[CANCER RESEARCH 42, 5106-5116, December 1982]
0008-5472/82/0042-0000$02.00
Induction of Cytoskeleton-associated Proteins during Differentiation
of Human Myeloid Leukemic Cell Lines1
Samuel D. Bernal2 and Lan Bo Chen3
Sidney Farber Cancer Institute and Departments of Medicine [S. D. B.I and Pathology [L. B. C], Harvard Medical School. Boston, Massachusetts
ABSTRACT
Alterations in the expression of proteins associated with the
cytoskeletal framework during differentiation of two human
myeloid leukemia cell lines were analyzed by two-dimensional
gel electrophoresis of Triton-insoluble cellular framework frac
tions. During in vitro differentiation of HL60 (human promyelocytic leukemia line) and U937 (human monocytoid leukemia
line), several new cytoskeleton-associated
(CSK) proteins are
induced. All of these CSK proteins are also present in freshly
isolated normal granulocytes and macrophages. One of these
differentiation-induced
proteins comigrates with vimentin.
There are several differentiation-sensitive
proteins, i.e., those
that are no longer synthesized upon differentiation.
The
changes in CSK protein synthesis during differentiation of
HL60 and U937 cells do not seem to be related to drug
treatment per se since exposure to conditioned medium from
phytohemagglutinin-stimulated
lymphocytes as well as to di
methyl sulfoxide and 12-O-tetradecanoylphorbol-13-acetate
results in the production of many similar proteins. In vitro
conditions that do not result in differentiation of HL60 and
U937, such as cultivation in serum-free medium, do not induce
the CSK proteins that we describe.
A notable finding in this study is that all of the qualitative
changes in the proteins synthesized during differentiation are
detected in the cytoskeletal (Triton-insoluble) fraction, whereas
only minor quantitative alterations are observed in the Tritonsoluble extract. The changes in CSK protein components occur
in an orderly fashion. Vimentin, an intermediate-filament
pro
tein, is synthesized in large amounts prior to changes in cellular
morphology and the induction or loss of other CSK proteins.
Vimentin may play an important role in the reorganization of
the cytoskeleton to support the process of differentiation. The
other CSK proteins are synthesized sequentially along with the
morphological and functional changes during differentiation.
These model systems, therefore, present an opportunity to
investigate the role of specific cytoskeletal components in
cellular differentiation.
02115
develop the morphology of more mature myeloid cells: dimin
ished cell size; decreased nuclearcytoplasmic
ratio; increased
nuclear segmentation; and decreased cytoplasmic basophilia
(12, 28). In association with morphological differentiation,
these cells develop some of the biochemical and functional
characteristics of mature granulocytes. Treatment of HL60 with
a phorbol ester induces differentiation to macrophage-like cells
(17, 35). The cells become adherent to the substrate. The
cytoplasm becomes less basophilic, and the nucleus becomes
more kidney shaped.
U937, derived from a human histiocytic lymphoma, has the
characteristics of immature monocytoid cells (36). Like HL60,
the cells differentiate into macrophage-like cells that adhere to
the substrate after treatment with TPA. Conditioned medium
from a mixed-lymphocyte
culture or PHA-CM markedly in
creases the antibody-dependent cellular cytotoxicity and anti
body-dependent phagocytosis by U937 cells (20). Therefore,
the HL60 and U937 cell lines, although neoplastic, are useful
models for the study of differentiation of human myeloid cells
along either granulocytic or monocytic-macrophage
differentia
tion pathways.
The CSK proteins actin and myosin are major components
of macrophages, granulocytes, and myeloid leukemia cells (2,
15). Actin has also been reported to increase during differen
tiation of mouse myeloid cells in culture (16). In this study, we
have analyzed the protein changes during differentiation of
HL60 and U937 cells using a Triton extraction procedure that
leaves the CSK framework and surface lamina intact (1, 4, 25).
We have found that several proteins appear in the Tritoninsoluble (CSK) fraction of HL60 and U937 cells induced to
differentiate into mature myeloid cells or macrophages. One of
the major differentiation-induced
proteins is identified as vi
mentin, an intermediate-filament
protein (24). These induced
proteins are not present in the Triton-soluble fractions of these
cells. A number of proteins present in the Triton-insoluble
fractions of untreated
differentiation.
HL60 and U937 cells are lost during
INTRODUCTION
MATERIALS
Several human leukemia cell lines capable of in vitro differ
entiation have been described (6, 19, 26, 36). HL60, a human
myeloid leukemia cell line, consists primarily of promyelocytic
cells (6). After treatment with DMSO" or L-ethionine, these cells
Cells and Reagents. HL60 (human promyelocytic leukemia line) (6),
U937 (human histiocytic lymphoma line) (36), CEM (human lymphoblastic leukemia line) (8), OH-1 (human oat cell carcinoma line) (9),
1 This work has been supported by Grants CA29793. CA22659, and CA06943
from the National Cancer Institute.
2 To whom requests for reprints should be addressed.
3 Recipient of an American Cancer Society Faculty Research Award.
4 The abbreviations used are: DMSO, dimethyl sulfoxide; TPA, 12-O-tetradecanoylphorbol-13-acetate;
PHA-CM, conditioned medium from phytohemagglu
tinin-stimulated lymphocytes; CSK, cytoskeletal; SFCI, Sidney Farber Cancer
Institute; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl
fluoride.
Received May 5, 1982; accepted August 23, 1982.
5106
AND METHODS
and FS2 (human fibroblast line) were all grown in Roswell Park Me
morial Institute Medium 1640 with 10% fetal calf serum and 1 DIM
glutamine.
Purified granulocytes and monocytes from normal human volunteers
were provided by Dr. J. Griffin and Dr. R. Todd, SFCI. HL60 cells were
treated with DMSO (Fisher Scientific Co., Pittsburgh, Pa.) to a final
concentration of 1%. Both untreated HL60 and DMSO-treated HL60
were further purified by Isopaque-Ficoll (Pharmacia Fine Chemicals,
Piscataway,
preparations
N. J.) centrifugation
as described previously (3). The
of undifferentiated
HL60, differentiated
HL60, normal
CANCER
RESEARCH
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VOL. 42
Cytoskeleton,
monocytes, and normal granulocytes were all greater than 95% as
determined by Giemsa-stained smears. TPA, generously provided by
Dr. P. Fisher, Columbia University, was added to a final concentration
of 1.6 x 10~B M to cultures of HL60 and U937. PHA-CM was a gift
from Dr. T. Cotner, SFCI. PHA-CM was added to a final concentration
of 20% to U937 cultures. The SFCI Special Hematology Laboratory
performed the histochemical stains for naphthyl (nonspecific) esterase
(21) and myeloperoxidase (18).
Labeling of Cellular Proteins with [35S]Methionine.
Cells were
washed with PBS 3 times and then incubated
in methionine-deficient
Roswell Park Memorial Institute Medium 1640 supplemented with
dialyzed fetal calf serum and 1 mw glutamine. [35S]Methionine (Amersham/Searle Corp., Arlington Heights, III.; 400 Ci/mmol) was added
at 100 ftCi/ml. After 3 hr incubation, cells were transferred to centrifuge
tubes and washed 3 times with cold PBS. Attached cells were scraped
from the culture dish with a rubber policeman and washed 3 times with
cold PBS in centrifuge tubes. Approximately
1 x 106 trypan blueexcluding cells were used for each cytoskeleton extraction; however,
5 x 106 to 1 x 107 DMSO-treated HL60 and normal granulocytes
were required for adequate [35S]methionine incorporation.
CSK Extraction
and Resolution
by 2-Dimensional
Gel Electropho-
resis. The cells were suspended in CSK extraction buffer (CSK buffer)
which contains 1% Triton X-100, 100 mw 1,4-piperazinediethanesulfonic acid, 100 mw KCI, 300 mw sucrose, 2.3 mM MgCI2, 1 mw PMSF,
and 100 kallikrein inhibitor units/ml of aprotinin and kept at 4°for 3
min (5). The cell suspensions were gently swirled during the extraction.
After centrifugation, the supernatant (soluble fraction) was collected
and lyophilized. The pellet was washed twice with CSK buffer without
Triton. The supernatant and washes were combined and lyophilized
and were called the soluble fraction (SOL). The pellet was quickly
resuspended in a cold buffer (3 x volume of pellet) containing 10 mw
Tris-HCI (pH 7.4), 100 mw NaCI, 1.5 mw MgCI2, 1 mM PMSF. 0.1%
sodium dodecyl sulfate and 1% Triton X-100 and was immediately
centrifuged for 2 min at 2000 rpm to pellet the nuclei. The supernatant
was collected as the CSK fraction. After lyophilization, the residues
were dissolved in isoelectric focusing buffer (9.5 M urea, 2% Triton X100, and 2% ampholines; pH 3.5 to 10, pH 4 to 6, and pH 5 to 8).
Aliquots of approximately 100,000 cpm of CSK and soluble fractions
were analyzed by isoelectric focusing gels and a sodium dodecyl
sulfate-polyacrylamide
gel electrophoresis
system according to the
method of Garrels (14) and O'Farrel (33). After electrophoresis,
the
gels were fluorographed using Kodak SB-5 film at -70°. Isoelectric
points were determined by sectioning the gel and measuring the pH of
eluted ampholytes. Molecular weights were estimated from the mobility
of radioactive molecular weight markers (New England Nuclear, Bos
ton, Mass.).
Vimentin Purification and Immunofluorescence
Studies. FS2 (hu
man fibroblast line) was grown in [35S]methionine-containing
medium
for 3 hr. The cells were washed with PBS and homogenized in buffer
containing 0.6 M KCI, 20 mw Tris-HCI (pH 7.4), and 1% Triton X-100.
The insoluble fraction was pelleted by centrifugation at 10,000 x g for
20 min. The pellet was dissolved in isoelectric focusing buffer, and the
proteins were separated by 2-dimensional gel electrophoresis.
The
gels were stained with Coomassie blue (Bio-Rad Laboratories, Rich
mond, Calif.) in 30% methanol-10% acetic acid. The vimentin spots
were cut out of the gel and eluted. The purified [35S]methionine-labeled
vimentin was analyzed by 2-dimensional
gel electrophoresis.
Rabbit antiserum directed against vimentin was provided by Dr. I. C.
Summerhayes, SFCI. Untreated and treated HL60 and U937 cells were
air dried on coverslips and immersed in cold methanol for 10 min. The
coverslips were washed with PBS, covered with 20 n\ of anti-vimentin
serum, and incubated in a humidified chamber at 37°for 30 min. After
a washing with PBS, the coverslips were covered with a 1:10 dilution
of fluorescein-conjugated
goat anti-rabbit IgG (Meloy Laboratories,
Inc., Springfield, Va.) and incubated at 37°for 30 min. The coverslips
were washed with PBS, mounted, and examined in an epifluorescence
microscope.
DECEMBER
1982
Differentiation,
and Myeloid Leukemia
RESULTS
Morphological and Cytochemical
Changes during Differ
entiation of HL60 and U937 Cells. The untreated HL60 cells
have the morphology and staining characteristics of promyelocytes, as described previously (6). The cells are round and
grow as single cells in suspension (Fig. 1a). The cytoplasm is
basophilic with numerous azurophilic granules. More than 95%
of the cells resembled myeloblasts and promyelocytes. Five
days after treatment with DMSO, about 80% of the cells resem
bled mature myeloid cells by Wright-Giemsa stain. They ap
peared smaller
(Fig. 1b) and had a decreased
nuclearcytoplasmic
ratio and less basophilic cytoplasm. Fortyeight hr after treatment of HL60 with 1.6 x 1(T8 M TPA, 90%
of the cells became adherent to the plastic dish. The cells were
flat and had several long processes (Fig. 1c). Histochemical
stains showed an increase in nonspecific esterase and a de
crease in myeloperoxidase in 95% of adherent cells compared
to the untreated cells.
U937 cells are oval and grow as single cells in suspension
(Fig. 1d). They resemble immature monocytoid cells, with low
nonspecific esterase activity. U937 cells treated with 20%
PHA-CM became larger (Fig. 1e) and formed large floating
aggregates within 48 hr. By the histochemical stains, 80% of
the cells increased nonspecific esterase activity, inhibitable by
NaF. Twenty-four hr after exposure to 1.6 x 10~8 M TPA, 80%
of the U937 cells attached to the substrate, flattened, and
assumed macrophage-like morphology (Fig. 1 f). Like mature
macrophages, cytochemical stains show increased nonspecific
esterase activity, inhibitable by NaF.
CSK Proteins of HL60 and U937 Cells. After extraction of
these cells with CSK buffer, approximately 20% of the [35S]methionine incorporated into proteins is found in the CSK
buffer-insoluble fraction (CSK fraction). The 2-dimensional gel
analysis of the CSK fraction of these cells is shown in Fig. 2a.
Three prominent spots with molecular weights of 43,000 and
pi's of 6.0 comigrate with a-, ß-,and y-actin. Unlike actin,
which is present in both soluble and CSK fractions, most of the
major spots detected in the CSK fraction are absent or greatly
reduced in the CSK buffer-soluble fraction (Fig. 2b). These
other proteins, with a few exceptions to be discussed later,
have not been described previously as components of the
cytoskeleton. The CSK fraction of DMSO-treated HL60 (Fig.
2c) contains several proteins that are absent in untreated cells.
One of the induced proteins, P1, with a molecular weight of
55,000 and a pi of 5.3, comigrates with vimentin. In addition,
there are 6 other proteins [P2 (M, 200,000, pi 5.0), P3 (Mr
60,000, pi 5.0), P4 (M, 50,000, pi 6.5), P5 (M, 33,000, pi 5.0),
P15 (M, 38,000, pi 4.1), and P16 (M, 57,000, pi 3.8)] that are
present in the CSK fraction of DMSO-treated cells but not in
untreated cells. All of the proteins induced in DMSO-treated
HL60 are also present in the CSK fraction of normal human
granulocytes (Fig. 4a). Some proteins are lost during differen
tiation. P9 (M, 45,000, pi 4.3), P10 (M, 37,000, pi 4.8), P13
(Mr 40,000, pi 4.1 ), and P18 (M, 38,000, pi 7.5) are present in
the CSK fraction of untreated HL60 but not in the CSK fraction
of DMSO-treated cells. The (SOL) fraction of DMSO-treated
HL60 (Fig. 2d) shows no significant changes compared to the
SOL fraction of untreated HL60 (Fig. 2t>). The CSK fraction of
TPA-treated HL60 is shown in Fig. 2e. P1, the M, 55,000
protein that comigrates
with vimentin, is induced even more
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S. D. Bernal and L. B. Chen
dramatically than in DMSO-treated cells. This protein appears
to be a major component of the cytoskeleton when compared
with actin. A cascade of polypeptides (indicated by a bracket)
with molecular weights of 50,000 to 54,000 and pi's of 4.2 to
5.2 were shown previously to be degradation products of
vimentin (13). P2 (M, 200,000, pi 5.0) is another protein
induced in DMSO-treated HL60 which is also present in TPAtreated HL60. However, other proteins that are not present in
untreated and DMSO-treated HL60 appear in the CSK fraction
of TPA-treated HL60: P6 (MT 50,000, pi 5.3), P7 (M, 53,000,
pi 5.6), and P8 (M, 150,000, pi 6.0). All of these proteins
induced after TPA treatment of HL60 are also present in the
CSK fraction of freshly isolated normal human monocytes (Fig.
4b). None of these induced proteins are present in the soluble
fraction of TPA-treated HL60 (Fig. 1 f). P10 (M, 37,000, pi 4.8),
P12 (M, 35,000, pi 4.5), and P13 (M, 40,000, pi 4.1) are
present in untreated HL60 but are lost after TPA treatment.
Some proteins, such as P11 (M, 42,000, pi 6.8), are greatly
reduced but not lost after TPA treatment.
The CSK fraction of U937 cells, like untreated HL60 cells,
contains actin. P1 (vimentin-like protein) is not detected in the
CSK fractions, the soluble fraction, or the total lysate of un
treated U937 cells. Similar to HL60, most of the CSK proteins
are not found in the soluble fraction, except for actin (Fig. 3b).
After treatment of U937 cells with PHA-CM, the proteins P1,
P2, P6, P7, P14, and P16 are induced (Fig. 3c). All of these
induced proteins are also present in the CSK fraction of normal
human monocytes (Fig. 3b). These proteins induced in the
CSK fraction of PHA-CM-treated U937 are not present in the
soluble fraction of these cells (Fig. 3d). After TPA treatment,
P1, P2, P6, P7, P14, and P16 are also induced in the CSK
fraction of U937 cells (Fig. 3e). These proteins are not present
in the SOL fraction of TPA-treated U937 (Fig. 30. Exposure of
U937 cells to TPA results in the loss of P10 and P12 from the
CSK fraction.
The CSK proteins induced or lost during differentiation of
HL60 and U937 cells are summarized in Table 1. During
differentiation of DMSO-treated HL60 to cells resembling granulocytes, P1, P2, P3, P4, P5, P15, and P16 are induced. All of
these proteins are also present in the CSK fraction of normal
human granulocytes (Fig. 3a). All of the CSK proteins induced
during differentiation of HL60 and U937 cells into macrophagelike cells are also present in the CSK fraction of normal human
macrophages (Fig. 4b). However, P17, which is present in the
CSK fraction of normal macrophages, is not detected in the
CSK fractions of TPA-treated HL60 (Fig. 2e), PHA-CM-treated
U937 cells (Fig. 3c), or TPA-treated U937 (Fig. 3e). Actin (M,
43,000, pi 6.0) is present as a major component of the CSK
and soluble fractions of HL60 and U937 cells. During differ
entiation of HL60 and U937, we have not observed major
changes in the density of the actin spot in 2-dimensional gels
of either the CSK or soluble fractions.
Time Course and Specificity of the Synthesis of Differentiation-inducible
Proteins. The synthesis of differentiation-inducible CSK proteins at earlier times after TPA treatment was
investigated. P1 (comigrating with vimentin) was detected
Table 1
Alterations in expression of CSK proteins during differentiation of HL60 and U937 cells
Arbitrary values, from undetectable ( —¿)
to high density ( + -f +), were given to the densities of the autoradiographic spots on 2-dimensional
gels of the various CSK
fractions.
+
U937 +
10-"552006050335053150453742364062385710038Pi5.35.05.06.55.05.35.66.04.34.86.84.54.15.24.13.85.27.5HL60
X
HL60
DMSO
Macrophage+
HL60 + TPA
U937
PHA-CM
U937 + TPA
ProteinP1P2P3P4P5P6P7P8P9P10P11P12P13P14P15P16P17P18M,
++
+++ +
Granulocyte
+ ++
++ +
+++
+
++
++
+
++
+++
+
++++
++
+++
+
+
++
+
+++
++
++
+++
4-
+
++
+
-t-
+
+
+
++
+
+
+++
+ 4-
+++
++++
+
++
+++
+++
+
±
+
+
+
+
++
+++
+
++++
+
+++
++++
+
+
+
+
+
++
-
-
-
++
+
5108
+
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RESEARCH
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VOL. 42
Cytoskeleton,
within 3 hr after treatment with 1.6 x 1CT8 M TRA (Fig. 4d). By
this time, P10 and P12 were no longer detectable. At 6 hr, both
P1 and P2 became prominent; about 10% of the cells were
attached to the substrate at this time, but few had flattened and
assumed macrophage-like morphology. The majority of U937
cells have attached and spread on the substrate 24 hr after
TPA treatment. The CSK extract of these cells (Fig. 3e) con
tains additional proteins: P6, P7, P14, and P16. When attach
ment is prevented by incubating the cells in a rotating polyeth
ylene-coated centrifuge tube, the TPA-treated U937 cells in
suspension synthesize the same CSK proteins as attached
TPA-treated cells. The CSK proteins were not induced during
cultivation of HL60 or U937 cells in serum-free medium. No
new CSK proteins were induced when CEM (human lymphoblastic leukemia line) and OH-1 (human oat cell carcinoma line)
were treated with TPA, DMSO, or PHA-CM.5
Differentiation-induced
Proteins Ruled Out as Result of
Proteolysis during Detergent Extraction. Proteases may be
induced during differentiation of myeloid cells and after expo
sure to TPA (27, 37). Although the CSK extraction buffer
contains the protease inhibitors PMSF and aprotinin, it is still
possible that some of the induced proteins appearing during
differentiation are breakdown products of preexisting CSK
proteins acted upon by proteases during the extraction pro
cedure. To rule out this possibility, we mixed equal numbers of
[35S]methionine-labeled U937 cells and unlabeled TPA-treated
U937 cells and then performed the CSK extraction. The labeled
proteins in the CSK fraction, analyzed by 2-dimensional gel
electrophoresis, were the same as those of U937 cells alone;
none of the differentiation-induced
proteins were produced by
coextraction with TPA-treated U937 cells. The [35S]methioninelabeled CSK fraction of untreated U937 cells was also incu
bated with total lysate of TPA-treated U937 cells for 5 min at
4°. Two-dimensional gel analysis showed that none of the
differentiation-induced
proteins appeared as a result of this
incubation (Fig. 4c). Fig. 5a shows the 2-dimensional gel
pattern of [35S]methionine-labeled vimentin. When this purified
vimentin was added to the CSK extract from TPA-treated U937
cells, it comigrated with P1 (Fig. 5b).
Immunofluorescence
Staining with Anti-Vimentin
Anti
body. Immunofluorescence
with anti-vimentin antibody was
also used to monitor the expression of vimentin during differ
entiation. As shown in Table 2, very few untreated HL60 or
U937 cells were stainable with anti-vimentin antibody. Approx
imately 40% of DMSO-treated HL60 cells showed a significant
increase in fluorescence. In TPA-treated HL60 and in TPA- or
PHA-CM-treated U937, a dramatic increase in fluorescence of
60-70% of cells was detected (Fig. 6), reflecting the presence
of a large amount of vimentin. These results suggest that P1 is
vimentin and that, indeed, the amount of vimentin increases
dramatically during differentiation of HL60 and U937 into mac
rophage-like cells.
DISCUSSION
We have found that, during differentiation of HL60 and U937
cells to granulocytes and macrophage-like cells, several cel
lular proteins are induced. These differentiation-induced
pro
teins are specifically associated with the Triton-insoluble (CSK)
* Unpublished data.
DECEMBER
1982
Immunof/uorescent
Differentiation,
and Myeloid Leukemia
Table 2
staining of vimentin in HL60 and U937 cells
positiveHL60HL60
% vimentin
DMSOHL60
+
TPAU937U937
+
PHA-CMU937
+
+ TPA<54070<56060
fractions and are not detectable in the soluble (SOL) fractions.
There are also several differentiation-sensitive
proteins, i.e.,
those that are no longer synthesized upon differentiation into
granulocyte- and macrophage-like cells.
One of the proteins induced in DMSO-treated HL60 cells,
P1, has a molecular weight of 55,000 and a pi of 5.3 and
comigrates with vimentin, a known component of intermediate
filaments of fibroblasts and cultured cell lines. The other in
duced proteins include P2, P3, P4, P5, P15, and P16. Whether
these other proteins are related to other known CSK compo
nents remains to be determined. All of these proteins induced
during differentiation of HL60 are also present in the CSK
fraction of freshly isolated normal human granulocytes. It ap
pears, therefore, that as HL60 cells develop the morphology
and functional capacity of cells resembling mature granulo
cytes they acquire CSK proteins that are present in normal
mature granulocytes. During differentiation of HL60 and U937
into macrophage-like cells, a set of CSK proteins are induced
which resemble the CSK proteins of freshly isolated normal
human macrophages.
The CSK proteins are induced in a sequential, orderly fash
ion. P1 (vimentin-like protein), the first CSK protein induced in
TPA-treated U937 cells, is detected prior to the morphological
changes indicative of differentiaton. P2 is detected when some
cells have attached but have not assumed macrophage-like
morphology; P6, P7, P14, and P18 become prominent when
the cells have flattened and spread on the substrate.
The CSK fraction of HL60 and U937 cells induced to differ
entiate into macrophage-like cells, however, does not contain
all of the CSK proteins of normal macrophages. These findings
may indicate that the differentiation of HL60 and U937 induced
in vitro is not complete. Likewise, it is known that TPA-treated
HL60 cells do not acquire all of the ultrastructural and func
tional characteristics
of normal macrophages; they do not
contain the secondary lysosomes of mature stimulated mac
rophages (35), and they show no increase in respiratory burst
activity or complement secretion (30).
During differentiation of myeloid cells, the synthesis and
release of proteolytic enzymes may be stimulated, particularly
after exposure to phorbol esters (27, 37). Therefore, it is
important to consider the possibility that the proteins appearing
during differentiation might be breakdown products of preex
isting CSK proteins acted upon by proteolytic enzymes in vitro.
To rule out this possibility, we mixed equal numbers of un
treated U937 cells labeled with [35S]methionine and unlabeled
TPA-treated U937 cells and then performed the CSK extrac
tion. We found no 35S-labeled proteins in the Triton-insoluble
fraction that were gained or lost as a result of the coextraction.
Likewise, when the [35S]methionine-labeled CSK fraction of
untreated U937 cells was incubated with a total lysate of TPAtreated U937 cells, none of the differentiation-induced
proteins
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S. D. Bernal and L. B. Chen
appeared. The same results were obtained from similar exper
iments with HL60 and TPA-treated HL60 cells. Thus, it is
unlikely that any of the proteins appearing during differentiation
of U937 and HL60 are products of in vitro proteolysis of other
CSK proteins. These proteins are not the result of changes in
partitioning between the CSK, SOL, and nuclear fractions
during differentiation since they are not detectable in total cell
lysates of untreated HL60 and U937 cells.
New surface glycoproteins appear during differentiation of
HL60 and U937 cells. HL60 cells induced to differentiate into
granulocytic cells with DMSO acquire a M, 130,000 surface
protein (11) and lose M, 88,000 and M, 86,000 proteins (29).
TPA-treated HL60 acquire an M, 83,000 protein, reactive with
antimonocyte serum (29). The appearance of a H 130,000
glycoprotein was also associated with membrane differentiation
in several myeloid leukemic cell lines (10). U937 cells induced
to differentiate into macrophage-like cells gain surface proteins
with molecular weights of 180,000, 140,000 and 85,000 (32).
Since some outer membrane proteins are bound to the cytoskeleton (1), a few of the proteins in the CSK fraction that we
have described may be related to some of these cell surface
proteins.
Increased synthesis of vimentin in chicken cells (23) and of
keratin in mouse epidermal cells (22) has been observed after
treatment with TPA. However, the induction of vimentin and
other CSK proteins in HL60 and U937 cells is not related to
drug treatment per se since exposure to a variety of differen
tiating agents such as PHA-CM, DMSO, and TPA result in the
synthesis of similar proteins. The CSK proteins are not induced
during cultivation of HL60 and U937 in serum-free medium;
under these stressful in vitro conditions, cell growth is inhibited,
but there is no appreciable evidence of differentiation. When
GEM (human lymphoblastic leukemia line) and OH-1 (human
oat cell carcinoma line) are treated with PHA-CM, DMSO, or
TPA, these proteins are not induced. Thus, the CSK protein
changes that we have described are more likely to be related
to the process of differentiation than to drug treatment or
adverse in vitro conditions per se.
Although the most dramatic morphological change during
differentiation of U937 is adherence to the substrate and cell
flattening such as that induced by TPA, the appearance of
these new CSK proteins is not necessarily associated with
adherence and cell flattening, because the same proteins ap
pear after PHA-CM-induced differentiation of U937 cells into
phagocytic cells that form floating aggregates in culture. More
over, TPA-treated U937 cells the attachment of which is pre
vented by incubation in a rotating polyethylene-coated centri
fuge tube synthesize the same proteins as do attached TPAtreated U937 cells.
A number of functional properties are acquired by HL60 and
U937 cells in association with their morphological differentia
tion. These include the expression of Fc and C3 receptors (12,
32, 36), lysozyme secretion (32, 35, 36), increased respon
siveness to chemotactic factor (7), increased phagocytic activ
ity (12, 32, 35, 36), and increased cytotoxicity against tumor
cells (32, 38). It is possible that the CSK proteins induced
during differentiation of HL60 and U937 cells participate in the
expression of these various functions. The various CSK com
ponents may also play a pivotal role in mediating the coordi
nated changes in cell shape, surface structure, and protein
synthesis that occur during differentiation. The cytoskeleton,
5110
having intimate connections with nuclear structures and sur
face proteins, has been thought to be important in the mainte
nance of cell shape (34) and surface structure (31). Interme
diate filaments, including vimentin filaments, have been sug
gested to be mechanical integrators of cellular space (24).
Vimentin may also play a role in cell adhesion, spreading, and
motility and in maintaining the integrity of the cell nucleus (24).
In our study, we have found that the vimentin is the first protein
to be induced during differentiation of human myeloid leukemia
lines. The synthesis of large amounts of vimentin can be
detected prior to changes in cell morphology. Thus, vimentin
may play an important role in the early events of differentiation.
The addition of vimentin intermediate filaments may result in a
new CSK architecture that is needed for the expression of
differentiated functions.
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1982
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5111
S. D. Bernal and L. B. Chen
1a.HL60
1d. U937
Ib.
le.
HL60+DMSO
*
9 •¿:
le.
U937+CM
s,.«
^
HL60+TPA
If.
U937+TPA
v
Fig. 1. Phase microscopy of HL60 and U937 cells in culture. In a, HL60 cells are round and nonadherent to plastic. In b, DMSO-treated HL60 cells are smaller
with decreased cytoplasmic basophilia. In c. TPA-treated HL60 cells are flat and adherent to the plastic dish. Several long processes are noted. In d, U937 cells are
oval and grow as single cells in suspension. In e, PHA-CM (CM)-treated U937 cells are larger and rounder and often form clusters in suspension. A few cells adhere
to plastic. In Õ.TPA-treated U937 cells are flat, tightly adherent to plastic. The cells may have pseudopods but generally do not form long processes.
Fig. 2. Two-dimensional gel analysis of CSK fractions and Triton-soluble fractions (SOLÕof untreated and treated HL60 cells. HL60 cells were treated with 1%
DMSO for 5 days or with 1.6 x 1CT8 M TPA for 2 days. After labeling for 3 hr with [35S]methionine, the untreated HL60. DMSO-treated HL60, and TPA-treated HL60
were extracted with CSK buffer to collect the CSK and soluble fractions, as described in "Materials and Methods." A, actin spot: arrows, differentiation-induced
and
differentiation-sensitive
proteins. The arrow in each of the soluble fractions indicates the absence of P1 which comigrates with vimentin.
Fig. 3. Two-dimensional gel analysis of CSK fractions and Triton-soluble fractions (SOL) of untreated and treated U937 cells. U937 cells were treated with 20%
PHA-CM for 2 days or with 1.6 x 1CT8 M TPA for 2 days. After labeling for 3 hr with [35S]methionine, the untreated U937. PHA-CM-treated U937, and TPA-treated
U937 were extracted with CSK buffer to collect the CSK and soluble fractions, as described in "Materials and Methods."
51 12
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VOL.
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42
Cytoskeleton,
CSK
2a.
Differentiation,
and Myeloid Leukemia
SOL
HL60
2b.
HL 60
2d.
HL60
200-
100
80
16
60-
9->>«
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13-*»
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HL60+DMSO
+ DMSO
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200-
2
1 DO
SO
O)
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•¿X
•¿
•¿
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O
O
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12
2e.
HL60
+ TPA
2f.
HL60+TPA
200t
1oo
so
16
60
40
4.0
5.0
6.0
4.0
7.0
s'.O
e!o
7.0
Pi
DECEMBER
1982
5113
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S. D. Bernal and L. B. Chen
CSK
SOL
3a.U937
3b.
U937
200
1 00
80
16
60
-•¿'*
40
•¿AT
3c. U937
co
i
O
200
X
100
•¿>
•¿
•¿
».I
13
+ CM
3d. U937
+ CM
t
2
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80
OT
60
u
.. » , ».
18
'0
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- .,
et «A"
•¿
,
- •¿
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40-
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3
o
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+ TPA
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U937
+ TPA
t
2
10080-
"
. »..
16
60-
40-
3-*.
>-
e
4.0
5.0
6.0
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4.0
5.0
6.0
7.0
Pi
51 14
CANCER
RESEARCH
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VOL. 42
Cytoskeleton,
Granu
Macrophage—
locy t e
4b.
.
8î_
r16
80-60-09
1
*"13-1^
IO
40-T—X*-O)'55^5
. î
*:
i -
/3
...
¿i*1"* •¿
'
^
^
16
i
*
•¿'
.
and Myeloid Leukemia
CSK
CSK
200-100-
Differentiation,
A
•¿
14-j.
f-"""""""^'
L^1
•¿â€¢;-..
_ è
•¿
»y
*" 7
.'••.*
13-*--1
i
•¿124C.U937
•¿
(3Hrs.)1
CSK
4 Lysate
4d.
U937
+ TPA
20°-£
*^
1
100-CD
81
^t
".';'--
1
•¿'m
•¿'
60-40-4a.
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>•
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.,/•
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.
'A
^
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'
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9-*
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Fig. 4. Two-dimensional gel analysis of CSK fractions of granulocytes, macrophages, and TPA-treated U937 cells. In a. granulocytes isolated from the peripheral
blood of normal human volunteers were incubated with [35S]methionine for 3 hr and extracted with Triton buffer to collect the CSK fraction. In b, adherent
macrophages isolated from the mononuclear fraction of human peripheral blood were labeled with [35S]methionine and extracted with Triton buffer. In c, the [35SJmethionine-labeled CSK fraction of untreated U937 cells was incubated with a total lysate of TPA-treated U937 cells for 5 min at 4°.The mixture was then analyzed
on 2-dimensional gels. In d, U937 cells were incubated with 1.6 x 1CT8 M TRA and [35S]methionine for 3 hr. The cells were then harvested, and the CSK fraction was
collected.
DECEMBER
1982
5115
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S. D. Bernal and L. B. Chen
5a.
v ¡ment in
5b.
Fig. 5. a, Two-dimensional gel analysis of [35S]methionine-labeled
fraction from TPA-treated U937 cells.
+ CSK(U937+TPA)
vimentin, isolated from a human fibroblast
line, FS2: b, mixture of vimentin and labeled CSK
6a. HL60
6b. HL60 +TPA
6c. U937
6d. U937 +TPA
Fig. 6. Immunofluorescent
5116
vimentin
staining of vimentin in HL60 and U937 cells before (a, c) and after (b, d) TPA treatment.
CANCER
RESEARCH
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VOL. 42
Induction of Cytoskeleton-associated Proteins during
Differentiation of Human Myeloid Leukemic Cell Lines
Samuel D. Bernal and Lan Bo Chen
Cancer Res 1982;42:5106-5116.
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