[CANCER
RESEARCH
35. 554-559,
March
1975]
Subcellular Fate of Protein Antibiotic Neocarzinostatin in Culture of
a Lymphoid Cell Line from Burkitt's Lymphoma'
Hiroshi
Maeda,2
Departments
Shogo Aikawa,
ofMicrobiology
and Akira Yamashita
[H. M., S. A.] and Anatomy
[A. Y.], Kumamoto
SUMMARY
“C-Labeled protein antibiotic neocarzinostatin
(NCS)
was prepared efficiently by chemical modification. With the
use of lymphoma-derived
cell line P3HR-l, the subcellular
behavior of this antitumor antibiotic was studied by the
uptake and autoradiography
of isolated nuclei of radioac
tive NCS.
The antibiotic was taken up by the cells, reaching the
maximum value at 1.5 hr and decreasing in value at 4.0 hr to
the level at 0.5 hr. The silver grains in the autoradiograms
were also found in the isolated nuclei. The grain count in the
nuclei showed a tendency similar to the uptake of NCS by
the whole cells, i.e., a gradual increase at 0.5 hr, reaching
the maximum value at I .5 hr, and then decreasing after 4.0
hr to the level at 0.5 hr. These facts indicated that NCS
reached not only to cytosol but also into the nucleus, and/or
at least to the nuclear membrane of the lymphoid cell.
The number of NCS molecules incorporated into the cells
at I .5 hr was calculated to be about I x 106/cells at a
concentration of 3 sg NCS per ml of medium, which can be
extrapolated to I x l0@molecules per cell at the minimum
inhibitory concentration.
The number of molecules should
be even less within the nucleus.
In cell-free systems, the interaction of DNA and NCS,
which is an inhibitor of DNA synthesis, was investigated
with the use of a Sephadex G-l00 column, with negative
results. In the cell culture system, NCS molecules were
degraded into smaller polypeptides of certain sizes by
proteolysis
either by serum component(s)
or by cells
themselves.
An inactive isomer, pre-NCS, which is an antagonist of
NCS and a partially denatured homologous
molecule,
behaved similarly to NCS in all of these experiments.
Because the chemically modified NCS used in this study
retained biological activity essentially similar to that of
parental NCS, the results obtained here could be inter
preted as similar to those of parental NCS in vitro.
INTRODUCTION
NCS3 is a proteinous antitumor
culture filtrate of Streptomyces
1 A
part
of
this
investigation
was
2 To
3 The
whom
requests
abbreviation
for
used
reprints
is:
NCS,
by
be
Japan.
addressed.
neocarzinostatin.
Received June 19, 1974: accepted October
554
Special
of Education,
should
Medical School, Kumamoto,
Japan
single-chained polypeptide without carbohydrate.
Its amino
acid sequence has been reported recently (13, 14, 19), and it
was shown to have a molecular weight of about 10,700.
Clinical trials (4, 5, 24) have shown that NCS is of
considerable value in treating acute leukemia (4, 5).
The molecular mechanism of action of NCS in bacteria
and mammalian tumor cells is the arrest of DNA synthesis
and the initiation of degradation of preexisting cellular
DNA (6, 2 1, 22). This is comparable to the action of colicin
E2, or to the infection of T-even phages or some DNA
animal viruses, but is quite different from the mode of
action of mitomycin C, which inhibits replication of DNA.
However, the subcellular behavior and target of NCS are
unknown.
In the present investigation, we attempted to determine
whether the protein antibiotic NCS could penetrate the cell
membrane and what would eventually happen to NCS
molecules, with the use oflymphoid cellline P3HR-l, which
was originally obtained from Burkitt's lymphoma. Interpre
tations of the results were based on the assumption that the
derivative used and the parental NCS behaved in a similar
manner because of their similar biological activities, molec
ular sizes (about 2% increment), gross conformations,
and
very acidic nature (p1 approximately 3.0 versus 3.4) ( 11).
NCS was succinylated with [‘4C]succinic anhydride as
described previously ( 11, I2). There are only 2 free amino
groups in NCS, one at N-terminal alanyl residue and the
other at c-NH2 of lysine 20, both of which were acylated,
yielding bis-succinyl-NCS.
The [‘4C]bis-succinyl-NCS thus
obtained had a specific radioactivity of 3 to 7 x l0
cpm/mg (54 .tCi/.tmole). Two forms of derivatives were
obtained (I I, 12). One is an inactive derivative, derived from
a partially denatured form of isologous molecule of NCS,
called pre-NCS4 and designated as SUC-I: the other is ac
tive succinyl-NCS, designated as SUC-Il.
Evidence of uptake of [‘4C]NCS by the cells was
obtained. Furthermore,
autoradiographic
analysis of iso
lated nuclei revealed that NCS or its fragment existed on or
in the nuclei. Time-course study of the uptake and break
down of the protein was performed with [‘4C]NCSin the
cell culture system.
antibiotic obtained from
carzinostaticus.
It is a
supported
(901542 for 1974) from the Ministry
University
21, 1974.
Cancer
Grant
II
4 Pre-NCS
and
NCS
possess
identical
properties
in
amino
acid
analy
sis, amino-terminal
residue, molecular size, and peptide mapping, but they
differ from each other in the far-UV absorption spectra, circular dichroism
spectra (indicating a conformational
difference), elution profile in carbox
ymethylcellulose column chromatography,
and biological activity (16).
CANCER RESEARCH
VOL. 35
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Subce!!u!ar Fate of Protein A ntibiotic NCS
MATERIALS
AND METHODS
Chemicals. Carboxymethylcellulose (Whatman, CM-52),
Sephadex G-l5 and G-lOO, Tris (Trizima base), and May
Griinwald-Giemsa
solution were obtained from W. & R.
Balston Ltd., Maidstone, England; Pharmacia Fine Chemi
cals Inc., Uppsala, Sweden; Sigma Chemical Company,
St. Louis, Mo., and E. Merck AG, Darmstadt, Germany,
respectively.
NCS (Lot T-58) was supplied by Kayaku
An
tibiotic Research Laboratory, Tokyo, Japan. [l,4-'4C]Suc
cinic anhydride (27 mCi/mmole) was obtained from Daiichi
Pure Chemical Co., Ltd., Tokyo, Japan, as benzene solu
tion.
It was used for succinylation
of NCS after removing
benzene and drying it in a vacuum. All other chemicals,
unless specified separately, were obtained from standard
commercial sources.
Succinylation of NCS. A representativeexample is as fol
lows ( I I , I 2), 5.0 mg NCS, (0.4 Mmole), was dissolved in 0.4
ml of 0. 1 M NaC1 and the pH of the solution was adjusted to
8.0 by the dropwise addition of 0. 1 M Na2CO3 at 2°.Then
the solution was poured into a vial containing dry succinic
anhydride ( I .8 @zmoles).
The reaction was carried out at 5°
under constant stirring. The pH was maintained between 7.0
and 8.0 by appropriate additions of 5% NaHCO3. Then the
reaction was terminated after 30 mm by the addition of I ml
of 0. 1 M acetic acid and was followed by dialysis against I
mM acetic acid overnight
at 4°. At this stage, NCS had a
specific activity of about 6.7 x 106 cpm/mg protein. Then
the material was applied to carboxymethylcellulose chro
matography (column, 4.5 x I .5 cm) as described before.
Two fractions, SUC-I and SUC-Il, were obtained with a
ratio of SUC-l to SUC-I I of 3: 1. The rest remained, being
adsorbed in the column. The protein concentration deter
mined by the method of Lowry et a!. (10) paralleled the
radioactivity. Specific radioactivity was the same as that of
the unfractionated derivative.
Cell and Cell Culture. Lymphoid cell line P3HR-l was
originally derived from Burkitt's lymphoma (3), and the
cells were cultured in Eagle's minimum essential medium
(Grand Island Biological Co., Grand Island, N. Y.),
enriched with 10% bovine serum (obtained locally), in a
stationary floating state in rubber-stoppered
Roux bottles
or test tubes at 37°.Cells used in all experiments were at
their logarithmic stage and their cell density was 5 to 8 x
l05/ml. The cells were usually inoculated at about 2 x
105/ml with a doubling time of about 20 hr. Numbers of
cells were counted by a hemocytometer,
and cell viability
was checked by the trypan blue dye-exclusion test.
Drug
Treatment.
[‘4CJNCS was dissolved
to give 30
zg/ml for SUC-Il and 80 @zg/ml for SUC-I in 0.01 M
phosphate-buffered
0.15 M saline, pH 7.1, and this solution
was added to the cell culture or cell concentrates to give 10%
of the volume of medium reported in Table I . In the
preparation of nuclear and autoradiographic
analyses, 10
times more untreated cells, as a carrier, were added to the
NCS-treated cells after the incubation period.
Preparation of Nuclei. Nuclei were prepared by the
modified method of Naora (20) and Yamashita and Naora
(25). Briefly, the mixture ofdrug- and nontreated cells in 50
MARCH
ml was washed 3 times with 10 m@iTris-HC1 buffer solution
(pH 7.6) containing 0.25 M sucrose and 3 mM MgCl2 by
centrifugation at 250 x g for S mm at 4°.The volume of
washing buffer solution was 10 to 20 times that of packed
cells. Then 1 ml of chilled 1% Brij 58 (polyoxyethylene cetyl
alcohol ether) (Kao-Atlas Chemicals Ltd., Tokyo, Japan)
was added to 10 volumes of cell suspension in the above
Tris-sucrose
buffer solution and mixed well, then was
allowed to stand for 2 mm in an ice bath. Cells were
subsequently
homogenized
by a Teflon homogenizer
at
moderate speed (500 rpm) with 6 strokes in an ice bath. The
suspension of the homogenate was centrifuged to separate
pellets (nuclear fraction) and supernatant
(cytoplasmic
fraction) at 200 x g for 5 mm. The nuclear fraction was
washed 2 more times. The radioactivity of the cytoplas
mic pooled fraction was measured in a liquid scintillation
counter. The scintillation liquid used was a mixture of
toluene (3.0 liters), Triton
X-lOO (1.5 liters), PPO (15 g),
and dimethyl-POPOP (I .5 g), all obtained from either
Wako Pure Chemical Co. Ltd., Osaka, Japan, or Packard
Instrument Inc., Downers Grove, III. When necessary, ‘4C
was added as an internal standard. The counting efficiency
was more than 95% throughout.
The homogeneity of nuclear fractions was checked by the
method of Barer et a!. (I). It was found that the proportion
of whole cells and cytoplasmic contamination in all the nu
clear preparation was less than 0.5%, as revealed by re
fractometry.
Autoradiographic Technique. The autoradiograph was
prepared principally as described previously (25). One drop
of nuclear suspension was smeared on a gelatine-coated
slide glass and fixed with acetic acid:methanol:water
(1:89:10, v/v/v) for 20 mm at about 4°.Subsequently, the
smear was rinsed with 100% methanol for 30 mm at 0°,
and then with running tap water overnight. It was then
rinsed with distilled water and dried. The dried slide glass
was then dipped in Sakura autoradiography
emulsion NR
M2 (Sakura X-ray K.K., Tokyo, Japan) at 40°in darkness
and air dried. The emulsion-coated
slide glass was placed
in a sealed box with dry silica gel at 4°until development.
An exposure time of 19 days was found to be best. The
slides were stained with May-Grtinwald-Giemsa
solution,
following authoradiographic development.
The validity of the autoradiography wassupported by the
fact that the grain patterns and their distributions were
limited only in or over nuclei or cell fragments. No latent
image fading or chemography
in the autoradiographs
during the exposure time has been observed. The number of
silver grains over nuclei was counted microscopically with
an oil-immersion lens (x 1000). Only the grains directly
over nuclei were counted; those scattered around the
peripheral area were excluded. At least 400 nuclei were
counted for each sample.
Gel Filtration. Analysis of [‘4C]NCS or its breakdown
products in the culture medium was carried out with
Sephadex G- 15 ( I.5 x 43 cm), which was eluted with distilled
water;
phenol
red in the medium,
quenching of radioactivity,
system.
The approximate
which caused
was separated
size and amount
some
well in this
of the degra
1975
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1975 American Association for Cancer Research.
555
H. Maeda et a!.
dation products of NCS were analyzed by the radioactiv
ity and the elution volume.
Interaction
between DNA (calf thymus; Sigma) and
[14CJNCS was studied after both were mixed at isotonic
condition
(0.01 M phosphate-buffered
0.15 M saline) with
and without both 2 mM Mg2@ and I m@viCa2t DNA (I
mg) and NCS (20 zg), each dissolved in 0.5 ml of buffered
solution containing an appropriate salt concentration, were
applied to the Sephadex G- 100 column (1.5 x 48 cm) after
incubation at 37° for more than 3 hr. The elution was
carried out with 0.01 M phosphate-buffered
0. 15 M saline
or Hanks' balanced salt solution with Ca2@and Mg2@(2).
The eluates were monitored by radioactivity for NCS and
:
absorbance at 260 nm for DNA.
RESULTS
Table 2
Uptake of['4C]NCS
by cellularfractions
SUC-I and SUC'-!!
Each tube contained about 7 x 10' cells or naked nuclei.
Time
(hr)cpm/tubeCytoplasmic
fractionSUC-I0.5
1.5
4.082.5
9.5SUC-JI0.5
1.5
4.0
Control86.0
Uptake of [‘4CJNCS. The results are shown in Table I.
NCS molecules were taken up by the cells, and the numbers
of the molecules can be calculated from the following data:
180 dpm/7.0
x 10' cells, 2.2 x 1012 dpm/Ci, and 54
@iCi/@zmoleof NCS at 37°,yielding 1 x l0 molecules/cell.
Uptake for the heat-killed (56°, 1 hr) cells incubated with
NCS at 37°,and for the intact cells (with a viability of more
than 98%) incubated without serum at 4°,was 57 and 36%,
respectively.
Washing the cells 16 times with Hanks'
balanced salt solution (pH 7.4) by low-speed centrifugation
at 4° resulted in only a slight loss of radioactivity (not
shown). These data indicate that NCS molecules are at least
firmly bound to cells, and that active (viable) cell func
tions seem favorable for the uptake.
Uptake of 2 Isologous Derivatives of [‘4C]NCSinto
Subcellular Components. Cells were incubated with active or
inactive derivatives for 3 different periods of time (0.5, 1.5,
and 4.0 hr). As shown in Table 2, both of the derivatives
were incorporated
in a similar fashion into cytoplasma
fraction. The radioactivity of the subcellular fractions of
SUC-I treated was of almost the same magnitude as for
those of SUC-II, although the former was at a concentra
tion 2.7 times higher than the latter. This may indicate
either that the amount of NCS incorporated into cells
reaches the saturation point or that SUC-I is taken up less
effectively. However, there is evidence that pre-NCS, which
corresponds to SUC-I, can abolish the activity of NCS only
when pre-NCS
is given prior to NCS (unpublished
observation). Thus, the evidence supports the hypothesis
Table I
Uptake of['4C]NCS
by lymphocytic
Incubation with NCS: 2 zg/ml, 1.5 hr.
cells, P3HR-1
controlIntactcellsat3l°167.5±
Cell and incubationcpm/tube%
of
1.5°100Heat-killed
2.557.3Intact cells at 370bI
cells at 4°80.5
a Mean
b At
556
10.2 ±
±2.536.0
± S.D.
56°,
Background
±95a
227.5 ±4.5
106.0 ±7.5545@
9.5
47.5 ±0
64.0 ±
±9.5
232.5 ±9.5
99.0 ±9.5
33.5 ±046.5
±0
46.0 ±0
51.5±9.5
30.5 ± 1.5
AND DISCUSSION
a Mean
@
fractionNuclear
1
hr.
all
cells
were
cpm were about 31.5.
killed
[trypan
blue
staining
(99.9%)].
± S.D.
that pre-NCS reaches the same target as NCS, or its
neighborhood, and interferes with the activity of NCS.
The time-course study of [‘4C]NCS uptake (SUC-I and
SUC-Il) showed that the radioactivity of the cytoplasmic
fraction reached a maximum (peak) at I .5 hr and then
decreased to less than one-half of the maximal value within
4 hr (Table 2). This fact indicated that there might be a
possible excretion after degradation of NCS, as described
below.
Only a limited amount of radioactivity was detected in
the nuclear fraction (Table 2). This was further elaborated
by the autoradiographic
analysis as described below.
Degradation of NCS. The resultsof column chromatogra
phy of [‘4C]NCS-treated culture media with Sephadex G-l5
showed that there was little desuccinylation reaction (Chart
I), which would have yielded succinicacid with a molecular
weight of 118, if any. However, the existence of considerable
breakdown of the radioactive NCS to the medium size
molecular weight may imply the peptide bond cleavages. At
1.5 hr of incubation and at a cell density of 6 x 105/ml with
10%bovine serum, about 30%of the total radioactivity was
found in the region of molecular size lessthan 1000(Chart
1A, Peaks C and D) and about one-third of the activity still
remained in the region of medium-size polypeptides (arbi
trary molecular size, between 1500 and 2000) (Chart IA,
Peak B). However, at 22 hr of incubation, more than 80% of
NCS was found in the region of smaller peptides (Table 3,
Peaks B, C, and D). The magnitude of this degradation can
be reduced to about 25% by heating both cells and medium
at 56°for I hr, which kills all of the cells, although all
enzymes may not have been inactivated (Table 4). Further
more, it was also shown that in the serum-free system there
was a lesser breakdown of NCS, but it still occurred. These
facts imply that the degradation seems to be caused by
either cells or serum alone, and this process may be closely
related to its inactivation process.
Autoradiography of Isolated Nuclei and Distribution of
Silver Grains. As shown in Fig. I, grains were observed over
and in the vicinity of nuclei. This is attributed to the fact
that 14C emits
particles that reach within an average
radius of more than 5 @m,a size similar to or somewhat
CANCER RESEARCH
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VOL. 35
Subcellu!ar
@%
Fate of Protein Antibiotic
0
NCS
Table 3
Degradation
of [14C]NCS
in cell culture system and distribution
of
fragments
Peaks A to D corresponded to that of chromatography
on Sephadex G-15
in Chart lA . Percentages were obtained after extrapolation of each peak
and computing the reconstructed peak.
treatmentl.Shr22hrA
degradation of drug and time of
PeaksinG-l5%
54.1
21.5
35.7
29.7
4.717.9
B
C
6.8Total100.0100.0
D29.9
Table 4
Degradation of NCS in cell culture
All experiments except b were performed with a cell population
10'/ml and an incubation period of 1.5 hr at 37°.
of 6.8 x
ofpolypeptides
NCS and largerSmaller
peptides
of MW.MW.
1200,NCS>l300,PeaksA+BPeaksC+Dtreatment(%)(%)a61.039b7822c9010d8218
<
a Intact
0 Same
C Cell
cells
as
and
a,
and
except
complete
culture
incubation
culture
medium
Eagle's
minimal
medium.
temperature
were
pretreated
at
at
4°.
56°
for
I
hr.
All
cells
were
dead.
d Cells
10
20
30
TUBE
40
50
NO.
Chart I . Chromatographic
separation of breakdown products of NCS
by Sephadex G-15 as revealed by radioactivity. A, After an incubation
period of 1.5 hr. an aliquot (I ml) of the culture medium was applied to the
column. Peaks A and B have molecular weights (M. wt.) larger than 1500.
Peaks C and D have molecular weights less than 1000. Each tube contains 2
mi/tube. B, After an incubation period of 22 hr. about the same amount of
the culture medium was applied as in A . The reference standard molecular
weights are indicated on the top of B. Blue dextran (at V0, void volume),
bacitracin, actinomycin D, ascorbic acid, and acetic acid were used for this
purpose. Note that only a very small peak (Peak D) corresponds to the
molecular weight of succinic acid as desuccinylation product.
larger than the diameter of a lymphatic cell nucleus (Fig.
1). This finding suggested that the NCS molecule (or its
fragments, either active or inactive) attached to or entered
into the cell nucleus. The results of grain distribution in
nuclear samples after different incubation periods, using 2
isologous derivatives, are shown in Table 5. The results are
quite concordant with those of radioactivity (cpm) (Table
2). The number ofgrains over nuclei began to increase at 0.5
hr and reached a peak at I .5 hr, then fell at 4 hr to the level
MARCH
in
essential
medium
without
serum.
at 0.5 hr. If the smaller fragments penetrate into cells by
diffusion, the results should have been reversed, namely, the
4-hr fraction should have had larger grain counts than that
at 0.5 or I .5 hr. These facts favor the conclusion that NCS
or that derivative close to its original size were taken up by
the active cell function (e.g., pinocytosis) into cytosol. It
appears possible, therefore, that the location of NCS action,
in relation to DNA metabolism, may be the nucleus at
which replication of DNA proceeds, although it has been
shown in other instances that such an effect on DNA can be
controlled from outside the cell, on the cell membrane.
Interaction of DNA and NCS in Vitro. In Chart 2, the
results . of column chromatography
on Sephadex G- 100
indicate that NCS did not bind to DNA and thus separated
into 2 distinctive peaks. Therefore, the direct target of NCS
is more likely to be an apparatusthat involves replication of
DNA but not DNA itself.
This conclusion is based on the fact that NCS or its
derivative inhibits DNA synthesis of both tumorous and
normal cells (I I). Although the DNA used in this experi
ment was not obtained from P3HR- I cells, it was expected
to exhibit properties similar to that of P3HR-l for the
reason cited above.
1975
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1975 American Association for Cancer Research.
557
H. Maeda et al.
B
Fig. I. Autoradiography of [14C]NCS using isolated nuclei from
lymphoid cells. The nuclei derived from large (A) or small (B) lymphocytes
incorporated radioactive molecules similarly. Silver grains are visible as
dark dots in the vicinity of nuclei.
Table 5
Distribution of grains in the autoradiograms of isolated nuclei
No. of grains over nucleus* (%)
Incubation
time (hr)
Drug"
0.5
SUC-I
SUC-II
Av.
1.5
SUC-I
SUC-II
Av.
4.0
SUC-I
SUC-II
Av.
0-3
4-8
9-15
15
67.0
71.1
32.1
26.7
0.5
2.1
0.5
O
69.3
29.0
1.5
0.0
18.2
46.5
44.0
35.3
17.2
14.6
20.7
3.8
40.0
37.2
15.2
7.1
93.0
65.7
6.6
24.4
0.5
8.0
O
1.0
76.0
17.8
5.7
0.6
•¿
SUC-I and SUC-II represent inactive and active derivatives of NCS
(11); see text.
' As described in the text, only 10%of the population was treated with
the drug. Grain counts were carried out at x 1000 for more than 400 nuclei
for each experiment and a total of more than 6000 nuclei.
obtained by chemical modification. We had previously tried
tritiation by the Wilzbach method with tritium gas but with
only limited success (radioactivity was too low to use). The
present preparation exhibited biological activity at less than
0.2 /¿g/ml,and yet the covalently bonded succinyl group was
not readily desuccinylated.
It has been shown that NCS is very resistant to proteolytic enzymes (15, 18), perhaps due to its rigid overall
conformation ( 17) under experimental conditions, but it was
found in this work that NCS was readily degraded through
the process of proteolysis by serum and cell components in
cell culture system. The low-molecular peptide fragments
thus produced were distributed only at certain molecular
sizes and not at random. The detailed mechanism should be
further worked out, since this problem is of particular
importance for the clinical application of this antibiotic.
The observation of penetration of NCS into the cell is
rather different from that of another protein antibiotic,
macromomycin (8, 9) (M. W. 15,000), which exerts the
effect from outside the cell membrane. There are few
well-documented examples demonstrating that intact pro
teins or macromolecules cross cell membranes and reach the
cytosol while still functional. Relatively well-clarified exam
ples along this line are the toxin of diphtheria (23), and
perhaps antigen-antibody complexes (7), and other foreign
protein particles generally accepted in cytology. In these
instances, proteins or macromolecules in soluble or particulate forms are taken up into cytosol through a pinocytosis or
phagocytosis, which is usually followed by proteolytic
degradation within the endocytotic vesicles. The degrada
tion products or small peptides and amino acids then
traverse the membrane. This seems to be exactly what
happened with NCS.
Nuclei from both large and small lymphocytes similarly
incorporated [MC]NCS (Fig. 1).
Also, the inactive counterpart of NCS, i.e., pre-NCS,
seems to have subcellular behavior similar to that of NCS.
The approximate number of molecules bound to a cell
was calculated to be 1 x 10" molecules at 3 fig/ml, as
0.2 •¿
10
20
TUBE
Discussion
This Study showed
radioactive
derivative
558
that the preparation
of a protein
antibiotic
of a highly
was easily
30
40
50
NO.
Chart 2. Separation of NCS from DNA by Sephadex G-100 after
mixing and incubation. Peaks based on absorbance at 260 nm for DNA
and radioactivity for NCS were separated completely. The elution was
carried out at 2 ml/tube in 4 min by hydrostatic pressure.
CANCER RESEARCH
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VOL. 35
Subce!lu!ar
described. If extrapolation to the minimal effective concen
tration of parental NCS (about 0.02 zg/ml) is made, then
the number of molecules is 1 x 104/cell, and perhaps a
much smaller number may be needed in the nucleus.
ACKNOWLEDGMENTS
We thank Professor Y. Hinuma of the Department of Microbiology for
his support in these experiments, Dr. Y. Koyama of Kayaku Antibiotic
Research Laboratory for supplying NCS, and M. Fujii and K. Izumi for
typing the manuscript.
REFERENCES
I. Barer, R., Joseph, S., and Esnouf, M. P. Method for Distinguishing
Intact Cells from Free Nuclei. Science, 123: 24-25, 1956.
2. Hanks, J. H., and Wallace, R. R. Relation of Oxygen and Tempera
ture in the Preservation of Tissues by Refrigeration. Proc. Soc. Exptl.
Biol.Med.. 71: 196-200,1949.
3. Hinuma, Y., and Grace, J. T., Jr. Cloning of Immunoglobulin-produc
ing Human Leukemic and Lymphoma Cells in Long Term Culture.
Proc.Soc. Exptl.Biol.Med., 124:107-Ill,1967.
4. Hiraki, K., Kamimura. 0., Takahashi, I., Nagao, T., Kitajima, K.,
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559
Subcellular Fate of Protein Antibiotic Neocarzinostatin in
Culture of a Lymphoid Cell Line from Burkitt's Lymphoma
Hiroshi Maeda, Shogo Aikawa and Akira Yamashita
Cancer Res 1975;35:554-559.
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