Vol. 59, Issue 3, 462-469, March 2001
Dolastatin 11, a Marine Depsipeptide, Arrests Cells at
Cytokinesis and Induces Hyperpolymerization of Purified Actin
Ruoli
Bai,
Pascal
Verdier-Pinard,
Sanjeev
Gangwar,
Chad C.
Stessman,
Kelly J.
McClure,
Edward A.
Sausville,
George R.
Pettit,
Robert
B.
Bates, and
Ernest
Hamel
Screening Technologies Branch, Developmental Therapeutics Program,
Division of Cancer Treatment and Diagnosis, National Cancer Institute
at Frederick, National Institutes of Health, Frederick, Maryland (R.B.,
P.V.-P., E.H.); Department of Chemistry, University of Arizona, Tucson,
Arizona (S.G., C.C.S., K.J.M., R.B.B.); Developmental Therapeutics
Program, Division of Cancer Treatment and Diagnosis, National Cancer
Institute, National Institutes of Health, Rockville, Maryland (E.A.S.);
and Department of Chemistry and Biochemistry and Cancer Research
Institute, Arizona State University, Tempe, Arizona (G.R.P.)
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Abstract |
The successful synthesis of dolastatin 11, a depsipeptide originally
isolated from the mollusk Dolabella auricularia,
permitted us to study its effects on cells. The compound arrested cells at cytokinesis by causing a rapid and massive rearrangement of the
cellular actin filament network. In a dose-and time-dependent manner,
F-actin was rearranged into aggregates, and subsequently the cells
displayed dramatic cytoplasmic retraction. The effects of dolastatin 11 were most similar to those of the sponge-derived depsipeptide
jasplakinolide, but dolastatin 11 was about 3-fold more cytotoxic than
jasplakinolide in the cells studied. Like jasplakinolide, dolastatin 11 induced the hyperassembly of purified actin into filaments of
apparently normal morphology. Dolastatin 11 was qualitatively more
active than jasplakinolide and, in a quantitative assay we developed,
dolastatin 11 was twice as active as jasplakinolide and 4-fold more
active than phalloidin. However, in contrast to jasplakinolide and
phalloidin, dolastatin 11 did not inhibit the binding of a fluorescent
phalloidin derivative to actin polymer nor was it able to displace the
phalloidin derivative from polymer. Thus, despite its structural
similarity to other agents that induce actin assembly (all are peptides
or depsipeptides), dolastatin 11 may interact with actin polymers at a
distinct drug binding site.
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Introduction |
The
shell-less mollusk Dolabella auricularia has yielded a
number of cytotoxic peptides and depsipeptides [for a review, see Pettit (1997)
]. The most potent of these have been dolastatins 10 and
15 (Bai et al., 1990, 1992), both of which interact with tubulin and
arrest cells in mitosis. Although dolastatin 11 (Pettit et al., 1989)
(structure in Fig. 1) is not as potently
cytotoxic, we observed in flow cytometric studies an accumulation of
cells arrested at G2/M. However, dolastatin 11 neither arrested cells in mitosis nor reacted with tubulin or
microtubule protein. With some cell lines, treatment with dolastatin 11 caused a rapid shape change; ultimately, a large number of cells
became binucleated. Thus, the cells seemed to be arrested at
cytokinesis, suggesting actin or another component of the microfilament
network was the target of dolastatin 11 [similar findings with other
compounds described by Watabe et al. (1996) and Harrigan et al.
(1998)].
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Fig. 1.
Structural formulas of dolastatin 11 and related
structures, jasplakinolide, chondramide D, and phalloidin. The
stereochemistry shown for phalloidin is derived from Kessler and Wein
(1991).
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A detailed investigation of this possibility required an adequate
supply of dolastatin 11, and this was made possible by total synthesis
of the depsipeptide (Bates et al., 1997). We found that dolastatin 11 caused rapid, concentration-dependent disruption of the intracellular
microfilament network. These changes were similar to those observed
with the sponge-derived depsipeptide jasplakinolide (Senderowicz et
al., 1995; Spector et al., 1999; Bubb et al., 2000) and the
myxobacterium-derived depsipeptides known as the chondramides (Sasse et
al., 1998) (structures of jasplakinolide and chondramide D in Fig. 1).
When we evaluated the interaction of dolastatin 11 with purified actin,
we found that dolastatin 11, like jasplakinolide, the chondramides, and phalloidin (structure in Fig. 1) stimulated the assembly reaction. Furthermore, in a quantitative assay that we developed, dolastatin 11 was more potent than either jasplakinolide or phalloidin in stimulating
actin assembly. Despite its efficient induction of actin assembly,
however, dolastatin 11 differed from phalloidin, jasplakinolide, and
chondramides in being unable to inhibit the binding of a fluorescently
labeled phalloidin derivative to actin polymer.
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Experimental Procedures |
Materials.
Actin and pyrenyl-labeled actin from rabbit
muscle were obtained from Cytoskeleton (Denver, CO); jasplakinolide,
phalloidin, and Antifade Mounting Solution were obtained from Molecular
Probes (Eugene OR); latrunculin B was obtained from Calbiochem (San
Diego, CA); PtK1 cells (normal kidney cells of the kangaroo rat
Potorous tridactylis) were obtained from American Type
Culture Collection (Manassas, VA); DAPI, FITC-conjugated phalloidin,
and FITC-conjugated anti-
-actin monoclonal antibody were obtained
from Sigma (St. Louis MO); and the Chambered Coverglass System was
obtained from Nalge Nunc International (Naperville, IL). Natural and
synthetic dolastatin 11 were prepared as described previously (Pettit
et al., 1989; Bates et al., 1997). Majusculamide C was a generous gift
of Dr. R. E. Moore, University of Hawaii.
Methods.
PtK1 cells were maintained in culture as
recommended by the supplier. Drug effects on the growth of the cells
(increase in cell protein was the parameter measured) were evaluated as
described previously (Bai et al., 1993).
For immunofluorescence studies, PtK1 cells were grown to confluence,
disrupted by trypsinization, and seeded at about 10% confluence into
individual compartments of a Chambered Coverglass System. After 2 to 3 days of growth at 37° in a humidified 5% CO2
atmosphere, drugs, as indicated, were added [final dimethyl sulfoxide
concentration, 1% (v/v)], and cells were left for additional times at
37°, as indicated. Cells were washed twice with PBS, fixed with
methanol at 
20° for 10 min, and permeabilized with 20° acetone
for 1 min. The coverglass was washed twice with PBS; DAPI at 1.0 µg/ml and the FITC-conjugated anti--actin monoclonal antibody,
diluted 1:250 with PBS, were added in 150 µl to the coverglass, which
was left for 1 h at 22° in the dark. The coverglass was washed
twice with PBS, mounted on a slide with Antifade Mounting Solution, and
examined with a Nikon Model Eclipse E800 microscope equipped with
epifluorescence and appropriate filters. Images were captured with a
Spot digital camera, model 2.3.0, using version 3.0.2 software
(Diagnostic Instruments, Sterling Heights, MI). All images displayed
here were obtained with the 40× oil objective (N.A. 1.30).
Actin polymerization was evaluated by either a fluorometric or a
centrifugal assay. In the former method a fluorometer (Photon Technology International, Lawrenceville, NJ) was used, with FeliX for
Windows software. Before performing assembly assays, actin and
pyrenyl-labeled actin were diluted with AMB as a mixture to 12.5 and
1.0 µM, respectively, at 0°. In experiments in which assembly of
pyrenyl-labeled actin only was examined, the protein preparation was
diluted to 10 µM. After 1 h, the actin preparations were
centrifuged at 45,000 rpm at 4° in a Beckman Instruments (Palo Alto,
CA) Ti70 rotor. The supernatant was carefully removed and its protein
content determined by the Lowry assay.
When the actin/pyrenyl-labeled actin mixture was used, it was diluted
to 10 µM with AMB, and, for each assay, 100 µl of the final actin
solution was transferred to a fluorescence cuvette at 22°. Without
inducing salts, the fluorescence signal was referenced. Drug, if
present, was added at this point in 1 µl of dimethyl sulfoxide, and
fluorescence was monitored (excitation at 365 nm, emission at 407 nm).
With inducing salts, drug in 1 µl of dimethyl sulfoxide was added,
the fluorescence signal was referenced, 2 µl of PIB was added to the
cuvette, and fluorescence emission was followed.
When the pyrenyl-labeled actin alone was used, it was diluted to 0.6 µM with AMB. PIB (2 µl) was added to 100 µl of the
pyrenyl-labeled actin in the fluorescence cuvette at 22°. After about
4 min, drug in 5 µl of dimethyl sulfoxide was added. Fluorescence was
continuously monitored after addition of the polymerizing salts.
The centrifugal assay was developed (see below) as a means to obtain a
quantitative comparison of the effects of drugs inducing actin
assembly. The best data were obtained when the assembly inducing salts
were not added to the reaction mixture, and it also proved unnecessary
to perform the initial clarification centrifugation. In the presence of
inducing salts and the absence of drug, most of the actin (average,
76%) was pelleted under the centrifugal conditions used here. In
contrast, without inducing salts or drug, only 24% of the actin was
pelleted. For quantitative evaluation of drug effects, actin was
diluted to 25 µM with AMB, and the solution was left at 0° for
1 h. Various concentrations of drug in 5 µl of dimethyl
sulfoxide were added to 95 µl aliquots of the actin solution.
Reaction mixtures were incubated for 2 h at 22° and centrifuged
for 30 min at 45,000 rpm in a Beckman TA-45 rotor in a TL100
micro-ultracentrifuge. The protein content of the supernatant was
determined by the Lowry assay. The EC50 value for
a drug was defined as the concentration required to reduce the
supernatant actin concentration by 50% relative to control reaction
mixtures without drug.
The inhibition of the binding of FITC-phalloidin to actin polymer was
measured by removing polymer from the 100-µl reaction mixture by
centrifugation, followed by measurement of fluorescence at 517 nm
(excitation at 495 nm) of the supernatant. Several variations in order
of addition of reaction components were examined. 1) Actin was diluted
to 10 µM in AMB and incubated with or without potential inhibiting
drugs at various concentrations for 1 h at 22°. FITC-phalloidin
was added to 20 µM along with 2 µl of PIB (final dimethyl sulfoxide
concentration, 6%). After an additional 1 h at 22°, reaction
mixtures were centrifuged for 30 min at 40,000 rpm in a Beckman TA-45
rotor in a TL100 micro-ultracentrifuge. 2) Actin was incubated with or
without potential inhibitors at various concentrations in the presence
of 2 µl of PIB for 1 h at 22°. FITC-phalloidin (20 µM) was
added. After an additional 1 h at 22°, reaction mixtures were
centrifuged as in 1). 3) Actin was incubated with 20 µM
FITC-phalloidin and 2 µl of PIB for 1 h at 22°. Various
concentrations of the potential inhibitors were added to the reaction
mixtures. After an additional 1 h at 22°, reaction mixtures were
centrifuged as in 1).
For electron microscopy, 10-µl aliquots of reaction mixtures were
applied to carbon-coated, Formavar-treated, 200-mesh copper grids. The
sample droplet was immediately washed from the grid by 5 to 10 drops of
0.5% (w/v) uranyl acetate, and excess stain was wicked from the grid
with torn filter paper. The grids were examined in a Zeiss model 10CA
electron microscope. Reaction mixtures (50 µl) contained 10 µM
actin in AMB, drug at 10 µM as indicated, 1 µl of PIB if indicated,
and 5% dimethyl sulfoxide.
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Results |
Initial Studies.
Because of the precedents of dolastatins 10 and 15 (Bai et al., 1990, 1992), we initially assumed that dolastatin
11 would also inhibit mitosis through an interaction with tubulin.
However, despite the accumulation of cells at
G2/M by flow cytometric analysis, there was no
increase in the mitotic index, and the microtubules of cells treated
with the depsipeptide were intact. Moreover, the compound had no effect
on the polymerization of purified tubulin. When rat
C6 glial tumor cells were treated with dolastatin
11, within 15 to 30 min, virtually all cells in the culture underwent a
dramatic shape change, characterized by apparent extensive retraction of the cytoplasm. Many hours later, many of the cells became binucleate (data not shown). Similar observations with other drugs interacting with actin have been described in fibroblastic and smooth muscle cell
lines (Watabe et al., 1996; Harrigan et al., 1998). Finally, in a
variety of experiments comparing natural and synthetic dolastatin 11, we observed no significant difference in the effects of the two
preparations with either cells or purified actin. Therefore, unless
indicated otherwise, the studies presented here were performed with the
synthetic material.
Visualization of Intracellular Actin Filaments with a FITC-Labeled
Anti-Actin Antibody.
We evaluated the microfilament network of
PtK1 cells with both FITC-phalloidin and a FITC-conjugated antibody
directed against -actin. The staining patterns obtained were
identical, and only results with the antibody are presented here. We
wished to compare cells at defined equitoxic concentrations of drug,
and Table 1 summarizes
IC50 values for growth of PtK1 cells obtained
with dolastatin 11, jasplakinolide, and latrunculin B. We chose to study cells treated for varying time periods at these
IC50 values and at 10 times these concentrations
of the three drugs. Initial 24-h studies (data not shown) showed nearly
complete disruption of stress fibers after latrunculin B treatment,
confirming the findings of Spector et al. (1999). In contrast,
progressive and nearly indistinguishable aggregation of F-actin
occurred with dolastatin 11 and jasplakinolide treatment. In addition,
after 24 h with the latter two drugs, there was a much more
extensive retraction of the cytoplasm of
the PtK1 cells than occurred with latrunculin B.
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Fig. 2.
Comparison of the effects of dolastatin 11 (A-C) with
those of jasplakinolide (D-F) on PtK1 cells treated with the
IC50 concentrations of each drug for varying time periods.
Cells were grown for 30 min (A, D), 1 h (B, E), or 4 h (C, F)
in the presence of drug, as described in the text. F-actin stained with
the FITC-actin antibody is shown in green, nuclei stained with DAPI are
shown in blue.
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In Figs. 2 and 3, we present cells
treated at the lower and higher concentrations for shorter time periods
with dolastatin 11 and jasplakinolide. Cells treated with the
IC50 concentrations of the two drugs are shown in Fig. 2.
Perhaps because these concentrations are difficult to define
precisely, little difference was observed over time in the effects of
dolastatin 11 compared with those of jasplakinolide. Minor dissolution
of the microfilament network occurred with both drugs at 30 min, and
this became extensive, although incomplete, by 60 min. There was
perhaps somewhat greater clumping of F-actin structures with dolastatin
11 treatment (Fig. 2B) than with jasplakinolide treatment (Fig. 2E) at
60 min, but this was not striking when many fields of cells were
compared. By 4 h with both drugs, only F-actin clumps were visible
in the PtK1 cells. Cytoplasmic retraction was not prominent in cells treated at the IC50 concentrations of either
dolastatin 11 or jasplakinolide.
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Fig. 3.
Comparison of the effects of dolastatin 11 (panels
A-C) with those of jasplakinolide (panels D-F) on PtK1 cells treated
with 10 times the IC50 concentrations of each drug for
varying time periods. Cells were grown for 30 min (A, D), 1 h (B,
E), or 4 h (C, F) in the presence of drug, as described in the
text. F-actin stained with the FITC-actin antibody is shown in green,
nuclei stained with DAPI are shown in blue.
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In cells treated at 10 times the IC50
concentrations, there were noticeable differences between dolastatin 11 and jasplakinolide treatment as a function of time (Fig. 3). At 30 min,
there were few stress fibers remaining in dolastatin 11-treated cells
(Fig. 3A), but they were still relatively prominent in the
jasplakinolide-treated cells (Fig. 3D). By 60 min, no actin filaments
remained in the dolastatin 11-treated cells (Fig. 3B), whereas a few
persisted with jasplakinolide treatment (Fig. 3E). By 4 h,
however, cells treated with the two drugs were indistinguishable from
each other (compare Figure 3, C and F) and identical in appearance to
those treated for 24 h (not shown).
Effect of Dolastatin 11 on the Assembly of Purified Actin.
We
chose the coassembly of a tracer amount of pyrenyl-labeled actin with
unmodified actin as the system to study drug effects on actin
polymerization because of the readily measured enhancement of
fluorescence that occurs when the labeled actin is incorporated into
polymer (Cooper et al., 1983). Initially, we compared drugs at 10 µM
concentrations, with actin at the same concentration of 10 µM, using
a standard induction reaction condition (50 mM KCl/2 mM
MgCl2/1 mM ATP). At this actin concentration,
well above the critical concentration for the protein (Cooper et al.,
1983), we observed clear stimulatory effects with dolastatin 11 and
jasplakinolide and modest stimulation with phalloidin (Fig. 4A). In
contrast, latrunculin B completely inhibited actin assembly (data not
shown; compare Coué et al., 1987). It seems unlikely, however,
that the differences in fluorescence observed in the experiments of Fig. 4A are linearly related to extent of
assembly, because pellets formed without drug and with 10 µM
dolastatin 11 contained nearly identical amounts of actin. Moreover,
electron micrographs of similar reaction mixtures showed dense and
essentially identical networks of filaments, whether or not one of the
three drugs was included in the reaction mixture (see below). This
suggests the possibility that pyrenyl-labeled actin incorporated into
drug-containing polymers may fluoresce with different intensities
compared with control actin filaments.
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Fig. 4.
Drug effects on actin polymerization. A, with
inducing salts (50 mM KCl/2 mM MgCl2/1 mM ATP),
actin/pyrenyl-labeled actin at 10 µM, and drugs at 10 µM. Drug was
added at zero time. B, with inducing salts, pyrenyl-labeled actin at
0.6 µM, and drugs at 50 µM. The PIB solution was added at zero
time, and drug was added at the time indicated by the arrow. Reaction
mixtures contained drug, as follows: curve 1, none; curve 2, dolastatin
11; curve 3, jasplakinolide; curve 4, phalloidin. See text for
additional experimental details.
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We therefore examined drug effects on the assembly of pyrenyl-labeled
actin (without unlabeled actin in the reaction mixture), measured by
increase in fluorescence near the actin critical concentration (0.025 mg/ml, about 0.6 µM). As shown in Fig. 4B, there was little change in
fluorescence in the absence of drug, and only a small increase in
fluorescence upon addition of either 50 µM jasplakinolide or 50 µM
phalloidin. The increase in fluorescence was more dramatic with 50 µM
dolastatin 11. In additional experiments with a range of jasplakinolide
and dolastatin 11 concentrations, we observed progressive increase in
fluorescence as increasing amounts of drug were added in the 2 to 50 µM range, which seems to exclude hypernucleation as an explanation
for the relatively low fluorescence changes observed with low
concentrations of pyrenyl-labeled actin.
Because we could only examine one specimen at a time with the
fluorometer available to us, it seemed desirable to develop a
centrifugation assay to more readily compare effects of different drug
concentrations in enhancing actin assembly. We found that readily
measurable differences required eliminating K+
from the reaction mixtures. Moreover, the stimulatory effects of both
jasplakinolide (Bubb et al., 1994) and chondramides (Sasse et al.,
1998) were more readily demonstrated under "noninducing" reaction
conditions. Figure 5 compares the
fluorometrically followed reaction without drug to those with 10 µM
dolastatin 11, jasplakinolide, or phalloidin. There was no apparent
actin assembly in the absence of drug, and the relative activities of
the three stimulatory drugs became apparent (dolastatin 11 > jasplakinolide > phalloidin).
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Fig. 5.
Drug effects on actin polymerization in the absence
of inducing salts (residual ATP from AMB). Drug was added at zero time.
Reaction mixtures contained drug, as follows: curve 1, none; curve 2, 10 µM dolastatin 11; curve 3, 10 µM jasplakinolide; curve 4, 10 µM phalloidin. See text for additional experimental details.
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The differences between the compounds were even more readily apparent
when reaction mixtures were centrifuged. Figure
6 shows residual protein in the
supernatant after assembly with varying concentrations of these three
drugs. Average EC50 values obtained in at least
three independent experiments with each drug are shown in Table
2. The relative values obtained with
dolastatin 11 and jasplakinolide are consistent with their relative
cytotoxicity toward the PtK1 cells.
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Fig. 6.
Drug effects on actin polymerization without inducing
salts. Reaction mixtures contained 24 µM actin in AMB, 5% dimethyl
sulfoxide, and drugs, as indicated by the following symbols:  ,
dolastatin 11;  , jasplakinolide;  , phalloidin.
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TABLE 2
Relative drug effects on actin polymerization
The polymerization reaction was performed without polymerization
inducing salts, as described in the text.
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Dolastatin 11, Unlike Jasplakinolide, Does Not Inhibit the Binding
of FITC-Labeled Phalloidin to Polymer Formed from Purified Actin.
Jasplakinolide was previously shown to inhibit binding, in a manner
consistent with competitive inhibition, of fluorescently labeled
phalloidin to actin polymer (Bubb et al., 1994), and a comparable
result was obtained with chondramide A (Sasse et al., 1998). We
performed a similar study with dolastatin 11 in comparison with
jasplakinolide and phalloidin (Fig. 7),
anticipating that dolastatin 11, too, would inhibit binding of
FITC-phalloidin to actin fibers.
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Fig. 7.
Inhibition of binding of FITC-phalloidin to actin
polymer. Reaction mixtures contained drugs symbolized as follows: ,
dolastatin 11; , jasplakinolide; , phalloidin. Reaction
conditions were described in detail in the text. A, actin and potential
inhibiting drugs were preincubated before addition of FITC-phalloidin
and PIB. B, actin, PIB, and potential inhibiting drugs were
preincubated before addition of FITC-phalloidin. C, actin,
FITC-phalloidin, and PIB were preincubated before addition of the
potential inhibiting drugs.
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Quite unexpectedly, we could demonstrate no enhancement by dolastatin
11 of supernatant fluorescence after removal of actin polymer by
centrifugation. Many variations in experimental design were attempted,
both with and without polymerization-inducing salts, and three examples
with the salts are shown in Fig. 7. In the experiment shown in Fig. 7A,
actin and inhibitors were preincubated before induction of assembly in
the presence of FITC-phalloidin. In the experiment shown in Fig. 7B,
the preincubation of actin and potential inhibitors included the
inducing salts with subsequent addition of FITC-phalloidin. In the
experiment shown in Fig. 7C, FITC-phalloidin was preincubated with
actin and inducing salts before addition of potential inhibitors. Both
jasplakinolide and phalloidin had similar activity under all three
reaction conditions. In addition, the results shown in Fig. 7C
demonstrate that the binding of the FITC-phalloidin to actin filaments
is readily reversible.
Morphological Studies.
The inability of dolastatin 11 to
inhibit FITC-phalloidin binding to actin polymer led us to ask whether
the basic mechanism of action of dolastatin 11 differed from that of
the other peptides. For example, dolastatin 11 could induce formation
of a nonfilamentous polymer. The dolastatin 11-induced reaction would
thus be comparable with drug-induced aberrant polymerization reactions
that occur with tubulin [examples in Hamel et al. (1995) and Bai et
al. (1996)]. We therefore performed initial evaluations of polymer
morphology by negative stain electron microscopy of reaction mixtures
with 1.5 or 10 µM actin with 10 or 50 µM dolastatin 11, jasplakinolide, or phalloidin with the polymerization inducing salts,
and with 10 µM actin without the salts.
With the inducing salts at the higher actin concentration, the grids
were covered by a thick mat of actin filaments under all conditions,
including absence of drug. The number of filaments was much reduced at
the lower actin concentration. There were no significant morphological
differences in the filaments formed under any reaction condition, and
the width of the fibers observed was consistent with that of actin
filaments. Typical images are shown in Fig.
8, which presents low and high
magnification views of the polymer formed in the presence of 10 µM
dolastatin 11.
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Fig. 8.
Electron micrographs of actin polymer formed after
addition of PIB in the presence of 10 µM dolastatin 11. Incubation
was for 20 min at 22°. A, 24,000×. B, 185,000×.
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Without the inducing salts, the actin filaments were much sparser on
the grids, but they were identical in appearance to those observed with
the inducing salts. Figure 9 presents low
and high magnification views of the filaments formed with 10 µM
dolastatin 11.
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Fig. 9.
Electron micrographs of actin polymer formed in the
absence of polymerization-inducing salts in the presence of 10 µM
dolastatin 11. Incubation was for 20 min at 22°. A, 24,000×. B,
185,000×.
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Discussion |
Dolastatin 11 and related compounds represent the fourth
peptide/depsipeptide family that induces the assembly of actin in vitro, after phalloidin, jasplakinolide, and the chondramides. Dolastatin 11 is probably the most potent of these compounds. With the
exception of phalloidin, the anti-actin peptides are cytotoxic at
nanomolar concentrations and cause cells to arrest at cytokinesis. In
cells, however, the hyperassembly of actin is not associated with an
apparent increase in actin filaments, but with the rapid appearance of
clumped F-actin that stains both with FITC-phalloidin and with an
FITC-labeled antibody to -actin. Bubb et al. (2000) recently
proposed that this phenomenon is caused by formation of multiple actin
filament nuclei in jasplakinolide-treated cells, with fiber propagation
limited by the resulting shortage of monomeric actin in the cells. Such
a mechanism should also apply in the case of dolastatin 11, because we
find it to be more potent than jasplakinolide as an inducer of assembly
under the reaction conditions we have studied.
Harrigan et al. (1998) described dolastatin 12, lyngbyastatin 1, and
majusculamide C. In a number of cell lines, these three compounds all
had similar cytotoxic activity. Harrigan et al. (1998) also reported
disruption of the actin filament network in a smooth muscle cell line
treated with the dolastatin 12 and lyngbyastatin 1 preparations. The
apparent interpretation in this study was that these agents were
inhibitors of actin assembly. Although Harrigan et al. (1998) did not
characterize the in vitro interaction of dolastatin 12, lyngbyastatin
1, or majusculamide C with actin, the published image of the
lyngbyastatin 1-treated cells is similar to what we have described here
with dolastatin 11. We therefore conclude that this entire group of
compounds induces actin assembly in vitro (we have also directly
demonstrated assembly induction with majusculamide C; see Table 2).
Furthermore, dolastatin 11 has been 10 to 30 times more cytotoxic than
dolastatin 12 in several cell lines (Pettit et al., 1989; R. Bai and E. Hamel, unpublished observations), indicating it is the most
active member of the family yet isolated.
Dolastatin 11, despite its relative potency both against cells and as
an inducer of actin assembly, had negligible ability to inhibit the
binding of FITC-phalloidin to actin polymer. Dolastatin 11 was also
unable to displace prebound FITC-phalloidin from polymer. Such
inhibition or displacement was readily accomplished with jasplakinolide
and phalloidin, confirming published reports of similar experiments for
these compounds and for chondramide D (Bubb et al., 1994; Sasse et al.,
1998). This finding is highly suggestive that dolastatin 11 may bind at
a different site on actin polymer than the other peptides, but
definitive proof of this conclusion will require preparation of
radiolabeled dolastatin 11 or of a fluorescent active analog.
The possibility that dolastatin 11 and related compounds may bind to a
distinct site on actin polymer could be regarded as a positive feature
for development of this group of compounds as potential
chemotherapeutic agents. Although no anti-actin compound has a role in
cancer chemotherapy, jasplakinolide was under investigation at the
National Cancer Institute as a candidate for clinical development. Jasplakinolide, however, was found to have significant pulmonary toxicity in both rats and dogs (Schindler-Horvat et al., 1998), involving edema and hemorrhage; consequently, the compound is no longer
under active development. Whether pulmonary toxicity is universal for
anti-actin compounds, restricted to those that interfere with
phalloidin binding, or restricted to those that induce assembly is
unknown at the present time. Although a distinct actin binding site for
dolastatin 11 could be viewed as potentially avoiding the toxicity
observed with jasplakinolide, the study of Harrigan et al. (1998) is
not encouraging. They observed minimal antitumor activity with
dolastatin 12 or lyngbyastatin 1, but toxic effects of the compounds
included pulmonary hemorrhage.
Finally, it is perhaps worth pointing out a remarkable difference
between drugs inducing actin assembly and those inducing tubulin
assembly. Thus far only peptides and depsipeptides have had this
activity with actin. With tubulin, an increasingly diverse group of
molecules stabilize microtubules and induce hypernucleation of tubulin
assembly. The newest members of the class are steroid derivatives
(Mooberry et al., 2000; Verdier-Pinard et al., 2000).
| |
Acknowledgments |
We thank Drs. Kimberly Duncan and Adrian Senderowicz for
performing preliminary studies on the effects of natural dolastatin 11 on the cytoskeleton of cultured human tumor cells and J. F. Endlich for assistance with the electron microscopy.
| |
Footnotes |
Received August 17, 2000; Accepted November 20, 2000
Send reprint requests to: Dr. E. Hamel, P.O. Box B,
Building 469, Room 104, NCI-Frederick, Frederick, MD 21702. E-mail:
hamele{at}mail.nih.gov
| |
Abbreviations |
DAPI, 4',6-diamidino-2-phenylindole;
FITC, fluorescein isothiocyanate;
AMB, actin monomer buffer, containing 5 mM
Tris-HCl, pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, and 5 mM
dithiothreitol;
PIB, polymerization inducing buffer, containing 2.5 M
KCl, 100 mM MgCl2, and 50 mM ATP.
| |
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Copyright © 2001 by U.S. Government work not protected by U.S. copyright
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Abstract
Introduction
