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[C AN CER R ESEA RCH 4 3, 2 81 9-2 83 0, J un e 1 98 3]

B iochem ical Prophage Induction A ssay: A Rapid Test for A ntitumor AgentsThat Interact with DMA1R . K . Elespuru2 and R . J. W hite3B io lo gic al C ar cin og en es is P ro gr am [R. K . E .] a nd Chemoth er ap y F erment at io n P ro gr am [R. J . W .] , NC I- Fr ed er ic k C an ce r R es ea rc h F ac ilit y, F re de ric k, Ma ry la nd 2 17 01

ABSTRACTA b io chem ical ( co lo rime tr ie ) a ssay o f bact er io phage A induct io nw as utilized in the dete ction , identification, an d purification ofDMA-in te ra ctin g n atu ra l p ro du cts w ith p ote ntia l a ntitumor a ctiv

ity. A set of 142 standard antib iotics, com posed principally ofnatural prod ucts w ith esta blished antitum or activity an d/or defined mechanisms of action, was tested in the assay. As expected, m ost ind ucers w ere direct inhibitors of D NA synthesis.A few other types of inducer, w ith probable indirect effects onDNA s yn th es is , w ere fo un d a fte r p ro lo ng ed in cu ba tio n: o ne c la sso f R NA sy nth es is in hib ito r, a d ih yd ro fo la te re du cta se in hib ito r;a nd tw o in hib ito rs o f b ac te ria l c ell w all s yn th es is .The biochem ical induction assay w as sem iautom ated for u sea s a p re sc re en in th e s ea rc h fo r n ov el a ntitu mo r a ge nts in 1 0,7 24actinom ycete ferm entation broths. Approxim ately 1% of thecultures produced com pounds that were active in the assay;som e appear to be novel. None required m etabolic activation(v ia ra t liv er 8 9) fo r in du cin g a ctiv ity .The biochem ical indu ction assay w as a dapted for bioautog-

ra ph y (th e d ete ctio n o f in du cin g ch em ica ls ch roma to gra ph ed o nth in -la ye r p la te s) a nd fo r s tra in imp ro vemen t p ro grams (s ele ctio nof isola tes with enhanced inducing activity). The speed of theassay (2 to 5 hr) m ade it useful for m onitoring antitum or agentp ro du ctio n a nd p urific atio n. T he s en sitiv ity o f th e a ssa y c ou ld b evaried, depending on the length of the incubation period. M icrobe s, nutrients, and toxic so lvents did not u su ally in terferew ith th e d ete ctio n o f in du cin g a ctiv ity .INTRODUCTION

The a ctino mycetes an d fu ngi have proved to be a m ost fru itfuls ou rc e o f b io lo gic ally a ctiv e m eta bo lite s w ith th era pe utic u tilityand ha ve bee n heavily investigated in scre ening p ro gram s forthe last 40 years. Because animal models have proved to berather insensitive and expensive, it has been necessary to dev elo p in v itro te st s ys tems fo r a ntic an ce r a ge nts th at a re p re dictive of clin ical activity (44). S uch tests fu nctio n as a prescreen,w ith the object of selecting a sm aller nu mbe r of sam ples for te stin vivo a ga in st a tra nsp la nta ble ro de nt tumo r, s uc h a s th e murin eP388 leukemia. Cell culture cytotoxicity has been one of themost commonly used types of prescreen and is capable ofdetecting m ost, if not all, of the com pounds with establishedclinical utility. H ow ever, because cytotoxicity prescreens arenon sp ecific, i.e., capab le of detecting com pound s w ith a w idevarie ty of m echanism s of action, they can generate an overly1 Th is w ork w as su ppo rte d b y C ontract N 01 -C O-7 538 0, N atio nal C ance r Insti

t ut e, N IH , B e tn es da , M e t. 2 02 05 .' T o w hom re qu es ts fo r re pr in ts s ho uld b e a dd re ss ed , a t F er me nta tio n P ro gr am ,B ld g. 4 34 , F re de ri ck , M d . 2 17 01 .3 P r e se nt a dd re ss : L ed er le L ab or at or ie s, P e ar l R iv er , N . Y . 1 09 65 .

R ec eiv ed S ep te mb er 8 ,1 98 2; a cc ep te d M arc h 8 ,1 98 3.

large num ber of positive sam ples for in vivo screening. Otherm ore selective in vitro tests have bee n de vised that have sho wnp rom is e. O ne e xample is th e b ac te ria l a ss ay fo r a ntime ta bo lite sp io ne ere d b y H an ka e f a l. (1 7). A no th er is th e ly so ge nic in du ctio nassay developed during the last 2 decades by Endo et a l. (10),F le ck (1 1), G eis sle r (1 4), H ein em an n e f a l. (1 9, 2 6), a nd D ev ore t(32). This assay detects com pounds that interact w ith DNA orinterfere w ith D NA syn thesis, and a goo d correlation betw eenin du cin g a nd a ntic an ce r a ctivity h as b ee n re po rte d (1 ,1 8, 3 5).In its c la ss ic al fo rm , th e ly so ge nic in du ctio n a ss ay m ea su re sth e num ber of b acterio phages prod uce d subseq uent to incub ation w ith the test sam ple. R ecently, a strain of E scherichia colilysogenic for a X-lacZ fusion phage was constructed for use ina BIA4 (9). Induction of the prophage is measured by the appearance of /i-galactosidase, product of the lacZ gene, in acolorim etrie assay. This assay was designed to be of greaterutility than are classical prophage induction assays by beingfaster, easier, more sensitive, and adaptable for a variety ofpurposes. In this paper, we describe the use of the BIA as a testsystem for the detection of a standard set of antitum or agentsand m odel com po unds; as a pre screen for indu ce rs in m icrob ialfe rm en ta tio n b ro th s; a nd a s a n a id in th e a na ly sis, p urific atio n,and production of natu ral p ro ducts w ith p ote ntia l antitum or activity.MATERIALS AND METHODSAn ti tumor Agen ts

B le om yc in w as th e g ift o f D r. W illia m B ra dn er, B ris to l L ab ora to rie s;p la tin um -c on ta in in g c ompo un ds w ere o bta in ed fr om D r. R on ald O . R ah n,O ak R idge National Laboratories; and IC R com pounds w ere from D r.R ichard P eck, Institute for C ancer R esearch. O ther antitum or agentsw ere s up plie d b y D r. J oh n D ou ro s, N atio na l C an ce r In stitu te.BacteriaE . c oli stra in B R5 13 is (A p /a cZ cl*P Rf 7) p ro-lac A uvrB A envA azi thi

rp sL g al (9 ).Reagents

Bacteriological m edia are from D lfco, D etroit, M ich.; inorganic andorganic salts are from Fisher Scientific, Silver Spring, M d.; sodiumamp ic illin (P oly cillin -N ) is fro m B ris to l L ab ora to rie s, S yra cu se , N . Y .; o -n it ro ph en yl- |8 -D -g ala cto py ra no sid e, BNG, F as t B lu e RR s alt , N ADP mon -o so dium s alt, g lu co se 6 -p ho sp ha te , c hlo ramp he nic ol, /3 -m e rc ap to et ha -nol, and T ris (as T rizm a B ase) are from S igm a C hem ical C o., S t. Louis,Mo.Med ia a nd B uffe rs

L BE . T he m ed ium c on ta in s (p er lite r) 1 0 g B ac to -try pto ne , 5 g y ea stextract, 10 g sodium chloride, and 5 ml 1 M Tris. After autoclaving,

4 T h e a bb re via ti on s u se d a re : B IA , b io ch em ic al in du ct io n a ss ay ; BNG, 6 -b romo-2-naphthyl -0-D-galactopyranoskJe; BCNU, 1,3-bis(2-chloroethyl )-1-ni trosourea.

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R . K . E lespu ru and R . J. W hitemedium is supplemented w ith 4 m l of 50x Medium E (43) and 10 ml of2 0% g lu co se . F or L BE am p a ga r, 15 g a ga r a re a dd ed b efo re a uto cla vin g,and 1 m l of a freshly prepared solution of sodium am picillin (10 m g/m l)is added just before pouring plates . P lates should be poured on a levelsurface.ZCM Buffe r. T he b uffer c on ta in s (p er lite r) 1 6.1 g N a2 HPO4 -7 H2 O (o r8.5 g Na2H PO 4 anhydrous); 5.5 g N aH2PO4-H2O , 0.75 g KCI, 0.246 gMgSO4 -7 H2O ( or 0 .1 2 g MgSO4 a nh yd ro us ), 2 .7 m l /i-m e rc ap to et ha no l,and 25 m g c hloram phenicol. Adjust to pH 7.O . D o not autoclave. S torecold.M edium A. This m edium contains (per liter) 10.5 g K2H PO4, 4.5 gKH2PO4, 1 g (NH4)2 SO 4, a n d 0 .5 g s od ium c it ra te -2 H2O. A ut oc la ve .Ac ti va ti ng Enzymes

R at liv er (s tra in u ns pec ifie d) 9 00 0 x g p os tm ito ch on dria l s up ern ata ntw as from Litton B ionetics, K ensington, M d. A ctivation m ix, freshly prep are d, w as 0 .5 m l ra t liv er S 9, 0 .4 m l c ofa cto r s olu tio n (2 5-m g/m l p ortio nsea ch o f N ADP, g lu co se 6 -p ho sp hate , M gC I2 ), a nd 2 .1 m l 0 .1 M p ho sp ha tebu ffe r, p H 7 .4 .Labora tory Supplies

Polystyrene plastic tubes (16 x 125 or 17 x 100 m m) are No. 2025 orN o. 2057 tubes, respectively, from F alcon; large (243- x 243- x 18-m m)bioassay plates are from A/S Nunc, Kamstrupvej 90, Kamstrup, DK-4000 R oskilde, D enm ark, distributed in the U nited S tates by V angardIn te rn atio na l, In c. , N ep tu ne , N . J .; g rid de d "m in ip la te s" (1 00 mm squ are )a re N o. 1 01 2 fr om Falc on ( Fis he r S cie nt if ic ).BIA Spot Test (Chart 1; Fig. 1)

A fresh overnight culture of BR513 was diluted ~ 100-fold (to ASM0.05) into LB E m edium and grow n at 37 for approxim ately 3 hr to A oo

0.4. B acteria w ere distributed in 25-m l aliquots into centrifuge tubes,pelleted (4000 to 5000 rpm , 3 m in), and gently resuspended in 1 m l LB Em edium . In som e cases, 3 m l rat liver S9 activation m ix were added tothe tube. T wenty-five m l m elted soft (1% ) agar at 45 w ere added to thetubes, and the contents w ere poured onto w arm (37 ) bioassay plates(243 x 243 mm) containing approxim ately 330 m l (solid) LB Eam p agar.A fte r t he to p la ye r s olid ifie d, c hem ic al s olu tio ns a nd fe rm e nta tio n b ro th sw ere s po tte d d ire ctly o nto th e p la te s u sin g P as te ur o r c ap illa ry p ip ets o ra m ultis am ple a pp lic ato r (s ee b elo w). P la te s w ere k ep t o n a s lid e w arm erset at 38 during the tim e required for spotting. T he plates w ere thenincubated at 38 for 3 hr, after w hich another agar layer containingsubstrate was added. For each plate, 60 mg Fast Blue RR salt and 10m g B NG w ere com bined w ith 1.0 m l dim ethyl sulfoxide just prior to use;a Pasteur pipet w as used to aid solution of the m ixture. Twenty-five m lm elted soft agar at 45w ere added to the substrate m ixture and thenpoured onto the plate. C olor developm ent w as com plete in 10 to 15 m in.Proportions of m aterials for standard sm all (100-m m) Petri dishes areo ne -t en th o f t he amou nts d es cr ib ed .L arg e-S ca le S cr ee nin g (F ig . 2 ). T he fe rm en ta tio n b ro th s s cre en edfo r in du cin g a ctiv ity w ere p re pa re d fro m is ola te s o f a v arie ty o f d iffe ren tgenera of the Actinom ycetales w hich were supplied as part of a cooperative venture by Dr. C. N ash of the Sm ith, K line and French Laboratories. Each organism was grown in at least 4 different ferm entationm ed ia . S am ple s fro m fe rm en tation b ro th s w ere s potte d d ire ctly o nto B IAassay plates by hand or w ith a specially constructed m ultisam ple applicator that could sim ultaneously load up to 144 sam ples on glass rods.[T his a pp ara tu s is s im ila r in p rin cip al to a m ultip oin t in oc ula to r (4 2)]. T hero ds w ere d ip pe d in to v ia ls c on ta in in g fe rm en ta tio n b ro th s a nd lo we re dto the agar surface of a 243-m m bioassay plate for spotting. The assayw as conducted w ith and w ithout rat liver S9 activation m ix. A ll culturesthat caused induction in at least one ferm entation m edium w ere refer-m en te d a nd te ste d a ga in .B io au to gr aphy (F ig . 3 ). P ur ifi ed c hem ic al s ta nd ar ds , fe rm e nt ati on

Cha rt 1 . B IA s po t te st a pp lic atio ns .Bact er ia l col on ieson agar plugs

Log phase BR5131) Centr ifuge2) Resuspend in soft agar,p ou r into p etri dish

T hin la ye rc hromatogram (TLC )'.. O . . P ., O. ./">.. Jv ./ ) / // // / /! // // // // )Mu lti-p oin t s amp leapplicator

3) 3 hr, 374) rem ove p lu gs5 ) s ub stra te o ve rla y

3) 3 hr, 374) rem ove TLC5 ) s ub str ate o ve rla y

3) 3 hr, 374 ) s ub stra te o ve rla y

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B IA fo r Ant itumor Agentsbro ths , o r ex tr ac ts were ch romatog raphed on s il ica ge l t hin- layer p la tes(Bake r F le x 1B -F ). A p ie ce o f we t fil te r paper was p la cedon the sur fa ceo f a 243 -mm b ioassay p la te con ta in in g LBEamp aga r and smoo thed toremove w ri nk le s. The th in -la ye r p la te s we re then p la ced f ace down onth e filte r p ap er (s lowly , to a vo id th e e ntra inmen t o f a ir b ub ble s) a ndincuba ted for 2 to 3 h r a t 38.A f te r i ncuba ti on , t he thin- laye rp la tes andfilte r p ap er w ere remov ed from th e a ga r s urfa ce . B ac te ria g rown a sd es crib ed a bo ve w ere p ou re d in s oft L BE amp aga r o nto th e w armedb ioassay p la te . The p la te was f ur th e r in cuba ted a t 38o r 3 h r to a llowinduct ion and deve lopedby addi ti on o f subs tr ate over lay .Th is me thod a ll ow s t he time o f d iff us io n o f chem ical fr om t hin -la ye rp late in to agar to be contro lled. Severa lo ther var ia t ionson th is techniquea re poss ib le . I n one va riat ion , t he thin- layer p la te may be p laced d ir ec tl yo n to p o f a b ac te ria l l aw n, a s sh ow n in C ha rt 1 . T h e p la te is remo ve dprior t o add it ion o f subst ra te over lay . I n ano the r va ri at ion , t he bacter ia llaw n is poured directly on top of a thin-la ye r pla te p laced face up.S ub stra te o ve rla y is a dd ed o n to p. T he la tte r m eth od a llo ws g oo daera ti on o f t he bac te ri a, bu t t he thin- layer p la te canno t be recove red .IndividualColonyVar iants (Fig. 4) . For the identi ficationof naturalva rian ts o f p roduce r o rgan isms , aga r p lugs (about 8 mm in d iamete r)o fin di vi dual bac te ria l c olon ie s we re cut w ith a cor k bor er and p la ced onthe p la tes p repared as for t he spot tes t. P lugswere removedbe fo re theadd it ion o f t he Fast B lue RR sa lt -BNG subs tr ate over lay .L iq uid In cuba tio n B IA (Q ua nti ta tiv e) (C ha rt 2 )Dose-ResponseAssay. Bacter iagrownas for the B IA spot test toAeo o0 .4 w ere d ilu te d 1 0-fo ld in to LBEamp and d is trib ute d in 0 .5 -m la li quo ts into p las ti c t ubes containing 50 n \ o f t he desi red concent ra ti ono f l argomyc in F ll ( aqueous so lu ti ons ). Tubes were incuba ted for 3 h r a t38w ith shaki ng and t hen chille d w it h t he add itio n o f 4 .5 m l cold ZCMbu ff er . A fte r th e m ix tu re was wa rmed t o 28,t he enzyme assay wasin it ia ted by the addit ion of 1.0 ml o-n it rophenyl -i i-D-ga lactopyranosideinMed ium A (4 mg/ml ). The reac ti on was term ina ted a fter su ff ic ien t co lo rd ev elo pment ( 10 m in to 3 h r) b y th e a dd itio n o f 2 .5 m l c old 1 M sod iumcar bonate . The A2oas r ead in a Bausch and Lomb Spec tr on ic 20 byd ir ec t i nse rt ion o f t he p las ti c i ncuba ti on tubes . The spect ropho tomete rwas adju st ed to zer o using a con tr ol t ube w ith out bacte ria . Enzyme i sc alc ula te d a s 1 00 A2o /f,h ere f is th e c olo r d ev elo pment tim e in h r(Chart 2a) .K ine tic Assay . The assaywas as for P rocedur e1 except bact er iaa nd c hem ic al w ere c omb in ed in a s in gle -s ha ke fla sk . A t a pp ro pria tetim e s, a liq uo ts o f 0 .5 m l w ere remov ed to tu be s a nd th en c hille d w itht he add it io n o f 4 .5 m l cold ZCM bu ff er (Char t 20 ).RESULTSTest o f S tandard Compounds .A schematicdiagramof theme thodolo gy u sed fo r 3 v aria tio ns o f th e B IA spo t te st is s hownin Char t 1 . Spot test r esul ts w it h 17 we ll- known na tu ra l p roduc tsare sh ow n in F ig. 1a an d T able 1. E ach comp ou nd w as testeda t 2 co ncen tratio ns d ifferin g b y a fa ctor of 1 0, de pen ding onsolu bility a nd b io lo gic al a ctiv ity . T he in te ns itie s o f th e s po tsre flec t relat ive inducing ac tivi ties . Observa tion o f the spot pa ir s

prov ides information regarding relat ive potenciesand the locat ionof the op tim um d ose rang e for ind uction, w hich m ay the n beu ti li zed in the quan ti ta tiv e assay . Fo r examp le , in F ig . 1a, P la te1 ,Row B , Spots 1and 6 a re near t he op timum dose fo r i nducti on ;Spof 2 is below it; and Spofs 3, 4, and 5, showing zones oftoxi city , a re above t he op timum dose . F ig 1u il lu str ates t he samep rin cip le w ith comp ou nd s d ilu te d to th e e nd p oin t. S olu tio nss how in g good a ctiv ity in th e s po t te st, when d ilu te d 10-fo ld in tothe bac te rial suspension , a re general ly in the correc t concen trati on r ange fo r opt imum inducti on in the quan ti ta tiv e tube assay.T his is seen to be th e ca se for larg om ycin F ll, sp otted fro m a

6C ha rt 2 . Q ua ntita tiv e a ss ay o f ly so ge nic in du ctio n b y la rg om yc in F ll. a , d os e-response assay after 3 hr (in the presence of am picillin). b, kinetic curves ofin du cti on a t th e c on ce nt ra ti on s s hown ( *i g/ml) ( in t he p re se nc e o f ampic illin ).

s olu tio n o f 1 00 Mg /m l [ 1 Mg /s po t (F ig . 1 a)], g iv in g a pea k doseo f 10 / tg /m l in the quan ti ta tiv e assay (Cha rt 2a ).T he re su lts o f th e te st o f 1 42 c ompo un ds in th e sp ot te st a resummarize d in T able 2. In T able 2, w e have trie d to co rre la tei nducing acti vit y (B IA ) w ith the mechani sm o f act ion (DMA inte ract ion ) and an ti cance r po ten tia l ( 2, 8 , 16, 24 , 34, 38 ) o f a d ive rseset o f compounds. We purpose ly i nc luded compounds tha t we ren ot e xp ec te d to in du ce b ut w ere re pre se nta tiv e o f a v arie ty o fchem ical types and modes o f acti on . The s tudy i s b iased towardnatu ra l p ro du cts a va ila ble in o ur la bo ra to ry , s ome o f whic h a renot we ll s tud ied as t o mode o f act ion . Fo r t h is r eason , chem icalsare grouped by relat ive induc ing capac it y (as de te rm ined by spo tin te ns ity ), ra th er th an b y s tru ctu ra l ty pe o r mode o f a ctio n. T heamoun t o f compound r equ ir ed fo r i nduction var ie s over 6 o rde rsof m agnitude. This quantity is indicated in Table 2 for eachinducer.

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R . K . E lespuru and R . J. W hiteTab le 1Key to Chem ica ls spo tted in F ig . Jaand resu ltsob tained

Compound*StreptovarcinDRifamycinSVActinomycinDNeocarzinostatinHedamycinRufochromomycinBruceantinMacromomycinBleomycinDaunorubicinRubiflavinLargomycinStreptonigr inV

acidHjOMethanolAflatoxin

B,Quantity

(g/spot)60.20.020.20.020.20.0210.10.20.020.20.020.20.0210.110.110.10.20.0210.10.20.0210.10.20.021

id2pl0.010.001Spot

locationA123456B123456C123456DI23456E123456FI23456Platel:noampicillinNoS9tcttt+++++++R+++R+++R+++_-++++++++R++R+-+++R++++++++++

2: N oampici l l in+S9ttt++++++R+++R+++R+++_+++++R++R+++--H-+3: +ampicillinNoS9ttttt+++++++R+++R-H-+R+++_++++++++

4: +ampici l l in+89-ttt++++++R+++R+++R+++_

* So lv en t a s i nT ab le 2 .0 Chemic al s olu tio ns mad e a t 1 0 a nd 1 00 >g /m \;o lume s po tte d d ep en de nt o n s olv en t (s ee T ab le 2 ,Footnoteb) .0 - , n o in du ctio n; t, to xic , n o in du ct io n; ;e qu iv oc al; + , + +, + ++ , w ea k, m od er ate , s tr on g in du ctio n,r espec t ive ly , as de te rm ined by spot co lo r i ntens it y; R , r ing o f i nduct ion (d if fu s ion zone ) su rr ound ing tox iccenter.

More than one-third of the components tested (56 of 142)were detected as inducers. The great m ajority of the stronginducers a re co mpo unds kn ow n to inte ra ct w ith D NA , includingman y s ta nd ard a ntitumor a ge nts s uc h a s A driamyc in , b le omy cin ,BC NU , IC R com pounds, a nd platinum com ple xe s. Th e inducingactivity of piperazin edione su pports the sug gestion tha t it is ana lk yla tin g a ge nt (5 ). Mo re d eta ile d s tu die s o f s tru ctu re -a ctiv ityrelationships for som e of these groups of com pounds w ill bepresented elsew here. O ther inducers, generally classified asw ea k, h av e in dire ct e ffe cts o n DNA , o fte n b y in hib itio n o f s pe cificenzym es involved in D NA b iosynth esis. E xam ples include a za -

serine (4, 37), trim ethop rim (7), no vo biocin (41), nalidixic acid( 15 ), 5 -flu o rour ac il a nd 2 '- deoxy -5 -f lu or ou rid in e ( 6) . 2 '- Deoxy- 5-b romou rid in e, o n th e o th er h an d, is e xte ns iv ely in co rp ora te d in toDNA in place of thym ine (12). It did not induce the phage. Ase xp ec te d, in hib ito rs o f p ro te in s yn th es is a nd m ic ro tu bu le a ss embly did not induce. Cysteine was negative, in contrast to thep os itiv e e ffe ct re po rte d in a no th er X -p ha ge in du ctio n a ss ay (3 5).Chemica ls w ith apparently sim ilar m odes of action oftenshowed quite large differences in BIA activity. For exam ple,a ctin om yc in D , a nth ra ce ne dio ne s, IC R c om po un ds , a nd d au no -ru bic in a re a ll in te rc ala tin g a ge nts ; h ow ev er, a ctin omy cin is n eg -2822 CANCER RESEARCH VOL. 43


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1a

No ampB

+ S92

O'"-V

F ig . 1 . C olo rim etrie s po t te st fo r ly so ge nicin du ctio n b y a ntitu mo r a ge nts a nd m od el c omp oun ds. C he mica l so lu tio ns w ere sp otted d ir ec tly o nto g rid de d 1 00 -mm p la te s c on ta in in gb ac te ria , w ith o r w ith ou t a rn pic illin (a mp ) a ndra t live r 8 9 activ atio n m ix. A fter in cu ba tio n a t3 0 , pla te s w ere d ev elo pe d b y th e a dd itio n o fa chromogenic substrate for the enzyme -g ala cto sid as e. a , 3 h r in cu ba tio n. T ab le 1 p rov id es a k ey t o t he c hem ic als s po tte d a nd r es ult so bta in ed , b , s er ia l 2 -f ol d d ilu tio n f or d et ermin ation of m inim al inducing doses. A differentch em ica l is sp otte d in e ach ro w, w ith th e h ig hest dose (/ig/spot) at the left m argin. R ow 1,b te om yc in (0 .1 ^ 9) ; " w 2 , la rg om yc in F ll ( 0.5n g); R ow 3 , rifa mp ic in ( 1 ( g );R ow 4 , a za se rin e(100 xg); Row 5, BCN U (100 M9); R ow 6, c/s-d ic hl or od iammin ep la tin um ( II ) ( 10 t ig ). S o lv en tis dim ethyl su lfo xid e fo r B CN U; a ll o th er so lutions are in w ater. See legend to Table 2 fordetails.

amp

1b No23

amp1 45amp2

6

2 hrABCDE

Jt

r l O* *v

5 hr

x

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f.K . E lespuru and R . J. W hiteT ab le 2

T es i o f a ntitu mo r a ge nts a nd m od el c om po un ds in th e B IA s po t te stC hemic al s olu tio ns w ere s po t-te ste d d ire ctly o n E . c oli s tra in B R5 13 ( Ap /a cZ ) p ou re d in a ga r c on ta in in g a mp ic illin (1 0>


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B IA fo r Anti tumor AgentsTabl e 2Con ti nued

CompoundCyaneinCyclomycincomplexDuramycinEnteromycinFusarubinGliotoxinKasugamycinMikamycinMithramycinNarangomycinOxytetracycl inePactamycinProdigiosinR

salicylateStreptorubinViridogriseinViundrymycinDNA

Interaction"olventMethanolMethanolWaterWaterMethanolMethanolWaterMethanol+

MethanolMethanolMethanolMethanolMethanolMethanolWaterMethanolMethanolMethano

appliedngf115511510.01-0.211111510.006-0.211Minimalin du cin g A ntitu mo ramount (^g/spot)0ctivi ty0-+++++++++Compounds

w ith n octivityAmicetinCAMPAlanosineAminopterinAnguidineAnisomycinAnthracenedionesNSC

7833NSC15367NSC72 1NS C278467NSC7957NSC295560(Anthracenedione)NSC28751NS C1485(Mitoxantrone)NSC

279836Azacolutincomplex5-AzacytidineAzotomycinBlasticidinSBruceantin5-Bromo-2'-deoxyuridineCandicidinCordycepinCysteine

(DL)Cystine(DL)Cytovir inDuazomycinAFlammulinFormycinAFormycinBFumagil lmGougerotinGriseofulvinHadacidinMethotrexateMitogi l linMitosperMycorhodinNebular inOligomycinOosporinPeptinoganPlatinum

compoundstrans-PtAm2CI2[PtAm3CI]CIK2[PtCI]PuromycinPyrazomycinRestr ict

ocinSangivamycinSaramycetinSeptacidinSistomycosinSparsomycinMethanolWaterWaterWaterMethanol

Methano lDMSODMSODMSO+

DMSODMSODMSO- t -

DMSODMSO+

DMSODMSOWaterMethanolWater-

MethanolDMSOWaterWaterWaterWaterWaterWaterWaterMethanolWaterMethanolWaterMethanol

0 .1 5 MaCI0.15 MaCI0.15 MaCIWaterWaterWaterMethanolMethanolWaterWaterMethanol155-2501-10010.1-10.1-1

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R . K . E lespu ru an d R . J. W hiteTabt e 2Con ti nued

CompoundStatalonStreptolydiginThiosangivamycinThreomycinTrienineVe rr uc ar in AVinblastineDNA

Interaction8SolventMethanol

WaterMethanolWaterMethanolMethanolWaterAmount

appliedMgr11-1015110.1-1Minimal

in du cin g A ntitum ora mo un t (/ig /s po t)c a ctiv ity ''+

8 W here kn ow n (R efs. 4, 7, a nd 12 ; an d see te xt).6 A bs olu te a mo un t a pp lie d in ~ 10 n \ H 20 o r DMSO a nd 2 1 m eth an ol (s urfa ce te ns io n o f th e s olv en ts d iffe rs ); s olu tio nc on ce ntr atio ns in n g/m \ th er efo re a re 1 00 o r 5 00 tim es th e q ua ntity s po tte d.' A s de term ine d b y th e ap pea ra nce of a visib le co lo re d sp ot a fte r 3 h r (5 h r) in cub atio n.d A ntitu mo r a ctiv ity in e xp er im en ta l a nim al s ys tems w ith tra ns pla nte d tumo rs ( 2, 8 ,1 6, 2 4, 3 3, 3 7) .* + , in te ra ctio n; -, n o in te ra ctio n.'A bb re vi at io ns u se d: DMSO. d im e th yl s ul fo xi de ; ND , n ot d et ermin ed ; c /s -P tAm zC t, c ;s -d ic hl or od iammin ep la tin um (ll) ;K [PtAmCI3 ], po tass ium t r ich lo roamminepla ti num( ll ); DON, 6 -d iazc -5 -oxo -L-no rl euc ine ; [P tAm4]CI2 , te traamminep la t inum( ll )c hl or id e ; cAMP, c yc li c adenosi ne 3 ': 5'-monophosphat e; ra n s-P tAm jC l z, f ra n s- di ch lo rod iamm inepl at in um (l l) ; [ PtAm3CI]C I,ch lo ro tr iamminep la t inum( ll ) ch lo r ide ; ^ [P tCUJ . po tass ium te trach loroamminepla ti num( ll ).9 R ep ro du cib le + o nly w he n b ac te ria a re g ro wn in th e p re se nc e o f N -a ce ty lg lu co sa min e ( in du cib le tr an sp ort s ys te m)." + only on less rich m edium (TB *) (9).

ative; ICR 170 is a strong inducer while ICR 191 is weak;a nthracen edion es are dete cted poorly or not at all; a nd d auno-ru bic in is d ete cte d q uite w ell. O f th e s ev era l in hib ito rs o f n ucle o-tid e synthesis tested, m ost are w eak inducers, w hile azaserineis uncharacteristically strong. A closer look at such factors asp erm ea bility, toxicity, and specific D NA interactions provid essome explanation for these differences, but these will not beconsider ed he re .In the case of a few com pounds, the expected correlationbetween inducing capacity and effects on DNA was not obse rv ed . A fe w DNA -in te ra ctin g c om po un ds w hich p re fe re ntia llyin hib it R IA s yn th es is ra th er th an DNA s yn th es is (a ctin om ycin D ,m ithramycin, and cinerubin B) (7, 12) were not detected asin du ce rs . T his is n ot s urp ris in g, s in ce R NA sy nth es is is re qu ire dfo r th e a ssa y o f in du ctio n. S tre pto zo to cin fa ile d to re pro du cib lycause induction unless A /-acetylglucosa mine w as a dded to induce the transport system co ntrolling its uptake (27). T his w asunexpe cted in vie w of the activity reporte d in an othe r pro phageinduction assay (35). W hile azaserine showed good inducingactivity, other glutam ine analog ues (inhibiting purine bio synth es is ) (4 ) s uc h a s 6 -d ia zo -5 -o xo -L -n orie uc in e, a zo tomyc in , a ndduazom ycin showed little or no activity. The absence of zoneso f to xic ity fo r th e la tte r 2 c om po un ds in dic ate s th at th ey p ro ba blydid no t perm eate the bacteria. A lkylating agents such as B CN Uan d cyclop hospham ide w ere detecte d only at high concentrations.On the other hand, induction by the group of structura llyre la te d a ns am yc in s (s tre pto va ric in s a nd rifa my cin s) th at a re inhibitors of DNA-dependent RNA polym erase is som ewhat surprising (F ig. 2o). It is possible that induction is related to anin dire ct in hib itio n o f D NA s yn th es is (b y in hib itio n o f R NA p rim erfo rma tio n) a t c on ce ntra tio ns s till a llo win g re sid ua l tra ns crip tio nof the lacZ gen e (30).F os fomy cin a nd amp ic illin , in hib ito rs o f b ac te ria l c ell w all s ynthesis (25), were found to be w eak inducers. The loss of structural integrity of the cell wall m ay affect the DNA-m em branecom plex in this case. In com mon w ith other agents that appearto w ork v ia in dire ct m ec ha nisms , th ere is a s ig nific an t la g b efo rein du ctio n o cc urs , g en era lly re qu irin g a 5 -h r in cu ba tio n (F ig . 1 b).Therefore, the BIA can be m ade m ore selective for agents thatd ire ctly in te ra ct w ith D NA b y lim itin g th e in du ctio n p erio d to 3 h r.T he e ffe ct o n in du ctio n o f lo w c on ce ntra tio ns o f amp ic illin , ra tliver m icrosom al e nzym es, and incubation tim e is show n in F ig.

1. Table 1 shows our scoring m ethod and lists the chem icalsa nd am ounts tested . W hile m ost inducers w ere d etected after a3 -h r in cu ba tio n p erio d, th e a ns amy cin s re qu ire d 5 h r fo r in du ctio n(F ig . 1 6). O th er c hem ic als w ere d ete cte d a t lowe r c on ce ntra tio nsa fte r th e lo ng er in cu ba tio n tim e (T ab le 2 ). T his is c on sis te nt w ithty pic al in du ctio n k in etic s (C ha rt 2 b).Am picillin w as routinely ad ded to th e a ssay as an inh ibitor o fbackgroun d cell grow th (3 2) before its e ffect as an ind ucer w asknown. (Induction is not seen, however, at the concentrationpresent in the pla tes.) H ow ever, induction by som e chem icals,in clu din g rifamyc in SV, s tre pto va ric in D , a nd fo sfomyc in a pp ea rsto b e in hib ite d b y ampic illin .W hereas only cyclophospham ide and aflatoxin B, (used as ac on tro l) s howed a n a bs olu te re qu iremen t fo r me ta bo lic a ctiv atio nby rat liver S9 fraction, rubiflavin, and those com pounds withp ro te in c om po ne nts , n eo ca rz in os ta tin , m acromomy cin , a nd la r-gomycin were inactivated by the S9 fraction, the la ttermostcompound to the extent of more than 90% [loss of both 1.0-and 0.1-//g spots (Fig. 1a); therefore, less than 10% of the 1.0-n g sp ot is rema in in g]. O n th e o th er h an d, d au no ru bic in , a c la ss icinducer in the absence of m etabolic activation , w as d ete cted atlower concentra tions in the presence of the S9 fraction, ina gre em en t w ith p re vio us re su lts (1 ).BIA as a Prescreen. We have used the BIA spot test as ap re screen (F ig. 2) fo r the de tection of p ote ntial antitum or a ntib iotics in a total of 10 ,72 4 m icrobial ferm enta tio n b ro ths (from2,68 1 cultures) (1 3). A pproxim ate ly 1% of the cultures te stedgave a reproducib le positive result in the BIA. In no case wasinduction in the BIA dependent on S9 m icrosom al activation;how ever, inactivation of inducing activity by S9 w as n ote d w ithone culture. This culture (SK F 42) produces a m acrom oleculara ntib io tic th at is p ro ba bly g ly co pro te in in n atu re a nd a pp ea rs tob e d iffe re nt from o th er re la te d a ntib io tic s, s uc h a s la rg om ycin ,neoca rz in os ta ti n, a nd macr omomyc in .B IA -p os itiv e c ultu re s w ere fe rmente d a nd s ubm itte d fo r te stin gin m ice against the P388 leukem ia. Those cultures which gavein vivo activity (life sp an of trea ted /control, 3 =1 30) w ere refer-m ented and retested against P388. Ten of the 37 cultures haveconfirmed in vivo activity, while testing is incomplete on 7.Tw enty w ere nega tive. C ultures w ith confirm ed in vivo activityh av e b ee n te nta tiv ely id en tifie d th us fa r a s p ro du cin g stre pto ni-g rin , a nth ra cy clin es , n eo ca rz in os ta tin , a nd s tre pto ru bin , a s w ella s a t le as t o ne n ov el c om po un d, 2 06 4A (4 3). S tru ctu ra l a na ly sis

2826 CANCER RESEARCH VOL. 43


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B IA f or Ant it umo r Agentsand further testing of the novel com pounds is currently beingundertaken.Id en tific atio n a nd P urific atio n o f In du cin g A ctiv itie s. F ermen ta tio n b ro th s th at w ere b io lo gic ally a ctiv e w ere fu rth er a nalyzed to determ ine whether they contained novel or knownactivities (a p ro ce dure kn ow n as "dereplication"). A sm all numb er o f c om po un ds e xh ib ite d sp ot c ha ra cte ris tic s th at a re e ss entially diag nostic (Fig. 1). F or e xa mple, la rg om ycin Fll w as comp le te ly in ac tiv ated by mammali an enzymes (S9 ) a t concentr at io n sbelow 100 /ng/m l. S treptovaricins C and D and rifam ycins prod uc ed a c ha ra cte ristic to xic c en te r s urro un de d b y a h alo o f w ea kinduction over a w id e co nce ntration ran ge (solid spots of induction were never seen). Inducers that were very toxic (e.g .,rubiflavin) or very slow to diffuse (large m olecules such as theg ly co pro te in la rg om yc in ) s ho we d v ery n arro w rin gs o f in du ctio nw ith to xic c en te rs a t h ig h c on ce ntra tio ns .F erm en ta tio n b ro th s a nd e xtra cts from in du cin g c ultu re s w erec hroma to gra ph ed o n th in -la ye r c hroma to gra ph y p la te s. Z on esof activity on the plates w ere determ ined by bioautography (20)as described in "M ate rials and M ethods" and C ha rt 1. R ( value sc an b e u se d to h elp c la ss ify a nd id en tify u nk no wn a ctivitie s (2 3).B io au to gra ph y o f a d au no ru bic in fe rm en ta tio n b ro th a nd s ev era le xtra cts is illu stra te d in F ig . 3 .T he re la tiv e su cc es s o f a ntib io tic p urific atio n p ro ce du re s w asdeterm ined by direct spotting of extracts onto prepared BIAplates. Extracts of ferm entation broths in water, m ethanol,ethanol, butyl alcohol, isobutyl alcohol, or acetone, but notchloroform , could be spotted without adverse effects on theassay. D im ethyl sulfoxide , acetonitrile, hexane , and other solv en ts m ay a ls o b e s po tte d d ire ctly o n th e p la te (d ata n ot sh ow n).Quanti fi ca tion of Induc ing Activi ti es . Induc ing activi ti es wereq ua ntifie d u sin g e ith er th e B IA sp ot te st o r th e liq uid in cu ba tio nassay. In the spot test, the m inim al inducing dose of a ferm entation broth or standard solution was determ ined in sam plesdilute d to the en d point (T able 2; F ig. 1b ). Th e liquid incubationa ss ay a llo ws th e d ire ct m ea su reme nt o f in du ce d e nz ym e (C ha rt2). If a standard ca libration curve is first derived (C hart 2 a), th eam ount of inducer present in a ferm entation broth can be estim ated . A kinetic curve of induction, such as th at sho wn in C hart2b, can be used to find the optimal induction time for thesensitivity desired in any given assay. An expression tim e of 2hr or less is su fficient fo r strong indu ce rs, w hile lon ger tim es o fup to 5 or 6 hr w ill a llow the detection of weak inducers (e.g.,p ip era zin ed io ne ), to xic c ompou nd s (e .g ., BCNU), c hem ic als th atre ac t s lo wly (e .g ., p la tin um c omple xe s), a nd in du ce rs p re se nt a tlo w c on ce ntra tio n (T ab le 1 ; C ha rt 2 b).Sele ctio n o f In div id ua l Is ola te s w ith In du cin g A ctiv ity . T hep ro du ctio n o f in du cin g a ctiv ity b y in div id ua l c olo nie s g ro win g o nsolid m edia can be d eterm ine d b y transferrin g aga r plugs ontostandard BIA spot test plates. Th is procedure can be useful ins ev era l c irc umsta nc es : s ele ctio n o f a ctiv e c lo ne s from m ix tu re sof produ cing and no nproducin g natu ral varian ts of an inducingc ultu re (s ee F ig . 4 ); s ele ctio n o f h ig he r-y ie ld in g mu ta nts in s tra inim provem ent program s (22); and selection of active culturesd urin g th e in itia l s cre en in g o f s oil is ola te s (p re lim in ary d ata in o urla bo ra to rie s in dic ate a n e nc ou ra gin g c orre la tio n b etw ee n p rod uc tio n o f in du cin g a ctiv ity o n so lid m ed ia a nd in liq uid c ultu re ).DISCUSSION

A b io ch em ica l p ro ph ag e in du ctio n a ss ay (B IA ) h as b ee n u se d

to scre en, analyze, a nd m onitor production of natural productsw ith p ote ntia l a ntitu mo r a ctiv ity . T he in du ctio n o f X -p ro ph ag e inE . coli is one of a group of coordinate ly regulated phenom enath at h av e b ee n c olle ctiv ely te rm ed SOS fu nctio ns (3 6,4 6). T he sefunctions are presum ed to be induced in response to a threat toth e s urv iv al o f th e c ell (a nd b ac te rio ph ag e) c au se d b y a n in hib itio nof D NA replication. This can be the re su lt of a varie ty of events,includ ing direct dam age to DNA (caused by interaction withc hem ic als o r ra dia tio n), a nd in dire ct e ffe cts o n DNA me ta bo lism,e.g., thym ine starvation, and in hibition of D NA gyrase (15 , 29,40, 41, 46). As discussed under "Results," the majority ofcompounds reported as inducers in Table 2 are known, orc on sid ere d, to in te ra ct d ire ctly w ith D NA . O th er in du ce rs d o n otin te ra ct w ith D NA b ut a re kn ow n to b e sp ec ific e nz ym e in hib ito rsw hich ultim ate ly affect D NA synthesis (4, 7, 12 ). Induction by afew com pounds w as unexpe cted, becau se their m ode of actionis no t co nsidered to involve D NA .In general, our resu lts agree with those using classical prophag e induction assays (1 ,10,14,18,19,26, 32, 3 5), w ith m inore xc ep tio ns n ote d p re vio us ly . H ow ev er, th e n ov el a ntib io tic fo un din B ro th 2 06 4, g ilv oc arc in V (4 4), w as n ot d ete cte d a s a p ro ph ag einducer in another laboratory (3), for reasons m ost probablyrelated to sensi ti vi ty .T ho se in du ce rs w ith n o d em on stra te d a ntitu mo r a ctiv ity (rifam yc in s, amp ic illin , fo sfomy cin , n alid ix ic a cid , n ov ob io cin , a ndtrim e th op rim ) a re a ll k nown to b e s pe cific in hib ito rs o f p ro ka ry otice nzym es and a re essentially inactive in eukaryotic system s (7,12, 15, 2 5, 30, 38, 41). H ow ever, eukaryotic cou nte rp arts e xist(e .g ., me th otre xa te fo r trim e th op rim a s a n in hib ito r o f d ih yd ro fo l-a te reduc ase) (7, 12). T hese com pound s fa ll into the cla ss ofin direct inducers, rathe r th an those that in teract directly w ithDNA.The strain constructed for this assay, BR513, contains 2m utations allow ing the detection of lower concentrations ofin du ce rs , e nv A, e nh an cin g p erm ea bility , a nd u vrB , re du cin g th ea bility o f the strain to repair certa in typ es of dam age to D NA (9).These m utations result in the detection of som e com pounds atc on ce ntra tio ns u p to 1 00 tim es le ss th an th os e d ete cte d b y w ild -type bacteria (32, 33). We have found that the effect of thep erm eability m utation varie s w idely w ith differen t chem icals,h ow ever. For exam ple, b leom ycin is detected at 10-fold lo werd os es w ith th e m uta nt, w hile p la tin um c omple xe s a nd n itro so u-reas show no difference between the wild-type and the m utants tra in (d ata n ot s hown ).The qu estio n of sen sitivity is im portant because of th e ra therlow concentrations (on the order of 25 i*g/m \ or less) at whichs ec on da ry me ta bo lite s n orma lly o cc ur in m ic ro bia l fe rme nta tio nb ro th s. N atu ra l p ro du cts w ith a ntitu mo r a ctiv ity fo un d in fe rm entation broths that m igh t be m issed b y the B IA due to se nsitivitylimi ta tio n s a re azase ri ne , p ip er az ined ione , cha rt re us in , and i llu di nS. Adriam ycin and daunorubicin are not detected at 25 pg/m \,bu t related m etabolite s that are usually coprod uced, e.g., ba u-m yc in (3 1 ), a re d ete cte d b elo w th is c on ce ntra tio n (T ab le 2 ).The bacterial strain constructed for this assay contains am utation preven tin g the expressio n of fun ctions controlled bythe rightw ard prom otor of A . A s a conseq uence, the expressionof the lacZ gene and production of 0-galactosidase are nota ba te d b y tra nsc rip tio n c on tro ls o r p ha ge -in du ce d c ell ly sis (9 ).The length of the induction period (Chart 2b) may be used tovary the sensitivity of the induction assay. A 3-hr period isg en era lly o ptim um fo r th e d ete ctio n o f m ost in du ce rs ; h ow ev er,

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R . K . E lespuru and R . J. W hitesh orte r or lon ger tim es m ay b e use d for m onitoring strong orw ea k in du ce rs , re sp ec tiv ely . F or e xamp le , la rg om yc in FM, astro ng indu cer, co uld b e de tecte d at 10 i/g/m l in 1 .5 h r an d a t0 .05 g/m ln 6 h r (C ha rt 2 0). B ec au se o f th e s ho rt a ssa y tim e,the product ion o f largomyc in by fermentat ion cou ld be mon itoredu sin g th e B IA .A co lo r imetr ie assay o f p rophage induct ion has certain inheren tadvan tages ove r a p laque assay , e .g ., the p resence o f a un ifo rmbac kg ro und aga in st wh ic h low le ve ls o f in du ctio n may be seenin a v ery sma ll a re a, a s in a s po t te st o r o n a b io au to gra ph (Chart1 ). A lth ough sev era l o th er b io chem ic al a ss ay s o f p ro phage indu ctio n exist (21 , 28 , 3 9), n one m ay b e u tilize d in a sp ot test,the form of assay that we have found to be most useful fors cr eening , b ioautog raphy , and s tr ain selec tion . An unexpectedad va nta ge of the B IA sp ot te st is its re la tive immu nity to thed ele te rio us e ffe cts o f s olve nts. W e su rm ise th at th e h ig h co nc en tra tio n o f b ac te ria u sed in th e b io chem ic al a ss ay is re spons ib le for this p ro tec tive e ffec t.A t most, o nly 1 7 o f 3 7 B IA -p os itiv e fe rmenta tio n b ro th s wereac tive aga ins t P388 leukemiawhen tes ted in v ivo , perhapsowingto the l im its o f sensi tiv ity o f t he an ima lassay . Ac tive compoundsin fe rmenta ti on b ro ths a re fr equen tl y p resen t a t concen tr ationso f 0 .1% o r le ss o f th e to ta l s olid s. T he amount o f s olid s th at c anbe admin is te red (approx imate ly 400 mg/kg) l im i ts the sensi ti vi tyo f in v iv o te stin g (4 5); we a re a ttemptin g to purify (a nd c on cent ra te ) induc ing ac tivi ties prior t o repea t in v ivo tes ting .T he observ atio n th at o nly o ne o f th e natu ra l p ro du cts te ste d(a fla to xin B ,) s howe d a n a bs olu te re qu ireme nt fo r m eta bo licactivation is not surpr is ing, s ince ant itumor ant ib iotic d iscoveriesin th e p as t w ere m ad e w ith ou t th e u se o f a ctiv atin g e nz ym es.However , ou r scr een o f 10 ,000 fe rmen ta tion b ro ths d id not y ie ldany posi tiv e samp les r equ iri ng acti va ti on . Th is r esu lt i nd icat esth at in du ce rs such as a fla toxin are n ot comm on ly pre se nt infermentations.O ur e xp erie nc e w ith th e B IA h as m ad e u s awa re o f its lim itatio ns , th e ma jo r o ne bein g s en sitiv ity , d es pite th e p re senc e o fmuta tions wh ich a ffec t sensi ti vi ty to some ex ten t. Cer ta in c lasseso f comp ou nd s e xp ec te d to in te ra ct w ith DNA , su ch a s a nth ra -cenediones, a re no t de tect ed . In addi tion , cond iti ons tha t favorthe detection of one chem ical are often far from optim al ford ete ctio n o f o th er in du ce rs (F ig . 1 0). F or th is re as on , c omp rom ises m ust be m ade in pro tocols for op tim um de tection of av arie ty o f in du ce rs in a p re scre en . N ev erth ele ss , a s d emonstrate d here , the B IA fun ctio ns a s an effective a nd practicalm ea ns fo r d ete ctin g p ote ntia l a ntitumo r a ge nts o f a s pe cificmode of ac tion . Add it iona l genet ic man ipu la tions in the bacter ia ls tra in , in p ro gre ss , s hould in cre as e s en sitiv ity a nd fa cility o finduc tion fol lowing a variet y o f DNA-chemica l interac tions .ACKNOWLEDGMENTS

W e thank Robin Pennington and Ivan Lufriu for perform ing the tests. Dr.R aym on d R ud do n fo r valua ble sug ges tion s, a nd m an y colle ag ues in th e C he mot he ra py F erme nt at io n P ro gr am f or p ra ct ic al a dv ic e.REFERENCES1. A nderson. W . A ., M oreau, P . L, and D evoret. R . Induction of prophage \ byd au no ru bic in a nd d er iv ati ve s; c or re la tio n w it h a nt in eo pla st ic a ct iv it y. M u t t.Res. . 7 7 : 1 97- 208 ,1980 .2. A sz alo s. A ., a nd B rd y,J. List of th e k no wn n atu ra l a ntitu mo r co mp oun ds,a cco rd in g to che mica l typ es. In : A . A sza lo s (e d.), A ntitu mor C om po un ds o fN atu ra l O rig in : C hemis try a nd B io ch em is try , V ol. 1 . p p 1 -7 8. B oc a R ato n, F la .:

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19. H einem ann, B ., and H ow ard. A . J. Interaction of lam bda bacteriophageE sch er ch ia coli a s a scre en in g test fo r p ote ntia l a ntitu mor a gen ts. AM ic ro bio l. . 7 2. 2 34 -2 39 . 1 96 4.2 0. H ein em an n, B ., H ow ar d, A . J ., A ln a, J .. a nd H ollis te r, Z . J . A pp lic atio n o f pc hr om ato gra ms to th e s tu dy o f n du ce rs of X b ac te rio ph ag e in E sc he ricc oli. A pp i. M ic ro bio l., 7 5: 7 23 -7 25 , 1 96 7.21. H o. Y . L., and H o, S . K . T he induction of a m utant prophage \ in E schericc oli: a r ap id s cre en in g te st fo r c ar cin og en s. V ir olo gy , 9 9: 2 57 -2 64 , 1 97 9.2 2. Ic hik aw a, T ., D ate , M ., Is hik ura , T ., a nd O za ki, A . Im pro ve me nt o f k as ug ac in -p ro du cin g s tr ain b y th e a ga r p ie ce m eth od a nd th e p ro to ty pe m eth od . FM ic ro bio l. . 7 6: 2 18 -2 24 , 1 97 1.23. Issaq, H. J., Barr, E . W ., W ei, T., Meyers, C. I., and Aszalos, A. Thin lCh roma tog raphi e c la s si fi ca ti on o f an ti bi ot ic s e x hi bi ti ng an ti tumo r p roper ti esC hr om ato gr., 7 33 : 2 91 -3 01 , 1 97 7.24 . Jo hn so n. R . K ., Z ee-C hen g, R . K .-Y ., L ee, W . W ., A cton , E . M ., H enry. D .a nd C he ng . C . C . E xp erim en ta l a ntitu mo r a ctiv ity o f a min oa nth ra qu in onCa nc er T re at . R e p. , 6 3: 4 25 -4 39 ,1 97 9.25 Kahan, F. J., Kahan, J. S., Cassidy, P. J., and Kropp, J. The mechanisma ctio n o f fo sfo my sin (p ho sp ho nomy cin ). A nn . N Y . A ca d. S ci. 2 35 : 3 64 -31974.2 6. L ein , J ., H ein em an n, B ., a nd G ou re vitc h, A . In du ctio n o f ly so ge nic b ac te riaa m eth od o f d ete ctin g p ote ntia l a ntitu mo r a ge nts . N atu re ( Lo nd .) , 7 96 : 77 86 , 1 96 2.27 . Lenge ll er , J . Anal ys is o f t he phys io lo g ic a l e ff ec ts o f t he an ti bi ot ic S t re p to zo toon E sch eric hia co li K 12 a nd o the r sen sitiv e ba cte ria . A rch . M icro bio l.,1 96 -2 03 , 1 98 0.2 8. L ev in e, A ., M or ea u, P . L . , S ed gw ic k, S . G ., A dh ya . S ., G otte sm an , M ., D ev oR ., and Das, A. Expression of a bacteria l gene turned on by a poc ar cin og en . M u t t. R es ., 5 0: 2 9- 35 , 1 97 8.29. Little , J. W ., and H anaw alt, P. C . Induction of prote in X in E scherichiaM ol. G en . G en et., 7 50 : 2 37 -2 48 , 1 97 7.3 0. M cC lu re , W . R ., a nd C ec h, C . L. O n th e m echa nism of rifam picin in hib itioRNA s yn th es is . J . B io l. C hem., 2 53 : 8 94 9-8 95 6. 1 97 8.

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31. M cG uire, J. C ., T hom as, M . C ., Stroshane, R . M ., and H am ilton, B. K., andWh it e, R . J . B io sy nt he sis o f d au no ru bic in g ly co sid es : t he r ol e o f e -r ho dom yc i-n on e. A ntim ic ro bia l A ge nts C hemo th er., 1 3: 4 54 -4 64 , 1 98 0.3 2. M ore au , P ., B ailo ne , A ., an d D evo re t, R . P ro pha ge \ ind uctio n in E sch erichiaco li K 12 en vA u vrB : a h ig hly se nsitive te st fo r p oten tia l ca rcino ge ns. P ro c.Na ti. A ca d. S ei . U . S . A ., 7 3: 3 70 0- 37 04 ,1 97 6.33 . N orm ark, S ., B om an, H . G ., a nd M atsso n, E . M uta nts of E sch erich ia co li w itha nomalo us c ell d iv is io n a nd a bilit y t o d ec re as e e pi somally a nd c hr omo somallym ed ia te d re sis ta nc e to a mp ic illin a nd s ev er al o th er a ntib io tic s. J . B ac te rio l.,97 : 1334-1342 ,1969.3 4. P ec k, R . M ., a nd O 'C on nell, A . P . M ixed b ifu nctio na lity. 4 . A ntitum or a ctivityo f a lk yla tin g d eriv ativ es o f p oly cy clic a ro ma tic h yd ro ca rb on s a s a fu nc tio n o fstru cture a nd o f ve hicle . J. M ed . C hem ., 8 : 6 8-7 0, 1 97 2.35. P rice, K . E ., B uck, R . E ., and Lein, J. S ystem for detecting inducers of lyso-g en ic E sc he ric hia c oli W 17 09 (A ) a n d its a pp lic ab ility a s a s cr ee n fo r a ntin eo -p la st ic an ti bi ot ic s. Appi . M i cr ob io l. , 1 2 : 4 28- 435 ,1964 .3 6. R ad ma n, M . P he no men olo gy o f an in duc ible m uta gen ic D MA re pair p athw ayin E sch erich ia co li: S OS rep air h yp oth esis. In : L . P ra ka sh , F . S he rm an , M .M ille r, C . L aw re nce, a nd H . W . T abo r (e ds.), M ole cular an d E nviron me ntalA sp ects o f M utag ene sis , pp . 12 8-1 42 . S pring fie ld , III.: C ha rle s C T ho ma s,Publ is her , 1 974 .37. Sartorelli, A. C ., and Johns, D. G . Inhibition of the synthesis of thym inen uc le otid es b y a za se rin e. M ol. P ha rm ac ol., 3 : 7 1-8 0, 1 96 7.3 8. S eth i, V . S . A ns am yc in s. In : A . A sz alo s (e d.) . A ntitu mo r C om po un ds o f N atu ra l

B IA f or Ant it umo r AgentsO rig in : C hemis tr y a nd B io ch em is try , V ol. 1 , p p. 5 9- 85 . B oc a R ato n, F la .: C RCP re ss , I nc ., 1 98 1.39 . S mith , C . L ., a nd O ish i, M . T he m ole cular m ec ha nism o f virus in du ction , I. Ap ro ce du re fo r th e b io ch em ic al a ss ay o f p ro ph ag e in du ctio n. M ol. G en . G en et.,748:131-138,1976.40. S mith, C . L., and O ishi, M . E arly events and m echanism s in the induction ofb ac te ria l SOS f un cti on s: a na ly si s o f t he p ha ge r ep re ss or in ac ti va ti on p ro ce ssin vivo. P roc. N ati. A ca d. S ei. U . S . A ., 75 : 1 657 -1 661 , 1 97 8.41 . S ug in o, A ., H ig gins, N . P ., B ro wn , P . 0 ., P ee bles, C . L., a nd C ozza re lli, N . R .E nergy cou plin g in D NA g yra se an d the m ech anism of a ctio n of n ov ob io cin .P ro c. N ati. A ca d. S ei. U . S . A ., 7 5: 4 838 -4 84 2, 1 97 8.4 2. T ro tm an , R .E . T ec hn olo gic al A id s to M ic ro bio lo gy , p p. 1 7-2 1. L on do n: E dw ardA rn old , L td ., 1 97 8.43 . V og el, M . J., an d B onn er, D . M . A ce ty lorn ithina se of E sche rich ia coli: p artia lp urific atio n a nd s om e p ro pe rtie s. J . B io l. C hem., 2 78 : 9 7-1 06 , 1 95 6.44. W ei, T. T., C han, J. A., Roller, P . P., W eiss, U., S troshane, R. M ., W hite,R . J., and Byrne, K. M. Detection of gilvocarcin antitumor com plex by ab io ch em ic al i nd uc tio n a ss ay ( BIA ). J . A nt ib io t. ( To ky o) , 3 5: 5 29 -5 32 , 1 98 2.4 5. W hite , R . J. In vitro presc re en s. In: R . J. W hite an d I. J. F id le r (e ds.), D esigno f M od els fo r T estin g C ance r T he ra pe utic A gen ts, p p. 1-1 1. N ew Y ork: V anNo st ra nd , 1 98 0.4 6. W itk in , E . M . U ltr av io le t m uta ge ne sis a nd in du cib le DNA re pa ir in E sc he ric hiac oli. B ac te rio l. R ev ., 4 0: 8 63 -9 07 , 1 97 6.

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o.O* | Origin Lane 1234S6 123456*

F ig . 2 . C oto rim etr ic s po t te st fo r in du cin g a ctiv ity nm ic ro bia l fe rm en ta tio nb ro th s. F er me nta tio n b ro th s o f a ctin om yc ete s w er e s po tte d o n 2 43 -mm b io as sa yp la te s c on ta in in g b ac te ria a nd amp ic illin . In cu ba tio n p er io d, 3 h r. D ark rin gs a nds po ts a re in du ctio n p os itiv e; w hite a re as a re z on es o f to xic ity . T his s et o f s ele cte db ro th s c on ta in s ma ny i nd uc tio n p os it iv es .

F ig . 3 . B ioa utog ra phy o f e xtra cts o f a d au norub icin ferm enta tio n b roth ch ro -m atographed in 2 solvent system s. Lane 7, w hole broth; Lane 2, butyl alcohol-e xtra cte d a qu eo us ; la ne 3 , a cid h yd ro ly sis p ro du ct p ur ifie d ( da un oru bic in HC I);La ne 4 . b utyl alco ho l e xtra ct a fte r to lu en e:eth yl a ce tate p artition ; L ane 5, b utyla lc oh ol e xtra ct o f w ho le b ro th ; L an e 6 . to lu en e:e th yl a ce ta te e xtr ac t o f a cid ifie dw ho le b ro th . A mou nts sp otte d w ere a djuste d to avo id u nde r- or o ve rlo ad ing an dd o n ot refle ct relative in du cin g a ctivity p er sa mp le . S olve nt s ystem s: left h alf,ch lo ro fo rm:methano l:ace t ic ac id (80 :20 :2) ; r igh t ha lf , hep tane :ch lo ro fo rm:methano l(5:5:1).

F ig . 4 . In du cin g a ctiv ity p ro du ce d b y in div id ua l c olo nie s. A s po re s us pe ns io n o fF CRC -3 24 , a n u ns pe cia te d s tre pto my ce te th at p ro du ce s n eo ca rz in os ta tin , w asp la te d o nto a c om ple x n utrie nt a ga r; w he n c olo nie s w ere ju st v is ib le ( ab ou t 3 d ay sat 28 ), they w ere picked off on separate agar plugs. A fter 6 days of furtherinc ub ation , th e p lu gs w ere tra ns fe rre d to a p rep are d 2 43 -m m B IA p la te . A fte r 3 h ra t 3 8,t he p lu gs we re r emo ve d, a nd t he p la te s we re d ev elo pe d w it h a c hr omo ge ni cs ub st ra te . C le ar a re as , t ox ic w ith n o in du cti on ; s po ts , p os iti ve f or in du cti on ; r in gs ,toxic center w ith diffusion zone positive for induction. The relative am ount ofind ucin g su bstan ce is lo w. m od era te, an d h ig h fo r c lea r zo ne s, spo ts, an d ring s,respectively.

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Anti Tumor

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