[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
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 Õ7) 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
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.
JUNE
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.
1983
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2819
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
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 45°were 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 38°during 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 45°were 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
buffer, pH 7.4.
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 37°for approximately 3 hr to A«oo
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
(TLC)
3) 3 hr, 37°
3) 3 hr, 37°
3) 3 hr, 37°
4) remove plugs
5) substrate overlay
4) remove TLC
5) substrate overlay
4) substrate overlay
Variants
2820
Thin layer
chromatogram
'.. O. . P., O. ./">.. J
v./) ///// //!////////)
Multi-point sample
applicator
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 identificationof 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 A»2o
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 A»2o/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
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.
JUNE 1983
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2821
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*StreptovarÃ-cin
tionA123456B123456C123456DI23456E123456FI23456Platel:
NoS9tc—ttt—+++++++R+++R+++R+++_-++++++++R++R+-+++R++++++++++R+++_-—â
S9t—t—t—++++++R+++R+++R+++_—++±+++R++R+++--H-+-—+++Râ€
NoS9t—tttt+++++++R+++R-H-+R+++_—+++++++++R+++R+-++
89-—t—tt++++++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-
2822
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-
CANCER
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VOL. 43
1a
+ S9
2
B
O
No amp
'"-
V
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 ßgalactosidase. 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
1
2
23
A
B
2 hr
45
6
Jt
C
D
O*
r l
*
E
v
x
5 hr
JUNE 1983
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2823
fì.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
A,/A2BCNUBleomycinCopiamycin
Water+
MethanolDMSO'+
(0.06)25(10)0.05500.0030.060.60.00030.20.060.060.25-(0.05)1
Methanol+
DMSO+
WaterMethanol+
(acetyl)DaunorubicinHedamycinICR
453ICR
170ICR
292ICR
486Illudin
Slyomycin
complexLargomycin
FllMacromomycinMitomycin
CNalidixic
acidNeocarzinostatinPA
Water-1Methanol+
Water+
Water+
Water+
DMSO+
WaterWater+
Water+
Water+
MethanolDMSO+
(0.2)ND0.1
(0.03)0.050.01<10.10.010.006(1.0)(50)0.050.0300.0010.002
WaterWater+
147PhleomycinPlatinum
Water+
compoundscis-PtAm2CI2K[PtAmCI3]PorfiromycinRoseolic
0.1
NaCI0.15
5M
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+
bromide5-FluorouracilFosfomycin2
DMSO+
DMSODMSOWaterDMSO+
-Deoxy-5-fluorouridineICR
'
191LuteoskyrinNogalomycinNovobiocinPiperazinedionePlatinum
Water+
Methanol+
MethanolDMSO+
([PtAmJCyQuinine
compounds
sulfateQuinomycin
ARifampicinRifamycin
(0.25)10(2.5)(50)100(0.6)(0.06-0.1)(0.1)0.0001(0.2)10Antitumor
Water0.15
NaCI+
M
Water+
MethanolWaterMethanolMethanolMethanolWaterToxic
SVStreptovaricin
CStreptovaricin
DTrimethoprimActinobolinActinomycin
noninducersMethanol+
DActinorubinAzalomycin
complexBluensomycin
F
sulfateCinerubin
BCinnamycinDNA
2824
MethanolWaterMethanolWater+
MethanolMethanolAmount
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43
BIA for Antitumor Agents
Table 2—Continued
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
DMSODMSODMSO-t-
3NSC28751
1485(Mitoxantrone)NSC
DMSODMSO+
279836Azacolutin
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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2825
R. K. Elespuru and R. J. White
Tabte 2—Continued
CompoundStatalon
Inter
action8SolventMethanol
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
ap
(Mgr1
plied
inducing
amount (/ig/spot)c
Antitumor
activity''+
1-10
1
5
1
1
0.1-1Minimal
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;
Ã-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).
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
(inducible transport system).
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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VOL.
43
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
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 reducÃ-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,
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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.
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
anandìì:
gilvocarcins V. M, and E. J. Antibiot. (Tokyo). 34: 1544-1555, 1981.
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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.
26.
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t
o.O*
Origin
|
Lane
1234S6
123456
*
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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VOL. 43
Biochemical Prophage Induction Assay: A Rapid Test for
Antitumor Agents That Interact with DNA
R. K. Elespuru and R. J. White
Cancer Res 1983;43:2819-2830.
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