(CANCER RESEARCH 35. 3636-3641, December 1975]
Functional Properties of Mitochondria Isolated from Murine L5178
Lymphoblasts Grown in Cell Culture'
Ugo Carpentieri and Louis A. Sordahl
Division ofHematology. Department ofPediatrics [U. C.], and Division ofBiochemistrv,
A . S.], University of Texas Medical Branch, Galveston, Texas 77550
Department oflluman
Biological Chemistry and Genetics [L.
SUMMARY
the measurement of a number of energy-linked functions in
mitochondria.
Many of these studies (10) indicated that
Mitochondria were isolated from lymphoblasts grown for
mitochondria have the ability to transport rapidly various
5 days in cell culture. Measurement
of mitochondrial
cations, such as potassium
and calcium. It has been
respiratory activity revealed poor response to adenosine
suggested that mitochondria may function within the cell to
5'-diphosphate with reduced nicotinamide adenine dinucleo
regulate the ionic environment and thereby control meta
tide-linked substrates but well-coupled active respiration
bolic processes (4, 11, 17). Bygrave et a!. (2—4,11, 19) have
with succinate as substrate. These mitochondria also exhib
proposed that mitochondria may regulate certain metabolic
ited rapid initial rates of respiration-supported
calcium
activities ofthe cell via their control ofcalcium metabolism.
uptake as measured by dual-beam
spectrophotometry.
Recently, Thorne and Bygrave (19) demonstrated
that
H@/2e and Ca2@/2e ratios were in normal limits for the
mitochondria from Ehrlich ascites tumor cells have calcium
lymphoblast mitochondria
during calcium uptake in the
transport functions quite different from those of normal
presence of phosphate. In the absence of phosphate no liver cell mitochondria. Further, Reynafarje and Lehninger
calcium uptake, W ejection, or stimulation
of oxygen
have reported unusual calcium transport
properties in
mitochondria isolated from mouse ascites tumor cells (14)
consumption was observed. However, the lack of discharge
and normal rat liver (15).
of the accumulated calcium from the lymphoblast mito
Several studies have been conducted in an attempt to gain
chondria upon inhibition of respiration suggests possible
of mitochondrial
metabolism
in
different mechanisms
of cation transport
compared to a better understanding
leukocytes (6, 9). These studies have only been able to
mitochondria from normal, mammalian cell types. Electron
measure mitochondrial
respiratory functions in leukemic
microscopy of freshly prepared mitochondrial suspensions
lymphocytes and, in these instances, only by indirect means.
revealed preparations
with intact outer membranes and
However, there is evidence that establishes electron trans
abundant cristae and that were relatively free of other
port chain components and Krebs cycle activity in leukocyte
cellular structures. These studies demonstrate the feasibility
of obtaining intact respiring mitochondria
from cultured
mitochondria
(6, 9). In a recent review of the field,
lymphoid cells and indicate that active ion transport in these
Kirschner et a!. (9) gave some of the reasons why direct
mitochondria may be significantly different from “normal― measurement of mitochondrial
functions from leukocytes
cell mitochondria.
has been largely unsuccessful. These investigators indicated
that insufficiently purified suspensions of heterogeneous
white cells have been used, that salt solutions instead of
INTRODUCTION
serum were used as incubation media, that the influence of
the cell suspension densities on metabolic indices have been
A significant amount of research has been directed to ignored, and finally that the methods used for the quantita
mitochondrial
functions from a variety of tumor cell types
tive evaluations of the metabolic processes in these cells
(9, 17). These studies have been aimed at gaining a better
were not sufficiently refined (9). In this report we describe
understanding of the energy metabolism of malignant cells.
the successful isolation of intact mitochondria from murine
A number of years ago Warburg (20) suggested that
L5178Y lymphoblasts
grown in cell culture, the direct
mitochondrial respiratory impairment may be the primary
measurement of respiratory activity in these mitochondria,
mechanism
in the production
of malignant cell types;
and some of their calcium transport functions and ultra
however, definitive proof that mitochondria are a primary
structural characteristics.
mechanism in the etiology of carcinogenesis has never been
established.
MATERIALS AND METHODS
The development of highly sensitive spectrophotometric
and cationic-sensitive
electrode techniques has permitted
Murine L5178Y lymphoblasts were grown in Fischer's
medium (5, 12) plus 10% horse serum in an incubator at 37°
containing a gas mixture of 5% CO2 and 95% air. The cell
and Welfare Grant 5 SOI-RR-05427-l2.
Received August 12, 1974; accepted September 9, 1975.
suspensions were propagated in 500-mI bottles and aliquots
1 This
3636
work
was
supported
in
part
by
Department
of
Health,
Education
CANCER RESEARCH
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VOL.35
Functions
were removed for cell counts. The cell suspensions were
allowed to proliferate for 5 days and then were harvested.
Cell numbers were determined with a hemocytometer,
and
viable cell counts were performed according to the proce
dure of Phillips and Terryberry (13). Periodically, l0@cells
were injected i.p. into mice to test for carcinogenicity. Mean
survival time after injection was 21 . 1 days (1). Approxi
mately 520 to 660 x 10 cells were harvested for each
preparation with viabilities of 90 to 94%. The cells were
collected from the culture medium by low-speed centrifuga
tion (1600 x g for 15 mm). The collected pellets of cells
were washed in isolation medium (see below) and cen
trifuged at 300 x g for 10 mm to recollect the cells.
The cells were suspended as a 10% homogenate (w/v) in
of Lymphoblast
Mitochondria
Aliquots of standardized HCI were used to calibrate the H@
electrode system for each experiment and for each particu
lar assay medium. This is necessary si@e the H' electrode
response is different in the presence or absence of P1.
Isolated suspensions of mitochondria
electron
microscopy
by the method
were fixed for
of Spurr (18). Essen
tially, this involves fixation of pelleted mitochondria in
phosphate-buffered 2.5% glutaraldehyde and postfixed in
collidine-buffered 1% osmium tetroxide. Following dehy
dration,
tioning,
the material was embedded in Spurr. After sec
the material was stained with uranyl acetate and
lead citrate and viewed in a Philips 200 electron micro
scope. A sufficient number of fields were viewed to obtain
representative electron micrographs.
an isolation medium consisting of 0.225 M sucrose, 0.075 M
mannitol, 1 mM EGTA,2 and 0.5% bovine serum albumin
(Fraction V; Sigma Chemical Co., St. Louis, Mo.) at pH
7.2. This medium was used throughout the isolation proce
dure and final suspension of the mitochondrial pellet. The
suspended cells were homogenized with a Polytron PT-20
tissue processor at a rheostat setting of 4 for 5 sec. For
studies ofcalcium transport, EGTA was omitted in the final
mitochondrial
suspension medium. The total homogenate
was centrifuged at 600 x g for 10 mm to remove unbroken
cells, nuclei, and other cellular components. The resulting
supernatant was centrifuged at 12,000 x g for 10 mm. The
mitochondrial
pellet was then resuspended in isolation
medium and centrifuged at 12,000 x g for 10 mm. This
“wash―
procedure was repeated once. Finally, the mito
chondrial pellet was suspended in a small volume of
isolation medium to achieve an approximate protein con
centration of 7 to 12 mg mitochondrial
protein per ml.
Protein determinations
were done by a biuret method (8)
and cytochrome oxidase activity of the various homogenate
fractions was determined by a spectrophotometric
method
(21). Mitochondrial
respiratory activity was determined
polarographically
by previously described methods (17).
The respiration-supported,
rapid uptake of calcium by the
mitochondria was determined by either a dual-beam spec
trophotometric
method using the calcium-sensitive
dye
murexide (ammonium purpurate) or utilizing 45Ca rapid
Millipore filtration
and subsequent counting by liquid
scintillation techniques (16). Mitochondrial
oxygen con
sumption and H@ ejection in the presence of calcium were
monitored
with a vibrating platinum electrode (Gilson
Medical Electronics,
Middleton, Wis.) and a Beckman
combination electrode interfaced to a Beckman Research
pH meter (Beckman Instruments, Fullerton, Calif.) and
Sargent SRG recorder (Sargent-Welch
Scientific, Dallas,
Texas). The assay medium consisted of 0.2 M sucrose, 1.0
mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid
buffer, pH 7.0, 120 mM KCI, 2.5 mM succinate, 2.5 mM P1. 5
14
rotenone,
and
3.0 mg
mitochondrial
protein
in a total
volume of 3.0 ml. The assay system was maintained at 30°
with a water-jacketed
cell and 150 nmoles Ca2@ per mg
mitochondrial
protein were added in each experiment.
2 The
abbreviation
used
EGTA,
ethylenebis(oxyethylenenitrilotetraa
Chart I shows the distribution of cytochrome oxidase
activity (mitochondrial
marker enzyme) in the various
homogenate fractions, with the total activity of the whole
homogenate expressed as 100%. The percentage yield of
mitochondria (Chart I, M.F.) was particularly good, with
approximately 50% of the total cytochrome oxidase activity
in the mitochondrial
fraction. This percentage yield of
mitochondria
is considerably
higher than that usually
obtained with intact organ cell fractionation. This is proba
bly due to the isolated cell suspension itself, making
homogenization
relatively effective and complete. The ac
tual yield of mitochondrial protein was between 4 to 6 mg
mitochondrial
protein per g wet weight of cells. Chart 2
shows representative oxygen electrode tracings of mitochon
drial respiratory activity. It can be seen that NADH-Iinked
respiratory substrates (glutamate- or pyruvate-malate)
are
not well oxidized by these isolated mitochondrial prepara
4
U
2
U
T.H.
N.F.
N.E
PMS.
Chart I . Distribution of cytochrome oxidase activity in various ho
mogenate fractions of lymphoid cells. Assay procedure described in
“Materials and Methods.― The specific activity of each fraction was
determined and expressed in nmoles cytochrome c oxidized per mm per mg
protein. The specific activity of each fraction was multiplied by the total
protein to determine total activity. The specific activities for each fraction
were variable; the mitochondrial fractions (M.F.) exhibited a range of 190
to 320 with a mean of 254 ±43 (S.D.). However, for each preparation the
percentage yields were identical. Values expressed as percentage of total
homogenate
activity. T.H., total homogenate;
N.F.. nuclear fraction;
P.M.S.. postmitochondrial supernatant.
ceticacid).
DECEMBER
is:
RESULTS
1975
3637
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U. Carpentieri
and L . A . Sordahi
ADP/0.I 5
Rd
‘3.3
[02]
nonootoms
-
450
A NAOH
Li'*sd
ONP
B Succinote
- Linked
t(m@
Chart 2. Representative oxygen electrode traces of lymphoblast mito
chondrial respiratory activity. The basic assay medium (final concentra
tions) consisted of: 0.22 M sucrose/S m@ Tris-HCI, pH 7.2; 70 msi KC1;
8 msi P, ; 5 mM MgCI, and 2.0 mg mitochondrial protein in a total vol
ume of 2.0 ml. Temperature.
300. M@ , addition of mitochondria to the
medium; the downward deflection of the trace was the slow pre-State 3
rate of oxygen uptake. Addition of ADP (568 nmoles) produced an in
crease in the rate of oxygen uptake, which was the active State 3 phos
phorylating rate of respiration. Numbers to the left oftraces. State 3 traces
of oxygen consumption expressed as nanoatoms oxygen consumed per mm
per mg mitochondrial
protein. A, NADH-linked
mitochondrial
respira
tory activity; 5.0 mM glutamate/2.S
mM malate was the substrate. Addi
tion of ADP produced an increase of oxygen uptake that rapidly declined,
indicating a loss of respiratory control and phosphorylation
activity. Fur
ther addition of ADP had no effect. Addition of 2 @igrotenone (Rot.)
completely inhibited respiration. B. mitochondrial respiratory activity with
succinate (S mM) as substrate. Rotenone (2 @zg)was also included in the
succinate assay to inhibit any endogenous NADH-linked
respiratory activ
ity. After the 1st addition of ADP, addition of 2 @sgoligomycin (Oligo.)
followed by a 2nd addition of ADP caused no change in respiration. Sub
sequent addition of the uncoupling agent, 2,4-dinitrophenol
(DNP, lO
M) resulted in a marked
chondrial preparations (17).
Chart 3 is a representative spectrophotometric
tracing of
mitochondrial
calcium uptake measured by dual-beam
spectroscopy using the calcium-sensitive dye murexide. The
2 equal additions of calcium (upward deflection of the
tracing) produce a change in the absorbance spectra of the
dye. The subsequent
addition of respiratory
substrate
(succinate) initiates the rapid energy-dependent
uptake
(downward deflection) of calcium by the lymphoblast
mitochondria
(Chart 3). The number to the right of the
tracing indicates the initial rate of calcium uptake by the
mitochondrial
preparation
expressed in nmoles/min/mg
mitochondrial protein. In the various preparations used in
250
n mol@
increase in oxygen uptake that was effectively
inhibited by the electron transport chain inhibitor antimycmn A (Anti. A,
3 gig). A DP/O, nanoatoms of oxygen consumed in State 3 to phosphoryl
ate the amount of ADP (in nmoles) added. RCJ. respiratory control index
was the ratio of the rates of respiration in State 3 and State 4 and is an
index of the “tightness―
of respiratory coupling of the mitochondria.
tions (Chart 2A). Addition of ADP produces an apparent
increase in respiration,
but this respiratory rate rapidly
slows and a 2nd addition of ADP has no effect (Chart 2A).
Addition of rotenone completely inhibits the remaining
respiration (Chart 2A). Exogenously added NADH was not
oxidized by any of these mitochondrial preparations (data
not shown); however, the lymphoblast mitochondria exhib
ited good respiratory activity with succinate as a substrate
(Chart 2B). Upon addition of ADP, a marked transition
from the slow “pre-State 3―rate of respiration to the active
phosphorylating
State 3 rate of respiration is observed
(Chart 2B). When all of the ADP has been phosphorylated,
a return to the slower, resting State 4 respiration
is
observed. Addition of the phosphorylation
inhibitor, oh
gomycin, followed by another addition of ADP causes no
change in respiration (Chart 2B), as would be expected in
intact mitochondria. Subsequent addition of the uncoupling
3638
agent, 2,4-dinitrophenol,
produces a marked increase in
respiration (Chart 2B). Finally, the uncoupled respiration
can be blocked by the electron transport chain inhibitor,
antimycin A (Chart 2B). The rate of active phosphorylating
respiration in State 3 is indicative of the optimal respiratory
activity of mitochondria and is a measure of the amount of
active enzymatic protein in a given mitochondrial prepara
tion. The State 3 rates of succinate-supported
respiration in
the various lymphoblast mitochondrial preparations in this
study ranged from 48 to 136 nanoatoms of oxygen con
sumed per mm per mg mitochondrial protein. The ADP/O
ratios, which are an indication of phosphorylative
effi
ciency, had a range of 1.5 to 1.8, which is in normal
experimental limits with succinate as substrate. The respira
tory control indices are similar to values obtained in normal
cell mitochondrial preparations (17). This value is a reflec
tion of the “tightness―of respiratory coupling in mito
25
C
Chart 3. Representative
trace of mitochondrial
calcium uptake mea
sured by dual-beam spectroscopy. The chelometric dye murexide (ammo
nium purpurate) exhibits linear changes in absorbance at the wavelenth
pair 541-507 nm, when complexing with calcium. Two equal additions of
calcium (Ca'@, 125 nmoles/addition)
caused upward deflections of the
trace. Subsequent addition of succinate (Succ., 2.5 mM) produces respira
lion-supported
mitochondrial
calcium uptake (downward
deflection).
Breaks in traces, artifacts at the point of addition. Number to the right of
trace, initial rate of calcium uptake in nmoles/min/mg
mitochondrial
protein. The assay medium consists of: 0.2 M sucrose; 5 mM Tris-HCI: pH
7.2; 70 mM NaCI; 8 mM P1: SOpM murexide; 5 sg rotenone; and 3 mg
mitochondrial
protein in a total volume of 3.0 ml. Temperature,
30°.At
the point “Ca'@release― (- - -), preparations of mitochondria from normal
mammalian cell types would release the accumulated calcium either by
inhibition of the respiratory chain or by allowing the system to become
anaerobic. In the case of the lymphoblast mitochondria this did not occur.
as indicated by the continuing solid line at the bottom of the recording.
CANCER RESEARCH
VOL. 3S
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Functions
this study, a range of 55 to 75 nmoles calcium accumulated
per mm per mg mitochondrial protein was obtained. Under
these calcium uptake conditions, normal mammalian cell
mitochondria will release the accumulated calcium when a
mitochondrial
respiratory inhibitor is added or when the
system becomes anaerobic (Chart 3, dashed line). These
preparations of lymphoblast mitochondria did not release
the accumulated
calcium under any conditions. Chart 4
shows the oxygen uptake and H@ ejection by lymphoblast
mitochondria
during calcium uptake in the presence and
absence of phosphate. The Ca2@/2e and H@/2e ratios are
similar to those obtained with normal tissue mitochondria
in the presence of phosphate (Chart 4A ). No oxygen uptake
or H@ ejection occurred in the absence of phosphate when
calcium was added (Chart 4B). Separate measurements of
calcium uptake by either dual-beam spectrophotometry
or
with “Caindicated no active calcium uptake by these
mitochondria in the absence of phosphate (data not shown).
Fig. I shows electron micrographs of isolated suspensions
of lymphoblast mitochondrial preparations.
It can be seen
that the preparations
are relatively free of other cellular
contaminants
(Fig. IA) and that the mitochondria
have
intact outer membranes and substantial inner membrane
protein (Fig. 1B). The ultrastructural
configuration of the
inner membranes (cristae) of these mitochondria appear to
be what has been described as the condensed state by
Hackenbrock (7). This is typical of freshly prepared suspen
sions of normal liver mitochondria as well as mitochondria
from some tumors (17).
DISCUSSION
The above studies show that coupled, actively respiring
mitochondria can be obtained from leukemic lymphoblasts
grown in cell culture. These mitochondria appear to respire
best with succinate as respiratory substrate. The rates of
respiration during State 3 oxidation of substrate are some
what variable and are lower (48 to 136 nanoatoms oxygen
consumed per mm per mg) than those usually observed in
normal cell mitochondrial preparations (17). This may be
due to the fact that these mitochondria are derived from
tumor cells. It should be mentioned that actual comparisons
to normal lymphocyte mitochondrial values cannot be made
since appropriate controls were not run. Obtaining mito
chondria from normal lymphocytes or attempting to grow
these cells in culture is quite difficult. Studies are currently
in progress to develop a suitable “control―
cell preparation.
Preliminary studies of mitochondria isolated from cultures
of normal human lymphocytes indicate higher respiratory
and calcium uptake rates, as well as release of the accumu
lated calcium when these preparations
are blocked by
metabolic inhibitors or become anaerobic. However, the
“apparent―
low respiratory rates and lack of NADH-hinked
respiratory activity in the lymphoblast mitochondria
are
similar to results obtained from a number of tumor cell
types (17). It is also quite possible that, since these
mitochondria have been derived from tumor cells, a lack of
membrane-bound
pyridine nucleotide (NAD) results in the
poor rates of respiration with NADH-linked
substrates.
DECEMBER
1975
of Lymphoblast
Mitochondria
T
75
natoms 0@
I
t++
Ct
Co
‘
@@_±_!a@i_
8.—P,
I
300
notoms H@
I
$*.Imin-.@
Ccr/2.-
. .86
H@'/2e ‘ .76
4+
Co /2
H@/2e
: 0
:0
Chart 4. Representative
traces of oxygen uptake and H@ ejection by
lymphoblast mitochondria
during calcium uptake. Determinations
were
made independently under identical conditions. Details of assay described
in “Materials and Methods.― Calcium (Ca2@)was added at ISO nmoles/mg
mitochondrial
protein. Upper traces, oxygen consumption
(downward
deflection); lower traces, H@ ejection (upward deflection) in the presence of
calcium. A, with P1 =+P1); B, no P, (—P,).
Previous studies ( 17) with a variety of tumor cell mitochon
dna have shown that, in general, succinate-linked
respira
tory activity is better than NADH-linked
respiratory activ
ity. It is also possible that the bound mitochondrial NAD is
more labile in these preparations and is lost during isolation.
The effects of the various mitochondrial inhibitors (Chart 2)
also indicate that these lymphoblast mitochondria have all
the usual properties associated with intact, actively respiring
mitochondria.
The calcium uptake capabilities
(Chart 3) of these
mitochondria appear to be more consistent, in contrast to
the variable and slower rates of respiratory activity (Chart
2). This is of particular interest, since it indicates that
utilization of electron transport-generated
energy for cal
cium transport appears to be very efficient in these mito
chondria. A modified or more active calcium binding or
transport protein may be present in these mitochondrial
membranes. Reynafarje and Lehninger (14, 15) have shown
that mitochondria
from Ll210 mouse leukemic cells or
from normal liver tissue exhibit a superstoichiometry
of I-I@
ejection with respect to Ca2@ uptake when a permeant
anion such as phosphate is absent. In the studies reported
here we obtained the normal expected ratios for Ca2@/2e
and H@/2e with lymphoblast mitochondria in the presence
of phosphate (Chart 4A). In the absence of phosphate, no
calcium uptake was observed and no stimulation of oxygen
consumption
or W ejection occurred (Chart 4B). The
reason for this result is at present unclear. However, unlike
mitochondria
from normal mammalian cells, these mito
chondria from lymphoblastic
cells did not release the
accumulated calcium when the system became anaerobic or
when the electron transport chain was blocked by inhibitors.
Recently, Thorne and Bygrave (19) demonstrated a lack of
spontaneous release of accumulated Ca2―in mitochondria
3639
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U. Carpentieri
and L. A . Sordahl
from Ehrhich ascites cells. These investigators (19) have
suggested that the retention of Ca2@ in vivo could result in a
change in the intracellular Mg2@/Ca2@ ratio, resulting in
more rapid or possibly uncontrolled reaction rates of other
cellular metabolic processes. It is attractive to speculate that
the apparently unique cation transport properties of tumor
cell mitochondria may represent a mechanism leading to the
altered metabolic functions of tumor cells.
The electron micrographs revealed intact organelles with
substantial inner membranes (Fig. 1). This is in contrast to
the sparse amount of inner membrane (cristae) observed in a
number of other tumor cell mitochondria (17). Since the
respiratory rates were relatively “low―
(Chart 2) and are
considered to be indicative of active enzymatic protein
(inner membranes),
it is possible that these lymphoblast
mitochondria have lesser amounts of actual enzyme protein
(flavoproteins, cytochromes, etc.) in the inner membranes.
Analysis of actual cytochrome content as well as other
electron transport chain constituents remains to be done.
These studies have demonstrated the feasibility of obtain
ing intact respiring mitochondria from cultured lymphocyte
cell populations.
Experiments are currently underway to
reduce these procedures to microtechniques
and to obtain
purified lymphocytes from normal circulating human blood
as well as from leukemic patients. These studies will
hopefully provide additional insight into the energy-linked
functions of mitochondria in normal and abnormal white
cells.
ACKNOWLEDGM
ENTS
Our appreciation is extended to Dr. C. W. Abell and Dr. Tom Monahan
for supplying the lymphoblast cell cultures and to Dr. Norm Granholm and
Larry Thorpe in the preparation of the electron micrographs. The technical
assistance of Michael Stewart in these studies is gratefully appreciated.
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3640
CANCER RESEARCH
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VOL. 3S
Functions
of Lymphoblast
Mitochondria
L@.
-%\
@0'
@
.:@4
.@. . 4.
@-r
S
1
..1.
I
I
Fig. I. Representative electron micrographs of fresh suspensions of lymphoblast mitochondria. A , low-power view showing numerous intact
mitochondria with well-defined cristae. Some cellular debris is apparent. Stained with uranyl acetate and lead citrate. x 19,000. B, high-power view of
lymphoblast mitochondria showing intact outer membranes and significant amounts of inner membrane protein (cristae). The inner membranes are in a
condensed configuration. See text for further details. Stained with uranyl acetate and lead citrate. x 68,500.
DECEMBER 1975
3641
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1975 American Association for Cancer Research.
Functional Properties of Mitochondria Isolated from Murine
L5178 Lymphoblasts Grown in Cell Culture
Ugo Carpentieri and Louis A. Sordahl
Cancer Res 1975;35:3636-3641.
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1975 American Association for Cancer Research.