Department of Integrative Biology and Pharmacology, University of Texas
Medical School, Houston, Texas 77225, USA
* Present address: Department of Biology, San Diego State University, 5500
Campanile Drive, San Diego, CA 92182-4614, USA
Present address: Lexicon Genetics Inc., 4000 Research Forest Drive, The
Woodlands, TX 77381, USA
Author for correspondence (e-mail:
fcabral{at}uth.tmc.edu)
Accepted 8 June 2002
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Summary |
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Key words: Paclitaxel resistance, Tubulin synthesis, Autoregulation
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Introduction |
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Heterodimers of - and ß-tubulin are the building blocks for
microtubule assembly and, because essential cellular functions are mediated by
microtubules, it is anticipated that tubulin should be highly regulated. In
fact, production of tubulin appears to be controlled at several levels. The
- and ß- subunits are each encoded by a 6-7 member multigene
family in vertebrates, and the expression of those genes is regulated in a
tissue-specific manner (Sullivan,
1988
). For example, ßIII-tubulin production appears to be
largely restricted to brain and testes; but virtually all tissues contain the
more ubiquitous ßI-tubulin. Following transcription, tubulin mRNA
stability is affected by the state of microtubule assembly. When cells are
treated with colchicine, or other drugs that depolymerize microtubules,
degradation of tubulin mRNA increases and causes a corresponding decrease in
tubulin synthesis. In contrast, when cells are treated with paclitaxel, a drug
that enhances microtubule assembly, tubulin mRNA is stabilized and tubulin
synthesis increases (Ben-Ze'ev et al.,
1979
; Cleveland,
1989
). It has been proposed that this autoregulatory phenomenon is
sensitive to the level of free tubulin in the cell and acts to maintain a
steady supply of tubulin for microtubule assembly
(Theodorakis and Cleveland,
1992
).
Coordinate production of - and ß-tubulin subunits appears to be
regulated by a novel mechanism in which
-tubulin translation is
inhibited by free
-tubulin but not by
-tubulin that is complexed
with the ß-subunit (Gonzalez-Garay
and Cabral, 1996
). In this way,
-tubulin is synthesized
only when there are available ß-subunits to which it can bind. Finally,
tubulin levels can be controlled post-translationally through degradation.
Although tubulin heterodimers are very stable
(Spiegelman et al., 1977
),
excess free subunits (Gonzalez-Garay and
Cabral, 1995
) or defective tubulin proteins have a much shorter
half-life (Boggs and Cabral,
1987
; Kemphues et al.,
1982
).
Based on these observations, it is anticipated that tubulin mutations, or treatment of cells with antimitotic drugs, can elicit a complex response that may involve not only changes in microtubule assembly and dynamics, but changes in tubulin production as well. Despite its importance for understanding microtubule regulation, the relationship between tubulin assembly and synthesis is not fully understood. For example, it is not clear whether changes in microtubule assembly elicit a continuous or discontinuous change in tubulin synthesis; nor is it clear how large a change in microtubule assembly is needed to produce cytotoxicity. Answers to these questions are important not only for understanding the complex regulation of tubulin synthesis and assembly, but also for understanding mechanisms of action for the drugs that affect microtubule assembly.
Prior work from our laboratory demonstrated that a major mechanism by which
Chinese hamster ovary (CHO) cells in culture acquire resistance to paclitaxel
is through alterations in - and ß-tubulin that diminish the
capacity of microtubules to assemble
(Cabral, 2000
;
Cabral and Barlow, 1989
).
Despite the reduced microtubule assembly observed in paclitaxel-resistant
cells, little or no decrease in tubulin synthesis has been found in these
mutants, which suggests that the mechanism responsible for autoregulation of
tubulin synthesis through modulation of mRNA stability is not activated by
small changes in tubulin polymerization. We now report that a subset of
mutants that are not only paclitaxel-resistant, but also paclitaxel-dependent
for growth, have greatly reduced levels of polymerized tubulin and
significantly reduced tubulin synthesis. These studies establish the in vivo
limits of microtubule assembly that are consistent with normal microtubule
function and cell survival. The data further demonstrate that tubulin
synthesis is affected when microtubule assembly falls outside of these normal
limits.
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Materials and Methods |
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Measurement of drug resistance
A plating efficiency assay was used to measure the concentration of drug
required to inhibit the growth of each cell line. Approximately 100 cells were
seeded into replicate wells of a 24-well tissue culture dish containing
increasing drug concentrations. After 6 days, the medium was removed and the
cells were stained with 0.05% methylene blue as previously described
(Cabral et al., 1980).
Two-dimensional gel electrophoresis
The procedure used is described in detail elsewhere
(Cabral and Schatz, 1979).
Briefly, cells were metabolically labeled for 30 minutes with
[35S]methionine, lysed with SDS gel sample buffer
(Laemmli, 1970
), and proteins
were precipitated with five volumes of cold acetone as previously described
(Cabral and Gottesman, 1978
).
For isoelectric focusing, the protein pellet was resolubilized in urea sample
buffer (Cabral and Schatz,
1979
) and loaded onto cylindrical 4% polyacrylamide gels cast in
200 µl pipets. The isoelectric focusing gel contained 1% pH 5-7 and 0.2% pH
3-10 ampholytes (Crescent, Islandia, NY). A 7.5% polyacrylamide SDS gel was
used for the second dimension.
Tubulin polymerization
A previously described assay was used to measure the extent of tubulin
polymerization in various cell lines
(Minotti et al., 1991). The
procedure involves labeling cells to steady state (16-24 hours) with
[3H]methionine, lysing the cells in a microtubule stabilizing
buffer (20 mM Tris-HCl, pH 6.8, 0.14 M NaCl, 0.5% Nonidet P-40, 1 mM
MgCl2, 2 mM EGTA, 4 µg/ml paclitaxel), separating polymerized
from soluble tubulin by centrifugation at 12,000 g for 5
minutes, adding an [35S]methionine labeled wild-type CHO cell
extract to each fraction to act as an internal control, precipitating protein
from each fraction with cold acetone, separating proteins by 2D gel
electrophoresis, excising ß-tubulin from the gels, and calculating the
3H/35S ratio for tubulin from each fraction by liquid
scintillation counting. The percentage of total tubulin in the microtubule
fraction is then calculated as
{(3H/35S)p/[(3H/35S)s +
(3H/35S)p]} x 100%, where p is the pellet and s is
the supernatant.
Tubulin synthesis and accumulation
Measurements of tubulin synthesis and steady state accumulation have been
previously described (Gonzalez-Garay and
Cabral, 1995). For synthesis, cells were metabolically labeled
with [35S]methionine for 30 minutes, lysed in SDS sample buffer,
mixed with a [3H]methionine-labeled wild-type CHO cell extract,
acetone precipitated, resolubilized in urea sample buffer and resolved by 2D
gel electrophoresis. Relative levels of tubulin synthesis for each strain were
compared by measuring the 35S/3H ratio for
ß-tubulin excised from the gel and normalizing the value by dividing by a
similar ratio for actin from the same gel. For accumulation, a similar
procedure was followed except that cells were labeled to steady state (16-24
hours) with [3H]methionine, an [35S]methionine-labeled
wild-type CHO cell extract was added as an internal control, and
3H/35S ratios were used.
Isolation of RNA
Cells were grown in 100 mm dishes to about 80% confluence, the medium was
removed, and 2 ml of Ultraspec-II (Biotecx Laboratories, Houston, TX) was
added. The cells were scraped into this solution, transferred to a
17x100 mm polypropylene centrifuge tube, and 1/10 volume of chloroform
was added. The two phases were then mixed (Vortex Genie, setting 10),
incubated on ice for 10 minutes, and centrifuged at 12,000 g
for 15 minutes. The upper phase was collected, mixed with 0.5 volumes of
isopropanol and 0.05 volumes of RNAtrack resin, and centrifuged at 12,000
g for 2 minutes. The pellet was rinsed with 80% ethanol, air
dried and resolubilized in ribonuclease (RNase)-free water. Remaining glass
beads were then eliminated by centrifugation.
Ribonuclease protection assay
Labeled antisense riboprobes from CHO 1-tubulin and
ß1-tubulin were generated by linearizing the plasmids
pRC/HA
1 and pRC/HAß1 with the restriction enzymes CelII
and SapI respectively, and using them as templates for SP6 RNA
polymerase in the presence of
-[32P]UTP. For an internal
control, a DdeI linearized fragment of the gene encoding mouse
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Ambion, Austin, TX)
transcribed with SP6 RNA polymerase was used. The resulting riboprobes were
gel purified and used in the RNase protection analyses.
To measure relative levels of mRNA for 1-tubulin, ß1-tubulin,
and GAPDH, 10 mg of total RNA from wild-type or mutant CHO cell lines were
hybridized in solution (40 mM PIPES, pH 6.7, 0.4 M NaCl, 1 mM EDTA, and 80%
formamide) to either the
1-tubulin/GAPDH or ß1-tubulin/GAPDH
32P-labeled RNA probes described above and the resulting RNA-RNA
hybrids were digested in RNase digestion buffer (10 mM Tris-HCl, pH 7.5, 5 mM
EDTA and 300 mM NaCl) with RNase A and T1 for 2 hours at 37°C. The
digestion was stopped by the addition of SDS and proteinase K, and the
protected fragments were resolved on a 6% denaturing polyacrylamide gel. The
dried gel was exposed to X-ray film to obtain an image but was quantified
using a Storm phosphoimager (Molecular Dynamics, Sunnyvale, CA), or by cutting
out the bands and measuring radioactivity in a model LS2000 liquid
scintillation counter (Beckman Instruments, Fullerton, CA).
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Results |
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Some cell lines selected for resistance to drugs such as colcemid that
inhibit microtubule assembly have alterations in tubulin that increase
microtubule stability. For example, mutants CV 2-8
(Table 1) and Cmd 4
(Minotti et al., 1991) have an
increased fraction (
50%) of polymerized tubulin compared with wild-type
cells (
38%). By contrast, cells selected for resistance to paclitaxel, a
drug that promotes microtubule assembly
(Schiff and Horwitz, 1980
),
have alterations in tubulin that decrease microtubule stability. An example is
Tax 5-6 (Table 1), which has
only 28% of its tubulin assembled. All of these cell lines grow equally well
in the presence or absence of the selecting drug
(Fig. 1). Thus, CHO cells can
tolerate having as little as 28% and as much as 50% of their tubulin assembled
with no obvious effects on growth.
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|
To gain an insight into the limits for how little or how much tubulin
assembly is tolerated before cell growth is compromised, we examined mutants
that do not proliferate in the absence of the selecting drug. Tax-18 and Tax
2-4 are two previously described paclitaxel-dependent cell lines that are
unable to form a functional mitotic spindle, segregrate chromosomes, or
complete cell division unless paclitaxel is present in the growth medium, but
continue to synthesize protein and DNA at normal rates for at least 60 hours
following drug removal (Cabral,
1983; Schibler and Cabral,
1986
). As anticipated from the proposed mechanism of resistance,
drug-deprived paclitaxel-dependent mutants were found to contain significantly
less assembled tubulin (12-15%) than either wild-type cells (38%) or
paclitaxel-resistant, but not paclitaxel-dependent, mutants such as Tax 5-6
(28%, Table 1). Thus, 15%
assembly is clearly outside the range in which microtubules are able to
function, but 28% assembly is within the functional range.
In an attempt to identify the lowest level of polymerization consistent
with normal growth, we examined a cell line, Tax 9-5, that has partial
dependence on paclitaxel (Fig.
1). These cells can grow in the absence of drug, but grow better
when drug is present. Morphologically, the culture contains a mixture of
normal cells and large multinucleated cells that result from aberrant mitoses
when paclitaxel is absent (Fig.
2) (Cabral and Barlow,
1991). This mixed phenotype is seen in this and other similar
strains despite repeated subcloning, and suggests that a 21% extent of
microtubule assembly is near the transition from functional to non-functional
microtubules. Consistent with this interpretation, treatment of Tax 18 with
the minimum concentration of paclitaxel (50 ng/ml) that reverses the drug
dependence phenotype raised the level of polymerized tubulin to 22%. Moreover,
R3D, a revertant of Tax 18 that has lost paclitaxel dependence, but not
resistance (Fig. 3), has 27%
assembled tubulin.
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|
The upper limit for tubulin assembly consistent with cell survival has been
more difficult to obtain because colcemid-dependent cells have not been
directly selected. However, colcemid-dependent cells have been previously
created by transfecting DNA from colcemid-resistant Cmd 4 into wild-type CHO
cells (Whitfield et al.,
1986). One of the cell lines from this transfection, strain 11801,
was found to express elevated levels of altered ß-tubulin and to exhibit
poor growth unless colcemid was present in the medium. Analysis of this
partially dependent cell line indicated that 57% of the tubulin is assembled
(Table 1), suggesting an upper
limit to the normal range of tubulin assembly consistent with normal
microtubule function.
Together, these results indicate that microtubule function is maintained in
CHO cells when the extent of tubulin polymerization falls between
approximately 21% and 57% of total tubulin. Outside of these limits, cell
growth and survival are compromised. One possible caveat to this conclusion is
that the mutations could also be exerting secondary effects on the cells
because of changes in microtubule structure, or that mutant cells might
activate compensatory mechanisms to counteract the presence of the mutations.
To assess the effect of a tubulin reduction independently from the presence of
any significant amount of mutant tubulin subunits, we examined strain 6H2.
This cell line was derived by selecting revertants of Cmd 4, a
colcemid-resistant cell line with a mutation in one copy of its
ßI-tubulin gene (F.C., unpublished). Revertant 6H2 was found to contain a
further 12 amino acid deletion in the mutant protein (F.C., unpublished) that
prevented its polymerization into microtubules and dramatically increased its
degradation rate, a process we call `functional inactivation'
(Boggs and Cabral, 1987).
Because the two copies of the ßI-tubulin gene account for 70% of total
ß-tubulin production in CHO cells
(Sawada and Cabral, 1989
),
rapid degradation of altered tubulin from the mutant allele in 6H2 caused a
30% reduction in tubulin, a value close to the expected 35% decrease if no
gene product could accumulate at steady state. However, when analyzed for the
percentage of tubulin that is polymerized, 6H2 was found to be normal (38%,
Table 1), indicating that this
strain has 30% less microtubule polymer and 30% less soluble tubulin than
wild-type cells. Because this cell line grows normally, we conclude that a 30%
reduction in the level of tubulin in both the polymer and soluble pools is not
detrimental to the cells. This conclusion is consistent with the normal growth
of mutants such as Tax 5-6, which have also experienced a significant drop in
polymerized tubulin. It should be noted that attempts to functionally
inactivate a second ß-tubulin allele by genetic selection failed
repeatedly, suggesting that a 60-70% reduction in tubulin is too severe for
continued cell survival.
Paclitaxel-dependent CHO cells have reduced synthesis and
accumulation of tubulin
Autoregulation of tubulin synthesis has been proposed as a mechanism by
which cells sense the level of non-polymerized tubulin and adjust the rate of
synthesis to maintain a constant concentration of soluble tubulin for
microtubule assembly (Theodorakis and
Cleveland, 1992). The existence of mutant cell lines with altered
levels of tubulin in the polymer and soluble pools gave us the opportunity to
test whether small changes in those levels would affect tubulin synthesis or
accumulation. For these experiments, total steady state tubulin was quantified
by metabolically labeling cells overnight (1.5-2 generations) with
[3H]methionine, resolving tubulin from other cellular proteins on
2D gels, and measuring the radioactivity in ß-tubulin, actin and other
major proteins by liquid scintillation counting as previously described
(Gonzalez-Garay and Cabral,
1995
). A summary of the results for some representative cell lines
is presented in Table 2. Both
colcemid-resistant and paclitaxel-resistant mutants were found to have near
normal steady state tubulin levels; but cell lines with a
paclitaxel-dependence phenotype (Tax 2-4 and Tax 18) exhibited steady state
tubulin levels only about 60% as high as wild-type cells. As already
discussed, strain 6H2 exhibited a 31% reduction in steady state tubulin
consistent with the rapid degradation of an altered ß-tubulin subunit
that makes up 35% of the total tubulin
(Boggs and Cabral, 1987
).
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The same cell lines were used to measure rates of tubulin synthesis. For these experiments, cells were pulse-labeled with [35S]methionine for 30 minutes and ß-tubulin was quantified as before. The results summarized in Table 2 show that tubulin synthesis is reduced to an extent similar to that of its accumulation, indicating that the changes in steady state tubulin can be fully explained by reduced synthesis rather than enhanced degradation. The sole exception to this was strain 6H2, which had normal tubulin synthesis but reduced steady state accumulation because of enhanced degradation of mutant tubulin subunits.
Tubulin synthesis is restored by paclitaxel treatment
A number of investigators have shown that drug-induced disruption of
microtubule assembly affects tubulin synthesis by an autoregulatory mechanism
that alters the degradation kinetics of tubulin mRNA (for a review, see
Cleveland, 1989). The
decreased tubulin synthesis exhibited by paclitaxel-dependent cells could
potentially result from triggering this same mechanism, or it could result
from gene inactivation or other mechanisms that permanently reduce
transcription. To distinguish between these possibilities, we made use of the
well-known observation that paclitaxel enhances the rate of tubulin synthesis
by stabilizing (i.e. decreasing the degradation) of tubulin mRNA
(Cleveland, 1989
). We reasoned
that under conditions in which tubulin mRNA is maximally stabilized by
paclitaxel, cells that transcribe less tubulin because of inactivation of one
of their tubulin genes would continue to synthesize and accumulate less
tubulin than wild-type cells under similar conditions. However, cells that
have normal transcription but destabilized tubulin message should be capable
of a proportionally greater response to paclitaxel stabilization, which should
lead to levels of tubulin synthesis and accumulation that approach those of
wild-type cells.
To carry out this analysis, wild-type and mutant CHO cells were
metabolically labeled overnight with [3H]methionine in the presence
of various paclitaxel concentrations and the tubulin accumulated over that
period was quantified as already described. The results in
Fig. 4 show that wild-type CHO
cells exhibited a dose-dependent increase in tubulin accumulation to a maximum
of 160% of normal levels. This increase occurred at paclitaxel concentrations
that are minimally toxic (e.g. Fig.
1). Tax 5-6, a paclitaxel-resistant mutant with an altered
-tubulin, exhibited a similar increase in tubulin accumulation but the
dose response curve was shifted to the right as would be expected for
paclitaxel-resistant cells (Fig.
4B, inset). By contrast, Tax 2-4
(Schibler and Cabral, 1986
)
and Tax 18 (Cabral, 1983
),
paclitaxel-dependent mutants with altered ß-tubulins
(Gonzalez-Garay et al., 1999
),
started with less tubulin in the absence of drug (about 60% of the wild-type
level) in agreement with the data in Table
2, but increased to 240% of the initial value, reaching tubulin
levels comparable with those of wild-type cells at the higher paclitaxel
concentrations (Fig. 4A,B).
Similar results have been obtained with other paclitaxel-resistant and
paclitaxel-dependent mutants regardless of whether they carry alterations in
- or ß-tubulin (data not shown).
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The enhanced ability of paclitaxel-dependent cells to respond to the
tubulin-synthesis-promoting effects of paclitaxel, strongly argues that their
decreased content of tubulin is due to physiological modulation of tubulin
synthesis rather than to inactivation of a tubulin gene. Further evidence for
this interpretation came from examining the effects of paclitaxel on strain
6H2. As previously mentioned, this cell line has an alteration in one of the
ß-tubulin subunits that prevents assembly of that subunit into
microtubules and causes it to be rapidly degraded. As a result, total tubulin
synthesis is normal, but 6H2 has only 70% of wild-type tubulin levels at
steady state (Boggs and Cabral,
1987). An increased accumulation of tubulin in response to
paclitaxel treatment was also measured in this cell line
(Fig. 4); but, in contrast to
paclitaxel-dependent cell lines, the maximal increase in tubulin was only 160%
of normal and tubulin content never reached wild-type levels even at high
paclitaxel concentrations. These results are consistent with the view that
paclitaxel-dependent cells express a normal complement of tubulin genes, but
that cellular conditions favor more rapid degradation of the tubulin mRNA.
Paclitaxel-dependent cells have reduced levels of tubulin mRNA
To determine whether the decreased tubulin synthesis seen in
paclitaxel-dependent mutants resulted from lower steady state levels of
tubulin mRNA as would be predicted from the autoregulatory mechanism, total
RNA from wild-type CHO cells and two paclitaxel-dependent mutants (Tax 18 and
Tax 2-4) were analyzed for their ability to protect - and
ß-tubulin probes from RNase digestion. The
1-tubulin probe consisted of a 296 nucleotide antisense RNA
that produced a 218 bp fragment corresponding to nucleotides 1134-1351 of
1-tubulin, and the ß1-tubulin probe consisted of
a 250 nucleotide antisense RNA that produced a 203 bp fragment corresponding
to nucleotides 1128-1330 of ß1-tubulin. For each experiment a
150 nucleotide antisense RNA probe hybridizing to glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) mRNA was included as a control for quantifying the
relative amount of
- and ß-tubulin mRNA among different cell
lines. Following hybridization and RNase digestion, protected probes at the
expected sizes were observed and quantified. The results
(Fig. 5) indicated that both
paclitaxel-dependent cell lines contain lower steady state mRNA levels for
- and ß-tubulin compared with wild-type cells, thus accounting for
the lower synthesis of
- and ß-tubulin in paclitaxel-dependent
cell lines (Table 2) and
demonstrating that tubulin autoregulation was triggered in these cells.
|
Paclitaxel increases tubulin production and polymerization in a
dose-dependent manner
The ability of tubulin mutations to alter microtubule assembly and affect
tubulin synthesis led us to ask whether the effects could be replicated by
drug treatment. Wild-type CHO cells were treated overnight with varying doses
of paclitaxel and then analyzed for the extent of tubulin polymerization and
steady state tubulin levels as already described. The results in
Table 3 demonstrate that
increases in tubulin accumulation mirror increases in tubulin polymerization
induced by drug treatment and that those increases occur at the lowest
concentrations of paclitaxel that cause cytotoxicity
(Fig. 1A). However, unlike
tubulin mutations, paclitaxel treatment produces cytotoxicity and changes in
tubulin synthesis within the `normal range' of tubulin polymerization
established by mutant analysis.
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Discussion |
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Our own studies have used single step selections that give only low
resistance (2-4-fold) and have resulted in the isolation of a large series of
cell lines with a consistent phenotype
(Cabral et al., 1980;
Cabral, 1983
;
Schibler and Cabral, 1986
;
Schibler et al., 1989
).
Although we cannot rule out the possibility that multiple changes have
occurred in these cells, mutational frequencies, 2D gel analyses, genetic
reversion studies and direct transfection of mutant tubulin cDNAs all argue
that the indentified alterations in tubulin are sufficient to explain the
drug-resistance phenotype (Schibler and
Cabral, 1986
; Boggs and Cabral,
1987
; Gonzalez-Garay et al.,
1999
; Blade et al.,
1999
).
We previously proposed a model to explain the drug resistance mechanism in
these cells based on the concept that microtubules function normally within a
narrow range of microtubule assembly or stability
(Cabral et al., 1986). Tubulin
mutations that increase microtubule assembly or stability produce resistance
to drugs that inhibit assembly; whereas mutations that decrease tubulin
assembly or stability produce resistance to drugs that enhance assembly
(Fig. 6). Consistent with this
model, all paclitaxel-resistant cell lines we have examined have diminished
microtubule assembly, while all colcemid-resistant cell lines have increased
microtubule assembly (Minotti et al.,
1991
). We have now further demonstrated that paclitaxel-dependent
cells have even lower levels of microtubule assembly compared with cells that
are paclitaxel-resistant but not paclitaxel-dependent; and that a
colcemid-dependent cell line has higher levels of microtubule assembly than
cell lines that are resistant to the drug but not dependent. Thus,
drug-dependent cells contain tubulin mutations that exert greater effects on
microtubule assembly than the mutations present in resistant cell lines.
Indeed, mutations that confer drug-dependence affect microtubule assembly to
such an extent, that microtubule function is significantly disrupted and cells
are unable to survive unless the selecting drug is present to counteract the
effects of the mutation. By measuring relative amounts of microtubule polymer
in these mutant cells, we have, for the first time, quantified how severe
changes in microtubule assembly must be to produce a drug-dependent
phenotype.
|
The exact mechanism by which mutations in tubulin genes affect microtubule
assembly is not known. Many of the alterations in ß-tubulin that confer
paclitaxel resistance cluster in or near a loop that connects helices 6 and 7
of the protein (Gonzalez-Garay et al.,
1999). This loop is situated to potentially play a role in
longitudinal or lateral interactions between subunits forming the microtubule
lattice (Nogales et al.,
1999
). Other alterations occur in various regions of
- or
ß-tubulin subunits, but again could be causing structural changes that
perturb subunit interactions to strengthen (colcemid-resistance mutations) or
weaken (paclitaxel-resistance mutations) the microtubule lattice. Consistent
with this interpretation, immunofluorescence microscopy of
paclitaxel-dependent cells reveals a sparse microtubule network and defective
mitotic spindles (Cabral et al.,
1983
). Moreover, a recent study has demonstrated significantly
increased dynamic instability in a paclitaxel-dependent cell line
(Goncalves et al., 2001
).
Microtubule assembly and cell proliferation
The availability of drug-resistant and drug-dependent CHO tubulin mutants
has provided an opportunity to determine the tolerance limits for
perturbations in microtubule assembly in a mammalian cell line. Drug-resistant
cells have small changes in tubulin assembly that produce little or no effect
on cell growth and survival, whereas drug-dependent cells have larger changes
in assembly that produce clear defects in chromosome segregation and cell
division and ultimately cause cell death
(Cabral and Barlow, 1991).
Poised between drug resistance and drug dependence are cells with intermediate
alterations in microtubule assembly that produce partial drug dependence and a
significant decrease in cell survival. These latter cells, along with
paclitaxel-dependent cells rescued with a minimal concentration of the drug,
help to define the limits of how far microtubule assembly can be altered
before effects on cell division are encountered. Analysis of these various
cell lines suggests that microtubule function is maintained when tubulin
assembly is reduced or elevated by up to 45-50%, thereby defining a `normal
range' of microtubule assembly that is able to support cell proliferation.
Beyond those limits, microtubule function is compromised and cells fail to
survive.
Microtubule assembly and tubulin synthesis
These mutants have also allowed us to explore the relationship between
microtubule assembly and tubulin synthesis. It has long been known that
treating cells with drugs that depolymerize microtubules reduces tubulin
synthesis, but that treating with drugs that promote assembly increases
tubulin synthesis (Ben-Ze'ev et al.,
1979; Cleveland et al.,
1981
). These changes have been shown to arise from altered tubulin
message stability (Caron et al.,
1985b
; Pittenger and
Cleveland, 1985
). Although the mechanism by which changes in
microtubule assembly are transduced into alterations in tubulin message
stability remains unknown, it is believed that cells `sense' the level of
nonpolymerized tubulin and adjust message levels to maintain a constant pool
of the protein (reviewed by Cleveland,
1989
). Thus, it has been proposed that this autoregulatory process
acts as a fine-tuning mechanism to ensure appropriate levels of tubulin for
assembly (Theodorakis and Cleveland,
1992
).
Results from our mutant cell lines demonstrate that cells can tolerate considerable variation in free tubulin levels without evoking changes in tubulin synthesis. Table 4 summarizes the relative levels of polymerized and nonpolymerized (soluble) tubulin in wild-type and mutant CHO cells. In resistant cell populations, soluble tubulin concentrations can vary from 78% to 109% of normal without any accompanying change in tubulin synthesis. Moreover, tubulin synthesis is normal in strain 6H2 despite a 31% decrease in both soluble and polymerized tubulin. The only cell lines that experience a change in tubulin synthesis are Tax 18 and Tax 2-4, paclitaxel-dependent mutants that assemble too little microtubule polymer to survive when drug is absent. Therefore, mutant analysis argues that, contrary to the expectation for a fine-tuning mechanism designed to maintain a critical concentration of soluble tubulin, changes in tubulin synthesis do not occur despite significant alterations in the concentration of nonpolymerized tubulin. Instead, altered tubulin synthesis is seen only when microtubules become dysfunctional (as assayed by inability of cells to segregate chromosomes and divide).
|
Most studies using antimitotic drugs to modulate tubulin synthesis have
used very high drug concentrations that are clearly cytotoxic, but one study
(Caron et al., 1985a) used
lower concentrations of colcemid to show a correlation between microtubule
disassembly and decreased tubulin synthesis. We have extended these
observations to show that low concentrations of paclitaxel increase
microtubule assembly and increase tubulin synthesis in a parallel fashion
(Table 3). Although the authors
of the colcemid study concluded that tubulin autoregulation must be a
physiological response because it is triggered by small changes in tubulin
assembly, it is now recognized that antimitotic drugs are already cytotoxic at
concentrations that have minimal effects on microtubule assembly
(Jordan and Wilson, 1998
) and
we have directly shown that the lowest concentration of paclitaxel that
increases microtubule assembly and tubulin synthesis is cytotoxic (compare
Table 3 with
Fig. 1). Drug treatment differs
from mutant analysis in that it is able to elicit changes in tubulin synthesis
within the `normal range' of microtubule assembly
(Fig. 6). The reasons for this
difference are not yet clear, but we speculate that the binding of a large
organic molecule to tubulin may produce pleiotropic effects compared with a
more subtle amino acid substitution. Microtubule assembly and function are
determined by many factors including tubulin concentration, microtubule
dynamics, GTP hydrolysis, and participation of regulatory proteins. It is not
unreasonable to expect that drugs and mutations will have differential effects
on each of these factors. Drugs produce toxicity at concentrations that alter
microtubule assembly to a lesser extent than mutations, suggesting that they
may alter additional microtubule properties. In support of this notion, it has
been reported that drugs affect microtubule dynamics at very low
concentrations (Jordan and Wilson,
1998
). While drug treatment and tubulin mutations may differ
somewhat in the way they affect microtubule assembly, they both elicit changes
in tubulin synthesis only when they become cytotoxic.
The results are consistent with a mechanism in which loss of microtubule
function by drug treatment or by mutations produces a stress response that
alters the normal turnover of tubulin message. Perturbations that destabilize
microtubules also destabilize tubulin message, whereas perturbations that
stabilize microtubules stabilize tubulin message. This correlation suggests
the existence of a cellular factor that is able to monitor the state of
microtubule assembly, but the identity of the factor remains elusive. Early
studies suggested that free tubulin itself might be the trigger for changes in
mRNA stability (Ben-Ze'ev et al.,
1979). This interpretation was directly supported by an experiment
in which purified porcine brain tubulin was microinjected into cultured CHO
cells and decreased tubulin synthesis was subsequently measured
(Cleveland et al., 1983
).
Although the decreased synthesis was attributed to increased free tubulin, it
could also have resulted from altered microtubule assembly or some other
non-controlled factor. It is possible, for example, that the microinjected
tubulin contained modified subunits that assembled into, and adversely
affected the endogenous microtubule network. In support of this
interpretation, recent work in our laboratory indicates that expression of
brain-specific isoforms of ß-tubulin destabilizes the microtubule network
in CHO cells (M. Hari and F. Cabral, unpublished). Later studies from
Cleveland's laboratory showed no affinity of free tubulin for the
cis-regulatory sequence responsible for the autoregulation phenomenon, which
demonstrates that free tubulin alone is insufficient to trigger the response
(Theodorakis and Cleveland,
1992
). In fact, it is difficult to envision how small changes in
the pool of a highly abundant protein such as tubulin could trigger changes to
maintain the pool at a constant level. Moreover, we have demonstrated by
mutational analysis that a 30% change in the pool size of tubulin produces no
effect on cell growth or tubulin synthesis.
Although tubulin autoregulation is widespread among vertebrates and even
extends to non-vertebrate species, it's physiological function remains
unclear. Recently, for example, it was shown that pressure-induced hypertrophy
of cardiac tissue resulted in increased microtubule assembly and a persistent
upregulation of tubulin by a transcriptional mechanism; there was little or no
change in message stability in apparent contradiction to expectations if the
autoregulatory mechanism were operating
(Narishige et al., 1999).
However, the autoregulatory mechanism was shown to be intact and responsive to
colchicine treatment indicating that it simply was not activated during the
increased tubulin production accompanying hypertrophy. In simpler organisms,
Tetrahymena treated with drugs that either promote or destabilize microtubules
exhibit increased tubulin synthesis through a transcriptional mechanism
(Stargell et al., 1992
).
However, these cells are capable of modulating tubulin mRNA degradation as
demonstrated by the fact that tubulin mRNA is rapidly degraded following the
transcriptional upregulation of tubulin message that accompanies deciliation
of Tetrahymena (Seyfert et al.,
1987
). By contrast, changes in tubulin mRNA stability do appear to
be triggered during the normal course of embryogenesis in sea urchins
(Gong and Brandhorst, 1988
).
Also, it has been reported that tubulin mRNA is specifically degraded
following heat shock in Tetrahymena (Coias
et al., 1988
). This latter observation combined with the
observation that tubulin synthesis is altered in mammalian cells at the lowest
cytotoxic drug concentrations, or when the presence of mutant subunits alters
microtubule assembly to such an extent that they become non-functional,
suggests that autoregulation of tubulin synthesis is triggered by a stress
response that may intersect signaling pathways that are used during
development. How cells are able to sense microtubule dysfunction and activate
signals that affect microtubule message stability will be an interesting area
for further investigation.
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Acknowledgments |
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References |
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