[CANCER RESEARCH 30, 2832—2840, December 1970]
Isolation of Meth A Cell Surface Membranes Possessing
Tumor-specific Transplantation Antigen Activity
.
S
•
.
S
Duncan L. McCollester
Department of Surgery, FrancisDelafield Hospital, Collegeof Physiciansand Surgeonsof Columbia University, New York, New York 10032
SUMMARY
A method is described for isolating the surface membranes
from both transplanted Meth A ascites tumor cells in BALB/c
mice and the leukemic cells of AKR/J mice. These cells are
harvested
and washed
several times in a solution
of 150 mM
NaC1, 50 mM borate, 1.0 mM CaCl2 , and 1 mM MgCl2 , pH
7.2, at 0°. The cells are then extracted
at room temperature
in
large volumes of 0.2 mM EDTA and either 2.5 mM borate
(Meth A) or 20 mM borate (leukemic) at pH 9.2. After ex
traction, the further addition of 20 mM borate, pH 9.2, pre
cipitates out most of the intracellular structures. The mem
branes are freed of contaminating
particles
by washes in dilute
borate. The final product consists essentially of empty bags,
the surface membrane ghosts. Chemical and electron micro
scopic studies confirm the almost exclusive recovery of surface
membranes. The Meth A membranes possess tumor-specific
transplantation
antigens according to the transplantation
challenge test in BALB/c mice, the strain of origin of the Meth
A tumor. The leukemic cell membranes do not immunize
against
Meth A tumor.
The onset
of detectable
immunity
is
quite rapid, reminiscent of second set rejection of skin grafts
following i.p. sensitization with donor spleen cells. Isolated
Meth A surface membranes will be used for immunogenicity
studies of tumor transplantation antigens.
INTRODUCTION
This
paper
membranes
describes
a method
for isolating
the surface
Meth A tumor cells. These isolated membranes
possess TSTA2 or TSTA-like activity. TSTA are one of the
products of the malignant transformation
process. They are
present in tumors but not present in the tissues of animals in
which the tumors arise (19, 21, 30, 38). The Meth A tumor is
descended from a lymphosarcoma which Old et aL (3 1), using
methylcholanthrene,
induced in a member of their syngeneic
BALB/c colony. The tumor, in ascitic form, has been main
tamed
by serial
passage
in this colony.
Hitherto,
Meth
A
TSTA have been detected only in the living cell.
The in vivo detection of TSTA depends upon immunization
of an animal with tumor material so that the animal is able to
reject a subsequent challenging transplant of the same tumor, a
transplant which would be lethal had the animal not been
immunized.
In a serially transplanted
tumor, it is conceivable
that mutations
or viral infections subsequent
to the original
malignant transformation
may account for what appear to be
TSTA. This possibility must be reckoned with in the study to
be described. TSTA appear to behave as weak histocompati
bility
antigens
in
part
by
American
Cancer
Society
Grant
T-446
and
Damon Runyon Memorial Fund for Cancer Research Grant DRG-1023.
2 The
abbreviation
used
is:
TSTA,
tumor-specific
antigens.
Received March 12, 1970; accepted July 31, 1970.
2832
transplantation
they
are present
on the cell surface
allograft. An antigen-specific
form of immunological
paralysis
develops (1). Also, TSTA are extremely labile. Their activity
disappears with cell death or if the cell is treated with trypsin
or, in certain instances,
with high-dose
irradiation
(37).
Attempts to obtain TSTA in a subcellular state have had only
limited success (7, 15, 42, 45).
The lability of TSTA is reminiscent
of the lability of cell
surface membranes,
which lose their permeability
charac
teristics upon cell death (4, 17), treatment
with trypsin (2,
33), or exposure to irradiation (17). These similarities of
lability suggest that the proper isolation of the surface mem
brane as an intact structure might also permit the continued
existence of TSTA activity. This would then make possible the
recovery of TSTA in a subcellular form.
Good
methods
are available
for isolation
of surface
mem
branes from nonnucleated erythrocytes (e.g., Ref. 11). Because
there is no intracellular structure, these erythrocyte cells may
be readily extracted with hypotonic media. A nucleated cell,
on the other hand, possesses many intracellular structures
including cytoplasmic membranes, which resist extraction by
hypotonic solutions. Furthermore, when these cells are dis
rupted, the cytoplasmic membranes break down to form
particles
or vesicles, which are often indistinguishable
from the
particles derived from the disrupted surface membrane. This
difficulty can be partially surmounted by exacting centrifugal
separation of particle types (3, 8, 16) or by a method of cell
disruption
which enables the surface membrane to be
recovered as identifiable larger fragments (24, 28, 32, 36, 44).
However, these methods either are limited to a single cell type
or use harsh conditions, which might be expected to destroy
fragile membrane
components.
Consequently,
work in this
laboratory has been directed at development of a method of
isolating easily identifiable large fragments of cell surface
membranes,
one which
is applicable
to diverse cell types and
which uses mild physical and chemical conditions.
The method
1 Supported
in that
membrane and stimulate the development of TSTA-specific
cellular immunity (18). However, the response of a host to its
tumor does not appear to be identical with its response to an
to be described
is based upon
earlier
studies
(25, 26) involvingthe isolationof surfacemembranecom
ponents from segments of skeletal muscle cells. After appro
priate treatment, the contractile proteins of these segments
were readily
soluble
in water or in 0.6 M NaCl but were not
CANCER
RESEARCH
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30
Isolation of Meth A Cell Surface Membranes
soluble in 0.025 to 0.15 M NaCl. This behavior was unusual, mM CaCl2 , 250 mM MgCl2 , and 10 mM EDTA, adjusted to pH
for contractile proteins normally must first be extracted from 9.6 with NaOH pellets and 1.0 N NaOH.
A Wild M-20 binocular photo microscope with Wild-Fluotar
their in situ environment by prolonged exposure to strong salt
40X phase objective, lox eye piece, built-in illumination and
solutions before the aforementioned solubility characteristics
are observed (39). It was attributed to a cytoskeleton, prob 35-mm camera back was used for monitoring each preparation.
ably the endoplasmic reticulum (34), which must first be A 100-watt external light source was used for photo
broken down before the contractile proteins are soluble in microscopy.
For electron microscopy, membrane preparations were
water. Further studies on skeletal muscle indicated that the
cytoskeleton was stabilized by FAD. The removal of FAD by either fixed for 2 hr with 3% glutaraldehyde in phosphate
buffer at pH 7.6 followed by 1% osmium tetroxide, pH 7.6,
apo-D-amino acid oxidase resulted in cytoskeletal breakdown,
for 30 mm or they were fixed for 2 hr with 1.2% KMnO4 in
Le., the solubilization
of contractile
proteins
in situ by water.
A survey of inhibitors of FAD-dependent enzyme revealed 0.2 M Veronal-acetate, pH 7.6, buffer at 4°(23). The fixed
that borate and EDTA effectively break down the cyto membranes were dehydrated with increasing concentrations of
skeleton. These agents also transform other cell types to a ethanol, exposed to three 30-mn changes of propylene oxide,
water-extractable state, and this characteristic led to their use and left overnight in 1: 1 propylene oxide:Epon 8 12 mixture.
They were then transferred into capsules containing Epon 812
in the method to be described.
mixture and polymerized at 60°for 2 days. Thin sections were
prepared with a diamond knife on an LKB ultramicrotome III
MATh@ALS AND METHODS
(LKB InstrumentsInc.,Rockville,Md.),mountedon Formvar
carbon-coated grids, and stained with 2% aqueous uranyl
search, New York, N. Y., very kindly provided Millerton acetate for 30 mm followed by 0.4% lead citrate (43) for 5
Research Farms, Millerton, N. Y., with animals from his mm. They were examined at 75 kV with an Hitachi HU-HE
BALB/c
breeding stock. Through brother-sister mating, electron microscope.
For chemistries, membranes were first lyophilized. Lipids
Millerton Research Farms built up a colony, providing BALB/c
mice for use by this laboratory. The syngeneity of the BALB/c were extracted with chloroform:methanol (2: 1, v/v) (14). The
mouse colony was repeatedly confirmed by the acceptance of extract was evaporated off and the residue was extracted with
tail skin grafts between randomly selected female members of small volumes of chloroform. This extract was transferred to
foil boats and evaporated,
and the residue was
the colony. These grafts survived for at least 3 months. aluminum
Occasional histological examination confirmed the absence of weighed. Hexoses were extracted with hot 10% trichloracetic
an immune response against the grafts. Dr. E. Boyse also pro acid for 20 hr and estimated by the 3 cysteine H2SO4 reaction
Dr. E. Boyse
of Sloan-Kettering
vided his laboratory's
Institute
Meth A sarcoma,
for Cancer
Re
which was induced
and
has been maintained in their colony of BALB/c mice (31).
Since receipt in this laboratory, this tumor has been carried in
the ascitic form in our BALB/c females. Transfer into fresh
animals has been done at weekly intervals by the i.p. injection
of approximately 1 X 106 tumor cells. Six-month-old, retired
breeder AKR/J females were obtained from the Roscoe B.
Jackson
Memorial
Laboratory,
Bar Harbor,
the description
of 52/54-inch
For harvesting
centrifugations
and preextraction
Isolation of Meth A Cell Surface Membrane
From stock solutions are made 500 ml of harvesting solu
tion (150 mM NaCl, 50 mM borate, 1.0 mM CaC12 and I .0
mM MgCl2 , pH adjusted to 72 with 1.0 N NaOH), 800 ml of
extraction solution (2.5 mM borate, pH 9.6, and 0.2 mM
EDTA), and 750 ml of membrane wash solution (20 mM
borate, pH 9.6, and 1.0 mM EDTA). The pH of both of these
pH 9.6 borate
washes, the International
brane wash solutions are chilled in ice. The extraction solution
is kept at room temperature and equally divided between four
Co. clinical centrifuge was used. All subsequent
were performed
MEMBRANE ISOLATION METHODS
100% Super Nylon Tulle,
Quality 109, white.
Equipment
perchloric acid (29) and estimated by the diphenylamine (6)
and orcinol (29) reactions. Total carbohydrates were estimated
by the method of Devor (9) and nitrogen was estimated by the
microkjeldahl method (20).
Me. These animals
begin to develop leukemia at this age.
Harvesting and preextraction washes were done in 12-nil
graduated, glass centrifuge tubes and extractions were done in
300-nil glass Erlenmeyer flasks. The initial postextraction cen
trifugation was done in 250-ml Autoclear bottles (Inter
national Equipment Co., Boston, Mass.) and subsequently in
30-mi Autoclear tubes. Nylon mesh with holes about 0.8 mm
square was obtained from H. Bates Co., New York, N. Y.,
under
( 10). DNA and RNA were differentially extractedwith
with an International
Equip
ment Co. B-20 refrigerated centrifuge furnished with a 6-place,
No. 872, 250-nil, fixed-angle head and a 4-place, No. 940,
40-mi, horizontal swinging head. Distilled water, used exclu
sively, was prepared by a Loughborough 4-liter/hr glass still
(Beilco Glass, Vineland, N. J.) attached to tap water. Only
analytical grade reagents were used. Stock solutions consisted
300-mi
solutions
Erlenmeyer
falls to 9.2. The harvesting
flasks set up for stirring
on a magnetic
stirrer.
Step I. Between the 7th and 10th day after transplant, Meth
A tumor-carrying BALB/c mice are sacrificed by cervical dis
location, and the ascitic fluid is removed. The fluid is divided
between 4 graduated 12-ml centrifuge tubes packed in ice,
each containing approximately 7 ml of harvesting solution. It
is desirable to obtain sufficient ascitic fluid to yield 1.0- to
of 500 mM boric acid, 500 mM sodium borate (pH 9.6) (made
1.5-rn! cell pellets
fresh daily from boric acid and NaOH pellets), 1 M NaCl, 250
inverted several times to suspend the cells.
DECEMBER
and mem
at the time of extraction.
The tubes
1970
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1970 American Association for Cancer Research.
are
2833
Duncan L. McCollester
Step II. The tubes are centrifuged in a clinical centrifuge for
60 sec at 80% maximum speed.
Step III. The supernatants are decanted, and the pellets are
suspended in 10 ml of harvesting solution with a disposable
glass Pasteur pipet. Steps II and III are repeated twice.
Step IV. The pellet of each tube should be 1.0 to I .5 ml.
Each pellet is destined
to be extracted
in a separate
flask. The
magnetic stirrer of each flask is turned on to slow speed, pro
ducing up to a 1.5-cm vortex. The supernatants of the tubes
are decanted, and each pellet is rapidly suspended in approxi
mately 2 volumes of extraction solution taken from its flask.
Immediately, the suspension is added back to the flask.
Extraction proceeds for 10 mm.
Step V. Eight ml of 500 mM borate, pH 9.6, are quickly
added to each flask, and the magnetic stirrer continues to
operate for another 5 sec. Numerous small white clots form.
Step Vi. Each flask is decanted through 2 layers of nylon
mesh, previously moistened with water, into 250-mi plastic
centrifuge bottles.
Step VII. The bottles are centrifuged at 5000 rpm for 10
ruin at 10°.
Step VIII. The bottles are decanted rapidly, and 25 ml of
membrane wash solution are added. The bottles are capped
and shaken vigorously, bringing into suspension material on
the bottle walls and bottom.
Step IX. The suspensions in the bottles are transferred to an
equal number of 40-mi glass centrifuge tubes and centrifuged
on the clinical centrifuge at highest speed for 75 sec. This
removes additional clots as well as unextracted cells.
Step X. The glass tubes are decanted, each into a 30-mi
plastic centrifuge tube, and the supernatants are centrifuged at
5000 rpm for 10 mm.
Step XI. The tubes are decanted, and the pellets are sus
pended in 25 ml of membrane
wash solution.
Step XII. The suspensions are transferred to 40-mi glass
centrifuge tubes and centrifuged on the clinical centrifuge at
the highest speed for 45 sec.
Step XIII. The supernatants are centrifuged at 5000 rpm for
10 ruin and decanted, the pellets are resuspended in 25 ml of
membrane wash solution, and again the tubes are centrifuged
at 5000 rpm for 10 mm.
Step XIV. After decanting, the pellets are suspended in 1.5
ml of membrane wash solution and inspected.
Isolation
of AKR/J Leukemic
Cell Surface Membrane
Step I. Approximately 10 g of leukemic tissue, wet weight,
are removed with scissors from leukemic AKR/J mice and are
placed in a 100-mi beaker containing approximately 20 ml of
harvesting solution. The tissue is minced with scissors, and
then, with swirling and the aid of an additional 25 ml of
harvesting
solution,
the tissue suspension
is transferred
to a
50-rn! beaker, covered with 2 layers of nylon mesh affixed
with a rubber band, and pushed halfway down the beaker. A
pad of 6 or 8 layers of mesh is placed on top of the mesh, and
the beaker is packed in ice. The tissue is forced through the
nylon mesh with a pestle, consisting of a 12-mi test tube
packed
with ice and covered
with 2 layers of mesh. This is
essentially a variation of the method of Vaage (41). The crude
2834
suspension is then divided between 4 centrifuge tubes and
placed in the clinical centrifuge. The latter is brought to high
speed for 3 sec and then turned off. The supernatants are
decanted into graduated centrifuge tubes. Thereafter, the
exact procedure as described for the Meth A cells is followed,
beginning with Step II and with the following exceptions. In
Step II, centrifugation is for 75 sec at 80% maximum speed; in
Step V, the extraction solution consists of 20 mM borate and
0.2 mM EDTA; in Steps VII, X, and XIII, centrifugations are
at 7500 rpm for 10 ruin; in Step IX, centrifugation is for 2
mm; and in Step XII, centrifugation is for 1 ruin.
RESULTS
Gross Appearance of Membrane Pellets. The final pellets of
both Meth A and AKR/J leukemic ghosts are white. Any tinge
of brown is indicative of significant contamination (see
below). The pellets disperse easily to form a white turbid sus
pension.
Microscopic Events during Membrane Isolation. After the
final preextraction wash, the pellet may be resuspended in
harvesting
solution.
If a drop
under
a coverslip
is then
examined by phase microscopy at the same time as extraction
solution is drawn under one edge of the coverslip, the micro
scopic events during extraction can be observed. What occurs,
in sequence, is a slight swelling of the cell, the appearance of
Brownian movement of intracellular particles, and then either
a bursting of the cell with the nucleus being expelled and
floating away followed by intracellular particles or a bursting
of the cell and expulsion of the nucleus while the nucleus stays
attached to the cell ghost. Cells that do not burst or swell also
do not display Brownian movement of intracellular particles.
These cells have a shrunken appearance and are probably dead.
At the completion of extraction, the addition of borate pre
cipitates a great deal of material, apparently mostly DNA, with
a coalescence of the many completely extruded and very
hydrated nuclei. Those ghosts still attached to their nuclei are
coalesced with the nuclei and are never recovered. However,
the completely detached membrane ghosts are not coalesced
and are ultimately recovered. Thereafter, numerous cell
particles and fat droplets are progressively lost in the super
natants from Steps X and XIII. The latter may streak the inner
side of the centrifuge tubes or form a poorly defined layer on
the top of the tube. As the particles are lost, the pellet loses a
central brownish yellow zone and becomes pure white. Steps
Ix and XII remove unextracted cells, presumably mostly dead,
and nuclei.
Microscopic Appearance of Isolated Surface Membrane
Ghosts and Contaminants. The ghosts under 400X phase
magnification appear as structureless bags offering very little
contrast. Occasional ghosts contain partially or wholly ex
tracted
nuclei,
and many
contain
from 3 or 4 to up to 50
particles, presumably mitochondria. Many ghosts are free of all
visible structures. Virtually no extra ghost particles, whole
cells, or shreds of nuclear material are present. Fig. 1 and 2
show typical preparations
of isolated
and AKR/J leukemic cell membranes,
contrast
Meth A cell membranes
respectively. The ghosts
only faintly with the background,
the most prom
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Isolation ofMeth A Cell Surface Membranes
nent contrast being at the periphery or where the membrane
may be folded or torn.
Electron Microscopy. As depicted in Fig. 3, the electron
microscopic
study of KMnO4 -fixed membranes
discloses linear
elements with the trilamellar structure typical of biological
membranes (35). The membranes are about 80 A thick. In
over 50 photographs, no mitochondria or nuclei were seen.
Glu t a r al dehy de -and osmium tetroxide-fixed
membranes
revealed no additional structures. However, the contrast in the
trilamellar structure of the membrane was much less.
Basic Chemistries.
In batches,
a combined
total of 15 .2 ml
of packed Meth A cells was subjected to the membrane isolation
method. The resulting ghosts were pooled, centrifuged at
12,000 rpm for 0.5 hi, and decanted, and the ghost pellets
were lyophilized, yielding 11.I 0 rug of white, tacky material.
From this material, 0.240-, 0.360-, and 0.390-mg aliquots were
taken
for total nitrogen
analyses.
The remaining
material
was
refluxed for 6 hr with chloroform:methanol
(2: 1, v/v).
Unextracted material was recovered as a pellet and dried in a
vacuum over H2 504 , yielding 4.58 rug of grayish material,
which was divided into 3.10- and 1.48-mg aliquots for nucleic
acid and hexose extraction, respectively. The chloroform:
methanol extract was separated into 2 phases by the addition
of 0.2 volume of 0.05 M NaCl. Each was then dried in a vacuum
over H2 504 and reextracted with chloroform for total lipid
determination. Total carbohydrate was estimated for all of
these fractions and their residues. The results are summarized
in Table 1.
Yields. Estimates made from counts on recovered ghosts
and the numbers of whole cells just prior to extraction in
dicate that ghosts are recovered from 10 to 50% of the Meth A
cells and 50 to 70% of the AKR/J leukemic cells. From the
data
above,
it is evident
that
1 ml of packed
Meth A cells
yields about 0.73 mg of lyophilized material in isolated ghost
fraction.
Some Physical Characteristics of the Ghosts. The ghosts
tend to stick to glass. This makes photomicroscopy difficult,
for, when the ghosts adhere to the glass of a slide, they tend to
break up into particles and lose their identifying
Severe turbulence and shearing forces reduce
particles. Pipettings for suspension or transfer
tend to reduce the ghosts to a particulate state.
morphology.
the ghosts to
purposes also
The ghosts of
both Meth A and AKR/J cells may be transformed to a micro
scopically invisible state by 20 forceful passages with a syringe
through a No. 21 needle.
TSTA Activity of Isolated Meth A Membranes. After
preparation, isolated Meth A membranes were centrifuged to a
Table 1
Basicchemistriesof
membranesComponent%
isolatedMeth A cell surface
weightNitrogen
dry
(as protein)
Lipid (CHCI3 soluble)
Carbohydrate (CHCI3insoluble) (as glucose)
Carbohydrate (CHC1, soluble) (as glucose)
10%Trichloroacetic acid-extractable hexose (as galactose)
DNA
RNA41,44
Table 2
Immunogenicity ofmembrane ghost preparations againstMeth A
ascitestumor cells in BALB/c mice
40
4
0.5
0.9
0.2
0.3
Survivors/ImmunizationCeilsin
no.
challengetotal of
animalsMSD'Experiment
1bWholeMethAghosts5x
itT'10/10Whole
Meth A ghosts1
10'3/1017.1Whole
AKR/J leukemia ghosts5
10@0/iO19.6Whole
AKR/J leukemia ghosts1
10'0/iOi8.00.85%NaCisolution5
10@0/1019.20.85%NaClsolution1
10'0/1018.0Experiment
x
x
x
x
x
II@Whole
Meth A ghosts5
10@iO/i22i.0Whole
x
Meth A ghosts1
10'4/2320.2Disrupted
Meth A ghosts5
10@7/1221.2Disrupted
x
x
Meth A ghosts1
10'5/2420.70.85%
x
NaCl solution5
10@0/1219.50.85%
x
NaClsolution1
x 10'0/2417.4
aMST mean survival time of those animals succumbing to Meth A
ascites tumor.
bExpenment I immunization: 0.2 ml i.p. on Days 0, 7, and 14, with
i.p. challengeon Day 21.
CExperiment II immunization: 0.2 ml i.p. on Days 0 and I , with i.p.
challengeon Day 4.
pellet at 5000 rpm for 10 mm. The membrane
pellet was then
suspended in 150 mM NaCl to give a concentration of approxi
mately 1 x I @6ghosts/0.2 ml. Where indicated, isolated
AKR/J
leukemic
cell membranes
membrane preparations
preparation
were similarly treated.
were used for immunization,
These
a fresh
being made for each day of immunization.
In Experiment I, BALB/c female mice from 3 to 4 months
of age were immunized on Days 0, 7, and 14 with the i.p.
injection of 0.2 ml of membranes isolated from either Meth A
or AKR/J leukemic cells. The mice were challenged on Day 21
with either 50,000 or 100,000 freshly harvested, viable Meth
A cells. The number of animals surviving on Day 80, compared
with the number challenged is given in Table 2. The mean
survival times of those animals succumbing are also given.
These animals died with a massive Meth A tumor ascites.
In Experiment II, isolated Meth A membranes, approxi
mately 1 X 106 ghosts/0.2 ml in 150 mM NaCl, were divided
into 2 equal aliquots. One aliquot was not further treated. The
other aliquot (6 ml) was forcibly aspirated in and out of a
10-ml syringe via a No. 21 needle. This procedure fragmented
the membrane ghosts and reduced most of them to a sub
microscopic state. BALB/c females from 3 to 4 months of age
were immunized with one or the other preparation on Days 0
and 1. On Day 4, these mice were challenged with either
50,000 or 100,000 freshly harvested, viable Meth A cells. The
results taken on Day 60 are expressed as in Experiment I and
are summarized in Table 2.
DISCUSSION
The foregoing deals with the problem of surface membrane
isolation,
the nature of surface membranes,
and TSTA activity.
The isolation of surface membrane ghosts appears to depend
DECEMBER 1970
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2835
Duncan L. McCollester
practically the only cellular structure
recovered, these mem
branes
for the TSTA activity.
specifically upon borate. Several other buffers with pK's of 7
to 11 have been tried, i.e., imidazole, Tris, Veronal, EDTA,
inorganic phosphate, pyrophosphate, and bicarbonate. Despite
proper adjustments according to osmolarity or ionic strength,
very few ghosts were recovered, and these were covered with
particles (D. L. McCollester, unpublished). It seems evident
that borate not only promotes the extractability of cells by
hypotonic media but also prevents intracellular structures
from adhering to the separated surface membrane ghost. The
Proof of the specificity of this recovered TSTA activity was
attempted by 2 approaches, only 1 being successful. The 1st
approach was to prove that the immunization had to be done
with ghosts from the same cell type as the challenging cells,
i.e. , Meth A cells. For this reason, attempts were made, as in
Experiment I, to immunize BALB/c mice against a Meth A
tumor challenge with ghosts from an unrelated tumor, the
promotion
leukemic
of extractability
is presumably
via cytoskeletal
breakdown. Whether or not it is due to FAD extraction has
not been established. The prevention of particle adherence by
borate may be due to the combination of borate with carbo
hydrate residues (46) on the surface of both the isolated
surface membrane and the intracellular particles. This would
enhance the negative charge of each and hence favor mutual
repulsion as opposed to adherence.
The membrane isolation method conveniently provides
material for studies on the nature of cell surface membranes.
There can be no question that the isolation method permits
the recovery of surface membranes. All steps in the extraction
process can be followed microscopically. Cellular contents leave
the bag comprising the cell surface membrane. This morpho
logically identifiable and unique bag or ghost is the predomi
nant structure recovered. Electron microscopy of this material
reveals the presence of trilamellar structures about 80 A thick,
both features being characteristic of the electron microscopic
appearance of biological membranes (35). Chemical analyses
disclose that the material contains lipid, nitrogen (presumably
largely protein), and carbohydrates. This is consistent with the
present knowledge of cellular membrane composition (5 , 13,
22, 40). However, the key determinant in any assessment of
chemical data is the purity or homogeneity of the surface
membrane preparation. Phase microscopy shows the presence
of contaminating particles, presumably mitochondria. Electron
microscopy, however, does not reveal mitochondrial structure.
Conceivably, the particles observed by phase microscopy may
represent mitochondrial ghosts or possibly the breakdown
products of surface membrane ghosts. Although no nuclei have
been seen under the electron microscope, they have been seen
on rare occasions with the use of phase microscopy, and their
presence
must
be acknowledged.
If each
nucleus
contains
about 6.6 X l0@ rug of DNA (27), 1 nucleus for every 300
surface membrane
ghosts
detected
in the chemical
could account for the 0.2% DNA
analysis of the membranes.
This
assumes that the surface membrane is 80 A thick and has a
density of 1. The presence or absence of nuclear contamina
compatibility
immunization
raises questions
immunogenicity.
cellular
and to the
salt. An ash analysis to estimate
the latter was not done.
With respect to TSTA activity, it is evident that the isolated
Meth A ghost preparations are able to immunize BALB/c mice
against Meth A tumor cells. Because the isolated surface mem
brane ghosts comprise, by morphological and chemical criteria,
2836
The 2nd
approach
was to
antigens
response, albeit weak, is
et aL (12) on weak histo
and the production
of second set
fraction
with TSTA is undoubtedly
as to the factors
The isolation
should
important.
which might influence
This
TSTA
of TSTA activity in a sub
prove most useful in approaching
this
problem. These studies are in progress. Finally, as mentioned
under “Introduction,―the resolution between whether true
TSTA or TSTA-like activity are associated with the isolated
surface membrane ghosts must await experiment with autoch
thonous tumors. These studies are also in progress.
ACKNOWLEDGMENTS
I thank Dr. Davidson for doing skin graft tests, Drs. Z. Dische and C.
Rothschild for determining hexoses, Dr. M. Azzar and Mr. T. D. Pham
for the electron microscopy, and Mr. Zagle for skilled assistance.
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DECEMBER 1970
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2837
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2838
CANCER RESEARCH VOL. 30
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1970 American Association for Cancer Research.
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DECEMBER 1970
2839
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Duncan L. McCollester
3
Fig. 3. Electron microscopic appearance of KMnO4-fixed, isolated Meth A cell surface membrane ghosts. Note the trimellar structure in the
bottom photograph.
2840
CANCER RESEARCH VOL. 30
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Isolation of Meth A Cell Surface Membranes Possessing
Tumor-specific Transplantation Antigen Activity
Duncan L. McCollester
Cancer Res 1970;30:2832-2840.
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