|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3994-4005
Alteration of Actin Organization by Jaspamide Inhibits Ruffling, but
not Phagocytosis or Oxidative Burst, in HL-60 Cells and Human Monocytes
By
Ina Fabian,
Drora Halperin,
Smadar Lefter,
Leonid Mittelman,
Rom
T. Altstock,
Osnat Seaon, and
Ilan Tsarfaty
From the Departments of Cell Biology and Histology, Physiology, and
Human Microbiology, Sackler School of Medicine, Tel Aviv University,
Tel Aviv, Israel.
 |
ABSTRACT |
Jaspamide, a naturally occurring cyclic peptide isolated from the
marine sponge Hemiastrella minor, has fungicidal and
growth-inhibiting activities. Exposure of promyelocytic HL-60 cells and
human monocytes to jaspamide induces a dramatic reorganization of actin
from a typical fibrous network to focal aggregates. HL-60 cells exposed to 5 × 10 8 mol/L or 107 mol/L jaspamide
exhibited a reduced proliferation rate. In addition, 107
mol/L jaspamide induced maturation of HL-60 cells as indicated by the
appearance of a lobulated nucleus in 55% ± 5% of the cells and
immunophenotypic maturation of the leukemia cells (upregulation of CD16
and CD14 B antigens). Further characterization has shown that F-actin
is aggregated both in HL-60 cells and in human monocytes exposed to
107 mol/L jaspamide. Well-spread cultured human
monocytes contracted and adopted round shapes after treatment with
jaspamide. Moreover, a dose-dependent increase in both total actin and
de novo synthesized portions of the soluble actin was observed in
jaspamide-treated HL-60 cells. Jaspamide treatment inhibits ruffling
and intracellular movement in HL-60 cells and monocytes, but does not
affect phagocytic activity or respiratory burst activity. The
consequential effects of jaspamide-induced actin reorganization on
ruffling, versus its negligible effect on phagocytosis and oxidative
burst, may shed light on molecular mechanisms of actin involvement in
these processes. Jaspamide disrupts the actin cytoskeleton of normal and malignant mammalian cells with no significant effect on phagocytic activity and may, therefore, be considered as a novel therapeutic agent.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
ACTIN IS A ubiquitous eukaryotic
cytoskeletal protein, critical for many aspects of cell activity. In
addition to maintaining cell morphology, it is required for cell
motility, cell division, and intracellular transport.1
Actin reorganization is rapidly induced by many extracellular factors
and/or by adhesion to the extracellular matrix.1-3 Cellular
actin rapidly alternates between two forms: monomeric G-actin
(globular) and polymeric F-actin (fibrous). The dynamics of G-: F-actin
transition may be critical to many of its specialized cellular
functions including regulation of cell shape, motility, secretion,
intracellular transport, endocytosis, exocytosis, and cell
division.2,3
HL-60 human promyelocytic leukemia cells are a well-established model
for studying the effect of different physiologic and pharmacologic
agents on cell growth and maturation.4-6 Changes in
F-actin organization and actin-binding proteins have been observed during the differentiation of HL-60 cells along the granulocytic pathway.7 An increase in levels of actin and actin binding proteins has also been observed in both murine8 and
human9 differentiated myeloid leukemia cells.
In a recent study we have shown that jaspamide, a peptide isolated from
the marine sponge Hemiastrella minor, induces growth modulation
and differentiation in acute myeloid leukemia (AML) cells.10 We found jaspamide, similarly to cytosine
arabinoside, to be effective in suppressing leukemic blast colony
formation and inducing immunophenotypic maturation of three leukemic
cell lines (including HL-60 cells) and blasts of AML
patients.10
In the present study we examine the effects of jaspamide on cellular
growth and functional activity of HL-60 cells and human monocytes. We
show that jaspamide induces cellular maturation accompanied by a
dramatic reorganization of actin in both HL-60 cells and
human monocytes. Jaspamide-induced actin reorganization (aggregation)
inhibits intrinsic cell movement and ruffling but does not affect
phagocytosis or oxidative burst, biological activities that are
considered actin dependent.
| |
MATERIALS AND METHODS |
Cell Culture, Antibodies, and Treatments
HL-60 human promyelocytic leukemia cells were grown in 10% fetal
bovine serum (FBS) (Hyclone Laboratories, Logan, UT) with Iscove's
modified Dulbecco's medium (IMDM) (GIBCO, Grand Island, NY).10 Stock cell cultures were passaged twice weekly and
maintained at 37°C in a humidified atmosphere containing 5%
CO2. Cells (5 × 105/mL) were incubated
with different concentrations (108 to
106 mol/L) of jaspamide (Molecular Probes, Eugene,
OR) for 1 to 48 hours. A stock solution of 103 mol/L
jaspamide was prepared in dimethyl sulfoxide (DMSO) and stored at
 20°C. The drug was diluted before use with IMDM to the
appropriate concentrations. Experiments of cytochalasin treatment were
performed with cytochalasin D (3.94 µmol/L) (Sigma Chemical Co, St
Louis, MO) added to the cell cultures for 24 hours. The following
antibodies were used: CD16 (code no. 306) and CD14 B (code no. 253) (a
generous gift from Dr E. Gazit, Tel Aviv, Israel). These monoclonal
antibodies (MoAbs) were studied at the International Workshop on Human
Leukocyte Differentiation Antigens (Leukocyte typing III) in Oxford,
UK, September 21-26, 1986. Mouse anti-actin MoAb was
purchased from Boehringer Mannheim (Mannheim, Germany). Rabbit
anti-actin Ab was obtained from Sigma. Rhodamine-conjugated donkey
anti-mouse Ab and fluorescein isothiocyanate (FITC)-conjugated donkey
anti-mouse IgG were purchased from Jackson Immunoresearch Laboratories
(West Grove, PA). Anti-mouse IgG-HRP was purchased from Amersham
(Buckinghamshire, UK).
Human Blood Monocytes
Heparinized blood, obtained with informed consent from healthy donors,
was layered on Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala,
Sweden), centrifuged at 400g for 30 minutes at room
temperature; subsequently, the mononuclear cell layer was collected.
The cells were resuspended in IMDM containing 10% FBS and plated at a
concentration of 5 × 106/mL in 75-cm2
flasks. After a 24-hour incubation at 37°C, the cells were washed with phosphate-buffered saline (PBS) to remove nonadherent cells and
the adherent cells were detached by vigorous washing with cold
Ca2+Mg2+-free PBS, and resuspended in IMDM
containing 10% FBS as previously described.11 Monocyte
purity was greater than 85% as determined by morphology (Wright-Giemsa
staining) of cytocentrifugated specimens. The cells were plated
overnight at 106 cells/mL in flat-based tissue culture
tubes (Nunc, Roskilde, Denmark) containing coverslips, for F-actin
staining and phagocytosis studies. After incubation,
107 mol/L jaspamide or 0.01% DMSO was added to the
monocyte cultures, and treated cultures were incubated for an
additional 24 hours.
For NADPH-dependent O2
production studies, monocytes were plated in 96 flat-bottom well
tissue-culture plates at a concentration of 2.5 × 105
cells/100 µL/well and incubated at 37°C for 3 hours. After
incubation, 107 mol/L jaspamide, 0.01% DMSO, or
medium was added to appropriate wells and plates were incubated for an
additional 24 hours.
Cell Growth and Viability
104 HL-60 cells were aliquoted to 96-well plates in medium
with 108 to 107 mol/L jaspamide
at a final volume of 200 µL per well. Cells grown in the presence of
0.01% DMSO or medium were used as controls. After 24 or 48 hours,
cultured cells were stained by trypan blue and viable cells were
quantified by phase-contrast microscopy.
3H-thymidine incorporation experiments were carried out as
previously described.6 In brief, 104 HL-60
cells were cultured in 96-well plates in medium with
108 to 107 mol/L jaspamide at a
final volume of 200 µL per well. Cells grown in the presence of
0.01% DMSO or medium were used as controls. Cultures were pulsed on
days 1 and 2 with 1 µCi/mL 3H thymidine (1 mCi/mol/L;
Nuclear Research Center, Negev, Israel) and harvested 6 hours later
onto glass filters with an automated cell harvester under hypotonic
conditions. Filter discs were dried and counted in a Packard Tri-Carb
liquid scintillation counter (Downers Grove, IL).
Cell Differentiation
HL-60 cells were grown in liquid culture for 48 hours in the presence
of 107 mol/L jaspamide or 0.01% DMSO, and cell
differentiation was evaluated using cytospin (Shandon, Runcorn, UK)
slide preparations stained with Wright-Giemsa. Differential counts were
performed on at least 200 cells using a light microscope at
1,000× magnification.
Fluorescence-Activated Cell Sorter (FACS) Analysis of
Cell-Surface Antigens
Expression of the myeloid cell-surface antigens CD16 and CD14 B was
determined by FACS analysis of indirect immunofluorescence using MoAbs.
HL-60 cells grown in the presence of 107 mol/L
jaspamide or 0.01% DMSO for 48 hours were incubated with the CD16 or
CD14 B MoAb, and subsequently labeled with FITC-conjugated goat
anti-mouse IgG (Bio-Yead, Rehovot, Israel) as previously described.10 Fluorescent cells were detected by flow
cytometer (Becton Dickinson, San Jose, CA).
Actin Staining and Confocal Laser Scanning Microscopy Analyses
Total actin staining was performed on cytospin preparations of cells
fixed with ice-cold 100% acetone and 100% methanol (10 minutes each).
After washing, cells were blocked (1% normal donkey serum and 0.1%
bovine serum albumin [BSA] in PBS) for 1 hour; incubated with mouse
anti-actin MoAb (10 µg/mL) for 1 hour and subsequently labeled with
rhodamine-conjugated donkey anti-mouse-antibodies (diluted 1:100) for 1 hour. All steps were performed at room temperature. F-actin was
detected using rhodamine phalloidin (Rh-phalloidin) (Molecular Probes)
as previously described12 with slight modifications. After
incubation with the drug, cytospin preparations of HL-60 cells and
monocytes grown on coverslips in flat-based tissue-culture tubes were
fixed, permeabilized, and stained in a one-step procedure, as suggested
by the manufacturer (Molecular Probes; simultaneous fixation protocol).
In brief, treated and untreated cells were incubated for 20 minutes
with 3.7% formaldehyde containing 100 µg/mL lysophosphatidyl choline
(Sigma) and 0.3 µmol/L Rh-phalloidin and then washed. Slides and
coverslips were mounted using Gel Mount (Biomeda, Foster City, CA).
Staining analyses was performed using a Zeiss (Oberkochen, Germany)
confocal laser scanning microscope (CLSM). The Zeiss LSM
410 is equipped with a 25-mW Krypton-Argon laser and a 10-mW HeNe laser
(488, 543, and 633 maximum lines). Images were stored on an optical
disk drive and printed using a Codonics NP1600 printer (Codonics,
Middleburg Heights, OH). Competition experiments of jaspamide and
phalloidin were performed with HL-60 cells by exposure of untreated
cells to Rh-phalloidin in the presence of 107 mol/L
or 108 mol/L jaspamide for 30 minutes. In other
experiments, cells were incubated for 24 hours with
107 mol/L jaspamide, subsequently washed, and double
staining for F-actin and total actin was performed using Rh-phalloidin
(Molecular Probes; simultaneous fixation protocol) and anti-actin MoAb
followed by FITC-conjugated donkey anti-mouse antibodies (diluted
1:50). CLSM analysis was performed as described above.
Analysis of Actin
Sample preparation for total actin analysis.
HL-60 cells were exposed to 107 mol/L jaspamide,
0.01% DMSO, or medium for 24 hours. Cells were then washed
twice with PBS and lysed (20 mmol/L Tris-HCl, pH 7.8, 100 mmol/L NaCl,
50 mmol/L NaF, 1% NP40, 0.1% sodium dodecyl sulfate (SDS), 2 mmol/L
EDTA, 10% glycerol) with protease inhibitor cocktail (Boehringer
Mannheim). Protein concentrations were determined using Pierce BCA
assay (Pierce Chemical Co, Rockford, IL). Cell lysates were solubilized in X2 loading buffer (20% glycerol, 0.2% bromophenol blue,
4% SDS, 100 mmol/L Tris-Cl and 2%  -mercaptoethanol) and boiled at 100°C for 5 minutes.
Sample preparation for soluble, insoluble, and total actin analysis.
Cellular soluble, insoluble, and total fractions of actin were prepared
as described by Hallows et al with slight modifications.13 In short, an equal number of HL-60 cells were placed in three tubes (A,
B, and C); 100 µL of lysis buffer was added to each tube. The lysed cells were centrifuged 15,000g for
5 minutes. To obtain soluble actin, 100 µL of the supernatant from
tube A was transferred to a new tube. The supernatant from tube B was discarded, the precipitate saved, and 100 µL of fresh lysis buffer was added to tube B. Tube C contained total actin, where both supernatant and precipitate were saved. Boiling X2 loading buffer was
subsequently added to the three tubes.
Coomassie staining of SDS-polyacrylamide gel electrophoresis (PAGE)
gels.
Cell lysates in X2 loading buffer were boiled for 15 minutes,
then loaded and electrophoresed on 12% polyacrylamide gel. Gels were
stained for 2 to 6 hours with Coomassie brilliant blue R250 stain
(Bio-Rad, Hercules, CA; 0.1% dissolved in 50% methanol and 10%
acetic acid) and then destained in a 10% methanol 7% acetic acid
solution. The gel was subsequently scanned using a Microtek scanner.
Western blot analysis of actin.
Cell lysates were loaded onto a 10% SDS-PAGE, electrophoresed, and
transferred onto nitrocellulose. The nitrocellulose membrane blots were
blocked (2% BSA in PBS, pH 7.4), rinsed in PBS-T (0.2% Tween-20 in
PBS), and incubated with mouse anti-actin MoAb at 1:1,500 dilution in
PBS-BT (1% BSA, 0.1% Tween 20 in PBS) for 1 hour at room temperature.
The blots were washed three times with PBS-T and incubated with
anti-mouse IgG-HRP diluted 1:5,000 in PBS-BT for 1 hour at room
temperature. Blots were washed, incubated with enhanced
chemiluminescence peroxidase substrates (Amersham, Arlington Heights,
IL), and exposed to Fuji X-ray film RX (Fuji, Tokyo, Japan) for varying
exposure times.
Actin Immunoprecipitation of
[35S]-Cysteine-Methionine-Labeled HL-60 Cells
HL-60 cells were metabolically labeled with 0.3 µCi/mL
[35S]-cysteine-methionine (Amersham, Buckinghamshire, UK)
in supplemented cysteine-methionine-free DMEM (GIBCO) and exposed to
107 mol/L jaspamide or 0.01% DMSO for 1 hours.
Labeled cells were extracted in 1 mL lysis buffer (as described above).
Clarified cell lysates (2 × 107 cpm) were
immunoprecipitated with rabbit anti-actin Ab overnight at 4°C.
Immunocomplexes were collected on protein A-sepharose (Pharmacia, Uppsala, Sweden). Each sample was microfuged (10,000 rpm, 5 minutes), the supernatants were decanted, and the pellets were washed
three times, resuspended in loading buffer and boiled at 100°C for
10 minutes. Supernatants were collected from microfuged samples,
analyzed on 10% SDS-PAGE, and quantified using phosphor-imager (Molecular Dynamics, Sunnyvale, CA) or exposed to Fuji X-ray film RX
for varying exposure times.
Phagocytosis of Candida albicans
C albicans was grown in Sabouraud's dextrose broth for 5 days,
washed twice in saline, and resuspended in Hanks' Balanced Salt
Solution (HBSS) to a concentration of 2 × 107 yeast
particles/mL as previously described.6 Two types of experiments were performed:
(1) To determine the percentage of phagocytosing cells and the
phagocytosis index, HL-60 cells or monocytes were treated for 24 hours
with 107 mol/L jaspamide, DMSO or medium, washed,
and resuspended in HBSS. One hundred microliters of HL-60 cells
(containing 106 cells) were placed in capped plastic tubes
(12 × 75 mm) with 100 µL AB serum, 200 µL yeast
suspension, and HBSS (final volume, 500 µL). Tubes were incubated in
a shaking water bath at 37°C for 60 minutes. Cells were
then diluted 1:5 with cold HBSS, and cytocentrifuge preparations were
collected and stained with Giemsa. At least 200 phagocytosing cells
were surveyed to calculate the mean phagocytosing yeast/HL-60 cell
(phagocytosing index). We also determined the percentage of cells
phagocytosing the C albicans.6
Monocytes were grown on coverslips; 600 µL HBSS, 200 µL AB serum,
and 200 µL yeast suspension were added (final volume, 1 mL). The
tubes were incubated for 60 minutes. After incubation, cells were
washed, stained with Giemsa, and examined as described above.
(2) Fluorescence labeling of F-actin and phagocytosed C
albicans in experiments requiring simultaneous visualization were performed in two stages: C albicans (2 × 107)
were centrifuged at 2,000 rpm for 10 minutes and the yeast pellet was
stained with PKH2 green fluorescent general cell linker kit (Sigma)
according to the manufacturer's instructions. HL-60 cells or monocytes
preincubated with 107 mol/L jaspamide were incubated
with the stained C albicans as described above for 30 minutes.
After incubation, the cells were washed with HBSS and stained with
Rh-phalloidin as described above.
Electron Microscopy
Monocytes preincubated with 107 mol/L jaspamide were
incubated with C albicans for 30 minutes as described above.
After incubation, the cells were rinsed three times with PBS and fixed
in 2% glutaraldehyde for 2 hours at 4°C. The samples were rapidly
rinsed three times in 0.1 mol/L cacodylate buffer, pH 7.4, postfixed in osmium tetroxide for 2 hours, and then washed
extensively in cacodylate buffer. Samples were dehydrated through a
graded series of ethanols, then treated with propylene oxide for
1 hour and embedded in araldite. Thin sections were cut with a diamond
knife, stained with uranyl acetate and lead citrate, and examined in a
JEOL 100B electron microscope (JEOL, Tokyo, Japan).
Assay of O2 Production by HL-60
Cells and Monocytes
HL-60 cells.
Generation of O2 was measured by
determining the rate of superoxide dismutase-inhibitable
ferricytochrome C reduction as previously described.6 In
brief, HL-60 cells (5 × 105/mL) were grown for 24 hours in the presence of 107 mol/L jaspamide, 0.01%
DMSO, or medium. After incubation, the cells were washed twice in HBSS
containing 5% FBS, and suspended in a 160 µmol/L solution of
ferricytochrome C (Sigma) in HBSS. The cells (2.5 × 106 cells/mL) were plated in 96-well flat-bottom
tissue-culture plates, 100 µL per well. Phorbol myristate acetate
(PMA) (Consolidated Midland Corp, Brewster, NY) (50 mmol/L) was added
and the cells were incubated for various periods of time (30 to 90 minutes) at 37°C. Subsequently, the plates were transferred to a
Multiskan apparatus (Flow Laboratories, McLean, VA) and the absorbence
read at 500 nm against a blank of cytochrome C incubated for the
indicated time at 37°C in the absence of cells. The amount of
O2 produced was calculated using the
equation:
|
|
Results were expressed as nanomoles of
O2 per 106 cells per minute.
Specificity of cytochrome C reduction was verified by its elimination
in the presence of 300 U/mL of superoxide dismutase (Sigma).
Monocytes.
Monocytes (2.5 × 106 cells/mL) were plated in 96-well
flat-bottom tissue-culture plates, 100 µL per well, and exposed to
107 mol/L jaspamide, DMSO, or medium for 24 hours.
After incubation, the cells were washed twice with HBSS containing 5%
FBS, and O2 production was determined as
described for HL-60 cells.
Statistical Analyses
Statistical analyses were performed using Student's t-test.
| |
RESULTS |
Effect of Jaspamide on Cell Proliferation, Differentiation, and
Cell-Surface Antigen Expression
We have previously shown that jaspamide suppressed primary colony
formation in agar and the recovery of clonogenic cells from suspension
cultures of leukemic cell lines in a dose-dependent manner.10 In this study we show that incubation of HL-60
cells with 5 × 108 mol/L jaspamide for 24 or
48 hours resulted in 30% and 38% proliferation inhibition,
respectively (P < .05) as determined by
3H-thymidine incorporation
(Table 1). Incubation with
107 mol/L jaspamide for 24 or 48 hours
resulted in 46% and 77% inhibition, respectively (P < .05)
(Table 1). The inhibitory effect of 108 mol/L
jaspamide was not statistically significant. These results indicate
that jaspamide inhibits HL-60 cell proliferation in a dose- and
time-dependent manner.
To study the effect of jaspamide on cell differentiation, HL-60 cells
were treated with 107 mol/L jaspamide for 48 hours and examined for changes in cell morphology and surface
differentiation markers. DMSO-treated cells served as control. After
jaspamide treatment, 55% ± 5% (n = 3) of the cells exhibited
myeloid differentiation as indicated by a lobulated nucleus
(Fig 1B). This lobulation was not apparent in the DMSO-treated cells (Fig 1A). FACS analysis of surface antigen CD16 shows 53% upregulation of this differentiation marker in jaspamide-treated cells (Table 2 and Fig
1C). Treatment with jaspamide induced 26.6% upregulation in CD14B
expression (Table 2). These results indicate that jaspamide induced
maturation of HL-60 cells to the granulocyte monocyte lineage.
View larger version (91K):
[in this window]
[in a new window]
| Fig 1.
Jaspamide induces differentiation in HL-60 cells. HL-60
cells were grown for 48 hours with (A) 0.01% DMSO, or (B)
107 mol/L jaspamide. Cell differentiation was determined
on Wright-Giemsa-stained cytocentrifuge preparations. (C) FACS
analysis and overlay histograms of fluorescence intensity showing the
upregulation of the differentiation antigen CD16 in HL-60 cells after
exposure of the cells for 48 hours to 107 mol/L
jaspamide (thick line) or 0.01% DMSO (broken thin line). An irrelevant
antibody was used as background control (dotted line). M1
line defines the positive intensities range (A, B: original
magnification × 475).
|
|
Effects of Jaspamide on Actin Organization
The jaspamide-induced reorganization of actin in HL-60 cells was
examined using anti-actin MoAb staining and CLSM analysis. Exposure of
HL-60 cells to jaspamide for 24 hours resulted in a dramatic
reorganization of actin from the short filamentous actin network
ordinarily found in HL-60 cells (Fig
2A) to focal aggregates (Fig 2B, C, C'). This effect was dose
dependent: detectable at 108 mol/L (Fig 2B) and
observed to be most prominent at 107 mol/L jaspamide
(Fig 2C and C'). Jaspamide was toxic at concentrations of
106 mol/L (data not shown).
View larger version (105K):
[in this window]
[in a new window]
| Fig 2.
Jaspamide induces actin aggregation in HL-60 cells in a
dose- and time-dependent manner. HL-60 cells were treated with
jaspamide and stained for actin (with mouse anti-actin MoAb labeled
with rhodamine-conjugated donkey anti-mouse antibody) and analyzed by
CLSM. (A) Untreated cells. (B) Cells grown for 24 hours with
108 mol/L jaspamide. (C, C') Cells grown for 24 hours
with 107 mol/L jaspamide; in C' the actin staining
(red) is overlaid on the Nomarski image (gray). (D) Cells grown for 48 hours in the presence of 108 mol/L jaspamide. (E) Cells
grown for 48 hours in the presence of 107 mol/L
jaspamide (A through E: original magnification × 250) (C': original
magnification × 2,100).
|
|
To evaluate the time-course of jaspamide-induced actin reorganization,
HL-60 cells were incubated with 107 mol/L jaspamide
for 1, 4, 24, and 48 hours. At 1 and 4 hours, approximately 10% and
50%, respectively, of the HL-60 cells exhibited actin aggregation
(data not shown). The percentage of cells showing actin aggregation
increased over time, with greater than 80% of the cells exhibiting
large focal aggregates of actin at 24 hours (Fig 2C). Similar large
focal aggregates of actin were observed upon incubation of HL-60 cells
for 48 hours (Fig 2E). HL-60 cells exposed to 107
mol/L jaspamide for 24 hours, followed by extensive washing and subsequent examination after an additional 48 hours in media without jaspamide, exhibited no reversal of the jaspamide-induced actin aggregation (data not shown).
Previous studies have shown that jaspamide inhibits binding of
phalloidin to purified F-actin.14 Jaspamide is capable of entering cells unfacilitated.15,16 It was previously shown that phalloidin does not penetrate living cells without cell
permeabilization.17,18 However, it was recently shown that
phalloidin can penetrate living cells.19 We did not observe
significant penetration of phalloidin in nonpereablized cells (data not
shown). We proceeded to investigate whether jaspamide also inhibits
phalloidin binding to actin in permeabilized cells. Phalloidin staining
of HL-60 cells was performed in the presence of 108
mol/L (Fig 3B) or 107
mol/L jaspamide (Fig 3C) and compared with phalloidin staining in the
absence of jaspamide (Fig 3A). These experiments show that 108 mol/L jaspamide partially reduced Rh-phalloidin
staining whereas 107 mol/L jaspamide further reduced
Rh-phalloidin labeling. Conversely, HL-60 cells, treated with
107 mol/L jaspamide for 24 hours followed by their
extensive washing and then Rh-phalloidin staining, did not exhibit
reduced actin labeling by Rh-phalloidin, compared with untreated cells.
Moreover, these cells exhibited aggregated staining of actin similar to that observed in treated cells labeled with anti-actin MoAb (Fig 3E).
From these results we conclude that jaspamide competes with phalloidin
binding to actin in cells only if present in the labeling reaction. To
show that the aggregated phalloidin staining depicts actin
rearrangement, we performed double staining with Rh-phalloidin and
anti-actin MoAb (Fig 3G through I). CLSM analysis of double-labeled HL-60 cells (treated with 107 mol/L jaspamide for 24 hours) is shown in Fig 3G (green, MoAb staining) and Fig 3H (red,
Rh-phalloidin staining). The overlay of red and green images shows that
most of the anti-actin MoAb and Rh-phalloidin colocalize (yellow) (Fig
3I). These results demonstrate that jaspamide induces cellular F-actin
rearrangement in HL-60 cells.
View larger version (48K):
[in this window]
[in a new window]
| Fig 3.
Competitive binding studies between jaspamide and
Rh-phalloidin. Jaspamide induces F-actin aggregation in HL-60 cells and
in human monocytes. Competitive studies (A through C). HL-60 cells were
stained with Rh-phalloidin in the presence of (A) medium
(control). (B) 108 mol/L jaspamide and (C)
107 mol/L jaspamide; analyzed by CLSM. No actin
aggregation is seen. Aggregation of F-actin in HL-60 cells (D through
F). HL-60 cells were incubated for 24 hours with (D) medium. (E)
107 mol/L jaspamide subsequently washed and stained with
Rh-phalloidin (no jaspamide added during staining with Rh-phalloidin).
Aggregation of F-actin is seen in (E). (F) HL-60
cells grown for 24 hours in the presence of 3.94 µmol/L CD were
stained with Rh-phalloidin (arrows indicate cytochalasin-induced small
focal F-actin clusters). Costaining of HL-60 cells with Rh-phalloidin
and anti-actin MoAb (G through I). HL-60 cells grown for 24 hours in
the presence of 107 mol/L jaspamide were costained for
F-actin (Rh-phalloidin) and total actin (anti-actin MoAb labeled with
anti-mouse-FITC). Colocalization analysis was performed: (G)
Actin labeled with anti-actin MoAb (green), (H) F-actin
labeled with Rh-phalloidin (red), (I) colocalization of the total actin
and F-actin staining is observed (yellow). Effect of jaspamide on
monocytes (J through Q). Freshly isolated monocytes were grown for 24 hours in IMDM containing 10% FBS. The cells were incubated for
additional 24 hours with the following: (J through L, P) medium; (M
through O, Q) 107 mol/L jaspamide. (J, M, P, and Q)
Monocytes stained with Rh-phalloidin to show the distribution of
F-actin. In (P) and (Q) the actin staining (red) is overlaid on the
Nomarski image (gray) phase microscopy of the same cells. (L and O)
Monocytes stained with Giemsa to show the changes in cell morphology.
(A through F: original magnification × 250; G through H: original
magnification × 750; J through O: original magnification × 250; P
and Q: original magnification × 2,000).
|
|
Cytochalasin D (CD), a fungal toxin, inhibits microfilament function
and polymerization by blocking monomer addition at the rapidly growing
ends of F-actin filaments. Incubation of HL-60 cells with 3.94 µmol/L
CD for 24 hours resulted in the appearance of small actin clusters
distributed throughout the cytoplasm (Fig 3F), compared with the large
bulky aggregation induced by jaspamide (Fig 3E and H).
Jaspamide also induced dramatic changes in the cellular morphology of
human monocytes. Well-spread cultured monocytes (Fig 3J through L)
treated with 107 mol/L jaspamide for 24 hours
drastically contracted and adopted round shapes (Fig 3M through O). To
study the effect of jaspamide on F-actin organization in these cells,
jaspamide-treated monocytes and untreated control cells were stained
with Rh-phalloidin. In untreated monocytes F-actin is organized in a
network and is condensed in focal adhesion regions (Fig 3J, see arrow
and P). In jaspamide-treated cells, F-actin was dramatically rearranged
to form aggregated clumps (Fig 3M and Q). The clustering of F-actin in
jaspamide-treated human monocytes was similar to the actin
reorganization observed in jaspamide-treated HL-60 cells (Fig 3E and H).
Effect of Jaspamide on Total Actin Content and Synthesis in HL-60
Cells
The effects of jaspamide treatment on total cellular actin content and
de novo synthesis of soluble actin in HL-60 cells were determined using
SDS-PAGE, Western blot, and immunoprecipitation studies. Exposure of
HL-60 cells to 107 mol/L jaspamide for 48 hours
induces a dramatic increase in a 42-kD band corresponding
with actin, compared with untreated cells as determined by SDS-PAGE
(Fig 4Aa, lanes 2 and 1, respectively). Western blot analysis with anti-actin MoAb verified a
specific dose-dependent increase in total actin content after
incubation of the cells with 108 to
107 mol/L jaspamide for 24 hours, as
compared with DMSO (Fig 4Ab, lanes 4, 3, and 2, respectively). To study the time course of jaspamide-induced total actin accumulation, we measured total actin in
HL-60 cells after their exposure to 107 mol/L
jaspamide for various periods of time (1 to 24 hours) (Fig 4B). A
slight increase in total actin was observed after 4 hours of incubation
with jaspamide compared to DMSO (Fig 4B, lanes 5 and 4, respectively).
A further increase was observed after 24 hours of exposure to jaspamide
compared with DMSO (Fig 4B, lanes 7 and 6, respectively). No
significant increase in total actin was observed after a 1-hour
incubation of the cells with the drug compared with DMSO (Fig 4B, lanes
3 and 2, respectively).
View larger version (23K):
[in this window]
[in a new window]
| Fig 4.
Jaspamide induces increase in total cellular
actin and de novo actin synthesis in HL-60 cells. (Aa)
Coomassie-stained 12% SDS-PAGE gel showing total cellular protein of
HL-60 cells exposed for 48 hours to DMSO 0.01% (lane 1) or jaspamide
107 mol/L (lane 2). (Ab) Dose-dependent actin
accumulation: HL-60 cell extracts obtained from cells exposed for 24 hours to medium (lane 1), DMSO 0.01% (lane 2), jaspamide
107 mol/L (lane 3), jaspamide 108 mol/L
(lane 4) were resolved on SDS-PAGE, and analyzed by Western blot using
anti-actin MoAb. (B) Time course of actin accumulation. HL-60 cell
extracts obtained from cells exposed to medium (lane 1), DMSO 0.01%
(lanes 2, 4, and 6), or jaspamide 107 mol/L (lanes 3, 5, and 7) for 1 hour (lanes 2 and 3), 4 hours (lanes 4 and 5), or 24 hours
(lanes 6 and 7) were resolved on SDS-PAGE, and analyzed by Western blot
using anti-actin MoAb. (C) Effect of jaspamide on soluble and insoluble
actin: Soluble, insoluble, and total actin fractions were prepared as
described in Materials and Methods. (a) Coomassie blue and (b)
immunoblot with anti-actin MoAb studies were performed (the lanes
indicated are for both methods; ie, Fig 4Ca and Fig 4Cb). HL-60 cells
treated with DMSO (lanes 1 through 3) or jaspamide 107
mol/L for 24 hours (lanes 4 through 6) were examined for total (lanes 1 and 4) soluble (lanes 2 and 5) insoluble (lanes 3 and 6) actin. (D) De
novo actin synthesis. (a) Effect of jaspamide on de novo actin
synthesis. [S35]-cystein-methionine-labeled HL-60 cells
were exposed to medium (lane 1), DMSO (lane 2), or to jaspamide
107 mol/L (lane 3) for 1 hour, immunoprecipitated with
rabbit anti-actin Ab, and the immunoprecipitates were resolved on
SDS-PAGE and exposed to Fuji X-ray film. (b) Coomassie-stained SDS-PAGE
of HL-60 cells treated for 1 hour with DMSO 0.01% (lanes 1 through 3)
or jaspamide 107 mol/L (lanes 4 through 6) showing total
(lanes 1 and 4) soluble (lanes 2 and 5) and insoluble (lanes 3 and 6)
actin.
|
|
HL-60 cell actin is considered primarily ( 75%) to be composed of
"Triton-soluble" fraction.13 We proceeded to
determine the effect of jaspamide on the soluble and insoluble actin
fractions. Coomassie staining of SDS-PAGE (Fig 4Ca) and Western blot
analysis (Fig 4Cb) both show a significant shift of actin from the
soluble to the insoluble fraction in jaspamide-treated cells (Fig 4Cb, lanes 5 and 6, respectively) compared with DMSO-treated cells (lanes 2 and 3, respectively).
The identification of newly synthesized actin using immunoprecipitation
is a complicated experiment due to the fact that portions of actin are
insoluble. However, we proceeded to analyze de novo actin synthesis by
a standard immunoprecipitation procedure using short-term (1 hour)
labeling to limit the effect of an increase in the insoluble fraction
in treated cells. Results show a fivefold increase in short-term
35S-labeled immunoprecipitation actin. We verified the
limited increase in insoluble actin during the 1-hour treatment by
SDS-PAGE (Fig 4Da and b). These results indicate that a
significant portion of the increase in total actin levels is caused by
de novo synthesis.
Effect of Jaspamide on Cellular Morphology, Intracellular Movement,
and Ruffling
To study jaspamide's effect on cellular movement, we compared CLSM
time-lapse photography (1-second interval) of monocytes treated for 1 hour with 107 mol/L jaspamide and untreated cells.
The untreated monocytes moved extensively and spread on the substrate
(Fig 5A through C, see arrow) while the
jaspamide-treated cells moved significantly less and pseudopodia
formation was rarely detected (Fig 5D through F).
View larger version (119K):
[in this window]
[in a new window]
| Fig 5.
Effect of jaspamide on intracellular movement and
ruffling in monocytes. Nomarski time-lapse photography of monocytes
treated for 1 hour with (D through F and J through L)
107 mol/L jaspamide compared with (A through C, G
through I) untreated cells. Cellular movement (A through F). Nomarski
images of a 1-second interval of the (A through C) untreated cells
exhibiting movement on the substrate (arrow indicating spreading
region); (D through F) jaspamide-treated cells that exhibited
no significant movement. Ruffling and intracellular movement: (G
through L) 0.05-second interval of treated and untreated cells,
after (G and J) 10 minutes and (H and K) 10 minutes + 0.05 seconds
were analyzed. Using the CLSM program, we determined the cellular
movement that occurred in 0.05 seconds by subtracting the 10-minute
images from the images of 10 minute + 0.05 seconds (images 5H minus
5G and images 5K minus 5J) for untreated and treated cells,
respectively. The resulting calculation of movement is shown as red
areas superimposed on 10-minute + 0.05-second untreated and treated
images (I and L, respectively) (original magnification × 1,400).
|
|
To analyze the effect of jaspamide on membrane ruffling, we compared
rapid CLSM time-lapse photography (0.05-second interval) of untreated
and treated monocytes. CLSM images of 107 mol/L
jaspamide-treated and untreated cells after 10 minutes (Fig 5G and J,
respectively) and 10 minutes + 0.05 seconds were acquired (Fig 5H and
K, respectively). Using the CLSM program we subtracted the 10-minute
images from the 10-minute + 0.05-second images (Fig 5H minus 5G and 5K
minus 5J). The differences between images were calculated, and then
areas where changes occurred were superimposed on the corresponding
10-minute time image. These areas (shown in red Fig 5I and Fig 5L)
represent positional changes of cellular organelle and membrane, thus
indicating that movement took place. The movement and ruffling in
untreated cells were extensive as shown by the significant amount of
red in Fig 5I. Furthermore, untreated cells show extensive formation of
motile cell-surface protrusions-ruffling. No significant ruffling or intracellular movement was observed in the jaspamide-treated cells; therefore, the image scarcely features any red (Fig 5L).
Moreover, the internal movement of organelles is dramatically inhibited (Fig 5L). We performed 10 experiments to determine number of cells exhibiting membrane ruffling for at least 50 cells per experiment. Ninety-five percent of the untreated monocytes exhibit membrane ruffling while only 4% of the jaspamide-treated cells exhibited ruffling. None of the cells with significant actin aggregation was
observed to exhibit ruffling. These results suggest that
proper F-actin organization is essential for overall motility of the cell.
Effects of Jaspamide on Phagocytosis and
O2 Production
The actin network plays a major role in chemotaxis and phagocytosis. To
study the effects of jaspamide on phagocytosis, HL-60 cells and
monocytes were exposed for 24 hours to 107 mol/L
jaspamide, incubated with C albicans for 60 minutes, and examined for phagocytosis. Jaspamide did not affect the percentage of
phagocytosing cells or the number of yeast particles phagocytosed by
each cell (Table 3). To ascertain that
aggregated actin clusters did not affect phagocytosis,
fluorescein-labeled C albicans was incubated with
jaspamide-treated HL-60 cells and monocytes. The cells were fixed,
Rh-phalloidin stained, and analyzed by CLSM. HL-60 cells (Fig 6A and C
[see page 3998]) and human monocytes (Fig 6B and D [see page 3998])
that show jaspamide-induced actin aggregates phagocytose C
albicans. Electron microscopy analysis of jaspamide-treated
monocytes incubated with C albicans shows that the C
albicans was phagocytosed and is located inside the cell
(Fig 7).
View larger version (104K):
[in this window]
[in a new window]
| Fig 6.
Effect of jaspamide on phagocytosis of C albicans
by HL-60 cells and monocytes. (A and C) HL-60 cells or (B and D)
monocytes were grown for 24 hours in the presence of jaspamide
107 mol/L and exposed to fluorescence-stained C
albicans for 30 minutes. Cells were stained with Rh-phalloidin and
analyzed by CLSM for (A and B) fluorescence or (C and D) Nomarski
(original magnification × 1,700).
|
|
View larger version (174K):
[in this window]
[in a new window]
| Fig 7.
Electron migrograph of a monocyte that phagocytosed C
albicans. A thin section through monocytes following their growth
for 24 hours in the presence of 107 mol/L jaspamide and
their exposure for 30 minutes to C albicans (original
magnification × 9,250).
|
|
Superoxide anion production during the respiratory burst in monocytes
is presumed to be actin-dependent.20 We examined
O2 production by HL-60 cells and by
monocytes following their exposure to 107 mol/L
jaspamide for 24 hours. Jaspamide did not affect release of superoxide
anion upon PMA stimulation as indicated by the SOD-inhibitable cytochrome C reduction assay (Table 4).
Overall production of O2 by HL-60 cells
was very low, compared with monocytes.
View this table:
[in this window]
[in a new window]
|
Table 4.
Effect of Jaspamide on Production of Superoxide by
Monocytes and HL-60 Cells Incubated in the Absence or Presence of
PMA
|
|
| |
DISCUSSION |
HL-60 cells have been used as a model to study morphological,
functional, and biochemical changes after differentiation induced by
various agents.4,5,21 Previous studies have shown that actin synthesis undergoes changes during induced myeloid
maturation,9 and that actin polymerization affects a
variety of cell functions including locomotion, phagocytosis, and
maturation in a wide range of cells including HL-60
cells.22
In recent years, a number of molecules have been shown to affect actin
organization and cell functions.23,24 One such molecule, jaspamide, has been shown to directly interact with purified actin to
induce actin polymerization.14 In a previous study, we have shown that jaspamide induces growth modulation and differentiation of
AML cells.10 The present study was undertaken to
investigate the effects of jaspamide on cellular actin levels
and organization along with its effect on membrane ruffling, oxidative
burst, and phagocytic activity in both HL-60 cells and monocytes.
It has been previously shown that jaspamide has antiproliferative
activity and a cytotoxic effect on a number of tumor cell lines,
including breast and prostatic cancer cells.15,16 Here we
show that jaspamide inhibits the growth of human promyelocytic leukemic
HL-60 cells, induces differentiation, and acts on the actin-based
cytoskeleton. It is not clear whether growth inhibition by jaspamide is
directly induced by actin aggregation or indirectly by affecting other
proteins that induce growth inhibition. A recent report showing that
jasplakinolide inhibited bombesin-stimulated phosphorylation of FAK and
inhibited PC-3 cell growth25 offers support to the indirect
inhibition hypothesis. Moreover, these investigators suggest that
additional actin-disrupting agents can block FAK signal transduction,
which may be critical to their antitumor activity in prostate
carcinoma.25
Jaspamide has been recently shown to also markedly influence the
morphogenetic process in the green alga Micrasterias when used
in concentrations higher than 3 µmol/L. Development of
Micrasterias was found to be inhibited or strongly retarded,
and malformations occurred and large vacuoles were formed. At the
ultrastructural level, dense abnormal accumulations of filamentous
structures have been found, indicating actin filament polymerizing
activities of the drug in situ.26
The present data show that jaspamide induces actin accumulation in
HL-60 cells in a dose- and time-dependent manner. This increase in
total actin is accompanied by a shift of the major portion of actin
from the soluble to the insoluble form in jaspamide-treated HL-60
cells. We proceeded to measure de novo actin synthesis using 35S-labeling and immunoprecipitation. Although this method
does not take into account newly synthesized actin that was immediately polymerized to insoluble actin, we suggest that these results are a
good indication that jaspamide treatment increases actin synthesis in
HL-60 cells. This is based on the comparatively short labeling time
experiments and the assumption that the rate of actin polymerization to
insoluble form is greater in the jaspamide-treated cells as suggested
by the results of SDS-PAGE and Western blot of different actin
fractions (Fig 4C).
The increase in actin synthesis can be attributed to actin clustering
that may signal for jaspamide-induced reduction of the cellular actin
pool. Our results are in line with previous studies showing that HL-60
cells induced toward myeloid maturation by a 5-day exposure to
dimethylformamide contain about twice as much actin per milligram
protein as do uninduced HL-60 cells.9 An increase in the
content of total cellular actin has been reported in differentiated
myeloid leukemia cells compared with their nondifferentiated counterparts.8,27-29
We show that jaspamide not only induces actin accumulation but also
induces the clustering of actin in HL-60 cells in a dose-and time-dependent manner. Our results also demonstrate that jaspamide inhibits phalloidin binding to F-actin in cells if present in the
labeling mix. However, after a 24-hour incubation and extensive washing, jaspamide did not inhibit phalloidin biding to F-actin. This
loss of inhibition was probably caused by reduced jaspamide concentration resulting from the washing or its instability. Other drugs known to affect actin organization, as well as cell function, are
the cytochalasins.30 Cytochalasins disrupt actin
organization, inhibit various cell movements,31 bind to the
growing end of F-actin filaments, and block all association and
dissociation reactions at those ends.32 We compared the
effects of jaspamide in HL-60 cells to those of cytochalasin D and have
shown that while jaspamide induced large F-actin aggregates,
cytochalasin D induced the formation of only small F-actin clusters
distributed throughout the HL-60 cytoplasm. The different effect of
these compounds on actin organization indicates that the mode of action of the two classes of drugs is different. Our study also shows that
jaspamide has similar effects on the F-actin organization in human monocytes.
In membrane ruffling areas, the actin filaments are tightly associated
with the plasma membrane and a number of proteins have been shown to be
associated with these sites.33 The present results show
that aggregation of actin inhibits cell ruffling. A similar inhibition
is induced by other factors such as cytochalasin D.34 These
results indicate that ruffling, which is a well-coordinated membrane
cytoskeleton procedure, requires the presence of an organized membrane-bound actin cytoskeleton. Jasplakinolide was recently shown to
cause actin polymerization within neutrophils and an increase in
association of actin with the Triton-insoluble cytoskeleton. Actin
polymerization in neutrophils induce marked increase in rigidity and
affect adhesive and mechanical properties of the cells.35,36
In the present study we have shown that jaspamide does not affect
phagocytosis of C albicans by HL-60 cells or by monocytes. The
functions of mature neutrophils, such as phagocytosis,37 and expression of the oxidase involved in respiratory
burst38 have all been shown to be associated with F-actin
and the actin-binding protein network. Recent studies have shown that
although fibronectin enhances actin polymerization in neutrophils,
monocytes, and macrophages,39 it promotes phagocytosis of
unopsonized Staphylococci, IgG-opsonized bacteria,40 IgA-opsonized bacteria,41 and
complement receptor-mediated phagocytosis42 by phagocytes.
The role of actin in microorganism phagocytosis is still not fully
understood. Several works have shown that there is a dramatic change of
actin organization upon phagocytosis, but it is not clear if the
phagocytic process is dependent on actin polymerization. For example,
double-immunofluorescence staining of gonococci and actin
filaments in infected cells showed bacterium-associated accumulations
of F-actin as an early signal of bacterial entry. The recruitment of
F-actin was transient and disappeared once the bacteria were inside the
cells. Cytochalasin D disrupted the actin cytoskeleton architecture but
did not prevent the recruitment of F-actin by the
bacteria.43 It was also shown that changes in the
organization of actin occurred during the C albicans
internalization to fibroblastic cells.44 Other studies have
shown that endothelial cell microfilaments polymerized around C
albicans after phagocytosis of the organisms, suggesting that
condensation of actin filaments around the organisms is required for
C albicans phagocytosis.45 There is not a
conclusive answer to the question if actin is directly involved and is
necessary for phagocytosis. The results shown in this report suggest
that normal filamentous arrangement of actin is not essential for phagocytosis.
Phagocytosis is usually accompanied by respiratory burst activity.
Recent evidence suggests that the respiratory burst oxidase is
associated with the cytoskeleton.38 Studies have shown that fibronectin has an adverse effect on superoxide production by neutrophils and macrophages compared with monocytes. Precoating microwells with fibronectin significantly suppressed neutrophil and
macrophage release of O2 in response to
fMLP while moderately enhancing O2
production by monocytes.39 Our study indicates that
jaspamide did not affect O2 production
by HL-60 cells or monocytes. Possible explanations for these
observations are either that the residual actin filaments that are not
clustered could participate in the oxidative burst process or, in
contrast, that the clustered actin can provide the functional support.
A remote possibility is that actin is not obligatory for the oxidative
burst and other cytoskeletal components can replace its function.
In summary, the present study shows that jaspamide has a potent
antiproliferative effect on HL-60 cells and that it induces phenotype
differentiation. This antiproliferative activity correlates with
reorganization of the actin cytoskeleton with no significant effect on
phagocytic activity of HL-60 cells or monocytes. Taken together, our
previous10 and current studies suggest that jaspamide may
be considered as a novel therapeutic agent for the treatment of acute
myeloid leukemia patients.
| |
ACKNOWLEDGMENT |
The authors thank Rina Socher for excellent technical assistance in
conducting the electron microscopy studies and Michal Firon for
reviewing the article.
| |
FOOTNOTES |
Submitted June 4, 1998; accepted January 22, 1999.
Supported in part by the Tel Aviv University Basic Research Fund and
the Israel Science Foundation founded by the Israel Academy of Sciences
& Humanities.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Ina Fabian, PhD, Department of Cell Biology
and Histology, Sackler School of Medicine, University of Tel Aviv,
Ramat Aviv, Tel Aviv 69978, Israel.
| |
REFERENCES |
1.
Ridley AJ:
Rho-related proteins: Actin cytoskeleton and cell cycle.
Curr Opin Genet Dev
5:24, 1995[Medline]
[Order article via Infotrieve]
2.
Wessells NK, Spooner BS, Ash JF, Bradley MO, Luduena MA, Taylor EL, Wrenn JT, Yamaa K:
Microfilaments in cellular and developmental processes.
Science
171:135, 1971[Abstract/Free Full Text]
3.
Pollard TD, Cooper JA:
Actin and actin-binding proteins. A critical evaluation of mechanisms and functions.
Annu Rev Biochem
55:987, 1986[Medline]
[Order article via Infotrieve]
4.
Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC:
Normal functional characteristics of cultured human promyelocytic leukemia cells (HL-60) after induction of differentiation by dimethylsulfoxide.
J Exp Med
149:969, 1979[Abstract/Free Full Text]
5.
Collins SJ:
The HL-60 promyelocytic leukemia cell line: Proliferation, differentiation, and cellular oncogene expression.
Blood
70:1233, 1987[Abstract/Free Full Text]
6.
Fabian I, Lass M, Kletter Y, Golde DW:
Differentiation and functional activity of human eosinophilic cells from an eosinophil HL-60 subline: Response to recombinant hematopoietic growth factors.
Blood
80:788, 1992[Abstract/Free Full Text]
7.
Leung MF, Lin TS, Sartorelli AC:
Changes in actin and actin-binding proteins during the differentiation of HL-60 leukemia cells.
Cancer Res
52:3063, 1992[Abstract/Free Full Text]
8.
Nagata K, Sagara J, Ichikawa Y:
Changes in contractile proteins during differentiation of myeloid leukemia cells. I. Polymerization of actin.
J Cell Biol
85:273, 1980[Abstract/Free Full Text]
9.
Meyer WH, Howard TH:
Changes in actin content during induced myeloid maturation of human promyelocytes.
Blood
62:308, 1983[Abstract/Free Full Text]
10.
Fabian I, Shur I, Bleiberg I, Rudi A, Kashman Y, Lishner M:
Growth modulation and differentiation of acute myeloid leukemia cells by jaspamide.
Exp Hematol
23:583, 1995[Medline]
[Order article via Infotrieve]
11.
Mor S, Nagler A, Barak V, Handzel ZT, Geller Bernstein C, Fabian I:
Histamine enhances granulocyte-macrophage colony-stimulating factor and interleukin-6 production by human peripheral blood mononuclear cells.
J Leukoc Biol
58:445, 1995[Abstract]
12.
Sham RL, Packman CH, Abboud CN, Lichtman MA:
Signal transduction and the regulation of actin conformation during myeloid maturation: Studies in HL60 cells.
Blood
77:363, 1991[Abstract/Free Full Text]
13.
Hallows KR, Law FY, Packman CH, Knauf PA:
Changes in cytoskeletal actin content, F-actin distribution, and surface morphology during HL-60 cell volume regulation.
J Cell Physiol
167:60, 1996[Medline]
[Order article via Infotrieve]
14.
Bubb MR, Senderowicz AM, Sausville EA, Duncan KL, Korn ED:
Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin.
J Biol Chem
269:14869, 1994[Abstract/Free Full Text]
15.
Senderowicz AMJ, Kaur G, Sainz E, Laing C, Inman WD, Rodriguez J, Crews P, Malspeis L, Grever MR, Sausville EA, Duncan KLK:
Jasplakinolide's inhibition of the growth of prostate carcinoma cells in vitro with disruption of the actin cytoskeleton.
J Natl Cancer Inst
87:46, 1995[Abstract/Free Full Text]
16.
Stingl J, Andersen RJ, Emerman JT:
In vitro screening of crude extracts and pure metabolites obtained from marine invertebrates for the treatment of breast cancer.
Cancer Chemother Pharmacol
30:401, 1992[Medline]
[Order article via Infotrieve]
17.
Joos L, Gicquaud C:
Effect of phalloidin and viroisin on Acanthamoeba castellanii after permeabilization of the cell.
Biochem Cell Biol
65:261, 1987[Medline]
[Order article via Infotrieve]
18.
Wehland J, Osborn M, Weber K:
Phalloidin associates with microfilaments after microinjection into tissue culture cells.
Eur J Cell Biol
21:188, 1980[Medline]
[Order article via Infotrieve]
19.
Piazzolla G, Tortorella C, Serrone M, Jirillo E, Antonaci S:
Modulation of cytoskeleton assembly capacity and oxidative response in aged neutrophils.
Immunopharmacol Immunotoxicol
20:251, 1998[Medline]
[Order article via Infotrieve]
20.
Quinn MT, Parkos CA, Jesaitis AJ:
The lateral organization of components of the membrane skeleton and superoxide generation in the plasma membrane of stimulated human neutrophils.
Biochim Biophys Acta
987:83, 1989[Medline]
[Order article via Infotrieve]
21.
Gallagher R, Collins S, Trujillo J, McCredie K, Ahearn M, Tsai S, Metzgar R, Aulakh G, Ting R, Ruscetti F, Gallo R:
Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia.
Blood
54:713, 1979[Abstract/Free Full Text]
22.
Meyer WH, Howard TH:
Actin polymerization and its relationship to locomotion and chemokinetic response in maturing human promyelocytic leukemia cells.
Blood
70:363, 1987[Abstract/Free Full Text]
23.
Oliveira CA, Kashman Y, Mantovani B:
Effects of latrunculin A on immunological phagocytosis and macrophage spreading-associated changes in the F-actin/G-actin content of the cells.
Chem Biol Interact
100:141, 1996[Medline]
[Order article via Infotrieve]
24.
Spector I, Shochet NR, Blasberger D, Kashman Y:
Latrunculins Novel marine macrolides that disrupt microfilament organization and affect cell growth: I. Comparison with cytochalasin D.
Cell Motil Cytoskeleton
13:127, 1989[Medline]
[Order article via Infotrieve]
25.
Duncan MD, Harmon JW, Duncan LK:
Actin disruption inhibits bombesin stimulation of focal adhesion kinase (pp125FAK) in prostate carcinoma.
J Surg Res
63:359, 1996[Medline]
[Order article via Infotrieve]
26.
Holzinger A, Meindl U:
Jasplakinolide, a novel actin targeting peptide, inhibits cell growth and induces actin filament polymerization in the green alga Micrasterias.
Cell Motil Cytoskeleton
38:365, 1997[Medline]
[Order article via Infotrieve]
27.
Hoffman Liebermann B, Sachs L:
Regulation of actin and other proteins in the differentiation of myeloid leukemic cells.
Cell
14:825, 1978[Medline]
[Order article via Infotrieve]
28.
Rao JY, Hurst RE, Bales WD, Jones PL, Bass RA, Archer LT, Bell PB, Hemstreet GPd:
Cellular F-actin levels as a marker for cellular transformation: Relationship to cell division and differentiation.
Cancer Res
50:2215, 1990[Abstract/Free Full Text]
29.
Sheth B, Banks P, Burton DR, Monk PN:
The regulation of actin polymerization in differentiating U937 cells correlates with increased membrane levels of the pertussis-toxin-sensitive G-protein Gi2.
Biochem J
275:809, 1991
30.
Cooper JA:
Effects of cytochalasin and phalloidin on actin.
J Cell Biol
105:1473, 1987[Free Full Text]
31.
Yahara I, Harada F, Sekita S, Yoshihira K, Natori S:
Correlation between effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro.
J Cell Biol
92:69, 1982[Abstract/Free Full Text]
32.
MacLean Fletcher S, Pollard TD:
Mechanism of action of cytochalasin B on actin.
Cell
20:329, 1980[Medline]
[Order article via Infotrieve]
33.
Sato N, Funayama N, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S:
A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites.
J Cell Sci
103:131, 1992[Abstract/Free Full Text]
34.
Ting Beall HP, Lee AS, Hochmuth RM:
Effect of cytochalasin D on the mechanical properties and morphology of passive human neutrophils.
Ann Biomed Eng
23:666, 1995[Medline]
[Order article via Infotrieve]
35.
Sheikh S, Nash GB:
Treatment of neutrophils with cytochalasins converts rolling to stationary adhesion on P-selectin.
J Cell Physiol
174:206, 1998[Medline]
[Order article via Infotrieve]
36.
Sheikh S, Gratzer WB, Pinder JC, Nash GB:
Actin polymerisation regulates integrin-mediated adhesion as well as rigidity of neutrophils.
Biochem Biophys Res Commun
238:910, 1997[Medline]
[Order article via Infotrieve]
37.
Moore PL, Bank HL, Brissie NT, Spicer SS:
Phagocytosis of bacteria by polymorphonuclear leukocytes. A freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure.
J Cell Biol
76:158, 1978[Abstract/Free Full Text]
38.
Woodman RC, Ruedi JM, Jesaitis AJ, Okamura N, Quinn MT, Smith RM, Curnutte JT, Babior BM:
Respiratory burst oxidase and three of four oxidase-related polypeptides are associated with the cytoskeleton of human neutrophils.
J Clin Invest
87:1345, 1991
39.
Yang KD, Augustine NH, Shaio MF, Bohnsack JF, Hill HR:
Effects of fibronectin on actin organization and respiratory burst activity in neutrophils, monocytes, and macrophages.
J Cell Physiol
158:347, 1994[Medline]
[Order article via Infotrieve]
40.
Yang KD, Augustine NH, Gonzalez LA, Bohnsack JF, Hill HR:
Effects of fibronectin on the interaction of polymorphonuclear leukocytes with unopsonized and antibody-opsonized bacteria.
J Infect Dis
158:823, 1988[Medline]
[Order article via Infotrieve]
41.
Yang KD, Bohnsack JF, Hawley MM, Augustine NH, Knape WA, Egan ML, Pritchard DG, Hill HR:
Effect of fibronectin on IgA-mediated uptake of type III group B streptococci by phagocytes.
J Infect Dis
161:236, 1990[Medline]
[Order article via Infotrieve]
42.
Wright SD, Silverstein SC:
Receptors for C3b and C3bi promote phagocytosis but not the release of toxic oxygen from human phagocytes.
J Exp Med
158:2016, 1983[Abstract/Free Full Text]
43.
Grassme HU, Ireland RM, van Putten JP:
Gonococcal opacity protein promotes bacterial entry-associated rearrangements of the epithelial cell actin cytoskeleton.
Infect Immun
64:1621, 1996[Abstract]
44.
Bodo M, Becchetti E, Baroni T, Mocci S, Merletti L, Giammarioli M, Calvitti M, Sbaraglia G:
Internalization of Candida albicans and cytoskeletal organization in macrophages and fibroblasts treated with concanavalin A.
Cell Mol Biol (Noisy-le-grand)
41:297, 1995[Medline]
[Order article via Infotrieve]
45.
Filler SG, Swerdloff JN, Hobbs C, Luckett PM:
Penetration and damage of endothelial cells by Candida albicans.
Infect Immun
63:976, 1995[Abstract]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
H. Hiruma, T. Katakura, S. Takahashi, T. Ichikawa, and T. Kawakami
Glutamate and Amyloid {beta}-Protein Rapidly Inhibit Fast Axonal Transport in Cultured Rat Hippocampal Neurons by Different Mechanisms
J. Neurosci.,
October 1, 2003;
23(26):
8967 - 8977.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Odaka, M. L. Sanders, and P. Crews
Jasplakinolide Induces Apoptosis in Various Transformed Cell Lines by a Caspase-3-Like Protease-Dependent Pathway
Clin. Vaccine Immunol.,
November 1, 2000;
7(6):
947 - 952.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION