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[CANCER RESEARCH 38, 3012-3018, September 1978)
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Biological and Biochemical Effects of Chartreusin on Mammalian Cells1
L. H. Li,2 T. D. Clark, L. L. Murch, J. M. Wooden, L. M. Pschigoda, and W. C. Krueger
Research Laboratories, The upjohn Company, Kalamazoo, Michigan 49001
ABSTRACT
Chartreusin (CT), an antibiotic produced by Strepto
myceschartreusls,was recentlyfoundto be activeagainst
experimental tumors B16 melanoma and L1210 and P388
leukemia. This report describes the correlation between
ftsbiologicaland biochemicaleffectson mouseL1210and
P388leukemIa.
CT was growthInhibitoryto L1210cells and other mam
malian cells tested (drug concentrationrequired for 50%
inhibition of cell growth in culture, between 0.22 and 0.67
@ig/ml).
The inhibitionof L1210cell growthwas rapid and
dose dependent. The inhibftioncould not be eliminated
after a 30-mmexposureto 2.5-@tg/mldrugdoses and was
not affected by the simultaneous addftlon of various
metabolites. CT lnhibfted RNA synthesis greater than It
did DNA synthesisand had least effect on proteinsynthe
sis. At 2.5 ag/mI It inhibited about 60% RNA synthesis,
and the inhibitioncould no longerbe reversedafter a 30mm exposure to the drug. Again, this was a dose- and
time-dependentphenomenon.These results closely cor
related to those of growth inhibItion, suggesting that
inhibition of polynucleotlde synthesis by CT played an
important role In its action against mouse leukemia.
CT showedno effect on deoxyrlbonucleosideor rlbonu
cleosidekinasesbut significantlyinhibitedDNA and RNA
polymerase isolated from L1210 leukemia in culture and
In vlvo. The inhibItion of highly purified DNA polymerase
logical and biochemical effects on L1210leukemia growing
in culture in an attempt to elucidate its mechanism of
action. The structure of CT is shown in Chart 1.
MATERIALS AND METHODS
Materials. CT was suppliedby The UpjohnCo., Kalama
zoo, Mich. [methy!-14C]dThd(53 @Ci/mmoI)
was obtained
from Amersham/Searle Corp., Arlington Heights, Ill.; [2,83H]adenosine (30 to 50 Ci/mmol), [5-3H]uridine (27 Ci/
mmol), [methyl-3H]dThd(2.2 Ci/mmol), [8,5-3H]dGTP(20to
50 Ci/mmol), [methyl-3H]dTTP (10 to 20 Ci/mmol), [53H]UTP(>20 Ci/mmol), and DL-[1-'@CJleucine
(40to 60 mCi/
mmol) were purchased from New England Nuclear, Boston,
Mass.; Escherichia coli, RNA (16S + 235 mixture), and
poly(dA-dT)were supplied by Miles Laboratories, Inc., Elk
hart, md.; poly(dG-dC)was purchased from P-L Biochemi
cals, Milwaukee, Wis.; calf Ihymus DNA and BSA were
obtained from Calbiochem, San Diego, Calif. , and Armour
Pharmaceutical Co., Kanakee, Ill., respectively. a-Amanitin
and yeast ANA were purchased from Sigma Chemical Co.,
St. Louis, Mo.; APMI 1629 and 1634 media were supplied
by Associated Biomedic Systems, Buffalo, N. V.
MammalianCell Growthin Cufture.The basalmediafor
growing mouse leukemia cells, human acute myelogenous
leukemia RPMI6410, and human normal lymphocyte RPMI
1788 were APMI 1634 with 5% fetal calf serum, RPMI 1634
with 10% fetal calf serum, and APMI 1629 with 10% fetal
a and RNA polymers..
II by CT varied wIth the template
calf serum and 1% human serum, respectively.A mixture of
used. The latter correlated well wIth the interactionbe penicillin (0.1 mg/mI of medium) and slreplomycin (50 @g/
tween CT and DNA polymers such as calf thymus, DNA, ml of medium) was added to each of the basal media.
poly(dA-dT)- poly(dT-dA), and poly(dG-dC)'(poly(dC-dG),
For growth studies 4 ml of cells (approximately 5 x 10@
demonstratedby circular dichroismmeasurements,sug cells/mi for L1210 cells and 1 x 10@cells/mi for either
gesting that the biochemicaland biologicalactivities of human acute myelogenous leukemia RPMI 6410 or human
CT were manifestedin part by its bindingto DNA. CT also lymphocyte RPMI 1788cells) were pipeled into each sterile
caused significantdamage to DNA.
plastic culture tube with screw cap (16 x 125 mm; Falcon
Plastics, Oxnard, Calif.). The experiment was initiated by
INTRODUCTION
the addition of 0.5 ml of medium, metaboliles, and/or drug.
Cell number was determined with a Couller counter (Coul
CT3(NSC5159), an antibiotic produced by Streptomyces ler Electronics, Hialeah, Fla.). Three days later, after incu
chartreusis (10), was recently found to be active against
balion at 37°,the ID50and drug concentration required for
several experimental tumors, such as B16 melanoma and 90% inhibition of cell growth were calculated (5).
L1210and P388leukemia (14), and is currently undergoing
For time-course studies logarithmically growing L1210
preclinical evaluation by the National Cancer Institute. This cells (approximately 5 x 10@cells/mi) were incubated with
report primarily describes the correlation between its bio
CT for a given time (30 to 120 mm). Al the termination of
each incubation, drug was removed by centrifugalion; cells
I This
investigation
was supported
in part by Contract
N01-CM-43753,
were
washed twice and then resuspendedin fresh medium
Division of Cancer Treatment, National Cancer Institute, NIH, Department of
at
a
concentration
of 5 x 10@
cells/mI. Fiveml of the washed
Health, Education, and Welfare.
2 To
whom
requests
for
reprints
should
be
addressed
, at
Cancer
Re
search, The Upjohn Co., Kalamazoo, Mich. 49001.
S The
abbreviations
dT), poly(dA-dT)
used
are:
CT,
chartreusln;
‘
poly(dT•dA); poly(dG-dC),
dThd,
poly(dG-dC).
thymidine;
poly(dA
poly(dC-dG);
BSA,
bovine serum albumin; RPMI, Roswell Park Memorial Institute; IDa,,drug
concentration required for 50% inhibition of cell growth in culture; CD,
circular dichroism.
Received November 5, 1976; accepted June 16, 1978.
3012
cellswere pipeledintoeach culturetubeand incubatedat
370
for
3
days.
Cell
number
was
determined.
Macromolecular Synthesis. Cells used in the incorpora
lion studies were in the logarithmic growth phase. Cells (23
ml)were incubatedat37°
simultaneously
with1 ml ofCT at
a given concentration and 1 ml of a labeled melabolile such
CANCER RESEARCH VOL. 38
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CT and Mammalian Cells
system are described in Table 2. The initial rate of enzy
H3C
HO
—0
0—
CH3
@
—
0
D-FUCOSE
/\
OH
@,
@
CH3
D-DIGITALOSE
HO..
maticreaction
(cpm/min)was obtainedfrom linearregres
sion analysis. The percentage of inhibition was calculated
by comparing the reaction rates with and without addition
of drug.
The preparation of cell-free extract from L1210 cells
maintainedinculturewas reported(12).Thisextractwas
used for assaying
activity
of either
DNA polymerase
or
nucleoside kinases. The assay procedures for DNA polym
Chart 1. Structure of CT (NSC 5159), C@H@O,4.
erase dThd, and uridine kinases were described elsewhere
(12, 13).
as deoxyribonucleoside, ribonucleoside (2.5 @Ci/12@moI/ The isolation of intact liver nuclei from either normal or
ml of cells), or DL-[i-'4Cjleucine (0.5 @Ci/200@moI/ml)
for leukemic (5 days after inoculation of 10@ascites cells/
up to 4 hr with gentle shaking. Aliquots (6 ml) removed at mouse) CD2F1mice used as source of ANA polymerasewas
different limes (1, 2, and 4 hr) were then pipeted into a essentially according to the procedure of Widnell and Tala
centrifuge lube containing 1 ml of a corresponding unla
(17) except that dithiolhreitol (1 @mol/ml)and spermine
beled melabolite at 10 mg/mI and kept at 4°.One-half ml of phosphate (0.25 @moi/mI)were added to the isolation
the aliquols was used for cell counting, and 2 ml were medium further to protect the nuclei. The ANA polymerase
applied onto a glass fiber paper disc (Whatman GF/C; 2.4 activity was determined by the method described by Bab
cm presoaked in saturated phosphate) supported by a cock and Aich (1). Actinomycin D was used as a positive
Millipore 3025sampling manifold (Millipore Corp., Bedford, control.
Mass.). Samples (in triplicate) were then washed 4 times
Purificationof DNA Polymerases.DNApolymerasea of
with 4 ml of ice-cold 10% trichloroacetic acid and 3 times cultured L1210 cells was purified according to the proce
with 4 ml of absolute ethanol. For DNAand ANAextraction, dure of Grahamet al. (7) with some modifications. Briefly,
the dried discs were placed individually in the counting 12 g of Li 210 cells were sonically dispersed and extracted
vials and were extracted with 0.5 ml of 0.5 Nperchloric acid twice with 0.8 M KCI buffer. After ammonium sulfate frac
at 70°
for 60 mm. After cooling, 15 ml of Diotol (Burdick & tionation (65%saturation), the aliquot was dialyzed against
Jackson Laboratories, Inc., Muskegon, Mich.) were added. buffer composed of 50 mM Tris-HCI (pH 7.8), 1 mM dithio
For protein uptake each disc was placed in a vial, 5 ml of threitol, 1 mM EDTA, and 10% glycerol and was passed
Diotol were added, and the radioactivity was determined through a DEAE-cellulose column that was eluled with
(ii). The specificactivityfor the incorporationof a given buffer containing 0.3 M KCI to remove nucleic acids. Frac
labeled metabolile was expressed as cpm/10 cells. The lions containing DNA polymerase activity were pooled,
initial
rateof incorporation
(cpm/108cells/mm)was ob
dialyzed, applied onto a phosphocellulose column, and
tamed from linear regression analysis of the specific aclivi
eluled with buffer containing 0 to 0.05 M KCI. After the
liesfor 30-,60- and 120-mm incubation.
The Pearson's removal of most of the nonenzymatic proteins, the column
coefficients (r) in this analysis were generally around 0.99. was further eluted with a buffered KCI gradient from 0.1 to
The percentage of inhibition was calculated from the initial 0.7 M. Although both DNA polymerases a and p were
rate of incorporation with and without addition of drug.
collected, only DNApolymerase a was used for subsequent
For lime-course studies 1 ml of solvent or CT at a given studies.
concentration was added to 99ml of L1210 cells (approxi
Assayof DNA Polymerasea ActivIty.The enzymeachy
malely 5 x 10@ cells/mi)
and was mixed well.Five-mi ity was assayed in 100-p.l volumes containing 5 @gof
aliquots were pipeled into each culture tube and incubated activated calf thymus DNAor 0.1 A2@unit of poly(dA-dT) or
at37°for30,60,and 120 mm. At thetermination
of each
poly(dG-dC); 50 mM Tris-HCI buffer (pH 7.8); 1.5 mM MgCI2;
incubation, drug was removed by centrifugation and cells
were washed twice with 5 ml of medium, and were resus
pended in 4 ml of medium (37°).One ml of a labeled
melabolite was added to the cells, and the aliquols were
then incubated at 37°for 40 mm. The cell pellet was
collected on paper discs, and the radioactivity was deter
25 mM KCI; 5 mM dithiothreitol; 80 mM each dATP, dCTP,
and dGTP; 7 @&M
[3H]TTP(7400 cpm/pmol); and 25 @l
of
DNA polymerase a. The reaction was incubated at 37°for
20 and 40 mm. After incubation acid-insoluble material was
collected on a glass fiber paper disc, and radioactivity was
determined as described previously.
mined.
Purificationof RNA Polymerases. ANA polymerasesI
Enzyme Assays. The isolationof DNA polymeraseof and II were purified from isolated normal mouse liver nuclei
ascites cells of BALB/c x DBA/2 F1(hereaftercalled CD2F1) (17) essentially according to the procedure of Blair (3).
mice carrying L1210 leukemia was essentially according to Briefly, nuclear ANA polymerase was solubilized by sonic
the procedure of Manlsavenos(13).The 105,000x g super extraction of the nuclei in buffer [50 mM Tris-HCI (pH 7.9),
natant was subjected to acid precipitation (pH 5) and 0.1 mM EDTA,0.5 mM dithiothreitol, 5 mM MgCl2,and 25%
ammonium sulfate fractionation. This enzymewas concen
glycerol] containing 0.3 M (NH@),SO4.
The enzyme was
trated in the fraction between 30 and 40% saturation. The precipitated with (NH@SO4(0.42g/ml of extract), dissolved
assay procedure was primarily that described by Mantsav in buffer containing 0.05 M (NH@SO4, and dialyzed with
enos; however, the concentrations of Tris-HCI buffer and buffer. After centrifugation at 105,000 x g for 1 hr, the
deoxyribonucleotide triphosphates were altered, and EDTA supernatant was applied onto DEAE-SephadexA-25column
was added to the reaction system. Details of the assay and eluted with a linear gradient of buffered (NH@SO4
SEPTEMBER
1978
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3013
L. H. Li et a!.
solution (0.05 to 0.5 M). Although both ANA polymerases I
and II were isolated, Enzyme I was relatively a minor
component in our enzyme preparation.
Assay of RNA PolymeraseII Activity.The enzymeachy
the drug for 3 days. Human acute myelogenous leukemia
RPMI 64i0 was slightly more inhibited by CT than was
human lymphocyte RPMI 1788. Similarly, mouse leukemia
P388 ascites
cells growing
in culture
in the presence
of
mercaptoethanol (iO @mol/ml)
appearedto be slightly more
thymus DNAor 0.2 A2ssunit of poly(dA-dT)or poly(dG-dC); sensitive to CT than were mouse leukemia Li2iO asciles
62.5 mM Tris-HCI (pH 7.9); 2 mM MnCI2;85 mM (NH@SO4; cells. The difference was, however, not dramatic.
0.25 mM each ATP, CTP, and GTP; 2.5 @M
[5-3HIUTP(20
The growth inhibition of cultured L12i0 cells by CT was
@Ci/pmol);and iO to 20 @lof ANA polymerase II. The not preventedor reversedby the simultaneous addition of a
reactionmixturewas incubatedat 37°for20 mm, acid purine-pyrimidine mixture (75 @g/ml
of each component),
insoluble fraction was collected, and the radioactivity was by tricarboxylic acid cycle intermediates (125 p.g/ml), by a
ity was assayed in 200-pi
volumes
containing
40 mg calf
determined.
vitamin
Interaction with Macromolecules.The interaction be
tween CT and calf lhymus DNA,yeast ANA,Escherichia co!i
RNA (i6 and 23S mixture), BSA, poly(dA-dT), or poly(dG
dC) was studied at 25°by absorbance measurementson a
Cary 15 speclropholometer and by CD measurementson a
Eagle's basal medium), or by an amino acid mixture (5-fold
concentration of Eagle's basal medium) (data not pre
senled).
When cultured Li2iO cells were exposed to different
Cary60 speclropolarimelerwith a Model6003CD attach
the drug was removed by cenlrifugation and washings, the
growth inhibition by CT could no longer be eliminated. The
phenomenonwas, however, dose related. Atypical result is
illustrated in Chart 2. Al iO pg/mI a marked inhibition
(approximately 60%) was observed after a mere 30-mm
exposure, but at i @g/mlthe inhibitory effect was not
menl (Varian Associates, Palo Alto, Calif.). The Cary 60 was
calibrated with 10-camphorsulfonic acid (9).
The interaction between CT and calf thymus, poly(dA-dT),
or poly(dG-dC)
was also determined
by the change of Tm of
DNA polymers in a recording Ihermospectrophotomeler
(Gilford Instruments Laboratories, Inc., Oberlin, Ohio).
DNA Damage. The DNAdamagepossiblycausedby CT
was determined according to the alkaline eluhionprocedure
of Swenberg et al. (16). Briefly, cultured Li2iO cells were
labeled with [‘4C]dThd
(0.01 to 0.04 @Ci/ml
of cells) at 37°
(and coenzyme)
mixture
(5-fold
concentration
of
concentrations
ofCT fora givenperiod(30to240 mm) and
detectedeven aftera 120-mm incubation
withcells.
DrugEffectson MacromolecularSynthesis.The macro
molecular synthesis was measured as the incorporation of
radioactive precursors into the acid-insoluble fraction. The
results (Chart 3) indicate that RNAsynthesis (measured as
for 24 hr,and the radioactive
medium was removed by the incorporation of adenosine or uridine) appeared to be
centrifugation at 800 x g for 5 mm. The cells were resus more inhibited by CT than by DNA synthesis (measured as
pended in regular medium, incubated for another 4 to 24 the incorporation of dThd). Protein synthesis(incorporation
hr,and thenexposed toCT fora givenperiod(30mm, 2 hr, of leucine) was least affected. If the percentageof inhibition
and 4 hr). Following that the cells were collected by cenlrif
was plotted against the logarithm of drug concentrations
ugahion, resuspended in 0.5 ml of medium (2 ho 4 x 10@ up to 10 pg/mI, the lD@'scould be calculated. The lD50'sfor
cells/mi), and subjected to alkaline elution analysis as adenosine and uridine incorporation were 2.3 and 2.1 @g/
describedelsewhere(16).
ml, respectively,
4.4 @g/mlfor dThd incorporation,
and
about10 @g/mlforleucineincorporation.
RESULTS
Effect of CT on Mammalian Cell Growthin Culture. CT
was growth inhibitory to several mammalian cells tested
(Table 1). The lD@valueof the drug ranged between0.22 to
0.67 @g/mlwhen cells were continuously in contact with
When Li2iO cells were exposed to CT (2.5 or iO p@g/mI)
fora shortperiod(30,60,and i20 mm) and thedrug was
removed by washing, the residual effect of CT was mea
Table 1
Inhibition of mammalian cell growth in culture by CT
90
(@cg/mI)Human Cellsso
(@g/ml)a,b
17880.622.9Human
lymphocyte RPMI
leukemia0.350.78RPMI
acute myelogenous
6410Mouse
L12100.512.65Mouse
leukemia
ascitesc0.67>1.0Mouse
leukemiaL12i0
leukemiaP388ascites@'0.220.9
a Cells (approximately 5 x i0@cells/mI for 3 mouse cell lines, 1
x i0@ cells/mI for 2 human cell lines) were incubated with CT at
various concentrations
at 37°for 3 days, and the cell number was
determined.
b 1090, drug
concentration
required
for a 90% inhibition
of cell
growth.
ascites
cells
P388 leukemia
C The
were
obtained
cultured
from
in basal
mice
carrying
medium
either
(RPMI
L1210
or
1634 and 5%
fetal calf serum) containing 2-mercaploethanol (10 @@moI/ml)
ac
cording to the procedure described by Day and Blowers (6).
3014
60
128
180
CONTACTTiME(Nil)AT 37•
Chart 2. Time-coursestudy of growth inhibition of in vitro L1210cells by
CT. Cells (approximately5 x 10@cells/mI) were incubated with drug of
different concentrationsfor a givenperiod(30to 240mm).At the termination
of each incubation,drug was removedby centrifugation;cells werewashed
twice and then resuspended in fresh medium at about 5 x 10@cells/mI. Five
ml of these washed cells were pipeted into each culture tube and incubated
at 37°for 3 days. Cell number was determined.
CANCER RESEARCH VOL. 38
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CT and Mammalian Cells
drug was removed,the DNAand ANAsynthesisdid not fully
recover 24 hr later. Inhibition was 30% lower (Chart 5),
which might reflect the irreversible damage, possibly cell
death, caused by the drug.
Inhibition of Enzymes by CT. In an attempt to understand
the mechanism of the inhibition of macromolecular synthe
sis, the effect of CT on several enzymes involved in DNA
and ANA synthesis was investigated. At up to 100 @g/ml,
CT was not inhibitory to either dThd or uridine kinase of
cultured L1210 cells (data not presented) but significantly
C
S
inhibited DNA polymerase of both cultured and ascites
Of., Cs.ceutratlsu(pg/mi)
Chart 3. Inhibition of macromolecular synthesis of L1210 cells in culture
by CT. Cells (approximately 5 x 10@cells/mI) were incubated at 37°simulta
neously with CT of different concentrations and a labeled metabolite for up
L12i0 leukemic cells and ANA polymerase of liver nuclei
isolated from either normal or leukemic CD2F1mice (Table
2). The percentage of inhibition of DNA polymerase of
cultured cells was derived from the initial rate of reaction
forup to120-mm incubation.
A typical
result
isillustrated
in
Chart 6. AT 25 @g/mlCT inhibited approximately 50 and
60%of DNApolymeraseof cultured and ascites L1210cells,
counting and radioactivity determination. Percentage of inhibition was
calculated from the initial rate of incorporation (cpm/10 cells).
respectively (Table 2). Twenty-eight % inhibition of DNA
polymeraseof ascites Li 210 cells was detected at a concen
tration as low as 5 @g/ml.CT also showed significant
inhibitory effect on ANA polymerase of liver nuclei isolated
from either normal CD2F1or L1210leukemic mice (Table 2).
At 50 p@g/mlCT inhibited about 30% of the enzyme activity
of normal mice and approximately 4O%of that from leu
kemic mice.
interactionbetweenCT and Macromolecules.The inter
action between CT and calf thymus DNA,heal-activatedcalf
thymus DNA, E. coli rANA, yeast lANA, BSA, poly(dA-dT),
or poly(dG-dC)was studied by absorption and CD measure
ments (Chart 7). Difference spectra were determined by
subtracting the spectra of CT and macromolecule in sepa
rate cells from the spectrum of the mixture of CT and
macromolecule
obtained in the same cell. Hence, a nega
CONTACTT@( (Me)AT 37
Chart 4. Time-coursestudyof the inhibition of macromolecularsynthesis hivedifference response means that the absorbance or CD
of L1210cells in culture by CT. Cells (approximately5 x 10 cells/mI)were of the mixture of CT and macromolecule is less than the
incubated with drug of different concentrations (2.5 and 10 @.cg/ml)
for 30 to
sum of the absorbance or CD of the components. Yeast
120mm and drug was removedby centrifugationand washings.Cellswere
and BSAat up ho3.5 mg/mI showed little
then suspendedin fresh mediumand pulse labeled(400mm) with a given lANA at 40 @g/ml
radioactive metabolite (thymidine, uridine, or leucine). The inhibition was
or no interaction with CT (1.54 x 10@M).The CDdifference
to 4 hr. Aliquots(6ml)wereremovedat differenttimes(1, 2. and4 hr)for cell
calculated from the specific activity of incorporation (cpm/10 cells) of drug
treated as compared to those of the controls (no drug).
spectra between 500 and 218 nm for both tANA and BSA
sured by pulse-labeling (40 mm) with the respective radio
active precursors (dThd, uridine, or leucine). These results
ONA CYNTHISIS
are shown in Chart 4. At 2.5 pg/mI the drug effect on
protein synthesis was not detected until CT was in contact
a
with cells for more than 1 hr, but its effect on RNAsynthesis
a
could no longer be eliminated after a 30—mm
contact period,
and an approximate 20% inhibition was observed. The
inhibition increased to 50%after a 2-hr incubation.
CHART0005NI
= 10pg/mI
Aesulls in Chart 4A indicate that at 2.5 pg/mI drug effects
on DNAand protein synthesis lagged behind those on RNA
synthesis. This was, however, a dose-related phenomenon.
Al 10 @g/ml(Chart 4B), the inhibition of protein synthesis
by CT also persisted after a mere 30-mm contact time.
IC
NOUNS
AflENTHENENOVAL
OF0000
Approximately 20% inhibition was noted, and the inhibition
Chart
5.
Recovery
of
DNA
and
RNA
synthesis
in vitro L1210 cells after
increased with time. A 50% inhibition was recorded at the the removalof CT. Cellswere Incubatedwith CTof(10
@g/ml)
at 37°
for 2 hr,
end of a 2-hr period. Again, ANA synthesis was more and drug was removedby centrifugation and washings. Cells were then
inhibited than DNA synthesis, and protein synthesis was suspendedin freshmediumand pulse-labeled(40mm)with (methyl-3H)dThd
or [‘H)urldlneat 0, 4, and 24 hr after the removal of drug. The inhibition was
least affected.
calculated from the specific activity of incorporation (cpm/10' cells) of drug
When Li2iO cells were exposed to CT for 2 hr and the treated as compared to those of the controls (no drug).
SEPTEMBER
1978
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L. H. Li et a!.
2
Inhibition oTable
CTDrug
f DNA and RNA polymerase of L1210 leukemia by
inhibitionDNA
of
poIymerase@° polymerasebNormal
RNA
con
centration
Cultured
(@g/ml)%
nuclei5 Li2lOcells
25
Ascites
Li210
cells
47-49
CD2F1
liver nuclei
Leukemic
Li210 liver
34
42
60-62
78
56-65
50
74-86
100
200
25028
79-92
96
a Percentage of inhibition of DNA polymerase was calculated
from the initial rate of incorporation of [3H)TTP. For the cultured
Li2iO cells,the rateof enzymereactionwaslinearupto 120mm.
The crude cell-free extract was used as the enzyme source, and
calf thymus DNA was used as the template. The assay procedure
wasdescribedelsewhere(ii). ForascitesLi2iO cellsthe rateof
enzymereaction was linear for up to 60 mm. The reaction mixture
(1 ml) contained 0.1 mmol Tris-HCIbuffer (pH8), 16 .tmol M9CI2,1
@mol2-mercaptoethanol,
4
@molEDTA, about 250
concentration by a factor of 2 over that given in Chart 7
(data not presented). The absorbance and CD difference
spectra of heat-activated DNA and CT as well as the CD
difference spectrum of E. coli rANA and CT were compara
ble to those of calf Ihymus DNA and CT (Chart 7), but the
absorbance difference spectrum of E. coli rRNA and CT
was less (data not presented).
The interaction between CT and calf thymus DNA,
poly(dA-dT), or poly(dG-dC) was also investigated by Tm
determination. Surprisingly, the Tmof these 3 macromole
cules were not altered in the presence of CT (iO pg/mI)
(data not presented).
The interaction between CT and DNAwas further investi
gated by determining the drug effect on DNA (or chromo
some) structure and integrity. After the incubation of
[“C]dThd-labeled
cultured Li2iO cells with CT for 30 mm,
2 hr, and 4 hr, possible chromosome damage was assessed
by alkaline elution method (16). The results (Table 3) mdi
cate that CT did cause DNA damage in cultured Li210 cells,
and the effects were dose and lime dependent. Al 2.5 @g/
ml CT caused little damage hoDNAafter a 30-mmexposure
@genzyme
to the cells, but it caused over 50% damage after a 4-hr
protein,0.1 @.smol
eachdATP,dCTP,and dGTP,and 0.03 @.smolexposure.
[3H]dTTP (167 mCi/mmol) were added when heat-activated calf
InhibItion of DNA Polymerase a and RNA Polymerase II
thymusDNA(50 @.sg)
wasusedasthetemplate;0.1 @tmol
dATPand
0.03 .tmol [3H]dTTP were added when poly(dA-dT) (50
used as template;
0.1
@g)was
@moldCTP and 0.3 @.smol
[3H]dGTP (87 mCi/
mmol) were added when poly(dG-dC) (50 @g)
was used as template.
b RNA polymerase
of liver nuclei
was assayed
according
to the
procedureof Babcockand Rich (1). The assaysystem(0.1 ml)
contained10 nmolTris-HCIbuffer (pH7.9);0.22mol MnCI2;0.1
by CT (Template Study).With calf IhymusDNA, heat-acti
vated calf-thymus DNA, poly(dA-dT), or poly(dG-dC) as the
template, the effects of CT on the highly purified DNA
polymerase a of cultured Li2iO cells as well as on RNA
polymerase II of mouse liver were compared.
For DNA
polymerase a, 2 representativeexperiments were reported
valekinase;10i.tgeachof ATP,CTP,andGTP;0.8nmol(0.5 @g) here, and the results (Table 4) indicate that the enzyme
nmol dithiothreitol;
0.5 nmol phosphoenolpyruvate; 0.5 @.tg
pyru
UTP and [3H]UTP (2 mCi/nmol); and was incubated with liver nuclei
(2.5 x [email protected] x i0)at37°fori0min.
1600
activity was inhibited about 20 to 30% by CT at 2.5 @ig/ml
and about 50% at 10 @g/ml.There appeared to be little
difference in inhibition whether calf thymus DNA,denatured
calf thymus DNA, or poly(dA-dT)was used as the template.
However, inhibition was somewhat lower when poly(dG-dC)
was used as the template.
0 pg/mI
The pattern of inhibition of RNApolymeraseII by CT was,
a
$00
however, different from that of DNA polymerase a. The
400
results (Table5) indicate that CT inhibited RNApolymerase
II hoa greater extent when poly(dG-dC)or poly(dA-dT)was
used than when calf Ihymus DNAwas used as the template.
At 2.5 pg/mI the percentage of inhibition was 5, 46, and 87
log pg/mi
I
30
$0
go
m
I
ISO
TINE (MIN) AT 37
with calf thymus DNA, poly(dA-dT), and poly(dG-dC) as the
template,
respectively.
Chart 6. Inhibition of DNA polymerase of in vitro leukemic L1210 cells by
CT. The assayprocedurewas describedelsewhere(7). The percentageof
inhibition was calculated from the initial rate of reaction of drug treated as
comparedto thoseof the control (nodrug).
DISCUSSION
Since CTwas inhibitory hoseveralmammaliancells tested
were zero within experimental error (i .0 millidegree elliptic
(Table 1), cultured L1210 leukemic cells were chosen for
ity), and the maximum absorbance difference response was
the subsequent studies. The inhibition of cultured L12i0
small, about 0.05 and 0.03 absorbance units for lANA and
BSA, respectively (data not presented). In contrast to the
behavior of these macromolecules, calf thymus DNA, heat
activated calf thymus DNA, E. coli rRNA, poly(dA-dT), or
poly(dG-dC)significantly altered the spectrum absorbance
and CDof CT. The results (Chart 7) showed that, in general,
poly(dG-dC) and poly(dA-dT) show a larger CD difference
spectrum than does calf thymus DNA on an equimolar
basis. No significant change in the difference spectra of
calf thymus DNA was detected by increasing the DNA
cell growth by CT could not be prevented or reversed by the
simultaneous addition of a variety of metabolihes. Hence,
no apparent antidote would be available for this drug. Time
course study (Chart 2) indicated that the action of CT was
rather rapid but required a higher concentration (>10 @g/
3016
ml) ho achieve complete inhibition of cell growth that
correlated closely to the results of cell kill by CT (i4).
CT appeared ho inhibit RNA synthesis more than DNA
synthesis, and protein synthesis was least affected (Chart
3). The incorporation
of radioactive-labeled
adenosine or
CANCER RESEARCH VOL. 38
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CT and Mammalian Cells
2
5.0cmCELLS
A
I
0
C-)
z
0
Cd)
>-
12
0.5 cm CELLS
-a
w
C-)
‘“4
“a
CD
@
-i
200
230
250
270
290
310
420
WAVELENGTH
14NM
WAVELENGTh
14NM
Chart7. InteractionbetweenCTand DNApolymers.Absorbance(upper)and circular dlchroismdifferencespectra(mixturespectrumminusspectrumof
separate components of CT (10 @.tg/mlor 1.54 x 10 t@1)
and calf thymus DNA (-.-). poly(dA-dT) (- - - -), and poly(dG-dC) (—), all 1.02 x 10' M for
nucleotides, in 0.01 N phosphate buffer (pH 7.2). Elliptlcity has been defined as arc tangent of the ratio of minor to major axes of the elliptical polarization.
Table3
DNAdamagestudies with CT
Inhibition of highly
2
1
L1210Drug polymerasea
purified
CDNA
ells by CTof
cultured
Inhi
centra
lion (@.tg/
age'@300(control)
ml)Experi
(mm)CTcon
% DNA
activity
dam
dam
re
re
tained@'ment age―Experitamned@'ment
44.216.7
Rates'
% in
(cpm/
hibi
tionmm)
hibi
46
127
54174 79
27
% in
(ILg/(cpm/
46.8
2.5
27.62400
10.063.3
DNA0
19(denatured)10.047
2.582
42Poly(dA-dT)0
46.126.6
2.5
10.079.6
a DNA damage
trationRate0
tionCalf
Templateml)mm)
3
thymus
114
DNA0
2.5
55Calfthymus
10.021 98
73.1
62.95.1
43.380.0
Experiment 1Experiment
% DNA
activity
64.9
2.5
21.31200
10.078.0
2cen
con
%radio
%radio
bi
tion
time
32.3
27.759.4
was assessed
40.5
37.350.2
65.281.3
by alkaline
elution
66
12
54.0
method
(16).
36PoIy(dG-dC)0
2.5
10.01
85
64
24
4370
49
45
30
The procedurewas describedbriefly in the text. Percentageof
radioactivity
@
retained
on the membrane
was calculated
cpmretainedonthe membrane
as follows:
cpm on membrane + cpm in filtrate
@
b
2.5
10.06.6
100
5.8
4.7
12
28
a The condition for the enzyme assay was described in the text.
Reaction was carried out at 37°for 20 and 40 mm. The initial rates
DNA damage = (1 —
% radioactivity retained on membranefrom drug-treated cells)
% radioactivity retainedon membranefrom control cells
x 100
uridine was inhibited more or less equally by CT, suggesting
that CT might block the final stages of polynucleotide
synthesis. This hypothesis was indirectly supported by the
evidence that neither thymidine kinase nor uridine kinase
activity was affected by CT at concentrations of up to 100
pg/mI. Time-course study showed that the inhibition pal
tern of macromolecular synthesis (Chart 4) paralleled that
of cell growth (Chart 2), suggesting that these biochemical
of reactionwereanalyzedbylinearre9ressionandcelllinespassed
throughtheorigin.ThePearsoncoefficients(r)werebetween0.99
and 1.00.
effects of CT are an important aspect in its action against
L1210 cells and possibly other mammalian cells.
Further studies indicated that CT significantly inhibited
DNA polymerase of both in vitro and in vivo ascites leukemic
L1210 cells and RNA polymerase of liver nuclei isolated
from either normal CDF1 or leukemic L1210 mice (Table 2).
In an attempt to understand the mechanism of inhibition of
DNAand RNApolymerase,the possibleinteractionbetween
SEPTEMBER 1978
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1978 American Association for Cancer Research.
3017
L. H. Li et a!.
poly(dA-dT) rather than when calf thymus DNA was used as
the template (Table 5). This suggested that the enzyme
inhibition
was inpartmediatedby CT bindingto DNA.
the text. Reactions were carried out at 37°for 20 mm.
As evidenced by the results obtained, the action of CT is
inhibitionCalf
certainly a complex one. In addition ho CT binding to E. co!i
CT concen
rRNA as described here and to reticulocyte ribosome as
thymusml)DNAPoIy(dA-dT)PoIy(dG-dC)2.5546±7°8710.01065±2210050.052±1379±
tration
(/Lg/%
reported by Gregg and Heintz (8), we also found that CT
interacted with a Variety of DNA polymers, causing consid
erable DNA (chromosome) damage (Table 3). The inhibition
of cell growth and cell kill (i4) by CT paralleled that of
9100a
polynucleotide synthesis, particularly RNA synthesis which,
Mean
± S.E.(3
to 5 experiments).
in turn,correlated
closelyho thatof highlypurified
RNA
polymerase
II.
The
enzyme
inhibition
correlated
with
the
CT and macromolecules was investigated by means of
ability
of
CT
to
bind
ho
different
DNA
polymers.
The
under
difference spectrum determination with spectrum absorp
line of these findings was that, although the mechanism of
lion and CD measurements. Although CT did not show
action of CT could not be precisely pinpointed, its ability ho
significant interaction with either yeast RNA or bovine
interact with DNA and its efffecls on the DNA structure and
serum albumin, it did show small but significant interaction
integrity would certainly play a major role in determining its
with E. COli rRNA. These results might support the findings
biochemical
and cytotoxic effects as well as its anhilumor
of Gregg and Heintz (8) that CT bound to the reticulocyte
ribosome and inhibited eukaryohic ammnoacyltransferase I activity.
Table 5
Inhibition of RNA polymerase II of mouse liver by CT
The condition for assaying RNA polymerase II was described in
activity. In addition, we found that CT also showed signifi
cant interaction with calf thymus DNA as well as poly(dG
dC) or poly(dA-dT) (Chart 7).
The interaction between CT and DNA polymers was also
evaluated with Tmdetermination. To our surprise no appar
ent effect on the Tm of calf Ihymus DNA, poly(dA-dT), or
poly (dG-dC) was observed . Later we found that the inter
action was thermally labile. For instance, the difference CD
band at435 nm decreasedupon heatingthe mixtureofCT
and the polymer and was restored upon cooling of the
mixture.
This result
might
explain
why the Tm'5 of these
DNA polymers were not significantly affected by CT. Appar
enhly, the binding of CT hothe DNA polymer was dissociated
beforetheTm'Softhesepolymerswere reached.Hence,the
interaction between DNA polymers and CT appeared ho be
relatively weak as compared to that with, for example,
actinomycin
0.
ACKNOWLEDGMENTS
WethankJ. W. Culp for her skillful technicalassistanceand K. Franzand
N. D. Likam for their help in the preparation of this paper.
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inhibited
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Current evidence suggests that RNA polymerase I was 12. Li, L. H., Fraser, T. J., Olin, E. J., and Bhuyan, B. K. Action of
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(i5),
only
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3018
CANCER RESEARCH VOL. 38
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Biological and Biochemical Effects of Chartreusin on
Mammalian Cells
L. H. Li, T. D. Clark, L. L. Murch, et al.
Cancer Res 1978;38:3012-3018.
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