[CANCER RESEARCH 26, 131-136,January 1966]
The Fragility of Normal and Leukemic Lymphocytes
Study of Cell Injury by Physical Agents1
ARNOLD
E. REIF AND JOAN
of AKR Mice: A
M. ALLEN
Biochemistry Section, Department of Surgery, Tufts University School of Medicine, and the First (Tufts) Surgical Service, Boston City
Hospital, Boston, Massachusetts
Summary
The resistance of single cell suspensions to osmotic and ther
mal injury has been determined by use of vital dye exclusion
to document the integrity of the cell surface.
Four newly derived leukemias possessed a resistance to injury
by these physical agents that was similar to that of normal
marrow cells, but higher than that of various types of normal
lymphocytes. On continued isotransplantation, all 4 leukemias
progressed to a higher resistance to such injury.
Introduction
Within the range of tonieities at which the cell surface mem
brane remains substantially intact, much work has been done on
the osmotic properties of single cell suspensions of normal and
tumor cells (2, 4, 14, 15, 19, 33). Outside this range, fragility
tests have long been used as an index of the susceptibility of
erythrocytes and lymphocytes to cytolysis when they are ex
posed to osmotic shock (3, 22, 36, 40). However, the osmotic
fragility of cancer cells appears to have been studied only by
Brues and Masters, who worked with Sarcoma 180 (2), and by
Magalini and Djerassi, whose results suggest that human leukemic lymphocytes are more resistant to hypotonie cytolysis than
are white blood cells (15).
With regard to resistance to other types of physical agents,
Morrow et al. reported that human leukemic leukocytes are
significantly more resistant to cytolysis by ultrasonic vibration
than are normal leukocytes (18). In a previous study, we found
that a relatively high concentration of antiserum was required
to effect immune cytolysis of certain mouse ascites tumor cells
(27). Possible explanations were that these tumor cells possessed
a deficiency in surface antigens (17) or else that their surface
membrane was relatively resistant to immune cytolysis. If the
latter were true, tumor cells might also be relatively resistant
to nonimmune cytolysis. The present investigation was under
taken to examine this possibility.
In this study, the resistance of cells to cytolysis by physical
agents has been documented by vital dye exclusion (21, 29, 30,
32, 37, 39). Cells have been exposed to both hypotonie and
hypertonic media, frozen in media that contained graded amounts
of glycerol, and heated for various time periods at 56°C. The
cells studied have included normal AKR lymphocytes, newly
1This investigation was supported by USPHS Research Grant
CA 044(i9-CK)AI
from the National Cancer Institute.
Received for publication March 15, 1905; revised July l(i, 1905.
derived AKR leukemias, long-transplanted AKR leukemias'
and certain other mouse leukemias and ascites tumors. A pre
liminary report has been made (25).
Materials
and Methods
MICE. All mice were obtained from the Jackson Laboratory.
Normal tissues were taken from mice of either sex, 2-4 months
of age.
NORMAL
AKR CELLS. Single cell suspensions of various types
of normal AKR lymphocytes were prepared in modified Locke's
buffer (23) as previously described (24). Standardization of
cell concentrations was not necessary, but sufficiently high con
centrations (10-30 million cells/ml) were used to facilitate the
subsequent classification under the microscope. Cells were
maintained at 3°Cand were used within 2 hr of their collection.
Red blood cells from AKR mice were collected in Alsever's
solution and washed twice with isotonic saline diluent (28). One
part cell suspension was lysed with 8 parts of distilled water.
The cell concentration was adjusted so that the lysate gave an
O.D.J41of 0.700 when read in 1.0-cm cuvets in the Beckman DU
spectrophotometer (28).
TUMORS. Four newly derived AKR leukemias were used—
namely RAÕ,RA3, RA4, and RA5. The origin of these leu
kemias, and of the long-transplanted AKR leukemias L4946
and S775, has been described in detail (24). Data that illus
trate the biologic progression of these leukemias have been
presented (26).
Five other long-transplanted ascites tumors were employed.
These were the leukemias L70429 and Gardner of C3H mice,
which were transplanted in the C3HeB/Fe substrain (low in
mammary tumors); Ehrlich ascites carcinoma, transplanted in
C57BL/6 mice; Sarcoma 1A, transplanted in A mice; and
E6496 teratoma, transplanted in C3HeB/Fe mice. The C3H
leukemias were kindly donated by I. Wodinski of Arthur D.
Little, Inc.; Ehrlich carcinoma was donated by C. L. Maddock
of the Children's Cancer Research Foundation, Boston; and
the 2 remaining tumors were obtained from A. B. Griffen of the
Jackson Laboratory.
Tumor cells were collected in modified Locke's buffer at a
time after transplantation when the blood content of the perito
neal eluate was still low. Cells were stored at 3°Cand were
used within 2 hr of collection.
OSMOTICCYTOLYSIS.A concentrated solution of modified
Locke's buffer (23) was diluted stepwise in tonicity from 5.0 to
0.028.
A dilution containing 289 m osmoles, as determined in
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131
Arnold E. Reif and Joan M. Allen
oÃ% GLYCEROL
TONICITY
TIME OF INCUBATION AT 56°C, min.
CHART1. Percentage of cells stained with vital dye in various cytolysis tests. The mouse cells used were red blood cells (RBC) thymic lymphocytes (lymphs), L4940 leukemia (L4940), Ehrlich carcinoma (EA CA), and Sarcoma IA (SA A4). Left, Osmotic cytolysis:
percentage of stained cells after incubation for 2 hr at 37°Cin buffer of varying tonicities. Middle, Cryoscopic cytolysis: percentage
of stained cells after freezing for 24 hrat —
20°Cin buffer containing various concent rat ions of glycerine. Right, Hyperthermic cytolysis:
percentage of stained cells after heating at 5(>°C
for different time periods.
TABLE 1
REPRODUCIBILITY
OF CYTOLYSISMEASUREMENTS»
typeRAÕRA5Transplant
Cell
generation22-26
ty6)0.156
(tonici
cytolysis
cytolysis
(tonicity*)1.26
cytolysis
(glycerol concen
%)5.1
tration,6
cyto-1
ysis (Time,''
min)5.2
±0.015
±0.08
1.07.8±
±1.1
22-27Hypotonie
1.50 ±0.07Cryoscopic ± 1.7Hyperthermic
0.152 ±0.007Hypertonie
10.8 ±0.6
" Each result represents the mean of 5 or more determinations ±S.D.
bTonicity, glycerol concentration, and time required to reach the end point of the assay, which cor
responds to 50% vital staining of the cells.
the Fiske osmometer (Advanced Instruments, Inc.), was taken
to be isotonic with serum and was assigned a tonicity of 1.00
(10). Aliquot«of 0.90 ml of buffer dilutions were pipetted into
a series of test tubes; for the lowest tonicities, tubes containing
0.90, 1.40, and 1.90 ml of water were prepared. Then 0.10 ml
of cell suspension in isotonic buffer was added to each tube, the
contents were mixed, and the tubes were set at 37°C. When
necessary, half quantities of all additions were used.
The percentage of viability of cells was determined by vital
dye exclusion (23, 29). The final result for each tube was the
stained cell count obtained after incubation for 2 hr (29). The
end point for the hypotonie cytolysis test was the tonicity below
1.00 required to produce vital staining of one-half of the viable
cells present in the tube that contained the highest percentage
of viable cells, as judged by vital dye exclusion. Hypertonie
cytolysis was determined similarly at tonioities above 1.00.
For red blood cells, aliquots of 0.50 ml of cell suspension were
incubated at 37°Cwith 4.5 ml of buffer dilutions. After 2 hr,
the tubes were centrifuged at 5°C,and the percentage of lysis
was determined by reading the O.D.Mi of the supernatant (29).
The question of whether the above assays are true fragility tests
is discussed later.
CRYOSCOPIC
CYTOLYSIS.The cytolysis caused by freezing
cells in glycerol suspension was determined as follows. Aliquots
of 0.90 ml of serial 2-fold dilutions of 20% glycerol in modified
isotonic Locke's buffer (23) were pipetted into test tubes. To
each tube was added 0.10 ml of cell suspension, and the contents
were mixed. After they had stood for 15 min at room tempera
ture, the tubes were frozen at -20°C. After 24 hr they were
132
thawed in ice water, and the viability of the cells standing at
0°Cwas determined by vital dye exclusion (23, 29). The end
point of the test was the final percentage of glycerol required to
preserve the integrity, as judged by vital dye exclusion, of
one-half of the cells that were viable before freezing.
For red blood cells, aliquots of 0.50 ml of cell suspension were
added to 4.5 ml of glycerol dilutions. After it was frozen as
described above, the cell suspension was centrifuged at 5°Cand
the O.D.Mi of the supernatant was determined (28).
HYPERTHERMIC
CYTOLYSIS.The cytolysis caused by heating
at 56°Cwas determined as follows. Tubes containing 0.2 ml of
cell suspension in isotonic buffer were placed at 56.0 ±0.1°C.
They were withdrawn at timed intervals and cooled by hand
for 10 sec. Then 0.2 ml of staining medium was added and the
stained cell count was determined (23). The end point of the
test was the time required for loss of integrity of the cell mem
brane, as judged by vital staining, of one-half of the viable cells
present before they were heated at 56°C.
For red blood cells, 0.3-ml aliquots of suspension were with
drawn at recorded intervals from the 56°Cwater bath into 2.7
ml of ice-cold isotonic saline diluent (28). The suspension was
centrifuged, and lysis was determined from the O.D.Mi of the
supernatant (28).
In all cytolysis tests, half quantities were used when the sup
ply of cells was limited. Alternatively, the procedure previously
employed for small-scale immune cytolytic assays (23) was
used. Vital staining was accomplished by sucking the superna
tant from a tube and adding 0.04 ml of staining medium.
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Fragility of Normal and Leukemic Lymphocytes
TABLE 2
CYTOLYSISMEASUREMENTSFOR NORMAL AND LEUKEMIC
AKR CELLS«
respectively, for red cells, L4946 leukemia, and Ehrlich carci
noma (mean results of 2 experiments).
In all cytolysis tests, the end point was chosen at 50% stained
cells plus one-half the percentage of stained cells in the control
tonic
tonie
thermic
cytolysis
tube (Chart 1). The reproducibility
plant cytolysis
cytolysis cytolysis
cytolysis
(glycerol
gener
ratio"3.33.14.34.611.46.27.38.17.214.35.310.23.85.86.99.911.810.412.9Cryoscopic
(Time,6
(tonic(tonicdetermined for 2 newly derived AKR
concentra
ation3722-26627-28226-2812322-27+200+200+200Hypoity6)0.500.510.330.370.210.570.240.1900.1560.290.1540.260.1540.370.260.200.1520.1500.260.155Hyper
ity»)1.641.591.421.702.41.491.381.262.12.21.391.581.421.501.381.501.772.72.0Osmotic
tion,6
%)15.1+
min)2.72.93.75.26.85.43.03.85.27.18.42.911.66.07.48.110.87.910.19.9
of these end points was
leukemias (Table 1). For
other cell types, replicate determinations
(Table 2) indicated
that experimental errors were of the same order of magnitude.
AKRcellsLymph
Normal
MEANINGor THE CYTOLYSISDATA. Opie has shown that the
level of electrolytes that is isotonic for normal cells of different
nodelympho
types varies over a range of concentrations
(20), and the same
holds
true
for
tumor
cells
(19).
Hence,
the
stated tonicities
cytesSplenic
relate only to serum, not to individual cell types.
To eliminate
lym
18+1818.04.75.35.26.25.1+187.96.96.412.714.215.17.83.712.514.4Hyperthis difficulty, the osmotic cytolysis ratio has been introduced
phocytesI.
in this study as an index of the range over which osmotic cytol
P. lympho
cytesThymocytesMarrow
ysis is resisted (Table 2). This index is independent
of the
absolute value of isotonicity for any cell type, since it is dimencellsRed
sionless. The higher the index, the higher the resistance to
bloodcellsB.
osmotic cytolysis.
For instance, L4946 leukemia cells in hypotonie solutions could
withstand much lower tonicities than thymic lymphocytes; yet
Newly de
both cell types behaved similarly in hypertonic solutions (Chart
AKRleukemiasR
rived
1). However, without knowledge of isotonicity for the 2 cell
types, it is not possible to judge the significance of the ability of
AlRAIRAIR
the leukemia cells to withstand lower tonicities.
Thus, the only
significant parameter is the range of resistance to osmotic cy
tolysis, and this is indicated by the osmotic cytolysis ratio.
A3R
In the case of cryoscopic cytolysis, glycerol protects cells from
A3R
cytolysis under the standardized
conditions of the test. The
lower the concentration of glycerol required to protect 50% of
A4RA4RASRA5RASRASC.
the cells from cytolysis, the higher the resistance to cryoscopic
cytolysis.
For hyperthermic
cytolysis, the longer cells can
resist the lethal effect of hyperthermia, the higiier their resistance
to it. The present tests represent forms of nonimmune cytolysis,
in contrast to tests (23, 29) that employ immune cytolysis.
NORMALAKR CELLS. Lymph node and splenic lymphocytes
had a lower osmotic cytolysis ratio than intraperitoneal
lympho
Long-trans
cytes and thymocytes (Table 2, A). This may be an artifact
plantedAKR
due to cell damage, since of all cell types studied only lymph
leu
node and splenic lymphocytes
were prepared by traumatic
kemiasL4946S775BW5147Trans
manipulation
of their respective tissues of origin. The lower
resistance of red blood cells to hypotonie lysis is well known (5).
Xo results for hypertonic lysis were obtained for red cells, since
hemoglobin appears to crystallize within the cells at high tonici
0 Each result represents the mean of 2 or more experiments.
ties and thereby prevents rapture and release of hemoglobin
6 Tonicity, glycerol concentration,
and time required to reach
(22). The most interesting finding was that, on the basis of
the end point of the assay, which corresponds to 50% vital
each of 3 cytolysis tests, marrow cells had a considerably higher
staining of the cells.
resistance to nonimmune cytolysis than any type of normal
c The osmotic cytolysis ratio = hypertonic cytolysis/hypolymphocyte that was studied.
tonic cytolysis.
NEWLYDERIVEDAKR LEUKEMIAS. Cytolysis tests were done
with 4 newly derived AKR leukemias within their 1st 6 trans
Results
plant generations (Table 2, B). None was more resistant to
cytolysis than were normal marrow cells (Table 2, A). How
EEPKODUciBiLiTY. In preliminary experiments, the need for
ever, on the basis of at least 1 of the tests, all 4 leukemias were
standardized test conditions became apparent.
For instance in
the cryoscopic cytolysis test, preincubation
of cells in glycerolmore resistant to cytolysis than the other types of normal
containing buffer for 20 min at 37°C,instead of for 15 min at
lymphocytes that were tested.
room temperature,
was investigated.
Under these conditions,
On continued isotransplantation,
the leukemias progressed to
the requirements for glycerine were reduced by 17, 29, and 9%,
higher resistance to cytolysis.
All 4 leukemias showed higher
typeA. Cell
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1966
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133
Arnold E. Reif and Joan M. Allen
TABLE 3
CYTOLYSISMEASUREMENTSFOISSOME LONG-TRANSPLANTED
MOUSE TUMOIIS"
study, cells that became permeable to vital stain in hypertonic
buffer appeared to remain discrete and to retain much of their
contents, in direct contrast to their behavior in hypotonie buffer.
This suggests that the type of injury suffered by the cell mem
brane in hypertonic and in hypotonie media differs. Since cell
thermic
cytolysis
tonic
cytolysis
(glycerol
cytolysis
typeL70429
Cell
cytolysis cytolysis
metabolism influences active transport of ions (35), it may be a
ratio.11.57.730.918.025.2Cryoscopic
(time,
concentra
(tonicity)0.1550.240.1230.0970.155Hyper(tonicity)1.791.843.81.753.9Osmotic
min)28.46.415.316.328.3
tion,
%)3.66.83.66.52.6Hyperrelevant factor in osmotic cytolysis.
However, the properties
of the cell membrane are probably most important.
These
leukemiaGardner
include elasticity, permeability, structure, and perhaps (7) even
leukemiaEhrlich
rate of repair.
carcinomaSarcoma
In the cryoscopic cytolysis test, the cell membrane appears to
1AE6496
be a major site of injury (12, 34). Glycerol probably protects
teratomaHypotonie
the phospholipid-protein
complexes of the cell membrane by
" See the footnotes to Table 2.
modifying the shape and decreasing the volume of ice crystals
within and without the cell, and also by buffering the salt content
of the concentrate
that forms as freezing takes place (11).
resistance to osmotic and to hyperthermio cytolysis, and 2 also
Hence,
a
low
requirement
for glycerol to attain protection may
showed higher resistance to cryoseopic cytolysis.
Compared to
be a valid index of the resistance of the cell membrane to the
normal marrow cells (Table 2, A), 3 of the 4 AKR leukemias in
shock of freezing; however, the rate of diffusion of glycerol into
transplant generations 22-28 had progressed to a higher resist
the cell (14) and its effect on the cell metabolism are also im
ance to cytolysis.
plicated.
LONG-TRANSPLANTKOAKR LEUKEMIAS. The 3 long-transThe water permeability of the cell membrane of living cells
planted AKR leukemias tested (Table 2, C) had a resistance to
increases with rise in temperature
(16) until at elevated tem
cytolysis that was similar to that of the newly derived AKR
peratures it drops sharply (20). This drop is probably caused
leukemias in transplant generations 22-26 (Table 2, B).
and coagulation of cellular proteins, and these
LONG-TRANSPLANTED
MOUSE TUMORS. Except in the case of by denaturation
factors may be of major importance in hyperthermic cytolysis.
the 2 C3H leukemias, L70429 and Gardner, the data obtained
In addition, all the factors enumerated for osmotic cytolysis
with other ascites tumors (Table 3) have far less relevance, since
appear to be relevant.
results for a normal tissue that would be suitable for comparison
Is it permissible to call the osmotic cytolysis tests fragility
are not available.
Nevertheless, 2 points are of interest.
These
tests? On the negative side, osmotic fragility denotes cell de
are the very high resistance of L70429 leukemia to hyperthermic
struction by passage of water across the cell membrane, and this
cytolysis and the ven" high resistance of Ehrlich carcinoma,
term may be inaccurate when applied to cells classified by vital
Sarcoma 1A, and E6496 teratoma to both osmotic and hyperstaining.
Further,
although osmotic fragility is extensively
thermic cytolysis, even relative to AKR leukemias.
used for red blood cells, both cells and test conditions are much
simpler, and the term is therefore more meaningful.
Discussion
On the positive side, tests of hypotonie cytolysis of normal
The present cytolysis tests measured the ability of living cells
leukocytes have long been called fragility tests (36). It is now
to withstand the stress of unphysiologic conditions.
However,
possible to quantify the release of intracellular constituents of
each test gave results that depended on various properties of leukocytes almost as directly as the release of hemoglobin from
red cells, with the use of chromium-51 as label (8, 31, 39). While
different cellular constituents.
This explains why the results
for various cell types did not always run parallel in different tests
labeled cells have so far not been used for osmotic cytolysis, the
(Table 2).
end point of immune cytolysis was similar whether determined
Injury to the cell membrane is indicated by increased perme
by vital staining or with labeled cells (39); some differences
ability to water (13), to vital dyes, and to intracellular compo
would be expected, since permeability
develops faster to vital
nents.
Integrity of the cell membrane appears to be adequately
dyes than to labeled cell constituents (39). Thus, use of differ
indicated by exclusion of vital dyes, since this involves an active
ent criteria to substantiate
cytolysis will change the absolute
function of the membrane.
It is not a drawback that vital dye
results, but it should not change the relative results of tests that
exclusion overestimates cell viability as measured by the ability
are well designed.
Fragility tests for red cells also suffer from a
to proliferate (29, 37), since present concern is with injury of the
lack of standardization
(22).
cell membrane and not with cell sterility.
Was this injury
Despite the reasons to the contrary, the present osmotic cy
primary, or was it secondar}- to injury of other cell components?
tolysis tests are therefore termed osmotic fragility tests.
It
With regard to osmotic cytolysis, slight changes in tonicity
will fall to future investigators to improve and standardize the
produce changes in cell volume that are almost entirely due to
experimental conditions.
It seems inappropriate
to apply the
the exchange of pure water (4). In strongly hypotonie media,
term fragility to the present thermal cytolysis tests.
cells swell owing to influx of water, then progress to admit salt
It is not yet possible to pinpoint differences in the ultimate
solution, and ultimately burst (12). In hypertonic media, cells
molecular structure of the cell membrane of different mammalian
shrink as water is lost, then swell as salt solution enters the
cell types (1, 9, 38). However, such differences must exist, since
damaged membrane, and finally return to their original volume
the surface properties differ considerably.
Thus, tumor cells
as equilibrium in salt concentration
is attained (4). In this
tend to show a lack of contact inhibition and a lowered adhesion,
134
CANCER
RESEARCH
VOL. 26
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Fragility of Xormai and Leukemic Lymphocytes
and appear to carry a higher negative charge and a lower con
tent of tissue-specific antigens, than do their normal counter
parts (1).
The 4 newly derived AKR leukemias studied possessed a
higher resistance to nonimmune cytolysis than normal AKR
lymphocytes other than marrow cells. Of these leukemias,
RA3, RA4, and RA5 were derived from thymus or immediate
precursor cells, while RAÕwas nonthymic in origin (24, 26).
However, to be truly valid, comparisons of thymus-derived leu
kemias would have to be made not with normal adult thymocytes, but with the normal precursor cells of the leukemias.
On continued isotransplantation, all 4 AKR leukemias pro
gressed to a higher resistance to nonimmune cytolysis. Data
obtained with immune cytolysis (26) suggest that, for RA5, the
progression of this resistance between transplant generations 1
and 3 was mainly due to an increase in the proportion of leukemic to normal lymphocytes in the peritoneum; since the per
centage of leukemic cells had reached almost 90% in generation
3, further progression of resistance to nonimmune cytolysis in
later generations cannot be explained on this basis. For 3 of
the 4 leukemias, this resistance finally exceeded that shown by
AKR marrow cells (Table 2).
Thus, the present long-term study of newly derived AKR leu
kemias has helped to establish (15, 18) 1 more difference in
properties (1) between normal and leukemic lymphocytes. Pres
ent results are inadequate (Table 3) to generalize these findings
to other tumors. However, the data (Table 2) illustrate a new-
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
facet of tumor progression (6).
23.
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CANCER RESEARCH VOL. 26
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The Fragility of Normal and Leukemic Lymphocytes of AKR
Mice: A Study of Cell Injury by Physical Agents
Arnold E. Reif and Joan M. Allen
Cancer Res 1966;26:131-136.
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