[CANCER RESEARCH 43, 2819-2830,

June 1983]

Biochemical Prophage Induction Assay: A Rapid Test for Antitumor Agents That Interact with DMA1
R. K. Elespuru2 and R. J. White3
Biological Carcinogenesis Program [R. K. E.] and Chemotherapy Fermentation Program [R. J. W.], NCI-Frederick Cancer Research Facility, Frederick, Maryland 21701

ABSTRACT A biochemical (colorimetrie) assay of bacteriophage A induction was utilized in the detection, identification, and purification of DMA-interacting natural products with potential antitumor activ ity. A set of 142 standard antibiotics, composed principally of natural products with established antitumor activity and/or de fined mechanisms of action, was tested in the assay. As ex pected, most inducers were direct inhibitors of DNA synthesis. A few other types of inducer, with probable indirect effects on DNA synthesis, were found after prolonged incubation: one class of RNA synthesis inhibitor, a dihydrofolate reductase inhibitor; and two inhibitors of bacterial cell wall synthesis. The biochemical induction assay was semiautomated for use as a prescreen in the search for novel antitumor agents in 10,724 actinomycete fermentation broths. Approximately 1% of the cultures produced compounds that were active in the assay; some appear to be novel. None required metabolic activation (via rat liver 89) for inducing activity. The biochemical induction assay was adapted for bioautography (the detection of inducing chemicals chromatographed on thin-layer plates) and for strain improvement programs (selection of isolates with enhanced inducing activity). The speed of the assay (2 to 5 hr) made it useful for monitoring antitumor agent production and purification. The sensitivity of the assay could be varied, depending on the length of the incubation period. Mi crobes, nutrients, and toxic solvents did not usually interfere with the detection of inducing activity. INTRODUCTION The actinomycetes and fungi have proved to be a most fruitful source of biologically active metabolites with therapeutic utility and have been heavily investigated in screening programs for the last 40 years. Because animal models have proved to be rather insensitive and expensive, it has been necessary to de velop in vitro test systems for anticancer agents that are predic tive of clinical activity (44). Such tests function as a prescreen, with the object of selecting a smaller number of samples for test in vivo against a transplantable rodent tumor, such as the murine P388 leukemia. Cell culture cytotoxicity has been one of the most commonly used types of prescreen and is capable of detecting most, if not all, of the compounds with established clinical utility. However, because cytotoxicity prescreens are nonspecific, i.e., capable of detecting compounds with a wide variety of mechanisms of action, they can generate an overly
1This work was supported by Contract N01-CO-75380, National Cancer Insti tute, NIH, Betnesda, Met. 20205. ' To whom requests for reprints should be addressed, at Fermentation Program, Bldg. 434, Frederick, Md. 21701. 3 Present address: Lederle Laboratories, Pearl River, N. Y. 10965. Received September 8,1982; accepted March 8,1983.

large number of positive samples for in vivo screening. Other more selective in vitro tests have been devised that have shown promise. One example is the bacterial assay for antimetabolites pioneered by Hanka ef al. (17). Another is the lysogenic induction assay developed during the last 2 decades by Endo et al. (10), Fleck (11), Geissler (14), Heinemann ef al. (19, 26), and Devoret (32). This assay detects compounds that interact with DNA or interfere with DNA synthesis, and a good correlation between inducing and anticancer activity has been reported (1,18, 35). In its classical form, the lysogenic induction assay measures the number of bacteriophages produced subsequent to incuba tion with the test sample. Recently, a strain of Escherichia coli lysogenic for a X-lacZ fusion phage was constructed for use in a BIA4 (9). Induction of the prophage is measured by the ap pearance of /i-galactosidase, product of the lacZ gene, in a colorimetrie assay. This assay was designed to be of greater utility than are classical prophage induction assays by being faster, easier, more sensitive, and adaptable for a variety of purposes. In this paper, we describe the use of the BIA as a test system for the detection of a standard set of antitumor agents and model compounds; as a prescreen for inducers in microbial fermentation broths; and as an aid in the analysis, purification, and production of natural products with potential antitumor ac tivity.
MATERIALS AND METHODS

Antitumor Agents Bleomycin was the gift of Dr. William Bradner, Bristol Laboratories; platinum-containing compounds were obtained from Dr. Ronald O. Rahn, Oak Ridge National Laboratories; and ICR compounds were from Dr. Richard Peck, Institute for Cancer Research. Other antitumor agents were supplied by Dr. John Douros, National Cancer Institute. Bacteria E. coli strain BR513 is (A p/acZ cl*PRf 7) pro-lac A uvrB A envA azi thi rpsL gal (9). Reagents Bacteriological media are from Dlfco, Detroit, Mich.; inorganic and organic salts are from Fisher Scientific, Silver Spring, Md.; sodium ampicillin (Polycillin-N) is from Bristol Laboratories, Syracuse, N. Y.; onitrophenyl-|8-D-galactopyranoside, BNG, Fast Blue RR salt, NADP monosodium salt, glucose 6-phosphate, chloramphenicol, /3-mercaptoethanol, and Tris (as Trizma Base) are from Sigma Chemical Co., St. Louis, Mo. Media and Buffers LBE. The medium contains (per liter) 10 g Bacto-tryptone, 5 g yeast extract, 10 g sodium chloride, and 5 ml 1 M Tris. After autoclaving,

4The abbreviations used are: BIA, biochemical induction assay; BNG, 6-bromo2-naphthyl-0-D-galactopyranoskJe; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea.

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1983

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R. K. Elespuru and R. J. White

medium is supplemented with 4 ml of 50x Medium E (43) and 10 ml of 20% glucose. For LBEamp agar, 15 g agar are added before autoclaving, and 1 ml of a freshly prepared solution of sodium ampicillin (10 mg/ml) is added just before pouring plates. Plates should be poured on a level surface. ZCM Buffer. The buffer contains (per liter) 16.1 g Na2HPO4-7H2O (or 8.5 g Na2HPO4 anhydrous); 5.5 g NaH2PO4-H2O, 0.75 g KCI, 0.246 g MgSO4-7H2O (or 0.12 g MgSO4 anhydrous), 2.7 ml /i-mercaptoethanol, and 25 mg chloramphenicol. Adjust to pH 7.O. Do not autoclave. Store cold. Medium A. This medium contains (per liter) 10.5 g K2HPO4, 4.5 g KH2PO4, 1 g (NH4)2SO4, and 0.5 g sodium citrate-2H2O. Autoclave. Activating Enzymes Rat liver (strain unspecified) 9000 x g postmitochondrial supernatant was from Litton Bionetics, Kensington, Md. Activation mix, freshly pre pared, was 0.5 ml rat liver S9, 0.4 ml cofactor solution (25-mg/ml portions each of NADP, glucose 6-phosphate, MgCI2), and 2.1 ml 0.1 M phosphate buffer, pH 7.4. 0.4. Bacteria were distributed in 25-ml aliquots into centrifuge tubes, pelleted (4000 to 5000 rpm, 3 min), and gently resuspended in 1 ml LBE medium. In some cases, 3 ml rat liver S9 activation mix were added to the tube. Twenty-five ml melted soft (1%) agar at 45were added to the tubes, and the contents were poured onto warm (37)bioassay plates (243 x 243 mm) containing approximately 330 ml (solid) LBEamp agar. After the top layer solidified, chemical solutions and fermentation broths were spotted directly onto the plates using Pasteur or capillary pipets or a multisample applicator (see below). Plates were kept on a slide warmer set at 38during the time required for spotting. The plates were then incubated at 38 for 3 hr, after which another agar layer containing substrate was added. For each plate, 60 mg Fast Blue RR salt and 10 mg BNG were combined with 1.0 ml dimethyl sulfoxide just prior to use; a Pasteur pipet was used to aid solution of the mixture. Twenty-five ml melted soft agar at 45were added to the substrate mixture and then poured onto the plate. Color development was complete in 10 to 15 min. Proportions of materials for standard small (100-mm) Petri dishes are one-tenth of the amounts described. Large-Scale Screening (Fig. 2). The fermentation broths screened

for inducing activity were prepared from isolates of a variety of different genera of the Actinomycetales which were supplied as part of a coop Laboratory Supplies erative venture by Dr. C. Nash of the Smith, Kline and French Labora tories. Each organism was grown in at least 4 different fermentation Polystyrene plastic tubes (16 x 125 or 17 x 100 mm) are No. 2025 or media. Samples from fermentation broths were spotted directly onto BIA No. 2057 tubes, respectively, from Falcon; large (243- x 243- x 18-mm) assay plates by hand or with a specially constructed multisample appli bioassay plates are from A/S Nunc, Kamstrupvej 90, Kamstrup, DKcator that could simultaneously load up to 144 samples on glass rods. 4000 Roskilde, Denmark, distributed in the United States by Vangard International, Inc., Neptune, N. J.; gridded "miniplates" (100 mm square) [This apparatus is similar in principal to a multipoint inoculator (42)]. The rods were dipped into vials containing fermentation broths and lowered are No. 1012 from Falcon (Fisher Scientific). to the agar surface of a 243-mm bioassay plate for spotting. The assay was conducted with and without rat liver S9 activation mix. All cultures BIA Spot Test (Chart 1; Fig. 1) that caused induction in at least one fermentation medium were referA fresh overnight culture of BR513 was diluted ~ 100-fold (to ASM mented and tested again. 0.05) into LBE medium and grown at 37for approximately 3 hr to Aoo Bioautography (Fig. 3). Purified chemical standards, fermentation

Log phase BR513 1) Centrifuge 2) Resuspend in soft agar, pour into petri dish

Chart 1. BIA spot test applications.

Bacterial colonies on agar plugs 3) 3 hr, 37

Thin layer chromatogram

(TLC)

'.. O. . P., O. ./">.. J v./) ///// //!////////) Multi-point sample applicator 3) 3 hr, 37 4) substrate overlay

3) 3 hr, 37
4) remove TLC 5) substrate overlay

4) remove plugs 5) substrate overlay

Variants
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Bioautography

Screening
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BIA for Antitumor Agents broths, or extracts were chromatographed on silica gel thin-layer plates (Baker Flex 1B-F). A piece of wet filter paper was placed on the surface of a 243-mm bioassay plate containing LBEamp agar and smoothed to remove wrinkles. The thin-layer plates were then placed face down on the filter paper (slowly, to avoid the entrainment of air bubbles) and incubated for 2 to 3 hr at 38. After incubation, the thin-layer plates and filter paper were removed from the agar surface. Bacteria grown as described above were poured in soft LBEamp agar onto the warmed bioassay plate. The plate was further incubated at 38 for 3 hr to allow induction and developed by addition of substrate overlay. This method allows the time of diffusion of chemical from thin-layer plate into agar to be controlled. Severalother variationson this technique are possible. In one variation, the thin-layer plate may be placed directly on top of a bacterial lawn, as shown in Chart 1. The plate is removed prior to addition of substrate overlay. In another variation, the bacterial lawn is poured directly on top of a thin-layer plate placed face up. Substrate overlay is added on top. The latter method allows good aeration of the bacteria, but the thin-layer plate cannot be recovered. IndividualColony Variants (Fig. 4). For the identification of natural variants of producer organisms, agar plugs (about 8 mm in diameter) of individual bacterial colonies were cut with a cork borer and placed on the plates prepared as for the spot test. Plugs were removed before the addition of the Fast Blue RR salt-BNG substrate overlay.
Liquid Incubation BIA (Quantitative) (Chart 2)

Dose-ResponseAssay. Bacteriagrown as for the BIA spot test to Aeoo0.4 were diluted 10-fold into LBEamp and distributed in 0.5-ml aliquots into plastic tubes containing 50 n\ of the desired concentration of largomycin Fll (aqueous solutions). Tubes were incubated for 3 hr at 38 with shaking and then chilled with the addition of 4.5 ml cold ZCM buffer. After the mixture was warmed to 28,the enzyme assay was initiated by the addition of 1.0 ml o-nitrophenyl-ii-D-galactopyranosidein Medium A (4 mg/ml). The reaction was terminated after sufficient color development (10 min to 3 hr) by the addition of 2.5 ml cold 1 M sodium carbonate. The A2o was read in a Bausch and Lomb Spectronic 20 by direct insertion of the plastic incubation tubes. The spectrophotometer was adjusted to zero using a control tube without bacteria. Enzyme is calculated as 100 A2o/f, where f is the color development time in hr (Chart 2a). Kinetic Assay. The assay was as for Procedure1 except bacteria and chemical were combined in a single-shake flask. At appropriate times, aliquots of 0.5 ml were removed to tubes and then chilled with the addition of 4.5 ml cold ZCM buffer (Chart 20).
6

RESULTS

Test of Standard Compounds. A schematicdiagramof the methodology used for 3 variations of the BIA spot test is shown in Chart 1. Spot test results with 17 well-known natural products are shown in Fig. 1a and Table 1. Each compound was tested at 2 concentrations differing by a factor of 10, depending on solubility and biological activity. The intensities of the spots reflect relative inducing activities. Observation of the spot pairs provides information regarding relative potencies and the location of the optimum dose range for induction, which may then be utilized in the quantitative assay. For example, in Fig. 1a, Plate 1,Row B, Spots 1 and 6 are near the optimum dose for induction; Spof 2 is below it; and Spofs 3, 4, and 5, showing zones of toxicity, are above the optimum dose. Fig 1u illustrates the same principle with compounds diluted to the end point. Solutions showing good activity in the spot test, when diluted 10-fold into the bacterial suspension, are generally in the correct concentra tion range for optimum induction in the quantitative tube assay. This is seen to be the case for largomycin Fll, spotted from a
JUNE 1983

Chart 2. Quantitative assay of lysogenic induction by largomycin Fll. a, doseresponse assay after 3 hr (in the presence of ampicillin). b, kinetic curves of induction at the concentrations shown (*ig/ml) (in the presence of ampicillin).

solution of 100 Mg/ml [1 Mg/spot (Fig. 1a)], giving a peak dose of 10 /tg/ml in the quantitative assay (Chart 2a). The results of the test of 142 compounds in the spot test are summarized in Table 2. In Table 2, we have tried to correlate inducing activity (BIA) with the mechanism of action (DMA inter action) and anticancer potential (2, 8, 16, 24, 34, 38) of a diverse set of compounds. We purposely included compounds that were not expected to induce but were representative of a variety of chemical types and modes of action. The study is biased toward natural products available in our laboratory, some of which are not well studied as to mode of action. For this reason, chemicals are grouped by relative inducing capacity (as determined by spot intensity), rather than by structural type or mode of action. The amount of compound required for induction varies over 6 orders of magnitude. This quantity is indicated in Table 2 for each inducer.
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R. K. Elespuru and R. J. White

Table 1 Key to Chemicalsspotted in Fig. Ja and results obtained

no 2: No 3: + 4: + ampicillin ampicillin + loca ampicillin ampicillin+ (g/spot)60.20.020.20.020.20.0210.10.20.020.20.020.20.0210.110.110.10.20.0210.10.20.0210.10.20.0210.110 Compound*Streptovar-cin tionA123456B123456C123456DI23456E123456FI23456Platel: NoS9tcttt+++++++R+++R+++R+++_-++++++++R++R+-+++R++++++++++R+++_- S9ttt++++++R+++R+++R+++_+++++R++R+++--H-+-+++R NoS9ttttt+++++++R+++R-H-+R+++_+++++++++R+++R+-++ 89-ttt++++++R+++R+++R+++_-H.+++R DRifamycin SVActinomycin

DNeocarzinostatinHedamycinRufochromomycinBruceantinMacromomycinBleomycinDaunorubicinRubiflavinLargomycinStreptonigrinVinblastineSte

acidHjOMethanolAflatoxin id2pl0.010.001Spot

B,Quantity * Solvent as in Table 2. 0 Chemical solutions made at 10 and 100 >g/m\; volume spotted dependent on solvent (see Table 2, Footnote b). 0 -, no induction; t, toxic, no induction; ;equivocal; +, ++, +++, weak, moderate, strong induction, respectively, as determined by spot color intensity; R, ring of induction (diffusion zone) surrounding toxic center.

More than one-third of the components tested (56 of 142) were detected as inducers. The great majority of the strong inducers are compounds known to interact with DNA, including many standard antitumor agents such as Adriamycin, bleomycin, BCNU, ICR compounds, and platinum complexes. The inducing activity of piperazinedione supports the suggestion that it is an alkylating agent (5). More detailed studies of structure-activity relationships for some of these groups of compounds will be presented elsewhere. Other inducers, generally classified as weak, have indirect effects on DNA, often by inhibition of specific enzymes involved in DNA biosynthesis. Examples include aza-

serine (4, 37), trimethoprim (7), novobiocin (41), nalidixic acid (15), 5-fluorouracil and 2'-deoxy-5-fluorouridine (6). 2'-Deoxy-5bromouridine, on the other hand, is extensively incorporated into DNA in place of thymine (12). It did not induce the phage. As expected, inhibitors of protein synthesis and microtubule assem bly did not induce. Cysteine was negative, in contrast to the positive effect reported in another X-phage induction assay (35). Chemicals with apparently similar modes of action often showed quite large differences in BIA activity. For example, actinomycin D, anthracenediones, ICR compounds, and daunorubicin are all intercalating agents; however, actinomycin is neg-

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1a

+ S9 2

B No amp

O
'"-

amp
Fig. 1. Colorimetrie spot test for lysogenic induction by antitumor agents and model com pounds. Chemical solutions were spotted di rectly onto gridded 100-mm plates containing bacteria, with or without arnpicillin (amp) and rat liver 89 activation mix. After incubation at 30,plates were developed by the addition of a chromogenic substrate for the enzyme galactosidase. a, 3 hr incubation. Table 1 pro vides a key to the chemicals spotted and results obtained, b, serial 2-fold dilution for determina tion of minimal inducing doses. A different chemical is spotted in each row, with the high est dose (/ig/spot) at the left margin. Row 1, bteomycin (0.1 ^9); "w 2, largomycin Fll (0.5 ng); Row 3, rifampicin (1 (g); Row 4, azaserine (100 xg); Row 5, BCNU (100 M9); Row 6, c/sdichlorodiammineplatinum (II) (10 tig). Solvent is dimethyl sulfoxide for BCNU; all other solu tions are in water. See legend to Table 2 for details.

1b

No amp

amp 2 6

1
23 A B 2 hr C D E Jt

45

r l
v

O*

5 hr

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f. K. Elespuru and R. J. White

Table 2 Tesi of antitumor agents and model compounds in the BIA spot test Chemical solutions were spot-tested directly on E. coli strain BR513 (Ap/acZ) poured in agar containing ampicillin (10 ><g/ml). After a 3-hr (5-hr) incubation at 38, plates were overlaid with a colorless .-galactoside Red spots appearing within 10 min indicated chemical induction of phage X as monitored by the presence of tf-galactosidase, the lacZ gene product under A control. Inducing activity (strong, weak, etc.) was determined by spot intensity. Toxicity is indicated where color is less than background (see Chart 1 and Fig. 1). CompoundAdriamycinAflatoxin Inter action8 SolventStrong

ap inducing (g)60.3-50.00001-0.10.1-11-1000.01-0.11-1000.0004-80.06-10.3-50.00002-0.20.05-1000.03-1000. plied amount (^g/spot)c0.60.00010.1 activity"+++++++++++++++++++++++++++++++++++++++

inducers+" Water+ B,AnthramycinAzaserineBaumycin Water+ MethanolDMSO'+ Methanol+ DMSO+ (0.06)25(10)0.05500.0030.060.60.00030.20.060.060.25-(0.05)1

A,/A2BCNUBleomycinCopiamycin

WaterMethanol+
(acetyl)DaunorubicinHedamycinICR Water-1Methanol+ Water+ Water+ Water+ DMSO+ WaterWater+ Water+ Water+ MethanolDMSO+ WaterWater+ 147PhleomycinPlatinum

453ICR 170ICR 292ICR 486Illudin Slyomycin complexLargomycin FllMacromomycinMitomycin CNalidixic acidNeocarzinostatinPA

(0.2)ND0.1 (0.03)0.050.01<10.10.010.006(1.0)(50)0.050.0300.0010.002

Water+ compoundscis-PtAm2CI2K[PtAmCI3]PorfiromycinRoseolic 0.1 5 M NaCI0.15 NaCI+ M Methanol-tWater+ acidRubiflavinRufochromomycinSibiromycinStreptonigrinStreptozotocinTomaymycinZorbamycinAmpicillinChartreusinCyclophosphamideDONEllipticineEthidium Methanol+ Methanol+ (0.0004)0.0150.001(0.3)90.25(0.1)0.006(10)0.15100(5)"(0.5)(10)(10)(10)(10)0.3(0. Methanol+ Methanol+ Methanol+ Methanol+ WaterWeak inducersWater+

Methanol+ DMSOWater+ DMSO+ DMSODMSOWaterDMSO+

bromide5-FluorouracilFosfomycin2 -Deoxy-5-fluorouridineICR '

191LuteoskyrinNogalomycinNovobiocinPiperazinedionePlatinum Water+ Methanol+ MethanolDMSO+

(0.25)10(2.5)(50)100(0.6)(0.06-0.1)(0.1)0.0001(0.2)10Antitumor

([PtAmJCyQuinine compounds sulfateQuinomycin ARifampicinRifamycin SVStreptovaricin CStreptovaricin DTrimethoprimActinobolinActinomycin

Water0.15 NaCI+ M Water+ MethanolWaterMethanolMethanolMethanolWaterToxic

noninducersMethanol+

DActinorubinAzalomycin complexBluensomycin F sulfateCinerubin BCinnamycinDNA

MethanolWaterMethanolWater+

MethanolMethanolAmount

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BIA for Antitumor Agents

Table 2Continued

CompoundCyaneinCyclomycin

Inter inducing Antitumor ap activity0-+++++++++Compounds amount (^g/spot)0 action" SolventMethanolMethanolWaterWaterMethanolMethanolWaterMethanol+ (ngf115511510.01-0.211111510.006-0.211Minimal plied

complexDuramycinEnteromycinFusarubinGliotoxinKasugamycinMikamycinMithramycinNarangomycinOxytetracyclinePactamycinProdigiosinRubradirinSa

MethanolMethanolMethanolMethanolMethanolMethanolWaterMethanolMethanolMethanolMethanolAm

salicylateStreptorubinViridogriseinViundrymycinDNA

activityAmicetinCAMPAlanosineAminopterinAnguidineAnisomycinAnthracenedionesNSC with no

MethanolDMSODMSODMSO+ 7833NSC 15367NSC

3NSC721 278467NSC 7957NSC 295560(Anthracenedione)NSC 3NSC28751 1485(Mitoxantrone)NSC 279836Azacolutin DMSODMSODMSO-t-

DMSODMSO+

DMSODMSOWaterMethanolWater-

complex5-AzacytidineAzotomycinBlasticidin

SBruceantin5-Bromo-2'-deoxyuridineCandicidinCordycepinCysteine MethanolDMSOWaterWaterWaterWaterWaterWaterWaterMethanolWaterMethanolWaterMethanolWaterWater

(DL)Cystine (DL)CytovirinDuazomycin AFlammulinFormycin AFormycin BFumagillmGougerotinGriseofulvinHadacidinMethotrexateMitogillinMitosperMycorhodinNebularinOligomycinOosporinPeptinoganPlatinum

compoundstrans-PtAm2CI2[PtAm3CI]CIK2[PtCI]PuromycinPyrazomycinRestrict 0.15 NaCI0.1 M NaCI0.1 5M NaCIWaterWaterWaterMethanolMethanolWaterWaterMethanol155-2501-10010.1-10.1-100.1-100.1-1 5M ocinSangivamycinSaramycetinSeptacidinSistomycosinSparsomycinMethanolWaterWaterWaterMethanol

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R. K. Elespuru and R. J. White

Tabte 2Continued CompoundStatalon Inter action8SolventMethanol ap (Mgr1 plied 1-10 1 5 1 1 0.1-1Minimal inducing amount (/ig/spot)c Antitumor activity''+

Streptolydigin Thiosangivamycin Threomycin Trienine Verrucarin A VinblastineDNA 8 Where known (Refs. 4, 7, and 12; and see text). 6 Absolute amount applied in ~10 n\ H20 or DMSO

Water Methanol Water Methanol Methanol WaterAmount

and 21 methanol (surface tension of the solvents differs); solution

concentrations in ng/m\ therefore are 100 or 500 times the quantity spotted. ' As determined by the appearance of a visible colored spot after 3 hr (5 hr) incubation. d Antitumor activity in experimental animal systems with transplanted tumors (2, 8,16, 24, 33, 37). * +, interaction; -, no interaction. 'Abbreviations used: DMSO. dimethyl sulfoxide; ND, not determined; c/s-PtAmzCt, c;s-dichlorodiammineplatinum(ll); K[PtAmCI3], potassium trichloroammineplatinum(ll); DON, 6-diazc-5-oxo-L-norleucine; [PtAm4]CI2, tetraammineplatinum(ll) chloride; cAMP, cyclic adenosine 3':5'-monophosphate; -rans-PtAmjClz, frans-dichlorodiammineplatinum(ll); [PtAm3CI]CI, chlorotriammineplatinum(ll) chloride; ^[PtCUJ. potassium tetrachloroammineplatinum(ll). 9 Reproducible + only when bacteria are grown in the presence of N-acetylglucosamine " + only on less rich medium (TB*) (9). (inducible transport system).

ative; ICR 170 is a strong inducer while ICR 191 is weak; anthracenediones are detected poorly or not at all; and daunorubicin is detected quite well. Of the several inhibitors of nucleotide synthesis tested, most are weak inducers, while azaserine is uncharacteristically strong. A closer look at such factors as permeability, toxicity, and specific DNA interactions provides some explanation for these differences, but these will not be considered here. In the case of a few compounds, the expected correlation between inducing capacity and effects on DNA was not ob served. A few DNA-interacting compounds which preferentially inhibit RIA synthesis rather than DNA synthesis (actinomycin D, mithramycin, and cinerubin B) (7, 12) were not detected as inducers. This is not surprising, since RNA synthesis is required for the assay of induction. Streptozotocin failed to reproducibly cause induction unless A/-acetylglucosamine was added to in duce the transport system controlling its uptake (27). This was unexpected in view of the activity reported in another prophage induction assay (35). While azaserine showed good inducing activity, other glutamine analogues (inhibiting purine biosyn thesis) (4) such as 6-diazo-5-oxo-L-norieucine, azotomycin, and duazomycin showed little or no activity. The absence of zones of toxicity for the latter 2 compounds indicates that they probably did not permeate the bacteria. Alkylating agents such as BCNU and cyclophosphamide were detected only at high concentra tions. On the other hand, induction by the group of structurally related ansamycins (streptovaricins and rifamycins) that are in hibitors of DNA-dependent RNA polymerase is somewhat sur prising (Fig. 2o). It is possible that induction is related to an indirect inhibition of DNA synthesis (by inhibition of RNA primer formation) at concentrations still allowing residual transcription of the lacZ gene (30). Fosfomycin and ampicillin, inhibitors of bacterial cell wall syn thesis (25), were found to be weak inducers. The loss of struc tural integrity of the cell wall may affect the DNA-membrane complex in this case. In common with other agents that appear to work via indirect mechanisms, there is a significant lag before induction occurs, generally requiring a 5-hr incubation (Fig. 1b). Therefore, the BIA can be made more selective for agents that directly interact with DNA by limiting the induction period to 3 hr. The effect on induction of low concentrations of ampicillin, rat liver microsomal enzymes, and incubation time is shown in Fig. 2826

1. Table 1 shows our scoring method and lists the chemicals and amounts tested. While most inducers were detected after a 3-hr incubation period, the ansamycins required 5 hr for induction (Fig. 16). Other chemicals were detected at lower concentrations after the longer incubation time (Table 2). This is consistent with typical induction kinetics (Chart 2b). Ampicillin was routinely added to the assay as an inhibitor of background cell growth (32) before its effect as an inducer was known. (Induction is not seen, however, at the concentration present in the plates.) However, induction by some chemicals, including rifamycin SV, streptovaricin D, and fosfomycin appears to be inhibited by ampicillin. Whereas only cyclophosphamide and aflatoxin B, (used as a control) showed an absolute requirement for metabolic activation by rat liver S9 fraction, rubiflavin, and those compounds with protein components, neocarzinostatin, macromomycin, and largomycin were inactivated by the S9 fraction, the lattermost compound to the extent of more than 90% [loss of both 1.0and 0.1-//g spots (Fig. 1a); therefore, less than 10% of the 1.0ng spot is remaining]. On the other hand, daunorubicin, a classic inducer in the absence of metabolic activation, was detected at lower concentrations in the presence of the S9 fraction, in agreement with previous results (1). BIA as a Prescreen. We have used the BIA spot test as a prescreen (Fig. 2) for the detection of potential antitumor anti biotics in a total of 10,724 microbial fermentation broths (from 2,681 cultures) (13). Approximately 1% of the cultures tested gave a reproducible positive result in the BIA. In no case was induction in the BIA dependent on S9 microsomal activation; however, inactivation of inducing activity by S9 was noted with one culture. This culture (SKF 42) produces a macromolecular antibiotic that is probably glycoprotein in nature and appears to be different from other related antibiotics, such as largomycin, neocarzinostatin, and macromomycin. BIA-positive cultures were fermented and submitted for testing in mice against the P388 leukemia. Those cultures which gave in vivo activity (lifespan of treated/control, 3=130) were refermented and retested against P388. Ten of the 37 cultures have confirmed in vivo activity, while testing is incomplete on 7. Twenty were negative. Cultures with confirmed in vivo activity have been tentatively identified thus far as producing streptonigrin, anthracyclines, neocarzinostatin, and streptorubin, as well as at least one novel compound, 2064A (43). Structural analysis
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BIA for Antitumor Agents and further testing of the novel compounds is currently being undertaken. Identification and Purification of Inducing Activities. Fer mentation broths that were biologically active were further ana lyzed to determine whether they contained novel or known activities (a procedure known as "dereplication"). A small num ber of compounds exhibited spot characteristics that are essen tially diagnostic (Fig. 1). For example, largomycin Fll was com pletely inactivated by mammalian enzymes (S9) at concentrations below 100 /ng/ml. Streptovaricins C and D and rifamycins pro duced a characteristic toxic center surrounded by a halo of weak induction over a wide concentration range (solid spots of induc tion were never seen). Inducers that were very toxic (e.g., rubiflavin) or very slow to diffuse (large molecules such as the glycoprotein largomycin) showed very narrow rings of induction with toxic centers at high concentrations. Fermentation broths and extracts from inducing cultures were chromatographed on thin-layer chromatography plates. Zones of activity on the plates were determined by bioautography (20) as described in "Materials and Methods" and Chart 1. R( values can be used to help classify and identify unknown activities (23). Bioautography of a daunorubicin fermentation broth and several extracts is illustrated in Fig. 3. The relative success of antibiotic purification procedures was determined by direct spotting of extracts onto prepared BIA plates. Extracts of fermentation broths in water, methanol, ethanol, butyl alcohol, isobutyl alcohol, or acetone, but not chloroform, could be spotted without adverse effects on the assay. Dimethyl sulfoxide, acetonitrile, hexane, and other sol vents may also be spotted directly on the plate (data not shown). Quantification of Inducing Activities. Inducing activities were quantified using either the BIA spot test or the liquid incubation assay. In the spot test, the minimal inducing dose of a fermen tation broth or standard solution was determined in samples diluted to the end point (Table 2; Fig. 1b). The liquid incubation assay allows the direct measurement of induced enzyme (Chart 2). If a standard calibration curve is first derived (Chart 2a), the amount of inducer present in a fermentation broth can be esti mated. A kinetic curve of induction, such as that shown in Chart 2b, can be used to find the optimal induction time for the sensitivity desired in any given assay. An expression time of 2 hr or less is sufficient for strong inducers, while longer times of up to 5 or 6 hr will allow the detection of weak inducers (e.g., piperazinedione), toxic compounds (e.g., BCNU), chemicals that react slowly (e.g., platinum complexes), and inducers present at low concentration (Table 1; Chart 2b). Selection of Individual Isolates with Inducing Activity. The production of inducing activity by individual colonies growing on solid media can be determined by transferring agar plugs onto standard BIA spot test plates. This procedure can be useful in several circumstances: selection of active clones from mixtures of producing and nonproducing natural variants of an inducing culture (see Fig. 4); selection of higher-yielding mutants in strain improvement programs (22); and selection of active cultures during the initial screening of soil isolates (preliminary data in our laboratories indicate an encouraging correlation between pro duction of inducing activity on solid media and in liquid culture). DISCUSSION A biochemical prophage induction assay (BIA) has been used
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to screen, analyze, and monitor production of natural products with potential antitumor activity. The induction of X-prophage in E. coli is one of a group of coordinately regulated phenomena that have been collectively termed SOS functions (36,46). These functions are presumed to be induced in response to a threat to the survival of the cell (and bacteriophage) caused by an inhibition of DNA replication. This can be the result of a variety of events, including direct damage to DNA (caused by interaction with chemicals or radiation), and indirect effects on DNA metabolism, e.g., thymine starvation, and inhibition of DNA gyrase (15, 29, 40, 41, 46). As discussed under "Results," the majority of compounds reported as inducers in Table 2 are known, or considered, to interact directly with DNA. Other inducers do not interact with DNA but are known to be specific enzyme inhibitors which ultimately affect DNA synthesis (4, 7, 12). Induction by a few compounds was unexpected, because their mode of action is not considered to involve DNA. In general, our results agree with those using classical pro phage induction assays (1,10,14,18,19,26, 32, 35), with minor exceptions noted previously. However, the novel antibiotic found in Broth 2064, gilvocarcin V (44), was not detected as a prophage inducer in another laboratory (3), for reasons most probably related to sensitivity. Those inducers with no demonstrated antitumor activity (rifa mycins, ampicillin, fosfomycin, nalidixic acid, novobiocin, and trimethoprim) are all known to be specific inhibitors of prokaryotic enzymes and are essentially inactive in eukaryotic systems (7, 12, 15, 25, 30, 38, 41). However, eukaryotic counterparts exist (e.g., methotrexate for trimethoprim as an inhibitor of dihydrofolate reduc-ase)(7, 12). These compounds fall into the class of indirect inducers, rather than those that interact directly with DNA. The strain constructed for this assay, BR513, contains 2 mutations allowing the detection of lower concentrations of inducers, envA, enhancing permeability, and uvrB, reducing the ability of the strain to repair certain types of damage to DNA (9). These mutations result in the detection of some compounds at concentrations up to 100 times less than those detected by wildtype bacteria (32, 33). We have found that the effect of the permeability mutation varies widely with different chemicals, however. For example, bleomycin is detected at 10-fold lower doses with the mutant, while platinum complexes and nitrosoureas show no difference between the wild-type and the mutant strain (data not shown). The question of sensitivity is important because of the rather low concentrations (on the order of 25 i*g/m\ or less) at which secondary metabolites normally occur in microbial fermentation broths. Natural products with antitumor activity found in fermen tation broths that might be missed by the BIA due to sensitivity limitations are azaserine, piperazinedione, chartreusin, and illudin S. Adriamycin and daunorubicin are not detected at 25 pg/m\, but related metabolites that are usually coproduced, e.g., baumycin (31), are detected below this concentration (Table 2). The bacterial strain constructed for this assay contains a mutation preventing the expression of functions controlled by the rightward promotor of A. As a consequence, the expression of the lacZ gene and production of 0-galactosidase are not abated by transcription controls or phage-induced cell lysis (9). The length of the induction period (Chart 2b) may be used to vary the sensitivity of the induction assay. A 3-hr period is generally optimum for the detection of most inducers; however, 2827

R. K. Elespuru and R. J. White

shorter or longer times may be used for monitoring strong or weak inducers, respectively. For example, largomycin FM, a strong inducer, could be detected at 10 i/g/ml in 1.5 hr and at 0.05 g/ml in 6 hr (Chart 20). Because of the short assay time, the production of largomycin by fermentation could be monitored using the BIA. A colorimetrie assay of prophage induction has certain inherent advantages over a plaque assay, e.g., the presence of a uniform background against which low levels of induction may be seen in a very small area, as in a spot test or on a bioautograph (Chart 1). Although several other biochemical assays of prophage in duction exist (21, 28, 39), none may be utilized in a spot test, the form of assay that we have found to be most useful for screening, bioautography, and strain selection. An unexpected advantage of the BIA spot test is its relative immunity to the deleterious effects of solvents. We surmise that the high con centration of bacteria used in the biochemical assay is respon sible for this protective effect. At most, only 17 of 37 BIA-positive fermentation broths were active against P388 leukemia when tested in vivo, perhaps owing to the limits of sensitivity of the animal assay. Active compounds in fermentation broths are frequently present at concentrations of 0.1% or less of the total solids. The amount of solids that can be administered (approximately 400 mg/kg) limits the sensitivity of in vivo testing (45); we are attempting to purify (and concen trate) inducing activities prior to repeat in vivo testing. The observation that only one of the natural products tested (aflatoxin B,) showed an absolute requirement for metabolic activation is not surprising, since antitumor antibiotic discoveries in the past were made without the use of activating enzymes. However, our screen of 10,000 fermentation broths did not yield any positive samples requiring activation. This result indicates that inducers such as aflatoxin are not commonly present in fermentations. Our experience with the BIA has made us aware of its limita tions, the major one being sensitivity, despite the presence of mutations which affect sensitivity to some extent. Certain classes of compounds expected to interact with DNA, such as anthracenediones, are not detected. In addition, conditions that favor the detection of one chemical are often far from optimal for detection of other inducers (Fig. 10). For this reason, compro mises must be made in protocols for optimum detection of a variety of inducers in a prescreen. Nevertheless, as demon strated here, the BIA functions as an effective and practical means for detecting potential antitumor agents of a specific mode of action. Additional genetic manipulations in the bacterial strain, in progress, should increase sensitivity and facility of induction following a variety of DNA-chemical interactions.
ACKNOWLEDGMENTS
We thank Robin Pennington and Ivan Lufriu for performing the tests. Dr. Raymond Ruddon for valuable suggestions, and many colleagues in the Chemo therapy Fermentation Program for practical advice.

CRC Press. Inc., 1981. 3. Balitz. D. M., O'Herron, F. A.. Bush, J., Vyas, D. M., Nettleton, D. E., Grulich, R. E., Bradner, W. T., and Doyle, T. W. Antitumor agents from Streptomyces anand: gilvocarcins V. M, and E. J. Antibiot. (Tokyo). 34: 1544-1555, 1981. 4. Bennett, L. L. Glutamine antagonists. In: A. Sai-torelli and B. A. Booth (eds.), Antineoplastic and Immunosuppressive Agents, Part II, pp. 484-511. Berlin: Springer-Verlag, 1975. 5. Brockman, R. W., Shaddix, S. C., Williams, M., and Struck, R. F. Studies with 2.5-piperazinedione, 3,6-bis[S-chloro-2-piperidylj-, dichloride. 2. Effects on macromolecular synthesis in cell culture and evidence for alkylating activity. Cancer Treat. Rep., 60. 1317-1324, 1976. 6. Cohen, S. S., Flaks, J. G., Barner, H. D., Loeb. M. R., and Lichtenstein, J. The mode of action of 5 fluoro-uracil and its derivatives. Proc. Nati. Acad. Sci. U. S. A., 44: 1004-1012, 1958. 7. Corcoran, J. W., and Hahn, F. E. (eds.). Antibiotics III. Mechanism of Action of Antimicrobial and Antitumor Agents. Berlin: Springer-Verlag, 1975. 8. Creech, H. J., Preston, R. K.. Peck, R. M., and O'Connell, A. P. Antitumor and mutagenic properties of a variety of heterocyclic nitrogen and sulfur mustards. J. Med. Chem., 75: 739-741. 1972. Elespuru, R. K., and Yarmolinsky. M. B. A colorimetrie assay of lysogenic induction designed for screening potential carcinogenic and carcinostatic agents. Environ. Mutagen., 1: 65-78, 1979. Endo, H., Ishizawa, M., Kamiya, T.. and Sonoda, S. Relation between tumoricidal and prophage-inducing action. Nature (Lond.), 798: 258-260, 1963. Fleck, W. F. Development of microbiological screening methods for detection of new antibiotics. Postepy Hig. Med. Dosw., 28: 479-498, 1974. Gale, E. F., Cundliffe. E.. Reynolds, P. E., Richmond, M. H., and Waring, M. J. (eds.). The Molecular Basis of Antibiotic Action. New York: John Wiley & Sons, Inc., 1972. Garretson, A. L., Elespuru, R. K.. Lufriu. l.. Warnick, D., Wei. T., and White. R. J. In vitro prescreens for the detection of antitumor agents. Dev. Ind. Microbiol.. 22: 211-218, 1981. Geissler, E. Lysogenie and virale Kanzerogenese. Arch. Geschwulstforsch., 29:355-372, 1967. Geliert. M.. Mizuuchi. K.. O'Dea, M. H., Itoh, T.. and Tomizawa, J. Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity. Proc. Nati. Acad. Sei. U. S. A., 74:4772-4776, 1977. Goldin. A., and Venduti, J. M. Retrospective and prospective approaches to screening and to comparative evaluation of analogs in the U.S.A. In: H. Umezawa. S K. Carter. A. Goldin, K. Kuretani, G. Math,Y. Sakurai. and S. Tsukagoshi (eds.), Advances in Cancer Chemotherapy, pp. 179-200. Balti more: University Park Press, 1978. Hanka, L. J., Martin, D. G., and Neil, G. L. In vitro methods used in detection and quantitation of antitumor drugs produced by microbial fermentations. Lloydia. 47:85-97, 1978. Heinemann, B. Prophage mducton in lysogenic bacteria as a method of detecting potential mutagenic, carcinogenic, carcinostatic and teratogenic agents. In: A. Hollaender (ed.). Chemical Mutagens: Principles and Methods for Their Detection, Vol. 1, pp. 235-266. New York: Plenum Publishing Corp., 1971. Heinemann, B., and Howard. A. J. Interaction of lambda bacteriophage in Escherchiacoli as a screening test for potential antitumor agents. Appi. Microbiol.. 72. 234-239. 1964. Heinemann, B., Howard, A. J., Alna, J.. and Hollister, Z. J. Application of paper chromatograms to the study of nducersof X bacteriophage in Escherichia coli. Appi. Microbiol., 75: 723-725, 1967. Ho. Y. L., and Ho, S. K. The induction of a mutant prophage \ in Escherichia coli: a rapid screening test for carcinogens. Virology, 99: 257-264, 1979. Ichikawa, T., Date, M., Ishikura, T., and Ozaki, A. Improvement of kasugamycin-producing strain by the agar piece method and the prototype method. Folia Microbiol.. 76: 218-224, 1971. Issaq, H. J., Barr, E. W., Wei, T., Meyers, C. I., and Aszalos, A. Thin layer Chromatographie classification of antibiotics exhibiting antitumor properties. J. Chromatogr., 733: 291-301, 1977. Johnson. R. K., Zee-Cheng, R. K.-Y., Lee, W. W., Acton, E. M., Henry. D. W., and Cheng. C. C. Experimental antitumor activity of aminoanthraquinones. Cancer Treat. Rep., 63: 425-439,1979. Kahan, F. J., Kahan, J. S., Cassidy, P. J., and Kropp, J. The mechanism of action of fosfomysin (phosphonomycin). Ann. N Y. Acad. Sci. 235: 364-386, 1974. Lein, J., Heinemann, B., and Gourevitch, A. Induction of lysogenic bacteria as a method of detecting potential antitumor agents. Nature (Lond.), 796: 783786, 1962. Lengeller, J. Analysis of the physiological effects of the antibiotic Streptozotocin on Escherichia coli K12 and other sensitive bacteria. Arch. Microbiol., 728: 196-203, 1980. Levine, A., Moreau, P. L., Sedgwick, S. G., Adhya. S., Gottesman, M., Devoret, R., and Das, A. Expression of a bacterial gene turned on by a potent carcinogen. Mutt.Res., 50: 29-35, 1978. Little, J. W., and Hanawalt, P. C. Induction of protein X in Escherichia coli. Mol. Gen. Genet., 750: 237-248, 1977. McClure, W. R., and Cech, C. L. On the mechanism of rifampicin inhibition of RNA synthesis. J. Biol. Chem., 253: 8949-8956. 1978.

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31. McGuire, J. C., Thomas, M. C., Stroshane, R. M., and Hamilton, B. K., and White, R. J. Biosynthesis of daunorubicin glycosides: the role of e-rhodomycinone. Antimicrobial Agents Chemother., 13: 454-464, 1980. 32. Moreau, P., Bailone, A., and Devoret, R. Prophage \ induction in Escherichia coli K12 envA uvrB: a highly sensitive test for potential carcinogens. Proc. Nati. Acad. Sei. U. S. A., 73: 3700-3704,1976. 33. Normark, S., Boman, H. G., and Matsson, E. Mutants of Escherichia coli with anomalous cell division and ability to decrease episomally and chromosomally mediated resistance to ampicillin and several other antibiotics. J. Bacteriol., 97: 1334-1342,1969. 34. Peck, R. M., and O'Connell, A. P. Mixed bifunctionality. 4. Antitumor activity of alkylating derivatives of polycyclic aromatic hydrocarbons as a function of structure and of vehicle. J. Med. Chem., 8: 68-70, 1972. Price, K. E., Buck, R. E., and Lein, J. System for detecting inducers of lysogenic Escherichia coli W1709 (A) and its applicability as a screen for antineoplastic antibiotics. Appi. Microbiol., 12: 428-435,1964. Radman, M. Phenomenology of an inducible mutagenic DMA repair pathway in Escherichia coli: SOS repair hypothesis. In: L. Prakash, F. Sherman, M. Miller, C. Lawrence, and H. W. Tabor (eds.), Molecular and Environmental Aspects of Mutagenesis, pp. 128-142. Springfield, III.: Charles C Thomas, Publisher, 1974. Sartorelli, A. C., and Johns, D. G. Inhibition of the synthesis of thymine nucleotides by azaserine. Mol. Pharmacol., 3: 71-80, 1967. Sethi, V. S. Ansamycins. In: A. Aszalos (ed.). Antitumor Compounds of Natural Origin: Chemistry and Biochemistry, Vol. 1, pp. 59-85. Boca Raton, Fla.: CRC Press, Inc., 1981. Smith, C. L., and Oishi, M. The molecular mechanism of virus induction, I. A procedure for the biochemical assay of prophage induction. Mol. Gen. Genet., 748:131-138,1976. Smith, C. L., and Oishi, M. Early events and mechanisms in the induction of bacterial SOS functions: analysis of the phage repressor inactivation process in vivo. Proc. Nati. Acad. Sei. U. S. A., 75: 1657-1661, 1978. Sugino, A., Higgins, N. P., Brown, P. 0., Peebles, C. L., and Cozzarelli, N. R. Energy coupling in DNA gyrase and the mechanism of action of novobiocin. Proc. Nati. Acad. Sei. U. S. A., 75: 4838-4842, 1978. Trotman, R.E. Technological Aids to Microbiology, pp. 17-21. London: Edward Arnold, Ltd., 1978. Vogel, M. J., and Bonner, D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem., 278: 97-106, 1956. Wei, T. T., Chan, J. A., Roller, P. P., Weiss, U., Stroshane, R. M., White, R. J., and Byrne, K. M. Detection of gilvocarcin antitumor complex by a biochemical induction assay (BIA). J. Antibiot. (Tokyo), 35: 529-532, 1982. White, R. J. In vitro prescreens. In: R. J. White and I. J. Fidler (eds.), Design of Models for Testing Cancer Therapeutic Agents, pp. 1-11. New York: Van Nostrand, 1980. Witkin, E. M. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol. Rev., 40: 863-907, 1976.

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o.O*
*

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Fig. 2. Cotorimetric spot test for inducing activity nmicrobial fermentation broths. Fermentation broths of actinomycetes were spotted on 243-mm bioassay plates containing bacteria and ampicillin. Incubation period, 3 hr. Dark rings and spots are induction positive; white areas are zones of toxicity. This set of selected broths contains many induction positives.

Fig. 3. Bioautography of extracts of a daunorubicin fermentation broth chromatographed in 2 solvent systems. Lane 7, whole broth; Lane 2, butyl alcoholextracted aqueous; lane 3, acid hydrolysis product purified (daunorubicin HCI); Lane 4. butyl alcohol extract after toluene:ethyl acetate partition; Lane 5, butyl alcohol extract of whole broth; Lane 6. toluene:ethyl acetate extract of acidified whole broth. Amounts spotted were adjusted to avoid under- or overloading and do not reflect relative inducing activity per sample. Solvent systems: left half, chloroform:methanol:acetic acid (80:20:2); right half, heptane:chloroform:methanol (5:5:1).

Fig. 4. Inducing activity produced by individual colonies. A spore suspension of FCRC-324, an unspeciated streptomycete that produces neocarzinostatin, was plated onto a complex nutrient agar; when colonies were just visible (about 3 days at 28), they were picked off on separate agar plugs. After 6 days of further incubation, the plugs were transferred to a prepared 243-mm BIA plate. After 3 hr at 38,the plugs were removed, and the plates were developed with a chromogenic substrate. Clear areas, toxic with no induction; spots, positive for induction; rings, toxic center with diffusion zone positive for induction. The relative amount of inducing substance is low. moderate, and high for clear zones, spots, and rings, respectively.

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