,.? '^CA “lo a&a.u!>o'x . i’I^’iiko^X'iodKfjsortqobXisje ticfXdrri afi'v seTuxxoTc.vd ,;iu/?€ u.id;;i^I. , srlasfM-iT^ia ©^ri'iuq ovchi Bb ^rU 'I'j o..* ■-: j ixecv'jjr^C 3;vi*!;jy<5' otsrE 4?fe 3ss«'io»i> ot bstuo'jL' v,ll :,jl'2£q qj gff ijXwfOw iXontitoo -'^•y^l. In rrox^'iitv’V-, •>cd. to ai’Ofltt# 9gIrto -«'T fi , iMUtf? .A/i •.;>r!,-. .M-- €*viTos oT saoii$^«d^ bna <1QUt^ ot ^■s2) , %s^.j'ow«N‘r'l ^ 0 aai-'oi^X;»w'jr^tSxil Y U^d. CJ b9 "•'ly -?..' ll-X'ts t>XxfOW «'l'«V4SiW,Od ,^(3in (H 1*' -rl .£;-»*:«vxo'ib'^n o ^40^te^»9■s:i4 tr.~> di AiT^ otfti baS'J'toqioortl -&VA.ro£ •?d'' AocCri eaot liati;«tsd{f fe#txoqi>T naacf . t .(»<5 mh:>:£Sfi!dii , zia~ •^’/T'oa »c rtito niiovailinkl ’i.i'tfl'iartJ' ona sXI#o fiid^Xw a^fatlo 101 &Y.d4f'lv‘:Sv leffaXy.d^w. Oi,^ 0^ -tTrv,rdd-x .*T£’ fcixjs 9ni'siiq-Xrr *i iwft iflo-xt OXStu at/oarx rtao r-y-Tov-ojCjuai ,u*5-Qaqxft wA ,-crsx^ivOfX.r^ To ?.;ro-alT*, aniJb -fti ,7td3-jri j ; a . • s cf eit TifT oyp*-? »i> J icfidni Tort bib tiuow ni7ovo3{4/6sl . tc-T.'^HOQ :c ,^C‘I i?.t yX^rtatlB li Jb»8B«id oxt ss ol.TsX:^TaiTv:d i>j of a<^ cron rfiXw tr:-:-rrt£-#T :►■!?• to noTt buw-f^ X£ uflloojrx;tal ni .*ii ^ovojfy®! , iiU/do^ H^tw atrXcf joxtiv «( ,mzlffJ'xoalY zl£f{f i/iitAavnq to *no»7aa#7,q ia'xifJ’owTta • : ■ tiiiT?r.iV isfut ylcfmacaB ««, o :■ ?,i vm rixlifc'i;:}’ bfoiod odr »i%fi i*afldqa,f»« rii acTaoTcxe ; .di>i’wl.4liy 43 form intracellular, crystalline precipitates resulting in cell death and lysis(48). Following cell death, nucleic acid breakdown and purine salvage occur and the increased concen¬ trations of liberated purines decrease de novo purine synthesis by feedback inhibition. Vincristine was found to inhibit de novo purine synthesis to 15.2^ of control. No increase in intracellular PRPP would result from treatment with vincristine because the salvaged purines would inhibit PRPP synthetase as well as amidophosphoribosyl transferase. Therefore cytotoxic synergism resulting from sequential treatment with vincristine and FUra would be unlikely, Actinomycin D is an antibiotic which non-covalently complexes with DNA preventing its template function in trans¬ cription. Actinomycin D*s phenoxazone ring intercalates between two G-C base pairs blocking RNA chain elongation by RNA polymerase(49). The non-functional DNA-actinomycin D complexes are rapidly degraded producing increased intracell¬ ular ATP and GTP levels from DNA and RNA breakdown. The ele¬ vated nucleotide levels feedback and inhibit de novo purine synthesis regulating both amidopohsphoribosyl transferase and PRPP synthetase. In one study, a 50% reduction in intracell¬ ular PRPP levels was found after incubating Chinese Hamster cells with actinomycin D for 2 hours, suggesting feedback in¬ hibition by a measured two-fold increase in intracellular purine nucleotides(32), In this study actinomycin D was found to be a potent inhibitor of de novo purine synthesis, reducing the rate to 8.7% of control. With the lowered PRPP levels that actinomycin D induces, less FUra would be converted to the u i ^*^^sd'iqioi»'i Ui.^ o'dJ;>U;; il^.j ^iwoI.XrJ^ . (84)ala-y;X tmiE'nt9*ti ££90 *.^•V.TOO v-v-orri fcoe "£i;*?fsi3 ^^y£/s6 ©ni^irq b^j .-twofcjUncf ;i^jo •.^•>'t-.e «.':--*,uq ovo^f '^b h9t&i9iSl£ to nnclf^t .=;"5i.^J* fcmroj! B'j'fli ^’irior£X*iOi'iiV' .rfoxi'ii^jtrtiTt iCaadb®#!: '^^d' lii ;»^s»:-\-.x. .i.o•iI^■■■.-^<^ to OJ (yvoff rnsiKaxie'tT rx^asT tli;<>v qw! ‘i^Xi/II*04rttni" »n ‘ .'r/F jidirfni al^'ow aeni-oi/q baF^tsvl^© 9^^ ©aufio^d r-.'o 1'' :x2o^iao/lq»orfqo£>tms aii Ilaw iiv .'■f/i*,/ t,7'--ni.r£9T:^ isic’-rx^uoga/iro^Tt ai9ls*rdrTlie . V.Xi?itXi/t ^ 4. y iraijsvcc-.w (^bidv.- el a ftioxf^crrlToA * .'iita*! X ai rtoiTony't eS-sXqme.t a.rx ^ifnevsiq A5!C litlw 8#x®Xq«oo aaffeIcoT*1 ftnci*#% .'■ larfj e*«l fi:ir‘"ij;ia3rt'id'qA .ffoXi'qito ■^vf .tpxi'o^rroXs aiMtxj ^niAooXd snisq 0-i: owt rtdevrted XJ rr r-z-v.-^iirtlToi;-Isnolterfl . (^U) ea*'»»09Y(Xoq AKH - i r-ioanrai beefia^oni ^\loiJtiO’xq aaxaXqaroo -^;a artT ,mraf!Ji^9^.,i AHB brts APtC ciotl 3ii\''aX feat ^fk 'islu axTiiijq ovon ao fMirlqi bna iloMdl^e'i &X^-,>3l ebid-oaXpua bsJ’jrv bi‘Tii 9a£-M7=;na-£5- X\c«c/f-iQrfqaA.'^qobX4aj iftM yti^yBiyaat •isorttn^a -Iirfn ■T'rtrtf nl nntrou^B't s »ao r.l ■'.ifXrii.ujr af«i!?jci/oni -yarti* biwol aaw sXeveX rmiu -nl 3ii>/ifjb09l ^tTtsB^^a tBiifoft S d rtiP^orU^^^iit ji^h* iXXeo laXwXIaijG'ii/'rix rti eB^^nPfii biot-ov^t baTjxajisw a ^ rroiqidirt bruot aarwr a nio^onlffoa eidt rtl (Sf ^eebiJoalayn eni-xwq anxoAi&aT Vsiat^dc^itYS erti’itrq e^voii tab to tnatoq M*9d of fBdf aXavaX b»-iawoX adir .Xo't^'iroo to 3^^ .8 ot o^-btc ort;f Biit of b^t^sfvtioo 9d blwow B«ai. .oBOtibcil Q, rrXo\ioo]Titoji 44 metabolically active fluoro-nucleotides. Cycloheximide is a protein synthesis inhibitor which binds to ribosomal RNA inhibiting the peptidyl transferase activity of the 60S ribosomal subunit(50). These faulty RNA molecules are rapidly degraded and the liberated purines are salvaged. As with actinomycin D, de novo purine synthesis is inhibited by these nucleotides made available by nucleic acid catabolism. Cycloheximide decreased de novo purine synthesis to 10.9^ of control in this stud>. Taylor et al. found a 50% reduction in PRPP levels following a 2 hour incubation with cycloheximide and a concomitant two-fold increase in purine nucleotides(32). Pretreatment of cultures with cycloheximide would antagonize the effects of FUra because the decreased PRPP levels would decrease activation of FUra, De Novo Purine Synthesis in relation to Cell Culture Density Studies examining the rate of de novo purine synthesis along the growth curve of L1210 cells revealed that the rate of de novo purine synthesis decreased steadily with increas¬ ing cell culture density, even during the exponential phase of growth. Possible causes for this decline in de novo purine synthesis in the face of logarithmic cell growth include: 1) increased salvage purine synthesis secondary to either 14 decreased availability of 1- C-glycine in older, more popu¬ lated cultures or to increased salvage pathway substrate availability, 2) a decreased rate of de novo purine synthesis secondary to a lengthening of the cell cycle along the growth curve, and 3) decreasing cell volume with less purine and \ xXXjrdiXod'^^^fli "•■yTjw 'loTidi.ir: c*:.:ii':ta si ttoiwJbtf'^ioIo'^D t> - >.T^3 (rh/^qoKT T^^nUMirini X/woaodi-t eJ' aJbftlcf .-.i-iri \ J-(Uii> . (0^ . tinwcjys X4i»i!a«r'XiT 80^ ♦fi(X to ^flyl^QM •i :’- r..:TJ‘^ I ^{^f^rT5s^^b ^Xbiqjsf ♦fji 8«Xuo$Xoar ■': ^txrzvq ov-.a •.‘■•^I..jrT ya ^^ineiisysf Jt^sr 5«bij-oeloi*n bw^Idltinl -.1 ^ i^r>:nvB ^r;l-ivj .vcr. ar ^eas^c-st #biffrX.xdi-toiov,o .Br8iX0’d4l;^•o .'V 3. *a.r - , cb-'-ni i'.O'l^'lOO "to Ot !7l^‘ o: "17. i'.:l *rvcjn ^ 5 rtii/woXXo'l ^tXu’^aX :'ti .zoJtfOiVt>0l -i bio'i-awu TrmqtaiosirtJ-s » lKT;a wbi*Xx«rfoXoX9 '-:r\c->':?,i..X:'..D n7i-.* liJv 'to AS.C ) i^*t>l^otloun . oqss«*!c*.*'- riU i ♦/!/ foXycw . 1,1 ic nvij dr^»tO'T^& •Xtv»X ••, 7w.i."rrY8 ■svon rfc '‘ ■ «t1:^ aoilj'ijyS ala f-,lT JiRfO b«»X4dV#Tt «Xlao Of SIX to j rfywo't^^ ^.oim btiw yCibae./'a b*iR8»'^io*b axasiltryyiit »ni'Yu if:^n«co-TX« odt '?,fii'src nnv ,<*.'Xsrt«Jli t-t(-y.Ijuo XXfo ani .r.-iuq ovon 9b ai iniXo^sb xot Mcwtfo frXcfi«ftoH to ^•bLXoni dTyiC't'^ il70 oln/ffi-ranoX to oX-t nfX tlBg/lJ’/rvit Tarti’ii' AT yubrtoeM siMmdSij'ts #^v«vXfio bo(i*o^o«X (l -•*j1oq onoa ,i9blQ Ai to 'c^iiiXsXiftv# bosBoicoeb yj»rrij*q 9f.*vIsM boBX.9iert! o;^ 10 e^^uilido b^^MS onl-sx’q ovorr s>fe >0 ^3-4:71 httfi-ontoab » ,’c3'iiitfnXiav« yimlB 9X0 r7; jX?0 to sairt9rfta«9X fi ot yTtJBbcrodoa -§i.x»iL I ^ '.xii h.‘ ][ 45 pyrimidine synthesis required to maintain stable intracellular nucleotide concentrations. The observed decrease in de novo purine synthesis along the growth curve represents a rate measurement because cell cultures at varying densities were 14 each incubated with 1- C-glycine for 2 hours before the amount of newly synthesized adenine and guanine was quantitated. Because the cell cultures were growing logarithmically during this study, the total amount of purine synthesis was necessarily increasing as the cell culture density increased along the growth curve. One explanantion for the decrease in de novo purine synthesis in the face of logarithmic culture growth is that salvage purine biosynthesis is supplying increasing amounts of purine nucleotides with increasing culture density. It was 14 demonstrated above that the amount of intracellular 1- C- glycine per 10 cells in old and new cultures with densities of 3*8 X 10-^cells/cc and 9.0 X lO^cells/cc, respectively, after 14 a 24-hour incubation with 1- C-glycine was not significantly different. Therefore, decreasing availability of this radio¬ active substrate at higher culture densities could not account for the observed decline in de novo purine synthesis. Increasing concentrations of exogenous hypoxanthine in the culture media led to a steady decrease in de novo purine synthesis, and with increasing concentrations of exogenous 14 . . C-hypoxanthine, proportionately greater amounts of adenine 14 and guanine were synthesized from C-hypoxanthine. These results reveal that L1210 cells possess hypoxanthine-guanine phosphoribosyltransferase, the purine salvage enzyme, and that j L' i 19 !!> L. ji u 9 i ^ 3T a ■ 11 Sir fr t im o7 p'e^ r i « art 'xi{q *’. rr bairt^^tfo • ssiol-J^s^ft^onov 9bl^09££>t/n rs'ir: 4 avxwo nr. lAfti^-xr.' ?£ E^nL-::^ :w'c XXe:.’ ^ami^rwBBwu !■.: ^-0>!..1 8-,i,..^ ■_ •io-*,s,TiavXS-:>*‘^-i itt-iw ^«J•«(fiJOfi. rios* -. j^i jj; ;v : bssI^^-^dTmis vXwen *5:0 ^rpjo'njt ,Tr*!, 3«rT^?£i/o Xlg-j fei^jiaeff ■i.T aev r', ?o .U^oj »y:hL/r». «.i/<;f t;-.. 5;vr«i5 i^'ao'LSirl b-.,j-Sx/o Zisa ytiaj*'iofli .iav*li;o rt^wo'ia ►•'C. :«'/')i( . .-1. : 9t loi rtoionC •■'i t-ic^lwri o.'/aT 1:o S.it ai Eiatri^rty;* . jv:Ti 5««n" \;iqci 3z aisaxiurr^a^tcJ 9/tliuq tgMvIea --■3'v . .yiXaj^i'ion,; i^v aabX^oaXoi/n anxiwq ■" -^^'^‘■-^rr_ ^ r:} unij>..»Tf^ ir:j q’g^T.?' dvo^jB batnfBTioasb .V. isv -.o-u.r.'ii/o w*n tio cts i-II^o^OX ’faq .XIsvij.^7®»T »>f _ vllti.o^f;: _X 0,^ lvtc :>o\8ll#o‘'^0i X 8,C to jor. ' -L - ^ ta ‘XiJO/t-,;^S 4 -oibi.'i :jz<\j lo V'-'‘'i'J^Xiav . iSi9.ilt . “i-ooos Ton ti.’oo tiji i fsrei%p .sxurlp? '*'9»lairt jtvX^oa ,aj.asf(r.Ty:a •nit/.q ovij.t sjo ax arriiodfe b^rzum^io 9fit lot •'rrrgxoq'^it euon«2ox» 'lo 2frJla£i#TL3»%I •»ni':L'q ovoa st a: ^jcio^vsa « ot b«X «Wll/2 ortJ- ai 9r.lr.9bM tc afrjjortia ' boj'J.c:-a-;iJa\£« rxaw anitiXiUi fcna •niA«y2-*i^^/X^fUiiCoqx<< aa»«ii|>q OXSXX a^Xwa#:i: .fufq 'r §24vl4« ^ai-juq <9.f..-i‘?4l.«iw?XX8o<3i'iOiiqeo/iq 46 hypoxanthine is the preferred substrate for purine synthesis. Given the choice, L1210 cell cultures will utilize the salvage rather than the de novo pathway for purine biosynthesis. The Kjyj of rat liver hypoxanthine-guanine phosphoribosyltransferase for PRPP is 5uM(52) and of adenine phosphoribosyltransferase is 6uM(53). This contrasts with Kj^ values of mammalian amidophos- phoribosyl transferase for PRPP of 86-600uM(54). Thus the salvage pathway may inhibit de novo synthesis not only by endproduct inhibition of amidophosphoribosyl transferase and PRPP synthetase, but also by preferential utilization of PRPP. In order for the purine salvage pathway to become in¬ creasingly active during logarithmic growth, cell death with reutilization of nucleic acids must also increase proportion¬ ately during log growth. The viability of L1210 suspension cultures as tested by trypan blue exclusion was found to be 99% during exponential growth. After 120 hours of growth in culture, viability was reduced to C 70%i7), It has been sug¬ gested that since cell lysis rapidly follows cell death, the trypan blue exclusion test may not accurately measure the total amount of cell death(55). But even if the percentage of dying cells was slightly greater than \% during log growth, it is unlikely that enough cell death and subsequent purine salvage could occur during the log phase to compen¬ sate for the observed decrease in de novo purine synthesis to 20% of control. In a suspension culture of human lymphoid cells, the percentage of cells in S phase, actively synthesiz¬ ing DNA, was 50% during exponential growth(55). Thus much more than 1% of the cells would have to undergo lysis along the i'i- .cfasrtrmca @1 mlsltcisxoqxfi 0:it ^zrlz.tif Xiiv. sr9^,ti4fc, XxCes OfiJlJ *«&iod3> #rit n#YiC V. ■■' cvVon «6 mdt nsttit ■ ' SionsrlJ-iX?:- eilii^xi:i>^-©iTirf5'ruxo<3^ ^M-% -..s^^rslan^-i .•! ,-’->'j.‘ c.Aus;->f(q-(St^)?4^c Bi lol : bxTia :iaiiMfft^rr'.yj'' rn aM-f^ 3iJ’s«TtfiOG airf? ';'iT 3^-z ' , ♦^^;y.i/00a*di5 ^ 'ro't 'if^a£rt*'tivi£^7 I^BO<^X'XOri(T^ vd '/Ixio ^or. ;:t.iW4Ty>? ovon 5ii ^Xdirlrt* ifjswili'sq Sf^av^Xss t-rrs 983191V''- .X iyboXX *o.r; jr rqoSxmiS xo noltliidnii toutc-zqibm 'J.: aoi j.Uv'iil ‘ jrx ;£iJr::'^i©‘:'j-*t>i y.X .d^ais tird , ■X'aj£.t9dd‘nv:a -rx -jxnoo^d zi i f..:xT'ju ,9,1d lio'l T©{?rte lal . t'’» !'Xi#o p.x-'^td.'^ . r.^^sol ^rtna^b ©vixojs ■yXStfiiaisa'io :.i »«89iDn- calA ;raLf*Ti ai5l«« oi^iawc is r^^itasilltu^'i i^Z'izrifzzt. . "irxivl ■^o y'tfXfdixv' ^tiXTuro^ fcX ^pitub i^Xd^a =»T --* i:nL'- 1 e»i«r ttoiri'iDxi avid tTjaq^;'5(t fc s^Tiutls/o :1 .''Xv.-•:^3 •■^0 aiuo.-: OST q©i'1:j\ ,,ldwoi^ £«iJ'lxa^roqxB ’;|rtXiiXb ^xr. . r- i-a Sod I .•^:.'b>ivi/bfj'i s-aw v^^iXldjtiv ttn/tXi/o •*•'" ,l^Ts‘t!:' -’w^xXoS YXMqs'i sia^X Ildfi ©orsi* tjeult b«t8»^ »rt7 ^losesn: ^lisc^-.i'ivipda ;^of£ yjRsr ♦bg.' rraiairfiBxe «i/XtX iTBq snX tj n*y» rtrS i’.Xi'd Tto ^'cmottiB LByo^" 30^ SffiiTvfc njtdy ***©X!iB*i3 yXy ,§iia £isw bIX*?® yti^fe lo ?-.-brtfi Llaa a'a^on^ tMdiit .%le:iil/vj gJt ti ^rfdwoT^ -O;?iqtcno oJ" saiadq jcX 5-rftf giFTXiqb •sdc/Od bXj^d® ©SBvXjBa opJt’Tuq o^” »ia*5d^xi‘^8 Bniioq ©vorr bj& ni ?|&i baviaarfo exi^ tol edaa biodqfrrvX nfifflvd to ^m^tluo ftblapsqs^'-. « nl .Xtnynoo to 3^02 -5^softt/T^a tXa'vi.to/si ,93sdq 2 qi iXidO to «;^Bd’n»oY#q « rfavoi tMriT lalTnmcqxG ^fUiub sjsw lAMQ yxl ernif s^oXm bzaxl o^rttthzz ox svad fcXvew aXXao Bftt Ho b(t njutt e'xom | 4? growth curve to allow the salvage pathway to significantly con¬ tribute to purine synthesis during log growth. During plateau phase growth, however, in a cell culture that is not nutrition¬ ally depleted, the percentage of cells in S phase is equal to the percentage of dying cells, thus maintaining the plateau phase steady state(55). In this situation, the salvage pathway of purine synthesis would be expected to substantially contrib¬ ute to total purine biosynthesis, A second possible explanation for the reduced rate of de novo purine synthesis during logarithmic growth is elonga¬ tion of the cell cycle resulting in a slower overall rate of de novo synthesis, not necessarily accompanied by a decrease in total de novo purine synthesis. The cell cycle is defined as the interval between the midpoint of mitosis and the mid¬ point of the subsequent mitosis of one or both daughter cells (56). It has been found that the control of population growth in a cell culture may be due to nutritional deficiency or to cell-cell interactions and density inhibition(55»5^), Holly and Kiernan demonstrated that the concentration of serum in culture media determined the final saturation density of cultured cells(57). fen et al,, however, have shown that for human lymphoid cells in suspension cultures at a concentration of 6,5-7.0 X lO^cells/cc, no increment in serum concentration would stimulate continued culture growth. This shows that at 7 X lO^cells/cc, culture density becomes the growth limiting mechanism for human lymphoid cells(55). L1210 cultures passing through the exponential and plateau phases of growth in the inoculate®s original media, are likely growth regulated by both or iwl't ^oU.m: «?.t *.v^3 dTwo^x^ '4 f^vVo-a;! oi* •vwcflfltd’ •■■■'■ .*: r:;n toa .?i ixj'o IXwo ii^ ai ,-r-Av^wo4 »8firfq -•.. af E.jji!ria 4i ni Pl^.^a ^o ♦,i>«^i»Xqtl> ^IXs Uwti.. -'•'. -„q s^ii- i5?’ jfTo:'^»^. tal *i^ * )•^;t!JB8^8 eaiaB^ig ..X- t . - . acwf or 0".''^v<^^;)ce &X'«/oy^ ^Asat^iJ'rnca ©ni^wq *lo ‘ »tiXstinq-iT^ijol/X i.Ti*?tfC|. ijir’o^ oS 9&U " '"< - .' £1 ifei: 'U cal': a f ■: r A'' -o,rnc ■i:? ai :•?>>.,, • * z '■ c .'{'*■£'az.-; <^. - 1;c ” -.r: .i. i £ .. 'i' *.' * CC'voia ■'0 4 3 « a/ 3f!irXi/:^r^ »>I«jv/. <• •. ^ *' r- »83'a id *, 9.‘r .qn-.K’Of; •-■d:lX 1 “r r *- 9 ioV-. "lO ailT aiatfr'i'rrii- dolii/q ovon »£« Xsito^ ni -iia ,> •• 'IS. jX i> J' -.' f'-- • ■■ a ; iO I £r 1 roc 'to ? T'J *'•- VT.';, r; ^ ■•■aq >0 Xxi'ixftci OX - ' 1 '.‘n •» ioxl* •• ^ ’H .'-^e .^c )noir :idrj ytlui^b pan ^loitscn^trU iX«o-XX»o cii mu-i?3 "io n''^irj;xjn«gaoo ori-t raqj tj#Js*xrf'3ndfT)i8& fca« .'■ xrianeb nexts'iu^b^ »n.t sif)».T; mi/fXi/o n : J5i.r -Avortb ,TD'/awor< ,.ii n^r ,<.7^^^XX#o o roirfii-'.-rocnGo e j-s annwrix-o ;toi9stoq».ua ni ali#3 i)if?;-etifaxl naflurri nroir^^tneonoo (nuT9a. £xi rfi«ia5T:xv/ti o ? .oo\*XX9'O^Oi X 0*7-5.,d ^o ta J-arir Hworia airt? ,.U'v»c*x® sTiftiuo faeimitjToa 9/sJjjMlts tluow t^m/fluo ,pG\aXi gni -.9jiq OXSXX lisaesi/il 'jol fflaifrarto»/n *d!t ni flXvo'rg iO i/£a:jBlq &na Iai;^^lefyoq*8 ena^ /t^wo'irO* *i^ocf ^cf b9f9li^^9% ^iXdjiiX *-ui ,AlhM LAol^ito aUtaXwooni 48 inhibitory mechanisms. The cells of cultures in which nutri¬ tional deficiencies develop over time progressively accumulate in the phase of the cell cycle(55»58), the interval between completion of mitosis and the onset of DNA synthesis(56). Rossow et al. have proposed that during C-^, an intracellular protein (R) is synthesized and when accumulated in sufficient quantities, allows for the initiation of DNA synthesis and for the completion of the cell cycle(58). Serum growth factors are believed to induce this "initiator protein". Thus in nutritionally depleted cultures, cells build up in an elonga¬ ted G^ phase presumably because more time is required for the initiator protein concentration to reach the critical levels. During logarithmic growth, the nutritive serum factors are gradually consumed resulting in elongated G^ phases and thus in a lengthening of the cell cycle along the growth curve. As increasing numbers of cells accumulate in the G^ phase, a de¬ creasing percentage of cells will be synthesizing DNA (S phase) as logarithmic growth progresses. This gradual decrease in the percentage of S phase cells along the growth curve has been demonstrated by many investigators(11,59). In L1210 suspension cultures, thymidine labelling indices decline nearly 10-fold throughout exponential and plateau phases of growth(ll). This decreasing percentage of S phase cells coupled with a lengthening cell cycle offers an explanation for the observed decreased rate of de novo purine synthesis in the face of exponential cell culture growth. Per unit time, fewer and fewer cells will be actively involved in de novo purine synthesis as logarithniic grov.’th progresses, although 'O.-- V.I= -t-jvo Ifliiolj" ’’' ^ ■ sXoVji: XI^o ad'J' to ^,0 trfj* ni ^ i-v-’-'V-Yri AITnf :^-3ro a<*t‘tx'.jsi s}4:9C'/i* to /ioi-;foXqmo'!* jav>.i,-r£ , .r;i js/fn" N^oqO’lq PV8d , £j tt woeisoj!' .1? Iw DrTjB- ba^i; ti (q) ps • •-jJ'oTCf Aysti r’-a' rXrii -irf.* lo'r ;i'/>'oXlji ♦ "-£ i : X^^.' orft to doi?«I : r-: V'.:r.?'' giffr t#o " -:x ■2: 9«i’ -■;o’7i i\.'-'■■J-^,l ''Idf'rtWJBO'iq 9Sj.iiq ^0 : ■"’.' c’-t toc:i&j"07q 'to.v , it ini ** ''•150 .j.,].- ,,tJ'Vo*i.3 aimdTf'Xjjgoi. ^ni*wQ ■~ •' -.sefff - ^'>w-,id 'i-j £?Sftit/;thO(. ^Xl£i/bJ9'rj| -•'r.t ^ ;I'»o adt to i^inr»i1-#^el a fti 'c -•3='‘ r o;/t :Xu.’T:/oq- 'jX tc .naoiT^ya 3rti8!it^9fii ■*^ '^-■'' 5' - . r< ; .^ii:*v Hir .-o to .&o~^ ^nJ^cta'io f?r;c’^i5, r-^HT . i^isaaeTjo't.a I'lTvifO?;^ 0 tiTTsttl'x.ie^oX ai '..E f fv-tijo .'•' fs 3*Xa_' 3 2i5dq ^ to tdf '^ - - - % r X > p/t - ': 4 1 • 3 ^’rr. ? vid , 9 * - * tenoma b 'rr:t* :-f js^oiittfii i XX , ttoTuths^ f7olBi?»<|jfii£/0 - • daa-j.'o , r.-Tilq rr - X^icn^rfcT/^ *vo-.\.iUo’zciS blot-Or o^^r/q 2 >0 8X^7" o-::.q ai/Jt ,'! if )f(a‘wo'i3 ■Toxtis^^alqra its ^Loxo li'y:! S|.-j * j xitiw baxqyoo ali'sritm o ^rtf"liijq ovoti ab to b^ii.x^oafc il>#3riftdb ♦ff?' xol I* J-.crfu Tol f'T;;7£va Xi»d iftiMaiXoqitf to aoit •^,:f nt ovon grft fcMOYXovaj '4X9vlt53 od XXiSr B-U^o towft fena lowsl .l^uortv-Xx ,8fc«*i#73o*iq 3iirirt3■i^i^3^J£ 8« aiaadtnva ift^iwq 49 the total amount of de novo purine synthesis will increase as culture doublings continue along the growth curve. Hahn et al. observed a gradual lengthening of the cell cycle in Chinese Hamster cells with 15 hours being the average cell cycle for early log phase cells and J>2 hours the average cycling time for plateau phase cells(60), The growth curve for L1210 cells in Figure B demonstrates this elongation of the cell cycle with 10, 22 and') 44 hours being required for successive cell culture doublings during exponential and plateau phase growth. A third phenomenon possibly contributing to the explan¬ ation for the decreased rate of de novo purine synthesis during logarithmic growth is a progressive decrease in cell volume along the growth curve thereby requiring fewer nucleotides per cell to maintain constant nucleotide concentrations. If cell volume gradually decreased, less total purine synthesis would have to occur to supply a growing culture of smaller cells with the purines needed to achieve the appropriate nucleotide concen¬ trations. Yen et al. demonstrated that the cells in nutrition¬ ally limited cultures gradually accumulate in and that their volumes progressively diminish(55)• C. Benz et al. showed that in L1210 suspension cultures, cell volumes decrease throughout the mid log, late log and plateau phases of growth, being reduced to 60fo of their initial volumes by the late log and plateau phases(7). Scanning electron microscopy of these smaller cells revealed them to be of G^/G^ appearance with loss of surface microvilli(6l). Benz et al. also demonstrated a pro¬ gressive decrease in intracellular deoxynucleotide triphosphate pools/lO^ cells with increasing culture density, reflecting the need for reduced nucleotide pools with decreasing cell volume(7). .;. _ .'.f rfilsr -;) giFroXfl (si^iinltitoo. 33ju(t,IXcfjJOb il'-r- ■srfJ’ ‘:o X.fiuis«ij| ^ fcov^sado Ur/V!? ..i-:,; «rt,? ^fjlw »ll»o Tdj-aiBjBH »-•'■** ;i‘i. .c\;o 2{icf. jS'^fcd S^;, bi;£ .gXXao ^o£ • '■--.wl . fOa )i?IIao aejQrfQ Uiji&^slq *io^ ’ J’ i'' *-• ^'V., ■' J 4'o:.wV^i^i 3 .At,:., ..^+f.'3»tt-(rto-r.vf» ni-, -M-. ^T:a.) aipod 44i ( brj>5 SS ,0X rf/iw ‘ti,. f?^^'itanoqx:? gircipx>'t> aartXCXuofe ^'UJ^tXuo .■ • ■: 1* ^.a.r.’ii ;.;r.iO rX'.^■Jsoq nonsro'>;it>jr(q ircirt# A .'9iijnVii ; OV-JC: -^l LO bid.3Mif^09b t(ft lOt ClOltM - - . *-' : I V .' n: : JV'.rjta ij^piq a Ri otm/itX'Sjs^ol .-i-i v*ji;Z- ■ '•■yn ^h. -.rtyv/o^ arlt yioli r.X«:* ' ' . i..oiJ5''-j,7'>onoo c un *tr;3wterroL' ot Xiao r fu'w -i, 3 i.,*::-!;."; ^,:.j'-fX '3S':3l. ,h-.y^r'9n:oab yllMUbert:^ amuXov ?'v n'iiro ■T<>XX^^': ' jrXwci'xji a viqquB oJ- njopo of ♦Y*rf : /...•.• ■• V^i-:s•:>Xc-Lr’.•! 9? .i ■sqctqqB 9(1^ avaXiioa OnT Jhabaafi aaaiiijq 9t1t ni a Xiao dPi i>?T«i,-iv'3noiirsb ,Lb fB jit«Y .Bixotfjant .', i-j r.v ij bi£ pO iiX a-:cXsif‘xvfluo brnflmll xXIb ; 7a . . (l^^jiULi jjalb v^»vi«aB't^OT:q gaotyXov ) iiU :-.£B'i\9b BBf:;tlQW ILbo ^nsruj^^iJi) n'^isnsqwJS OISXJ al \.Tisc; ,iiJ-wQta lo aaaariq Mb iol 9>aX ,acX binr •45’ tna goi vS-fil a/it '{4 eaent/Xov tiorfy lo od- beoubrx ..i^B a?Tiny lo i lo B8oi rfJ-iw «.OfiBTr»»qqji ^ ®>fXBav»*i aXXao -^"xq M tBTs^tzaoptBb o«Xji ila ine^ , (Z^)iXXlvoToXai apal'ii/n 9q>*.!q?o.'kii'3t 8bX^^otX.sAjaic>co#b iBXMiXaoa'idni nX aaBflaoafc aviesaa^ 3(1? >':itoeiT:flq ,'(;?i8/T»,6 BWTXii/o cffiw sXXao ^OxXaXooq }mtu£jr tiQo 47iw “Xooq BtlfOBioun b»oubn *xol baart ^ ■i 50 De novo purine synthesis will decraese in response to the need for reduced nucleotide pools which develop as cell volumes decrease during log growth. In examining possible causes for the decreased rate of de novo purine synthesis throughout log and plateau phase growth, it seems likely that increased salvage pathway purine synthesis, elongation of the cell cycle resulting in fewer S phase cells per unit time, and decreased cell volume along the growth curve all contribute to the reduced rate of de novo syn¬ thesis. Given that the rate of de novo purine synthesis in late log phase has decreased to^ 205^ of its early log phase value, the anti-purine effects of an anti-tumor agent must be inter¬ preted keeping this natural decrease in mind. As seen in Table 2, methotrexate's anti-purine effects could be underestim¬ ated if tested only with late log phase cells; methotrexate is a very potent anti-purine agent when incubated with early log cells which are synthesizing larger quantities of purines per unit time. Conversely, the anti-purine effects of a weakly inhibitory anti-tumor agent could be overrated if tested with late log phase cells which have decreased rates of de novo purine synthesis for reasons unrelated to the cytotoxic agento The anti-purine effects of drugs, therefore, must be studied utilizing control cultures with nutritional states and culture densities identical to those of the experimental cultures. 5-fluorouracil exhibits cytotoxic synergism with anti¬ purine agents which elevate intracellular levels of PRPP re¬ sulting in enhanced metabolic activation of the fluoropyrimi- dine. The synergism between, for example, methotrexate and -i.'.T rfl asss'ietis XX*w ^>ffi^cwq ovojct sG •1 *■'‘cv Bs qvl^v^b daldw sXooq ^fcX'Jrofiliivfr b^owfct'i ,'xo'i £-: 'ri'vb a.-icf.'it5> aeai/cJo ^InlifiAXQ *tl tsa.ja'iD&fc tr.t .:«•!/1 qinr r%| aXX^»o sa^dq '3-J'’ L'VM/£>*'t -anq. c;^ V;! .1/1 liirrioo XXs ftvtSJO tiTwot^ L 8 4-^4,i?-; ;.i -3' .'“. i ovan -'• ■. 'if' taufi! iCirit;‘rii'nB .la ‘io 5,'* .fcrffiiT rri 0>2-f#^t'Ob eirtJ gfriqtsi ■ -; n<^,7^rvf. ,-• rXwo'^ ;4;-ua“liU‘ -^vq-1tf»;« e’ tJftj^a-itort^drff fXdsT M .j 5,' eai.-'q ^oi in^‘ xZn ,.-*1*:^ b9^sd*. c'n^ ai'.v wrto^oq >fiav a ai r vc - :*>-.r: ‘lagiLBX *Tiaert^Ytxa »"!« doidw aXIao gol l-sbw 2?o«‘£l3 snXiLq.ttaa adt ,xX»ir£*TaoO ?Xm/ -leq iriM •>: [.-•tiTT'ijvo 9d EjXuoo tn>9gi :vc • ^*': 3j#7j>: baaea'i^^fi dsidw eZiao eeadq goX aJ«X *.'.^^s oi-co.T'.'^qv.o sd^ ot^ srtoaai^i toI: aiaadtoxa «rci'it/q b9iG*,:ra a.-* ^oc/m ,^"ia"t-na B"LUT'.uo fo:T.£ 2aJ«3-g Xanol4'i’zyi/r! n^Iw afcit.wJ'rira Xo^r^noo gni5lXx.ti/ . ts'it.: uL'o Xjt;l’at»«ii'TfH{4ca ar.>r lo aaod^ XaolJ^fpafti aaiJ'l anab -Jna xr.aZgTarf^ia o^xotQi^o «;jdXfbca XZoantwd -B’t !« aXaraX *ssluJ.L&-'is'i;rni> <=^v,«vaXa rfoidw aJ-nagis arriiuq "•i*3ii”rvqo'5a4ji^ ariq la fsoirayi.'aB ^ZXooa;)^* baonadna fii gnii'Xi/a ferra atajca'xtf'P'^iJ’am ^aXqr.Bxa Ttol inaa»/?^ad .caig'xarrx® i 51 FUra, however, is related to the anti-purine potency of metho¬ trexate because the degree of PRPP level elevation is propor¬ tional to the magnitude of methotrexate’s inhibition of de novo purine synthesis(9). Because intracellular PRPP levels also decrease during log growth paralleling the rate of de novo purine synthesis, the synergism between methotrexate and FUra would be blunted if methotrexate were incubated with a late log culture in which the rate of de novo purine synthesis was only 20% of the maximal early log rate.(7) Similarly, the synergistic effects of sequential treatment with methotrexate and FUra seen with early log phase cultures in vitro would probably not be as apparent when tested against a solid tumor mass in vivo, the center of which contains cells in the resting phase due to hypoxia and nutrient depletion(62). The cell cycle kinetics of these resting tumor cells are more comparable to those of plateau phase cells in a nutrient- depleted culture and it is likely that the rate of de novo purine syn¬ thesis within this cell population would not be substantial enough to allow for marked inhibition by methotrexate. With methotrexate powerless to significantly inhibit low baseline levels of de novo purine synthesis in slowly cycling tumor cells, the already low baseline levels of PRPP would be ele¬ vated only slightly and the basis for the synergism between methotrexate and FUra would be undermined. On the other hand, several investigations have re¬ vealed that some malignant tumors have an increased capacity for de novo purine synthesis. Measurments of amidophospho- ribosyl transferase in rat hepatomas demonstrated a 2 to 4-fold ■'■-Hid*' o.^ D«‘--i‘!3Xai''x »i; I't^vawort »jrrtr*| -rn.-jrru^ -4 .rO :. X^'V-^r 't,- #^5W/*0 • •-“f - -.f *,IR'lir'jT iX^wcrt!’^ '^X dQ.aa'ioab 03X® •-. - ■•-• iv.^-ro.iy«m i': »e’^crd^i trt'T eni'lwq * 'U - •: iw r 9V3»v-r9:r^x#i:J-ort^em 11 frarm/icf erf bli^ow - •)o.:^l/g rv-rr ... f'. la grfj- nd/r’^ ftl •^wtluo ^oI '. •.s.r.cri.S goi X.'ajilx.-i'rr ♦rtj 'ic 3fOS ^^‘lo a-Bw i'c.-"wo.'. i>: "J-.n. .sjc ■^n?viITo t'!*®#!!.® -^iJ’al^^icfrYe o:d-L2. ■-■■ • s,,« rq 30A Yi;'»'.fe9 bns •. *-/XvB a ’ .n;-. : I .'a:-,.' -.e.T: JH : - 7 .•Si^qa 4:5 SCT ?Ofr YXt^SdOTLq 0“ ■’ ■ * '-- ’ifivrioc -'• i-i'v lo ^dJ' tOVlv rf^ eeafls ^/j!: ^ ■ ?,0-?-1; •/ si7^t/n alx.oq\/i or #i/b dMftq 5ni«l*8®‘t Qd: dXX?»3 -romut -.."/s-j-: To aols-aniaC aXoYo IX®o ■'*■ •'' ■ rr.1 eXibo 3- ’,,',t To s^ori.t o? fX ' ' ' - ■ ‘''''^^- 9tax Ffrfr reA^t •! cJ*! fcu-tji tou.lXiio i .‘20u3 lon b/irw >7ai *aX«qoq X.X^*o niritivr a>Xa®Kt •'^ ■BA.1/a.7£<5 <^qX Ji-Jli-tfri a of (B^axariXoriXom •lo.Tju.T ^rlloyo Yl’'>'0i2 njf 5i3®rt,t-iYg ai^lri.q ovon ib To aldvsX 9-' tlL'o^ .'9»*v'X3cr »Jlt -xoi arfX iiftga ^XXrtg^lXa ^Xao toaXav . £oalin®Df_ ad Oliiow cna atax9dSoi1fm -♦T aaolXBjixitimi Xin®r»a -la/flo arix xtO YXioaq 0 fcroaaairofll ri^ avail g-tOiWUf l.iarriUKa ®mo« Xadx b«Xaov -crtqaodqocima to •Xr.tsur'ttraaoH «aiaor*X increase in activity compared to normal rat liver(63). This increased enzyme activity was found even in well-differentiated hepatomas with slow growth rates. Increased amidophosphorihosyl transferase activity has also been observed in transplantable rat kidney tumors relative to normal kidney cortex(64). AICAR (aminoimidazolecarboxamide ribonucleotide) transformylase and adenylosuccinate lyase, two other enzymes of the de novo purine synthesis pathway, have also been shown to have increased activ¬ ity in rat hepatomas as compared to controls(65,66), Some malignant tumors, therefore, appear to have an increased capa¬ city for de novo purine biosynthesis and, like early log phase cells in vitro, would likely be strongly inhibited by anti¬ purine agents and by sequential methotrexate-5-fluorouracil chemotherapy. In summary, it has been shown that many anti-tumor agents with widely varying mechanisms of action have the abil¬ ity to inhibit de novo purine synthesis. Even agents which do not directly interfere with the de novo purine synthesis pathway can profoundly affect purine biosynthesis. It is hoped that this knowledge of the anti-purine effects of the cytotoxic agents will be useful to other investigators in¬ volved in designing biochemically rational combination chemotherapy. . (r. - 'evil n't Lm'itia otf ni ^usaianl . t>-i.i:^w ni fi«V9 bnuoJ RjE'V £)9Bj9r®‘ioH’i ' .’ t-lto ^.^xnase'ionrl waXa titlyt asrSotjBqsif : ij^nc-Tw E/’r7'2;^' ni L-5V':*sd . - -'•-'iM^ok os snoinuy icenbijt xTin, .'•o *f -• 'll.'I .:iv ,,i 'jt 3.ff lo o-fft ,»®£\5i e.^anioayaolicnftda '■.•r! b:-ca=? ot ..voita r*'®9d O'tii! svsrf aiaaff'fni^a ta-TTn*’ .','k'. ; j .'ciiriJTfro oJ bs^'sqmoo ej :m'5to3'iKj9ri ni - -- . '' be-:s«.-.;nf .!a f'Vfca yi- laiotatartT i«'xodia/.r irfi«ic5&iLa« r. .m; ^Ci. I .t'nj rsiBt-x1?'n^5->i«5 ifniTi/q ovotr -^t “loJ, ictio -isr.B vc oe.-ibi-'au «cf bXjijj^w «Qn3-iv ni ellso . Q-.- TiL-':: iiiTfiigri/f^#© bfu; ^i^’J-Tuq ' rr^qe^arti-oradslrj "lerrc --.rr.: *5;'” nv/o.-ig £Td£*d BjJff il ,*-{*5aiROUy» nl - I’d!! <»1; ev.9'f .'.ei;®)! 1.^ af!tair5iii;‘D®>r ^Xtbtw jHtiw s^treas^a 'infii'.v . 3l a«?r ^*1 ,;a ixfq ovfwr 1’ldtnai ot ^njtrcuq ovon *5 ^rlr ‘cXiaa^Xb ob sX ) I .ai id" rsn: r^j-^ 'cXfianfiOtonq n^c ^jfwrftaq &.-.J lo 0 ,^5a P a HaX*iiJC-X*n.3 lo f^b»l¥for{)i airli fiaqori -f'i 'i^clto gj Iris a*; a noiifiniimoa Xaaoitfin V;XXi«piintr(ooifif anin^laab nl bavlov .^qs'i9fW'o«®ffo 53 REFERENCES 1. Sartorelli, A.C. Approaches to the combination chemother¬ apy of transplantable neoplasms. Progr. Exp. Tumor Res. 6: 228-288, 1965. 2. Chabner, B. and Johns, D. Folate Antagonists. In Cancer - A Comprehensive Treatise, (F.F. Becker, ed.). Plenum Press, N.Y., Vol. 5, PP. 363-377, 1977. 3. Cadman, E., Benz, C. and Heimer, R. The influence of methotrexate pretreatment on 5-Tluorouracil metabolism in L1210 cells. J. Biol. Chem.,256: 1695-1704, 1981. 4. Reyes, P. The synthesis of 5-Tluorouridine-5-phosphate by a pyrimidine phosphoribosyltransferase of mammalian origin. 1. Some prpoerties of the enzyme from P-1534J mouse leuk¬ emia cells. Biochemistry, 8: 2057-2062, I969. 5. Benz, C., Schoenberg, M., Choti, M. and Cadman, E. Schedule- dependent cytotoxicity of methotrexate and 5-51uorouracil in human colon and breast tumor cell lines. J. Clin. Invest., 66: II62-II65, 1980. 6. Benz, C. and Cadman, E. Modulation of 5-Tluorouracil met¬ abolism and cytotoxicity by antimetabolite pretreatment in human colorectal adenocarcinoma HCT-8. Cancer Research, 41: 994-999, 1981. 7. Benz, C. and Cadman, E. Biochemical alterations during un¬ perturbed suspension growth of L1210 cells. Cancer Res., 41: 157-163, 1981. 80 Hryniuk, W.M., Brox, L.W., Henderson, J.F. and Tamaoki, T. Consequences of methotrexate inhibition of purine biosyn¬ thesis in L5178Y cells. Cancer Res., 35*- 1427-1432, 1975. .:.9Ag noi'^5/ri f.*TOo 3fft ot ,?.A , ?!,t ' I-' . £h/- io;rT[, r ,cpr3 .'ijo'i? . litres Xcf 3 eri 1:o ,aa$^dss • •iS'.nsD nl .nyp.lno^AtnA .C »?r?r;of bna ,3 ^-idfKfjtrfO ,S »• V .■;.-:>- .^^91, .rtC-CAt . maixodsifdri liet'Tyo'ror 11-^ no «7n .:3PI .AO':.-?--‘or 1 g?S.. .osrtO .xol^ .l .aXX^o oxsrj rti Y*! lo ’e leortJAYa oilT ,3 ,2»y:»« .4 Si'xc n£iIa.Tifl!iKn 'tr ^aa-ra't8iTjrTJXY»o -.iDeX »3cc:r Liir?r-'^ rabit srrjYsns ^Ai lo moZ .X .PM^I Id ,.aXIao aiaia ,5 ,n£OTb90 fxT^ .M ,iiOit0 , ,.M , .0 ♦snsfl lioffTi/o'iouX ;-c ^Jc'xe'xtoflJ’am \:tioixo.iotYd ^nabnaqab .. LLj .1 ,8«iniX Xi«D zomut ta&a^A bnjs nolo^ muaitA (tl * . ^ I .oe^X ibb ,.t0«vnl -ram XiosTKO'ioyXl-^ Ic noltFi,vl>oM ,a[, »rtan!)»0 Ma »0 ,atna€ ,d inaajrBfiiJa-iq qiiloiXa^omitna l^ioXxo^o^x^ onm tumilo^s ..1ona»a«H naonaO .3-TDH astoaioiaoonaba XaJ’catdXoo fxwn/ri rri .X39r *X4i -AV ^ziivb analfa-retlM laoiaadooIH .S ,na«i)aO ficui .0 ,«naa ..saH dsonaO .allao OXSI^I lo Acl^^rt^qaisz baAzu/^teq .XS^X .T »iioa«aT beta .3.1, ,Ao«*Tabff»H ».W.J ,xo*ra ,.J>f.W ,3ii/layrxH ,8 -nxaoirf aniiuq lo noliXitfirtni aJhajcaTJ-orttafli to aaonai/pssnoO , ^SC^X-TS-iir lie «.aaH zaoaaZ .alX^o lX3^X?a rri aleaAS J 54 9. Cadman.Eo, Benz, C., Heimer, R. and 0'Shaughnessy, J. Effect of de novo'purine synthesis inhibitors on 5-fluor- ouracil metabolism and cytotoxicity. Biochemical Pharm¬ acology, 30:2469-2472, 1981. 10. Bagnara, A.S., Letter, A.A. and Henderson, J.F. Multiple mechanisms of regulation of purine biosynthesis de novo in intact tumor cells. 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' r':r.M .: TT-i , *4'; i-* txo.Tp^'ro «7sx&’T70'17ttai * I, ,■ •■ 5r . 5X1*1.11 9b «i99rtXnM0oIu oniTUct "Xo aoxiidirtnl -ixo; I = .Iji? .1 .o'tiiv ni miXso •xomixl »«7i3aB -nQff’tnA .11 a9r!i‘-7uqoiit?-A .W.;3.t>6XT fenA ,.'roart«J‘a^ ific ,".A^.l’T tz^. .«7.*isaA *vi»^r^qquaoni/«s:l ijrj oi^talq ''SC .qq . X.il ,''■'^(Ti.'iqS a.atB , atirtot. ,0.3 iirua ill#*!©^ <*•' 4eo4i oi.t3dXqo#ni7rrA ni a;^al^o^JSi‘(Tc •rti/nati/XO ..'il»,.J,J .l-tartfrafi .?X ^.tj0 .O.A)^!! ^*u?^ ♦»#ne^A aviagrxqquaofuyiaml brtiB .qq , ,'i t{,ab» ,bxu1oT» ,0,3 djTiaXnBitoaiG £«oi«*i b»iA ar.i(7*11^01:1^-^ na»wtt»cf aiexg'no^a ariX *xol ...'■>■ I'''! ^ .iXItia o6X awo^ix.jf® nl •fcXaotiXoiiiiorfi'x i*niT(uqo7qBp . B 55 Res., 32:2034-2041,1972. 17. Scholar, E.M., Brown, P.R. and Parks. R.E.,Jr. Synergistic effect of 6-mercaptopurine and 6-methylinercaptopurine ribo- nucleoside on the levels of adenine nucleotides of Sarcoma 180 cells. Cancer Res., 32:259-269, 1972. l8o Brockman, R.W. and Anderson, E.P. 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Academic Press, N.Y., p. 117,1973. 25. Howell, S.B. Thymidine as a chemotherapeutic agent: Sen¬ sitivity of human solid tumor and marrow progenitor cells in vitro. In Nucleosides and Cancer Treatment,(R.M.N. Tattersal and R.M. Fox, eds.). Academic Press, Austrailia^ ,x4os^^C03;»sc ,.66^ ^ .... .1,.'^, .e>i'Xi.' bfLs .fiA ♦fwo'ia >iutloiiaZ ■'• - ■’• bn-5 itunX-iii-Toj-qso'nsvs-a todlla *'nr - .if3Di'o? M.rj srri*r=3t)f; -to ale^f^i rto sfriao^Ioun '■''■‘v®! T»onA0 .aXX«.-p OGX T4»l ..* :c '. r't'':,*.i ,i\o£:4'bc{k br'.o .ftszn'iooiS ,8i • '.'’^-T,:: 'pi^. ,. r-^KfaiA ) I'v-. -i *•*■. r. i; .-'.((i.j'’.i2 5'» waj^iiinsiTiovM nfis^n.oO'iS • '*' , tZdH ’^cxibO ctl r^oni^.’bA ^aJ'no^a “ J-IO,. I -' * -iTi' _ .;’r^’i • r.7 ■ .a , .S ,0S -.7< •'■ liia F.x‘"xJ'.r fctU '•V ;.'■ , [ T ,•..t-rol •!bi?(;.©i,oj-’fr Xlo^s^ ’ :..-. 2r. /i ; I'T: froi T«iitjsXc[xS inoii’e^:?’ .u ..4.n ..-nifooia . ,M .'i? ,ei'i>;v5l , ".L inijt'a*! .IS ••''-■ ■ .'mX r,;:'Tiiv VI .Tol «i?€€ .?***< .sXBJifiH :. fi. s.^,r>:. rrin....,i t-.,- ??;i-qi5 .xi i j ^ a--y I’lo Od” .9^$: ,09C > . , .aeH 't«on«0 •V'r '.0 :::£i'r£r,f^.T -s.i* sxO , . .W ,.ou^ia Lr:^ .H .R«not-Oob^ .SS .aJX^x- - 5’k.o^ no IZoa-j'- oTouXl-l lo rroido* iBiid-fX .*'3^1 ,.j»a ^ •■ ,i9^gb^^e >0 ajX'v ’^Tioijcotbdyo "io ^oi7Bi9^*IoO ,ce?x,ooov-<'^9ei^2^s .loia lo .t .Ana otm -cJs-TsM ifcXJDuloi/W ."T.H.A oTor^&d-iTjf^ .'T.X, .nqis-iahn^H ,4iS .cTPr.sll ,q .asa-rt piiuatusoA •nolTowbo'ji’fil ^xb .ftraiX • ,'»2 • ojttuaqBxarltoflSBdo a aa «/iibiaiXi^T .E.3 «XXowoK .^S aIX©o ^o3•irt•ao^q woman baa tomyt AiXoa ftjaiitd lo Y^^ivid-ia ♦ K.W .H) ^drtaaiyatiT boa a#i?Xao9io;/il nl ♦oiJ’iv I'fl jjJiXi/rjd’HVA ♦BBaxIt c-isratiaDA «(,ab« .M*5l bna hUI 56 pp.229-231, I98I0 26. Spiegelman, S,, Sawyer, R,, Nayak, R., Ritzi, E,, Stolfi, R., and Martin, D, Improving the anti-tumor activity of 5-fluorouracil by increasing its incorporation into RNA via metabolic modulation. Proc. Nat*l Acad, Sci., 77' 4966-4970, 1980. 27. Carrico, C.K,, and Glazer, R.I. Augmentation by thymidine of the incorporation and distribution of 5-fluorouracil in ribosomal RNA. Biochemical and Biophysical Research Commun¬ ications, 87:664-670, 1979. 28. Cadman, E. and Benz, C. Uridine and Cytidine metabolism following inhibition of de novo pyrimidine synthesis by pyrazofurin. Biochimica and Biophysica Acta, 609:372-382, 1980. 29. Cadman, E.C., Dix, D.E. and Handschumacher, R.E. Clinical, biological, and biochemical effects of pyrazofurin. Cancer Res., 38: 682-688, 1978. 30. Moyer, J.D. and Handschumacher, R.F, Selective inhibition of pyrimidine synthesis and depletion of nucleotide pools by N-(phosphonacetyl)-L-aspartate. Cancer Res., 39: 3089- 3094, 1979. 31. 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' .._Xfiox^udgoi^ ft.'-ij Zsoi.r.s^oo.M .AJIH Xapoaodii 1 .o^v>t .anoXi’aoi ii'rx- f-vr: -rui or./6x*rU ,3 xnofi iinj ,3 .fijMiBilO .8S ^2?’” V . :!bi: XvdX * sb lo noitidliifti sniwoXXol xaisvrlqoid ba5 soImlnoaiS .nifiitosis'xvq .o8^r .5.^ , : - .:-:»orvi/.ro» .asH bfra .'3.3 ,xil ..0.3 .ruBobsO ,9S V .. .alik-ysB-riq to 3?:?o'l'te XsoiniBfiooid .ns , rjSOijoXoltJ ,B^9i ,a6L>-S8^ i3C ,.8«a rrolrdidri «vl^'j*3i9c .TBrtpaaa/.'XbabnxH bna .3.L #0^ xXo.'q -abi J’C'i9ir'L7] lo rvji X? Ofri; ais&fl^rria aniftimi^Yg to -^oji i9C ,.30/^ leoa^w' .*.tsX■^iigas-J-(Xx^»ftiar!ortqaoffq)-W .wx ,we " ’ ' "J"' .T!xiiir,rn&iM j snifcir^oass^ .A .jfarfiO bna *1# .yXaioV ,XC .^rorlT ,oijn!Tsd=i ,3XX»o n«iXBc£UEja fii aS-oon:* X.a^iaoXoid' bnM ,64X8-Cf0 *S .XoV .A , ,'A .xoO »,A.H .anivoJ ..8 .aXX^vXIO ,*ioIyaT .SC ovort 9b to fjoitaXu^BH .J ,rioiwotalwj5 bat .O.H , .H .!rta to ,L T^fsmsH aaonirtO ni aXtorf^’aYBoXX 9nI'Ujq ,S89£ 57 the New York Academy of Sciences, 186:292-301, 1972, 34, Sawyer, R.C, and Stolfi, R,L, Mechanism of cytotoxicity in 5-fluorouracil chemotherapy of two murine solid tumors. In Nucleosides and Cancer Treatment, (H.M.N, Tattersal and R,M, Fox, eds,). Academic Press, Austrailia, pp,309- 335, 1981, 35, Heidelberger, C, Fluorinated pyrimidines and their nucleo¬ sides, In Antineoplastic and Immunosuppressive Agents, Part II,, (A.C, Sartorelli and D,G. Johns, eds,). 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Metabolism of 5-fliiorour- acil in sensitive and resistant Novikoff hepatoma cells. to ^^4»6jaoA iitoX w^H *rlt ■■-•,/ •'f''',J .H bn.£' ,C^*H *4^ )• V I j .. -vit lo V;^)5i'i^4.TQ.lf0rfO. LlnSrSJO^Qul%->t .jlXi *>'.t >■ '."T.'t; >; 7s-cait0 tan 'SViblQQ^loi.sVi cil - r , ■*«. j'tS ch'.'jl’-’tcA ^x'o*? »M*K ftnis .ice ‘ '•.»£ 5‘4n b’lr'-.'q bc'XT ,IC oi^'asiqo^-nl rn A nl .Be5X» . Sbg .i;a.1oL -u.l ftfTS .3.A) ,,II ,r'^S-€9;»qq cT»' - ur ^ ' .13_. r- ’ IlueH br.a , .!: , titi tusd^ , ,S , r-xadxaH .dC 'li.'i? i ''f ■' ' "T-jw'^ .IT!/ , aaax oxmiTxq Ntafriiojjll rro --li: ‘.\X ».ci^.O w > .1 diXodfiJ-w ■^:''J.7-. 3 .-i-d':-? ' L'r-' .H ,'r»3'5.o'X?i , .<1.0 .aimiS .^C ' " •- -.rr.Lj»‘‘;3ai‘ -- T ,:il’/X *3«iti bJifriT^q bt*,*'4inxo'XwXt bl.i'LM'’- X' .-q.710 0 i .6'>: »2 »Y'TlBXrBa.ioai? •sasf'nrodjq •. r ' .. 'I i;f*toW ,'j-id-?frct^rtaoi^ .J.A tT^^ilncUKl .6C .^^9X qq tJi'ior n? cn.-^jf^lt-nua .Q , ri' .i-!i; lo .Tc i rs'loq'ToonX - no 9rti30jfilx?fc:»t> ^^3 •fiiaoni to j f ^ > a f jXx ...:nx D ''3-iioa*Ti&flTo-X7* .^d^X ,Xi6-'?Q3 i'lT no a^o.-'xn to dort#uXlAl iO.T »IX«H bn# .a tXeassat .Oi^ ,ii?^X-94 -‘tifOTOirXt-^ to pa8iXo ,aXI*o ItoKX /o-'; tnadaXa®': bn* av'i^Xenea nX Xioa .tj 58 J. of Biol. Chem., 252:IO5I-IO36, 1977. 42o Roy-Burman, P. Recent Results in Cancer Research; Analogues of Nucleic Acid Components, Springer-Verlag, New York, pp. 16-27, 1970. 43. Cadman, E,, Grant, S, and Benz, C, Biochemical Modulation as a guide to rational combination chemotherapy for the treatment of cancer. In Pediatric Oncology II, Martinus Nijhoff, Hingham, in press. 44. Streeter, D.G., Watkowski, J.T., Khare, G.P., Sidwell, R.W., Bauer, R,J., Robins, R.K. and Simon, L.N. Mechanism of action of 1-B-D- ribofuranosyl-1,2,4-triazole-3-carboxamide (Virazole), a new broad spectrum antiviral agent. Proc. Nat'1 Acad. Sci., 70:1174-1178, 1973. 45. Stryer, L. Bichemistry, W.H. Freeman and Co., San Francis¬ co, p.537, 1975. 46. Krakoff, J.H. , Brown, N.C, and Reichard, P. Inhibition of ribonucleoside diphosphate reductase by hydroxyurea. Cancer Res., 28:1559-1565, 1968. 47. Hakala, M.T. and Taylor, E. The ability of purine and thy¬ midine derivatives and of glycine to support the growth of mammalian cells in culture. J.of Biol. Chem,, 234: pp.126-128, 1959. 48. Goodman, L.S, and Gilman, A,, eds. The Pharmacological Basis of Therapeutics, MacMillan Publishing Co., Inc., New York, pp.1284-1287, 1975. 49. Sobell, H.M, How actinomycin D binds to DNA. Scientific American, August, 1974, pp.82-91. 50. Rowe, P.B,, McCairns, E., Madsen, G., Sauer, D. and Elliott,H, ,6co:-Xv10!’is<^s • « , : ■• ^ 160,'x,J a/:rn«o«J( ,'? ^nfionuS'-YoH V V* . i*i«V-iQ;5^i-ii'XqE ,a^iifnoqmoO fcloA oisXotfW lo £, •’ i^.'’ .'..* , • ’ ' ' ■'■* ■ ' •- ''^>vv{io iioiJmidiiioo^lsixoiti^'^ ot a «& ■-*■• --i-■>-'<^0 atX .“t^ofuBij jrseai^sd'it ^.a^eaq ni ,mjarf;v^4H ,ltoirttiH " • T-'-'.v :■;■ * ,/>i.>’vc,: ^‘'^^' v.D.Q *44 ■ _‘5,V ..-Offlia bn«i *rv;rrj;77 fo:v S , r-X^'?cnE'iulo,jiT ?:c noi^'OJ . c- ' .•^' . .1.1 .’..i'Ti.vi i'nt'":,; ‘.Jr? ba'Ctq w»/i s t ((i^XoJifnlV) ./'/‘.V: .av.i-4iVMiO" ..io2 ,6iioA /MuH *'-■ ' ■’-'* ‘ i’* 'f( ,'r:Traiffl0-ir,iS ,J ,ii\;T[;t2 100 ....‘? ■ .1 . ".i'/ji-'H biiiS ,D.VI ^.ywo'iii ,.K.l. .tloiji’X ,d4 ■'-‘ ’M ■i'.x.J'oijOeT ai'^ftqao/lqi^ ‘»oi .'30(»Iot/notfi’'j .3oy>i •.-j'lL. '■ '/Jlliia Bill .S .':eav.fiT Hem .aX^jlaH r- >vs.'2> q^L-'a 1 o eavi^ arviiefc t . I'2 I'd '*r. .b .HT.jJjuJO a£X«.0 ABiXMAtAA lO i X30i3G£-3CA3iTj3 ,.oal , ,oO j^nirtuil'.ffj^ nBlitHojaW ^uoituaqjs’xailT to tXasS .^V9I .^rSSi-lieSX.qq ,:f»¥oY w»H .AMQ 03- Bbrtld Q nio'yiffloniifQB woH ♦iiaeJoS .94 *I9'^S8.qq ,4^9X ,ytu^A ^txabJtsaA t. .ttoiXlS cn# .C ,n#efe&*< * ,5 .amijiOoiH ,»wo^l .0^ 59 De novo purine synthesis in avian liver, J, of Biol. Chem., 253:7711-7721, 1978. 51. Fox, I.H. and Kelley, W.N. Human phosphoribosylpyrophosphate synthetase. J, of Biol, Chem,, 247:2126-2131, 1972, 52, Wohlheuter, R.M. Hypoxanthine phosphorihosyltransferase activity in normal, developing and neoplastic tissues of the rat, Eur, J, Cancer, 11:463-472, 1975. 53. Atkinson, M.R. and Murray, A.W, Inhibition of purine phosphoribosyltransferases of Ehrlich ascites tumor cells by 6-mercaptopurine. Biochem, J,, 94:64-70, 1965. 54, Tsuda, M,, Katunuma, N, Morris, H.P. and Weber, G. Purifi¬ cation, properties and immunotitration of hepatoma gluta¬ mine phosphoribosylpyrophosphate amidotransferase(amido- phosphoribosyl transferase, EC 2.4.2,14), Cancer Res,,39: 305-311, 1979. 55. Yen, A,, Fried, J. and Clarkson, B. Alternative Modes of population growth inhibition in a human lymphoid cell line growing in suspension. Experimental Cell Research, 107: 325-3^1. 1977. 56, Baserga, R. Multiplication and Division in Mammalian Cells, Marcel Dekker, Inc,, New York, p.163,1976, 57. Holley, R.W. and Kiernan, J.A. "Contact inhibition" of cell division in 313 cells. Proc, Nat*l Acad. Scio,60: 300-304, 1968. 58, Rossow, P.W., Riddle, V.G.H. and Pardee, A.B. Synthesis of labile, serum-dependent protein in early G^ controls animal cell grow^th. In Readings in Mammalian Cell Culture, 2nd edition, (R. Pollack, ed,). Cold Spring Harbor Labora- . , .loiii 'to ,'w ctMivA fil. ii3«rtcfm:a ^ciltsjq ov-on sG ! ‘ ■' TQ'TT’^qixaooi'^odqaoriq fiBiWi/K »K»I fXoH .1^ ■ ftj\^"taa3't.tIv.«Qd’l'iodq30(fq ©nidtoaxoqxW .M.fl .Tta^iiedXrfoW *S^ •1 ae^'aai?- foen ijr^s gnlqolsvofc ,lJsanon ai • ,S^4-caii» I't .L .Tua »t«“s »r sniTjq "lo noisldlfUil .W.A brs .JI,M .noeniitA t.xL^o lo.niit afl;t’io3& rfoilirtS "lo •^?.JntlafiA^^i^ao<^l'loriqliOriq .iL^CI ,.G .ut^riooia .ani'kJuqotqAO’XWi-a' -Hi 11/*? *0 .T9'i’»W tna -i'l.H ,ali*xoM ,H ,firaifRi/i’aii »,M ,BfeW8T AT.otsqar. '0 nolJ'a'ili;}“ontrmmi bfta aa.l^'^t3qo^q \nolt»o •oblm£)'-srian^'\?,ryiSoljxmM 9ttftq90(f\qo'Viqixno(Si'tQtlq^otici anln linns') . (UX .<■: .ii.S :,>3 , XviaodXiOffqaortq ‘ .nc-^oc lt> sj^boM 5'.’u yai* ‘'A *H .rraa^'raXO fans .t, ,bai'xT ,.A ,rt§Y enil i^'V' bXodqor^X a rrl iiol^l^Xrfni titwcz'^ ctc>.ltAXtJUoq t'(i. »-o-iaaasH IX*'' IjR^naatiiaqpcS .noXafraqtsya iti Jirtiwoi^ ,X4^:-esc at noialvi'I btia aolT^oilqitlfjM ,M *d?. ,iol ,q ^i-ioY w*H , .anrX Itoialf ■=% to "noiiidijrtnX to/KfrcoO*" .A.t* ,a«irtaiii bnis ,W«H .V?: i0d,.i72 .bssA X*tBVI .001*? .eXXao t^C fil ftoisivifl XXao ’ ,8a‘$t .4J0C-00C to 5X«adi .frsi/^XyO XXtO ftiiX^ffwnuM ni itl IXao ijaminB 'xod’jjiii •^nt*iqZ blot' ,(,b» 1/ 6o tory, pPo421-425, 1981, 59, Nordenskjold, B.A., Skoog, L., Brovm, N.C. and Reichard, P, Deoxyribonucleotide pools and ribonucleotide pools in cultured mouse embryo cells. J, Biol, Chem,,245s5360- 5368, 1970. 60, Hahn, G.M,, Stewart, J.R,, Yang, S.J. and Parker, V, Chinese Hamster cell monolayer cultures, Exp, Cell Research, 49: 285-292, 1968, 61, Knutton, S,, Sumner, M.C.B, and Pasternak, C.A. 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