From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Signal Transduction and the Regulation of Actin Conformation During Myeloid
Maturation: Studies in HL60 Cells
By Ronald L. Sham, Charles H. Packman, Camille N. Abboud, and Marshall A. Lichtman
Maturation of human myeloid cells is associated with quantitative and qualitative changes in protein kinase C (PKC) and
increases in N-formyl-L-methionyl-L-leucyl-L-phenylalanine
(FMLP) receptors, actin, and actin regulatory proteins. We
have studied the actin responses and cell shape changes
caused by FMLP and its second messenger pathways in HLW
cells undergoing neutrophilic maturation. In uninduced cells,
the PKC activators 12-0-tetradecanoyl phorbol-13-acetate
(TPA), bryostatin, and 1-oleyl-2-acetylglycerol (OAG) resulted in 15% t o 30% decreases in F-actin, whereas FMLP
had no effect. lonomycin had no effect on actin but did cause
a 10-fold increase in intracellular calcium. Cells grown for 24
hours in 1% dimethyl sulfoxide (DMSO) acquired the ability
to polymerize actin in response t o FMLP and ionomycin. TPA
continued t o cause a decrease in F-actin at 24 hours, but
caused an increase in F-actin at 48 t o 72 hours of maturation.
The PKC inhibitor 1-5-isoquinolinesulfonyl P-methylpiperazine (H7) partially blockedthe F-actin increase caused by TPA
in induced cells, but had no effect on the decrease in F-actin
caused by TPA in uninduced cells or the increase in F-actin
seen in FMLP-treated neutrophils. F-actin rich pseudopods
developed following TPA or FMLP stimulation of induced
HLW cells; in uninduced cells neither agent caused pseudopod formation but TPA caused a dramatic loss of surface
ruffles. The ability of FMLP and ionomycin t o elicit a neutrophil-like actin response in HLW cells within 24 hours after
DMSO treatment shows that the actin regulatory mechanism
is mature by that time. The inability of ionomycin to increase
F-actin in uninduced cells supports the view that calcium
increases alone are insufficient for actin polymerization. The
longer maturation time required for HLW cells t o develop an
actin polymerization response t o TPA compared with FMLP,
coupled with the inability of H7 to block the FMLP-mediated
F-actin increase in neutrophils, suggests that the F-actin
increase caused by FMLP is not mediated solely by PKC.
Lastly, the TPA-induced F-actin decrease and shape changes
in uninduced HL60 cells, and the longer time required for a
”mature” responset o TPA, may reflect immaturity in the PKC
isoenzyme pattern rather than immaturity of the actin regulatory mechanism.
8 1991by The American Society of Hematology.
M
use this information to assess the role of calcium and PKC
in FMLP-mediated actin responses.
ATURATION OF human promyelocytic leukemia
(HMO) cells can be induced with a variety of
physiologic and pharmacologic agentslg and may proceed
along monocytic or neutrophilic pathways depending on the
inducing agent used. The acquisition of a more mature
phenotype is assessed by morphologic, immunocytochemical, physiologic, or functional studies. One of the several
capabilities of the normal mature neutrophil, chemotaxis,
develops in HMO cells after they are induced to mature by
exposure to dimethyl sulfoxide
The number of
chemotactic receptors for N-formyl-L-methionyl-L-leucylL-phenylalanine (FMLP)’ and the actin content6 increase
as HL60 cells mature. Chemotaxis is related to actin
function because cell motility requires reversible polymerThe
ization of actin when chemotactic stimuli are
biochemical pathways regulating actin polymerization are
not fully characterized but are known to be affected by the
two second messenger pathways, calcium9-” and protein
kinase C (PKC).9,12
PKC is a calcium and phospholipid-dependent intracellular enzyme that modulates many cellular responses and
undergoes both qualitative and quantitative changes with
HL60 cell maturation.” This enzyme is activated by 12-0tetradecanoyl phorbol-13-acetate (TPA),14 1-oleyl-2-acetylglycerol (OAG), and bryostatin,” and inhibited by sphin(H7),16
gosine, 1-5-isoquinolinesulfonyl2-methylpiperazine
and stauro~porine.’~
The calcium second messenger pathway can be examined using calcium ionophores such as
A23187 and ionomycin.’o.’8
We have studied the effects of PKC activators, ionomycin, and FMLP on actin polymerization in HMO cells
during their neutrophilic maturation. The purpose of this
study was twofold: (1) to evaluate the actin responses
caused by activation of the different second messenger
pathways throughout neutrophilic maturation and (2) to
Blood, Vol77, No 2 (January 15). 1991: pp 363-370
MATERIALS AND METHODS
Preparation of cells. A neutrophilic variant of the HL60 cell
linet9was grown in RPMI medium containing 10% fetal calf serum
(FCS). The cultures were initiated at 2 X lo5 cellslmL and
incubated at 37°C in air with 5% CO,. A final concentration of 1%
DMSO was added to the incubation when appropriate. Paired
samples of induced and uninduced cells were examined at desired
points in their maturation. Cell morphology was assessed using
Wright’s stain.
Neutrophils from normal volunteers were isolated using dextran
sedimentation followed by Ficoll-Hypaque centrifugation.
Activation of cells. “PA, FMLP (Sigma Chemical Corp, St
Louis, MO), bryostatin (kindly provided by W. Stratford May,
Baltimore, MD), OAG, and ionomycin (Calbiochem, San Diego,
CA) were stored in DMSO at -20°C. For use, they were thawed
and diluted in phosphate-buffered saline (PBS) so that 10 pL
added to 1 mL of cell suspension achieved the desired final
concentration. H7 (Seikagaku America Corp, St Petersburg, FL)
was refrigerated in pH 4.0 distilled water and used directly from
the stock solution. All experimentswere performed at 37T, except
for the neutrophil studies performed at room temperature. H7 was
From the Departments of Medicine and of Bwphysics, University of
Rochester Medical Center, Rochester, hY
Submitted March 23,1990; accepted September 19,1990.
Supported in part by Public Health Service (PHS) Grant POI
HL18208-15and PHS Training Grant HL 07152.
Address reprint requests to Ronald L. Sham, MD, Hematologv Unit,
Rochester General Hospital, 1425 Portland Ave, Rochester, NyI4621.
The publication costs of this article were &frayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with I8 U.S.C. section 1734 solely to
indicate thisfact.
0 I991 by The American Society of Hematology.
0006-4971l91l7702-om4$3.00/0
363
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
SHAM ET AL
364
added 15 minutes before the addition of TPA, FMLP, and
bryostatin. Aliquots of the cell suspension containing 1 x lo6
cells/200 pL were removed before and at appropriate times after
the addition of activating agents and inhibitors, and placed into 1
mL ice-cold 3.2% paraformaldehyde and refrigerated for 48 hours
to permeabilize the cells.
Flow cyfomeoy. Cells were prepared for flow cytometry using
previously described methods: Cells were washed with PBS
containing 0.1% bovine serum albumin (BSA) and incubated in the
dark with 0.1 mL (0.7 pmol/L) of the F-actin-specific probe,
7-nitrobenz-2-oxa-diazole
(NBD) phallacidin (Molecular Probes
Inc, Eugene, OR), for 45 minutes. The cells were rewashed and
filtered through 53 pm nylon mesh. The final volume of 0.5 mL
contained about 1 x lo6cells.
The F-actin content of cells was measured on an EPICS Profile
flow cytometer (Coulter Corp, Hialeah, FL). A 15-mW laser was
used for fluorescence excitation at 488-nm and green fluorescence
was measured at 525-nm as was forward angle light scatter. The log
green fluorescence was converted to a relative linear scale and
plotted against time. A total of 20,000 cells was collected for each
measurement.
Fluorescent microscopy. Rhodamine phalloidin, 10 p1 (Molecular Probes), was added to 150-pL aliquots of permeabilized cells
and incubated in the dark for 45 minutes. The cells were then
washed with PBS/O.l% BSA and cytospin preparations were made.
The cell preparations were studied with fluorescent microscopy
and representative cells were photographed.
__..
*-..
Scanning electron microscopy. Aliquots of fresh cell samples
taken at appropriate time points were pipetted into 1%glutaraldehyde in Sorensen's phosphate buffer and fixed for 1 hour. Cells
were then rinsed with Sorensen's buffer and attached to poly-Llysinexoated mica chips. They were postfixed in 1% osmium
tetroxide for 1hour and rinsed again. Samples were dehydrated in
a graded ethanol series and critical point dried. Cells were coated
with gold palladium and observed with a JEOL T330A (JEOL
U S A Inc, Peabody, MA) scanning electron microscope.
Measurement of cytoplasmic free calcium. HL60 cells were
loaded with the calcium-sensitive fluorescent probe fura-2-AM
(fura-2-tetraacetomethoxyester) (Molecular Probes), using previously described methods?' A suspension of 5 x lo6HL60 cells/mL
in Hanks Balanced Salt Solution (HBSS) was incubated at 37°C for
10 minutes in a shaking water bath. The cells were diluted sixfold
and incubated for 20 minutes. This suspension was further diluted
fivefold, centrifuged at 25% for 10 minutes, and resuspended at
2 X lo6 cells/mL in HBSS containing 1 mmol/L calcium and 1
m m o m magnesium for the calcium determinations. Fura-2 fluorescence was monitored continuously with a SPEX Fluorolog photofluorimeter (SPEX Industries, Edison, NJ) using dual exitation
monochromators. The intracellular ionized free calcium concentration was determined as follows: Ca? = k (R - R,,,&(Rmm- R).
R is the ratio of emission intensities at 505 nm on excitation at 340
and 380 nm. R,,,, was obtained by inducing cell lysis with 0.1%
Triton, which exposed the fura-2 to 1mmol/L external calcium, and
R,,,,, was determined at zero calcium upon addition of 6.25 mmol/L
-
Control
A
--t- TPA
* TPA+H7
1.21.1
-
B
__..e-Control
..
1.1
OAG12.5uM
OAG25uM
OAG50uM
1.04
0.8 0.9
0.7
. I . I . I
-15 -10
-5
0
.
I
5
.
I
10
.
*
15
C
1.3
.-..*...
--)-
Control
Bryostatin
1.1
:I
0.7
. , . , . ,
.
, .
,
0.7
0
2
4
6
8 1 0 1 2
0
2
4
6
8
10
Time (min)
Fig 1. Uninduced HL60 cells were used for these experiments. Samples were taken at the indicated times for determination of F-actin content
by flow cytometry. The conditionsfor each set of experiments were as follows. (A) Effect of TPA on F-actin content. Cells were incubated with or
without H7 200 pmol/Lfor 15 minutes. TPA (100 nmol/L) (or buffer)was added et time = 0. TPA resulted in a 20% decrease in F-actin content. H7
had no effect on the TPA-induced decrease in F-actin. Resultsshown are the mean of three experiments 5 SE. (B) Effect of OAG on F-actin content.
OAG (12.5 pmollL, 25 pmol/L. and 50 pmol/L) (or buffer) was added at time = 0. OAG resulted in a dose-dependent decrease in F-actin. Results
are the mean of four experiments ?SE. (C) Effect of bryostatin on F-actin content. Bryostatin (10 nmol/L) (or buffer) was added at time = 0.
Bryostatinresultedin a 20% decrease in F-actin content. Resultsshown are the mean of three experiments 2 SE. (D) Effect of ionomycin on F-actin
content. lonomycin (400nmol/L) (or buffer) was added at time = 0. lonomycin did not change the F-actin content of uninduced cells. Results
shown are the mean of nine experiments SE.
*
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
REGULATiON OF ACTIN CONFORMATION IN HL60 CELLS
ethyleneglycol-bis-(9-amino-ethylether) N,N'-tetra-acetic acid
(EGTA) to the lysed cellular suspension at pH 8.5. k is the product,
kd x (FJF,), where kd is the effective dissociation constant of
fura-2 for calcium (224 nmol/L), F, is the 380-nm excitation signal
in the absence of calcium, and F, is the 380-nm excitation signal in
the presence of calcium.
RESULTS
Effects of PKC activators,FMLP, and ionomycin on F-actin
content and cell shape in HL60 cells. To determine the
effects of the two second messenger pathways on actin
polymerization in HL60 cells, PKC activators, ionomycin,
and FMLP were studied. TPA (Fig 1A) and OAG (Fig 1B)
caused dose-dependent decreases in F-actin in uninduced
HL.60 cells within 5 minutes. TPA (100 nmol/L) and OAG
(50 pmol/L) caused a maximal effect with a 15% to 30%
decrease in F-actin. Bryostatin (10 nmol/L) resulted in a
similar rapid decrease in F-actin content (Fig 1C). The
inactive phorbol ester, 4 a phorbol, had no effect on HL.60
cell F-actin content (not shown). Fluorescence microscopy
using rhodamine phalloidin staining showed minimal differences in fluorescence between control and TPA-treated
cells. However, scanning electron microscopy of TPAtreated HL60 cells showed rounding and a dramatic and
consistent loss of the surface villi and ruffles when compared with control cells (Fig 2). This change was detectable
at 1 minute, coincident with the decrease in F-actin.
Ionomycin (400 nmol/L) caused no change in uninduced
HL60 cell F-actin content (Fig lD), despite a 10-fold
increase in intracellular calcium (see below). FMLP (1
Fig 2. Effect of TPA and FMLP
on F-actin distribution and cell
shape in u n i n d u d HL60 cells.
TPA (100 nmol/L) and 1 pmol/L
FMLP were added to HL60 cells
and samples were taken at 1
minute and preparedfor fluorascent microscopy and scanning
electron microscopy. (A and D)
show small surface projections
on resting cells. (B and E) show
rounding and the loss of surface
features following TPA treatment. (C and F) show no detectable change from resting cells
with FMLP treatment.
365
pmol/L) did not alter the F-actin content (not shown) or
the cell surface features of uninduced HL60 cells (Fig 2).
Effects of maturation on the FMLP, lonomycin, and PKC
activator response. Mature neutrophils are known to have
enhanced chemotaxis when compared with immature cells.
The effects of the two second messenger pathways on actin
polymerization in mature neutrophilic cells were evaluated
using the same methodology as above. HL60 cells were
induced to mature by culturing them in 1% DMSO for 4
days. Comparison of induced and uninduced cells using
light microscopy of Wright's-stained samples showed cytoplasmic maturation characterized by inconspicuous primary
granules, the formation of secondary granules, and a
decrease in cell size. Nuclear maturation was characterized
by a decrease in nuclear size, loss of nucleoli, and some
nuclear segmentation. The actin polymerization responses
of HL.60 cells matured with 1% DMSO for 4 days were
studied as follows:
Addition of 1 pmol/L FMLP to matured HL60 cells
resulted in a 50% increase in F-actin content by 1 minute
with a return to baseline within 10 minutes (Fig 3A). TPA
(100 nmol/L) (Fig 3B) caused a 35% increase in F-actin.
Bryostatin (10 nmol/L) (Fig 3C) caused a 15% increase in
F-actin. Ionomycin (400 nmol/L) (Fig 3D) caused a 30%
increase in F-actin content by 30 to 60 seconds with a return
to the baseline within 10 minutes.
Scanning electron microscopy of FMLP- and TPAtreated mature HMO cells showed one or multiple pseudopods. Fluorescent microscopy with rhodamine phalloidin
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
-'a;i
.
I
B
1.51.41.3-
____ *.--Control
-f-
Bryostatin
-
---e-Control
lonomycin
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
REGULATION OF ACTIN CONFORMATION IN HL60 CELLS
367
rb
Fig 4. Effect of TPA and FMLP on F-actin distribution and cell shape in induced HL80 cells (following 48 to 72 hours of 1% DMSO indudon).
TPA (100 nmol/L) and 1pmol/L FMLPwere added t o HUrOcells, and samples were taken at 1minute and preparedfor fluomcent mkmscopy and
scanning electron microscopy. (A and D) show small rutface projections on resting cells. (e and E) show pseudopod formation following TPA
treatment with marked concentration of F-actin in pseudopods. (C and F) show similar responsesfollowing FMLP treatment.
strated increase in F-actin content (Fig 3A). The decrease
in F-actin content observed in uninduced cells treated with
TPA (Fig 1A) or bryostatin (not shown) was not inhibited
by pretreatment with H7.
Neutrophils were also used to assess the role of PKC in
FMLP-induced actin polymerization. Neutrophils isolated
from normal volunteers were treated with 1 nmol/L FMLP
following preincubation with H7.The increase in F-actin
content seen after FMLP application was not inhibited by
H7 in doses ranging from 12.5 pmoVL to 200 pmol/L (Fig
1.61
7).
DISCUSSION
0.9
l 0
2
4
.
6
8
o10
Time (min)
Flg 5. Effect of DMSO induction thne on the F - d n mpome to
FMLP In HL60 cells. HUW) celh were cultured in the pmsence of 1%
DMSO and experiments were performed after 0,24,48, and 72 hours
of culture. FMLP (1 ~ o l / L was
)
added at time = 0 minutes and
samples were a n a m as above (mean of two experiments; muklple
other experiments confirmed this finding). The day 0 cells had no
change in F-actin content in response to FMLP. After 24 hours of
DMSO incubation, the cells displayed a 25.h increase in F-actinwithin
1 minute of FMLP addition. A more pronounced increase in F-actin
day 1; (4) day 2;
was seen at 48 hours and 72 hours. ( 4 3 day 0; (-0-1
(-A-1 day 3.
These experiments
~ evaluate the regulation of actin conformation in HL60 cells undergoing neutrophilic maturation. Total cellular actin content increases twofold as HL60
cells mature over several days and reflects new protein
synthesis: Throughout maturation an equilibrium exists
between G- and F-actin and the G:Fratio of approximately
2 1 is similar in induced and uninduced HL60 cells?' The
actin changes we measured occur within minutes, and result
from shifts in G- to F-actin. These processes involve the
interaction between actin-regulatory proteins and cellsignaling pathways. One of the more important physiologic
pathways is initiated at the FMLP receptor. The biochemical events which follow FMLP receptor occupancy are not
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
SHAM ET AL
368
0.”
, ,0
2
4
6
a
10
Time (min)
Fig 6. Effect of DMSO induction time on the F-actin response to
TPA in HL60 cells. HL60 cells were cultured in the presence of 1%
DMSO and experiments were performed after 0,24,48, and 72 hours
of culture. TPA (100 nmol/L) was added at time = 0 minutes and
samples were analyzed as above (mean of two experiments; multiple
other experiments confirmed this finding). The day 0 cells exhibited a
20% decrease in F-actin content in response to TPA. At 24 hours of
DMSO incubation, there was only a 10% decrease in F-actin content in
response to TPA addition. By 48 hours of maturation a small F-actin
increase was observed in response to TPA. By 72 hours of maturation
the cells exhibited a 20% increase in F-actin after TPA addition.
Symbols for days 0 through 3 are the same as in Fig 5.
fully characterized but are known to result in activation of
the phosphotidylinositol pathway with subsequent PKC
activation and calcium mobilization.” These two second
messenger systems were studied and compared with FMLP
to determine their relative roles in actin polymerization
responses.
Uninduced HL60 cells treated with TPA showed an
immediate decrease in F-actin content and dramatic changes
in cell shape and surface features. This effect is presumably
mediated by PKC because several dissimilar PKC activators
cause a similar F-actin decrease. Uninduced HL60 cells
treated with either ionomycin or FMLP exhibited no
change in F-actin content. The analysis of the actin polymerization responses as HL60 cells mature helps to define the
relationship between the FMLP receptor, second messenger pathways, and actin-regulatory proteins. Within 24
hours of maturation, HL60 cells have acquired the ability to
increase F-actin in response to both ionomycin and FMLP.
In contrast, TPA continued to result in a decrease in
F-actin at 24 hours. The 24-hour maturation time required
for an actin response to FMLP probably reflects time
required to synthesize FMLP receptors. FMLP receptors
are few in number in uninduced cells: and in other studies
maturation of HL60 cells parallels the ability to respond to
chemoattractant stimuli.43z’Similarly, quantitative changes
in actin-regulatory proteins occur with maturation?’ Gelsolin and actin-binding protein, for example, increase threefold within 24 hours of HL60 cell maturation?’ The 24-hour
maturation time required for an actin response to ionomycin may in part reflect synthesis of calcium-dependent
actin-regulatory proteins. This explanation is favored be-
cause ionomycin caused a dramatic increase in intracellular
calcium in uninduced cells and in those matured for 24
hours, yet actin polymerization occurred only in the matured cells. It is also possible that qualitative changes in
actin-regulatory proteins occur during HL60 cell maturation. As an example, the characteristics of actin-gelsolin
complexes vary depending on the state of activation of
neutrophils.” Nonetheless, qualitative or quantitative
changes in actin-regulatory proteins alone could not account for the persistent decrease in F-actin in response to
TPA at 24 hours of maturation because the cells are
capable of exhibiting a “mature” response to ionomycin
and FMLP within 24 hours of maturation. This discrepancy
raises the possibility that maturation-associated changes in
PKC could account for the observed TPA results.
The quantity, isoenzyme pattern, and cellular localization of PKC change with maturation of HL60 cell^''^^ and
may be causally linked to changes in functional abilities of
cells. An immature PKC isoenzyme pattern in uninduced
cells could account for the decrease in F-actin in response
to TPA in these cells. In matured HMO cells, the TPAinduced F-actin response is qualitatively similar to that of
normal blood neutrophils.9326The role of PKC in this
process is supported by similar changes in F-actin mediated
by chemically dissimilar PKC activators. Furthermore, H7
partially inhibited the TPA-induced actin polymerization in
the matured cells. The failure of H7 to inhibit the actin
decrease in uninduced cells may be a manifestation of an
immature PKC isoenzyme pattern and the associated differences in kinetics and substrate specificities of the various
isotypes.” PKC (Y and PKC p are the predominant isotypes
in uninduced HL60 cells. DMSO-induced maturation results in at least a twofold increase in both isotypes within 48
hours.I3 Also, PKC y, which is barely detectable in uninduced HL60 cells, increases significantly following matur-
1.2
1.1
0.9
-15
-10
-5
0
5
Time (min)
Fig 7. Effect of H7 on FMLP-induced F-actin increase in mature
neutrophils. Neutrophils were incubated with or without H7 (100
pmol/L) for 15 minutes. FMLP (1 nmol/L) (or buffer) was added at
time = 0 minutes. Samples were taken at the indicated times for
determinationof F-actin content by flow cytometry. FMLP resulted in
a 40% increase in F-actin content. H7 (100 pmol/L) did not inhibit the
FMLP response. The results shown are the mean of four experiments
Control; (4-)
FMLP; (-A-), FMLP H7.
&SE. (---0---)
+
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
REGULATION OF ACTIN CONFORMATION IN HL60 CELLS
369
ation.I3 The changes in actin responses to PKC activators
during HL60 cell maturation may be a function of PKC
evolution.
In neutrophils, the roles of the two second messenger
pathways in actin polymerization have been studied and
compared with FMLP.9Calcium ionophores can cause actin
polymerization in neutrophils, yet FMLP-induced actin
polymerization still occurs when intracellular calcium fluxes
are inhibited."." Several investigators have shown that
increases in intracellular calcium are not necessary to
initiate actin polymerization.28A new observation, in fact,
suggests that increases in intracellular calcium caused by
electropermeabilization of neutrophils in calcium containing buffer causes actin depolymerization." Our experiments
in uninduced HL60 cells provide further evidence that a
calcium increase alone is not sufficient for actin polymerization because ionomycin causes an increase in intracellular
calcium in uninduced HL60 cells without associated actin
polymerization.
Similarly, the role of PKC in the FMLP-mediated actin
polymerization response has been studied. In blood neutro-
phils, TPA and FMLP result in different changes in shape
and cytoplasmic streaming,29suggesting different effects on
the cytoskeletal apparatus. Quantitative differences in phosphorylation stimulated by TPA and FMLP in neutrophils
have been described:' and it has been postulated that 'ITA
and FMLP activate different forms of PKC. H7 blocks
neutrophil oxidative burst and degranulation induced by
TPA, but not that induced by FMLP?' We have also found
that H7 is unable to inhibit FMLP-induced actin polymerization in normal neutrophils. These data together with the
time course analysis of the actin polymerization response in
maturing HL60 cells suggest that neither activation of PKC
nor increases in intracellular calcium caused by FMLP
entirely account for actin polymerization in neutrophilic
cells.
ACKNOWLEDGMENT
The authors thank Rachel Goss, Maureen Kempski, and Carol
Kirch for expert technical assistance, and Karen Balta and Linda
Piedemonte for preparing the manuscript.
REFERENCES
1. Collins SJ, Ruscetti FW, Gallagher RE, Gallo R C Terminal
differentiation of human promyelocytic leukemia cells induced by
dimethyl sulfoxide and other polar compounds. Proc Natl Acad Sci
USA 75:2458,1978
2. Collins SJ, Ruscetti FW,Gallagher RE, Gallo RC: Normal
functional characteristics of cultured human promyelocytic leukemia cells (HL60) after induction of differentiation by dimethyl
sulfoxide. J Exp Med 149:969,1979
3. Leglise MC, Dent GA, Ayscue LH, Ross D W Leukemic cell
maturation: Phenotypic variability and oncogene expression in
HMO cells: A review. Blood Cells 13:319, 1988
4. Fontana JA, Wright DG, Schiffman E, Corcoran BA, Deisseroth AB: Development of chemotactic responsiveness in myeloid
precursor cells: Studies with a human leukemia cell line. Proc Natl
Acad Sci USA 77:3664,1980
5. Niedel J, Kahane I, Lachman L, Cuatrecasas P: A subpopulation of cultured human promyelocytic leukemia cells (HL60)
displays the formyl peptide chemotactic receptor. Proc Natl Acad
Sci USA 771000,1980
6. Meyer WH, Howard TH: Changes in actin content during
induced myeloid maturation of human promyelocytes. Blood
62308,1983
7. Howard TH, Meyer WH: Chemotactic peptide modulation of
actin assembly and locomotion in neutrophils. J Cell Biol98:1265,
1984
8. Wallace PJ, Wersto RP, Packman CH, Lichtman MA: Chemotactic peptide-induced changes in neutrophil actin conformation. J
Cell Biol99:1060, 1984
9. Howard TH, Wang D: Calcium ionophore, phorbol ester, and
chemotactic peptide-induced cytoskeleton reorganization in human neutrophils. J Clin Invest 79:1359, 1987
10. Sha'afi RI, Shefcyk J, Yassin R, Molski TFP, Volpi M,
Naccache PH, White JR, Feinstein MB, Becker E L Is a rise in
intracellular concentration of free calcium necessary or sufficient
for stimulated cytoskeletal-associated actin? J Cell Biol 1021459,
1986
11. Downey GP, Chan CK, Trudel S, Grinstein S: Actin assem-
bly in electropermeabilized neutrophils: Role of intracellular
calcium. J Cell Biol110:1975,1990
12. Nishizuka Y:Studies and perspectives of protein kinase C.
Science 233:305,1986
13. Makowske M, Ballester R, Cayre Y, Rosen OM: Immunochemical evidence that three protein kinase C isoenzymes increase
in abundance during HL60 differentiation induced by dimethyl
sulfoxide and retinoic acid. J Biol Chem 263:3402,1988
14. Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa V,
Nizhizuka Y: Direct activation of calcium-activated, phospholipiddependent, protein kinase by tumor-promoting phorbol esters. J
Biol Chem 2577847,1982
15. Berkow RL, Kraft AS: Bryostatin, a non-phorbol macrocyclic lactone, activates intact human polymorphonuclear leukocytes and binds to the phorbol ester receptor. Biochem Biophys
Res Commun 131:1109,1985
16. Hidaka H, Ingaki M, Kawmoto S, Sasaki Y: Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 235036,
1984
17. Okazaki T, Kat0 Y, Mochizuki T, Tashima M, Sawada H,
Uchino H: Staurosporine, a novel protein kinase inhibitor, enhances HL-60-cell differentiation induced by various compounds.
Exp Hematol16:42,1988
18. Korchak H, Vienne K, Weissmann G: Stimulus response
coupling in the human neutrophil. 11.Temporal analysis of changes
in cytosolic calcium and calcium efflux. J Biol Chem 259:4076,1984
19. Tomonaga M, Gasson JC, Quan SG, Golde D W Establishment of eosinophilic sublines from human promyelocytic leukemia
(HL-60) cells: Demonstration of multipotentiality and single lineage commitment of HL-60 stem cells. Blood 671433,1986
20. Cobbold PH, Rink TJ: Fluorescence and bioluminescence
measurement of cytoplasmic free calcium. Biochem J 48:313, 1987
21. Meyer WH, Howard TH: Actin polymerization and its
relationship to locomotion and chemokinetic response in maturing
human promyelocytic leukemia cells. Blood 70363,1987
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
370
22. Berridge MJ: Inositol triphosphates and diacylglycerol: Two
interacting second messengers. Ann Rev Biochem 56:159,1987
23. Kwiatkowski DJ: Predominant induction of gelsolin and
actin-binding protein during myeloid differentiation. J Biol Chem
263:13857,1988
24. Howard T, Chaponnier C, Yin H, Stossel T: Gelsolin-actin
interaction and actin polymerization in human neutrophils. J Cell
Biol110:1983,1990
25. Kiss Z, Deli E, Kuo J F Temporal changes in intracellular
distribution of protein kinase C during differentiation of human
leukemia HMO cells induced by phorbol ester. FEBS Lett 23:4146,
1988
26. Rao KMK Phorbol esters and retinoids induce actin polymerization in human leukocytes.Cancer Lett 28:253,1985
SHAM ET AL
27. Nishizuka Y: The molecular heterogeneity of protein kinase
C and its implicationsfor cellular regulation. Nature 334:661,1988
28. Naccache PH: Signals for actin polymerization in neutrophils. Biomed Pharmacother 41:297,1987
29. Roos FJ, Zimmerman A, Keller HU: Effect of phorbol
myristate acetate and the chemotacticpeptide fNLPNTL on shape
and movement of human neutrophils. J Cell Sci 88399,1987
30. Pontremoli S, Melloni E, Salamino F, Sparatore B, Michetti
M, Sacco 0, Horecker B L Phosphorylation of proteins in human
neutrophils activated with phorbol myristate acetate or with
chemotactic factor. Arch Biochem Biophys 25023,1986
31. Berkow RL, Dodson RW, Kraft AS: The effect of a protein
kinase C inhibitor, H-7, on a human neutrophil oxidative burst and
degranulation.J Leukoc Biol41:441,1987
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1991 77: 363-370
Signal transduction and the regulation of actin conformation during
myeloid maturation: studies in HL60 cells
RL Sham, CH Packman, CN Abboud and MA Lichtman
Updated information and services can be found at:
http://www.bloodjournal.org/content/77/2/363.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.