- Email: [email protected]
On the action mechanism of mitomycin C
Vol. 6, No. 5, 1961
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ON THE ACTION MECHANISMOF MITOMYCIN C
By Yozo Nakata, Kiyoi
Nakata and Yukiya Sakamoto
Department of Biochemistry and Institute for Cancer Research, Osaka University Medical School, Osaka, Japan.
Received
November
13, 1961
Since Shiba -et al
reported
the anti-tumor
mitomycin C (MC), to have selective synthesis
of bacterial
have
been
done.
this
antibiotic
(Shiba -et -1 al
DNA
Among these studies,
1960).
paper we report
studies
Crystalline Tokyo.
have also
We
from the standpoint
Reich -et al
Determination 19.55).
(Okazaki -et al,
of DNA was carried
Tokyo.
after
were
assay (Hoff-Jbrgensen,
with venom phosphatase
Institute
deficient
strains
of Bacillus
mutant) by DNA from nutritional out according
The strain
DNase was isolated
oscillation
out by the method of
were determined as deoxyribo-
hydrolysis
of biochemically
was carried
1958).
Dr. Yiuga Saito,
of MC,
from the Kyowa Hakko Kogyo Co.,
by the microbiological
(tryptophan-less
(Spizizen,
In this
1958).
Transformation
pendent strain
DNA -in vivo
Acid soluble deoxyribonucleosides
(thgmidine)
that
mechanism.
"Masked" deoxyribonucleosides
aubtilis
reported
had the same result.
of the molecular
measured as thymidine
nucleoside
on the bio-
many investigations
19581,
on the mechanism of the action
MC was obtained
Burton (Burton,
1952).
effects
causes the rapid break down of bacterial
(Reich -et -9 al
Ltd.,
inhibitory
antibiotic,
of B.subtilis
of Applied
to the method of Spizizen were obtained
coli
of
B with sonic
(10 KC, 10 minutes) and then centrifugation 339
from
University
Microbiology,
from Escherichia
inde-
(lOC,OCC x g,
Vol. 6, No. 5,196l
BIOCHEMICAL
30 minutes). activity
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The eupernatant
liquid
The
of DNase was determined by measuring the release of ultra-
violet
absorbing material
extracted
from E.coli
and of acid-soluble
deoxyribose.
B with lysozyme treatment,
.repeated chloroform-octanol
salt
DNA was
extraction
are as follows:
E. coli
B cultured
shaking at 37'C in glucose-Simmons medium, was harvested
the logarithmic
phase of growth,
at 660 mp = 0.3) (1 &ml)
in similar
and incubated
at certain
intervals,
measured.
It
and
treatment.
The methods and results with
was used as enzyme source.
then resuspended (optical
shaking.
Aliquots
were taken
and the DNA and deoxyribonucleosides that
MC breaks down bacterial
and accumulates the "maskedIt deoxyribonucleosides
0
density
fresh medium in the presence of MC
at 37OC rith
is evident
at
10
20
30
were
DNA in vivo,
in the medium (Fig.
40 50 (min .I
Accumulation of llmaskede Fig.1. deoxyribonucleoside. "Masked" deoxyribonucleoside (0 - 01, Deoxyribonucleoside (0 - 0). The transforming when the bacteria
activity
(B. subtilis,
strain 340
of the DNA was greatly 116) were incubated
impaired with
1).
BIOCHEMICAL
Vol. 6, No. 5,196l
MC (1 pg/ml)
before
the DNA was prepared.
occurred
at the frequency
principle
(20 pg DNA/ml),
of this
Because transformation
of intact
the frequency
solution
with ethanol,the
suggested that cells
transformation
of one in 104 with the intact
transforming
decreased to less than 10 % with MC (Fig.
is known to be prevented
DNase, these results
in salt
Although
in the course of incubation
value
treatment
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2).
by treatment
with
the MC-DNAwas degraded by
with MC. When DNA was treated
with MC
at O°C for 20 minutes and then washed repeatedly activity
of transforming
0
principle
I
I
I
30
60
90
was not impaired. -
PREINCUBATIONTIMES (min.) Fig.2. Time course of impairment of transformating DNA by MC treatment of whole cells. MC alone had no effect activity
on DNAand also no effect
--in vitro.
When E. coli the specific
on DNase
,I B cells
activity
were preincubated
of DNase extracted
up to 180 % (Table 1). 341
with MC for 30 minutes
from the cells
was elevated
Vol. 6, No. 5,196l
BIOCHEMICAL
Table 1.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DNase activities
of E. coli
B
Systems
DNase activities
With untreated + none
enzyme 100 100
+ MC (50 pg) + protamine sulfate + protamine sulfate MC-pretreated
With
(100 Ng) (100 pg) + MC (50 pg)
28
30
enzyme
+ none
180
+ protamine sulfate
(100 pg) + MC (50 pg)
47
'Without enzyme + MC (200 pg) incubated
0
for 30 minutes at 37'C with 20 ug of DNA and
10m2Mof MgS04 in Verona1 buffer,
In a preliminary incorporation
of H3-thymidine
high concentration effects
on the activities report
degradation
found the degradation sulfate.
into
indicates
Amos demonstrated that of RNA by ribonuclease
that
in -- vivo
(Amos, 1961).
by protamine
inhibited
(Fig. it
actions
on DNAat
References Amos, H., Biochem. Biophys. Res. Communs., 2, 1 (1961). Burton, K., Biochem. J., 62, 315 (1956). E.,
Biochem. J.,
50, 400 (1952). 342
the cell-
3).
of the DNase activity.
Hoff-Jbrgensen,
He also
(Table 11, and to restore
(20 pg/ml)
suggested that MC exerts
MC has no
protamine sulfate
protamine sulfate
of E. co1i.B -in vitro
even at
kinase and polymerase.
of DNA by DN ase to be protected
the impaired ?'!I synthesis These results
system the
DNA was not inhibited
of deoxyribonucleotide
We also observed that
free DNase activity
the level
with the cell-free
of MC. This finding
In a recent protected
experiment
pH 7.4.
BIOCHEMICAL
Vol. 6, No. 5,196l
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DNA ($1 120 100 a0 60 40
20 01 -20 15
30
(min. 1
Fig. 3. DNA synthesis of E. coli B under the influence of MC and/or protamine sulfate. l
I 0 0
Okazaki,
Control (no addition) + MC (1 pg/ml) + Protamine sulfate (20 un/ml) + MC (1 &ml) + Protamine sulfate (20 &ml) ,
R. and Okazaki,
T.,
“.
Biochem. Biophys.
Acta,
g,
470
(1958).
Reich, E., Shatkin, 5,
608
A, J. and Tatum, E. L.,
Biochem. Biophys.
Acta,
(1960).
Shiba, S., Teraaaki, A., Taguchi, T., and Kawamata, J., 1, 179 (1958). Spizizen, J., Proc. Natl. Acad. SC%., s, 423 (1958).
343
Biken's
J.,
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ON THE ACTION MECHANISMOF MITOMYCIN C
By Yozo Nakata, Kiyoi
Nakata and Yukiya Sakamoto
Department of Biochemistry and Institute for Cancer Research, Osaka University Medical School, Osaka, Japan.
Received
November
13, 1961
Since Shiba -et al
reported
the anti-tumor
mitomycin C (MC), to have selective synthesis
of bacterial
have
been
done.
this
antibiotic
(Shiba -et -1 al
DNA
Among these studies,
1960).
paper we report
studies
Crystalline Tokyo.
have also
We
from the standpoint
Reich -et al
Determination 19.55).
(Okazaki -et al,
of DNA was carried
Tokyo.
after
were
assay (Hoff-Jbrgensen,
with venom phosphatase
Institute
deficient
strains
of Bacillus
mutant) by DNA from nutritional out according
The strain
DNase was isolated
oscillation
out by the method of
were determined as deoxyribo-
hydrolysis
of biochemically
was carried
1958).
Dr. Yiuga Saito,
of MC,
from the Kyowa Hakko Kogyo Co.,
by the microbiological
(tryptophan-less
(Spizizen,
In this
1958).
Transformation
pendent strain
DNA -in vivo
Acid soluble deoxyribonucleosides
(thgmidine)
that
mechanism.
"Masked" deoxyribonucleosides
aubtilis
reported
had the same result.
of the molecular
measured as thymidine
nucleoside
on the bio-
many investigations
19581,
on the mechanism of the action
MC was obtained
Burton (Burton,
1952).
effects
causes the rapid break down of bacterial
(Reich -et -9 al
Ltd.,
inhibitory
antibiotic,
of B.subtilis
of Applied
to the method of Spizizen were obtained
coli
of
B with sonic
(10 KC, 10 minutes) and then centrifugation 339
from
University
Microbiology,
from Escherichia
inde-
(lOC,OCC x g,
Vol. 6, No. 5,196l
BIOCHEMICAL
30 minutes). activity
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The eupernatant
liquid
The
of DNase was determined by measuring the release of ultra-
violet
absorbing material
extracted
from E.coli
and of acid-soluble
deoxyribose.
B with lysozyme treatment,
.repeated chloroform-octanol
salt
DNA was
extraction
are as follows:
E. coli
B cultured
shaking at 37'C in glucose-Simmons medium, was harvested
the logarithmic
phase of growth,
at 660 mp = 0.3) (1 &ml)
in similar
and incubated
at certain
intervals,
measured.
It
and
treatment.
The methods and results with
was used as enzyme source.
then resuspended (optical
shaking.
Aliquots
were taken
and the DNA and deoxyribonucleosides that
MC breaks down bacterial
and accumulates the "maskedIt deoxyribonucleosides
0
density
fresh medium in the presence of MC
at 37OC rith
is evident
at
10
20
30
were
DNA in vivo,
in the medium (Fig.
40 50 (min .I
Accumulation of llmaskede Fig.1. deoxyribonucleoside. "Masked" deoxyribonucleoside (0 - 01, Deoxyribonucleoside (0 - 0). The transforming when the bacteria
activity
(B. subtilis,
strain 340
of the DNA was greatly 116) were incubated
impaired with
1).
BIOCHEMICAL
Vol. 6, No. 5,196l
MC (1 pg/ml)
before
the DNA was prepared.
occurred
at the frequency
principle
(20 pg DNA/ml),
of this
Because transformation
of intact
the frequency
solution
with ethanol,the
suggested that cells
transformation
of one in 104 with the intact
transforming
decreased to less than 10 % with MC (Fig.
is known to be prevented
DNase, these results
in salt
Although
in the course of incubation
value
treatment
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2).
by treatment
with
the MC-DNAwas degraded by
with MC. When DNA was treated
with MC
at O°C for 20 minutes and then washed repeatedly activity
of transforming
0
principle
I
I
I
30
60
90
was not impaired. -
PREINCUBATIONTIMES (min.) Fig.2. Time course of impairment of transformating DNA by MC treatment of whole cells. MC alone had no effect activity
on DNAand also no effect
--in vitro.
When E. coli the specific
on DNase
,I B cells
activity
were preincubated
of DNase extracted
up to 180 % (Table 1). 341
with MC for 30 minutes
from the cells
was elevated
Vol. 6, No. 5,196l
BIOCHEMICAL
Table 1.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DNase activities
of E. coli
B
Systems
DNase activities
With untreated + none
enzyme 100 100
+ MC (50 pg) + protamine sulfate + protamine sulfate MC-pretreated
With
(100 Ng) (100 pg) + MC (50 pg)
28
30
enzyme
+ none
180
+ protamine sulfate
(100 pg) + MC (50 pg)
47
'Without enzyme + MC (200 pg) incubated
0
for 30 minutes at 37'C with 20 ug of DNA and
10m2Mof MgS04 in Verona1 buffer,
In a preliminary incorporation
of H3-thymidine
high concentration effects
on the activities report
degradation
found the degradation sulfate.
into
indicates
Amos demonstrated that of RNA by ribonuclease
that
in -- vivo
(Amos, 1961).
by protamine
inhibited
(Fig. it
actions
on DNAat
References Amos, H., Biochem. Biophys. Res. Communs., 2, 1 (1961). Burton, K., Biochem. J., 62, 315 (1956). E.,
Biochem. J.,
50, 400 (1952). 342
the cell-
3).
of the DNase activity.
Hoff-Jbrgensen,
He also
(Table 11, and to restore
(20 pg/ml)
suggested that MC exerts
MC has no
protamine sulfate
protamine sulfate
of E. co1i.B -in vitro
even at
kinase and polymerase.
of DNA by DN ase to be protected
the impaired ?'!I synthesis These results
system the
DNA was not inhibited
of deoxyribonucleotide
We also observed that
free DNase activity
the level
with the cell-free
of MC. This finding
In a recent protected
experiment
pH 7.4.
BIOCHEMICAL
Vol. 6, No. 5,196l
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DNA ($1 120 100 a0 60 40
20 01 -20 15
30
(min. 1
Fig. 3. DNA synthesis of E. coli B under the influence of MC and/or protamine sulfate. l
I 0 0
Okazaki,
Control (no addition) + MC (1 pg/ml) + Protamine sulfate (20 un/ml) + MC (1 &ml) + Protamine sulfate (20 &ml) ,
R. and Okazaki,
T.,
“.
Biochem. Biophys.
Acta,
g,
470
(1958).
Reich, E., Shatkin, 5,
608
A, J. and Tatum, E. L.,
Biochem. Biophys.
Acta,
(1960).
Shiba, S., Teraaaki, A., Taguchi, T., and Kawamata, J., 1, 179 (1958). Spizizen, J., Proc. Natl. Acad. SC%., s, 423 (1958).
343
Biken's
J.,