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INTRODUCTION |
The natural polyamines putrescine, spermidine, and spermine are
essential for normal growth and cell proliferation (1-4). It has also
been demonstrated that polyamine synthesis is increased in many tumors
and tumor-derived cell lines (3). The polyamine pathway was therefore
thought to be an ideal target of chemotherapeutic intervention designed
to disrupt abnormal tumor cell growth (1, 5). However, the initial
attempts to exploit this target using inhibitors of the polyamine
synthetic enzymes ornithine decarboxylase and
S-adenosylmethionine decarboxylase produced disappointing results in vivo because of the compensatory cellular uptake
of polyamines to fulfill growth requirements (6, 7). To circumvent this
problem, structural polyamine analogues were designed using the
rationale that if the therapeutic agent closely resembled the essential
natural compound, then the cellular self-regulatory mechanisms
controlling uptake and synthesis might remain intact, but the
growth-affecting functions might be disrupted (5, 8, 9).
Some of the first polyamine analogues synthesized and tested as
antiproliferative agents were bis(ethyl) analogues of spermine, BE
3-4-3,1 BE 3-3-3, and BE
4-4-4-4 (5). Since that time, a wide variety of structural polyamine
analogues have been synthesized with modifications to the natural
polyamine structures that include small symmetrical terminal
substituents, large unsymmetrical terminal substituents, increased or
decreased internal carbon chain lengths, the introduction of sites of
unsaturation in the internal carbon chains, and even the linking
together of two or more of these altered structures (5, 10, 11).
Results from testing in cell systems also have been widely varied and,
unfortunately, few clear cut structure/function relationships have been
discernible. Despite that, several of the polyamine analogues have
shown therapeutic promise, and BE 3-3-3 is now in phase II clinical
trials with multiple tumor types (12). However, to actively design
better chemotherapeutic agents it is necessary to understand which
actions are essential to the cytotoxicity, and that is something
that has been difficult to state with certainty for the polyamine analogues.
Investigating the mechanisms by which cells become resistant to
drugs is one approach to determining the mechanism of action of the
drug. We have found that a point mutation in the SSAT gene led to a loss of sensitivity to BE 3-3-3 in the C55.7Res cell line,
which was selected for resistance to polyamine analogues (13). It had
been demonstrated that there is an apparent correlation between the
cytotoxic effects of some of the bis(ethyl)polyamine analogues and the
induction of SSAT activity (14, 15), but it had not been shown that
this was integral to the cytotoxicity of the analogue. We now report
the detailed characterization of the mutant L156F-SSAT protein both
in vitro and in C55.7Res cells. The results demonstrate that
this single amino acid change has ramifications for the enzyme activity
and binding with both natural polyamine substrates, spermidine and
spermine, and that the mutation renders the protein a less efficient
acetyltransferase than the wild-type SSAT protein. It is also now
demonstrated that the L156F change reduces interaction of the polyamine
analogue with the SSAT protein and that this is directly linked to the
resistance to the analogue that is exhibited by the C55.7Res cell line.
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EXPERIMENTAL PROCEDURES |
Materials--
[1-14C]acetyl-CoA (63 Ci/mol) was
obtained from ICN Biochemicals (Costa Mesa, CA). LipofectAMINE, Plus
Reagent, and geneticin were purchased from Invitrogen. BE 3-4-3 and BE 3-3-3 were kindly provided by Dr. Raymond Bergeron (University
of Florida, Gainesville, FL). Putrescine, MTT, IPTG, ATP, creatine
kinase, phosphocreatine, cycloheximide, and ubiquitin were purchased
from Sigma. Oligonucleotides were synthesized by Invitrogen or the
Pennsylvania State University College of Medicine Macromolecular Core
Facility (Hershey, PA). EasyTagTM
L-[35S]methionine was purchased from
PerkinElmer Life Sciences. Rabbit reticulocyte lysate prepared from
phenylhydrazine-treated New Zealand White rabbits was obtained from
Cocalico Biologicals (Reamstown, PA) for use in the degradation
studies. Ubal was purchased from Boston Biochem (Cambridge, MA). Talon
Superflow metal affinity resin was obtained from
Clontech. Restriction endonucleases were purchased
from New England Biolabs (Beverly, MA). MG132 was obtained from
Calbiochem. PD-10 desalting columns were purchased from Bio-Rad.
Cell Culture--
The CHO cell line C55.7 (16), a kind gift from
Dr. Immo Scheffler (University of California at San Diego, La Jolla,
CA), and its derivatives were maintained in minimum essential
-medium (Invitrogen) supplemented with 10% fetal bovine serum
(Atlanta Biological, Norcross, GA), 100 µM putrescine,
100 units/ml penicillin, and 100 units/ml streptomycin. Cultures were
incubated at 37 °C in a humidified 5% CO2 atmosphere
and passaged every 5-7 days to maintain exponential growth. For all
experiments, concentrated solutions of BE 3-3-3 and BE 3-4-3 (10 mM and 1 mM, respectively, in water, stored at
20°) were diluted with medium to the desired concentrations.
Transfection of pCMV-SSAT--
The transfection of
pCMV-L156F-SSAT into C55.7Res cells was accomplished using
LipofectAMINE and Plus reagent according to manufacturer's instructions.
Growth Inhibition Assay--
Exponentially growing cells were
plated in triplicate at 103 cells/cm2 in 100 µl of medium per well in 96-well plates. After a 12-18 h period for
the cells to attach, 200 µl of medium containing 1.5× the desired
final drug concentration was added. Cells were incubated in the absence
or presence of at least six drug concentrations for 144 h, at
which time the medium was aspirated, and 100 µl of 5 mg/ml MTT in
Optimem (Invitrogen) was added. The cells were incubated an additional
4-6 h at 37 °C, after which the medium was aspirated, and 100 µl
of 50% EtOH in Me2SO (v/v) was added to each well.
After 20 min, the A570 (a value directly
proportional to the number of viable cells (17)) was determined using a
Bio-Rad plate reader. IC50 values were determined from
plots of the percentage of A570 of untreated
control cells versus the logarithm of the drug
concentration. Each complete experiment was performed at least twice.
Analysis of SSAT Activity--
Exponentially growing cells were
plated in triplicate at 2-4× 104 cells/cm2.
Following attachment, the medium was changed, and cells were incubated
for the desired time. SSAT activity was determined in cell extracts by
an assay that measures the incorporation of radioactivity from
[1-14C]acetyl-CoA into
[1-14C]acetylspermidine in 10 min at 30 °C as
described previously (18). A standard assay mixture contained 50 mM Tris-HCl (pH 7.8), 3 mM spermidine, and 12.7 µM (63 mCi/mmol) [1-14C]acetyl-CoA in a
total volume of 100 µl. Using purified enzymes, the standard assay
was linear to at least 2 ng of wtSSAT and 20 ng of L156F-SSAT. For
assays to determine Km and
Vmax values, 0.5 ng of purified wtSSAT and 8 ng
of purified L156F-SSAT were used with nine different substrate
concentrations ([S]) ranging from 1 µM to 3 mM for spermidine and 0.1 µM to 1 mM for spermine. Km values were
determined from the linear regression of plots of [S]/SSAT activity
versus [S] (r2
0.993 for all regression
plots). Vmax values were determined from the
exponential curve fit of plots of SSAT activity versus [S]. Values of kcat were determined as
described by Fersht (19).
Analysis of Intracellular Polyamine Content--
Cells were
plated as described for SSAT activity determination and then harvested
and extracted with 10% (w/v) trichloroacetic acid. Aliquots were
assayed for polyamine content using ion-paired, reversed-phase high
performance liquid chromatography and post-derivatization with
o-phthalaldehyde as described previously (20).
Western Analysis--
Proteins present in cell extracts were
resolved by SDS-PAGE using a 15% gel. Electrotransfer to
polyvinylidene difluoride membrane (Micron Separations Inc.,
Waterborough, MA) was followed by hybridization with a polyclonal
anti-SSAT antibody (prepared as described previously (21)) and
detection using the Vistra Western blot detection kit (Amersham
Biosciences). An Amersham Biosciences FluorImager model 595 and
ImageQuant application software (Molecular Dynamics, Sunnyvale, CA)
were used for visualization and quantitation.
Construction of Plasmids--
The Chameleon double-stranded
site-directed mutagenesis kit (Stratagene) was used to change the
nucleotide sequence CTG coding for leucine 156 of human wtSSAT cDNA
in the plasmid pSAT9.3 (14) to TTC coding for phenylalanine. The
primers used were P-GGGTTGGAGATTCTTCAAGATCGAC (nucleotides
to achieve the mutation are shown in bold type) and the KpnI
primer included in the Chameleon kit. The resulting construct was
termed pSAT9.3L156F, and the SSAT coding region was sequenced to
confirm the presence of the correct codon for phenylalanine 156 and the
absence of any 2° mutations.
The plasmid pSAT9.3L156F was then used as a template for PCR to
introduce a BamHI restriction site 5' of the initiation
codon (5'-CAGGGGCCTGGATCCCAAAGGGAAG-3') (the
BamHI site is shown in bold type) and to change the existing
BamHI codon downstream of the stop codon to a unique
NheI site (5'-CTAGAACTAGTGGCTAGCCCGGGCTGC-3') (the NheI site is shown in bold type). The Expand high
fidelity PCR System (Roche Molecular Biochemicals) was used according
to manufacturer's instructions. The PCR product was digested with BamHI and NheI enzymes and then ligated into the
pCMV-Neo-Bam vector digested with the same enzymes, as described
previously (22). The SSAT coding region of the resulting plasmid,
termed pCMV-L156F-SSAT, was sequenced to confirm that no secondary
mutations were introduced during plasmid construction.
To construct a plasmid containing the cDNA coding for a His-tagged
SSAT-L156F mRNA, the BamHI/NheI fragment
described above was digested with SphI and
HindIII. The SphI/HindIII fragment was
then ligated into the same sites of the pQE30SSAT plasmid containing
the wtSSAT cDNA sequence with a His tag sequence present at the
amino-terminal end. The resulting plasmid, termed pQE30L156FSSAT, was
sequenced to confirm that the desired construct had been obtained.
The plasmid pSAT9.3L156F was also used as a template for PCR to make
L156F/E170stop-SSAT and L156F/MATEEAA-SSAT, which has two additional
alanine residues added to the carboxyl terminus. The antisense primer
used to change E170 to a stop codon (shown in bold type) was
5'-TCTACAGCAGGGATCCTCACTCTTATGTTGCCAT-3', and the one used
to introduce the two additional alanine residues (shown in bold type)
was 5'-GAATGGATCCCATCTACAGCAGTTATGCTGCCTCCTCTGTTGC-3'. The
sense primer used for both constructs was 5'-GGGAAGAAAAGCAAAAGACG-3'. The PCR products were digested with SphI and
BamH1 and ligated into the pSAT9.3L156F vector digested with
the same enzymes. The resulting plasmids were sequenced to confirm that
the desired sequence was present.
Expression and Degradation of SSAT and L156F-SSAT in
Vitro--
35S-labeled wtSSAT and L156F-SSAT proteins were
synthesized in vitro from the T7 promoters of the pSAT9.3
and PSAT9.3L156F plasmids, respectively, using the TNT
coupled transcription/translation system (Promega, Madison, WI).
The standard 200-µl assay for degradation studies contained 50 µl
of rabbit reticulocyte lysate, 40 mM Tris/HCl, pH 7.5, 2 mM dithiothreitol, 5 mM MgCl2, 0.5 mM ATP, 10 mM phosphocreatine, 0.05 mg/ml
creatine kinase, 0.1 mM cycloheximide, and
35S-labeled protein produced by the TNT
reaction used as substrate at 1-3% (v/v). Some of the degradation
assays also included one or more of the following; 0.1 mM
BE 3-3-3, 0.1 mM MG132, 5 µM Ubal, and 0.15 mM Ub. Assays were incubated at 37 °C, and 30-µl aliquots were removed at the desired times. Those aliquots were then
mixed with SDS sample buffer, boiled 5 min, and resolved on 15% (w/v)
polyacrylamide gels. The gels were fixed, dried, exposed to imaging
screens, and, following an appropriate period of time, the resulting
images were viewed and quantitated using an Amersham Biosciences
PhosphorImager SI and ImageQuant application software.
Protein Purification--
The His-tagged wtSSAT and L156F-SSAT
proteins were purified essentially as described previously (23) but
with the following modifications. Protein expression was induced in the
bacterial culture by the addition of IPTG to a final concentration of
300 µM, and, following harvest and cell lysis, the
soluble cell fraction was loaded onto a gravity flow column packed with
Talon Superflow metal affinity resin.
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RESULTS |
Activity of Purified L156F-SSAT--
Site-directed mutagenesis as
described under "Experimental Methods" was used to change the
nucleotide sequence of human SSAT to code for phenylalanine instead of
leucine at amino acid 156. Although this mutation was originally
identified in the Chinese hamster SSAT mRNA, we chose to study the
human SSAT because, between the human and Chinese Hamster sequences,
there is 96% homology in the 171 amino acids making up the SSAT
monomer, and this mutation occurs in a completely conserved region of
the protein. Additionally, many previous studies relevant to SSAT
activity and interactions with the analogues have been conducted using
the human SSAT protein. Purified His-tagged proteins were used to
compare the kinetic parameters of the mutant human L156F-SSAT protein
with that of the human wtSSAT with the two natural polyamine substrates
spermidine and spermine. L156F-SSAT Km values are
increased by 4.0-fold and 9.9-fold, respectively, for spermidine and
spermine as compared with the wild-type SSAT (Table
I), indicating that the affinity of the
mutant enzyme for both of the natural substrates is significantly reduced. The Km values reported here for wtSSAT are
in agreement with SSAT Km values in the literature,
which range from 55-140 µM for spermidine and 5-60
µM for spermine (1). The parameter
kcat, a measure of turnover of substrate to
product at the active site of the protein, indicates that significantly less product is produced by the L156F-SSAT than by the wild-type protein over any given time period by a given amount of protein (Table
I). An overall estimate of enzyme efficiency can be determined from the
kcat/Km value, which can be
considered a specificity constant. The
kcat/Km values indicate that
the efficiency of the mutant protein is significantly reduced, because
the values for L156F-SSAT are 56 and 78 times lower with spermidine and
spermine substrates, respectively, than those for the wtSSAT (Table
I).
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Table I
Kinetic parameters for purified L156F-SSAT and wtSSAT proteins
His-tagged L156F-SSAT and wtSSAT proteins were purified and assayed for
activity with spermidine or spermine as substrate as described under
"Experimental Procedures."
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Activity of L156F-SSAT in Cells--
To raise the activity of
L156F-SSAT in C55.7Res cells to levels that were significantly above
the limit of detection of the SSAT activity assay and could be studied,
pCMV-L156F-SSAT was transfected into the C55.7Res cells as described
under "Experimental Procedures." Only 6 of the 87 C55.7Res+L156F
clones selected following transfection exhibited SSAT activity of >100
pmol/min/mg protein, and the SSAT activity values of those clones
ranged from 106-186 pmol/min/mg protein. These values are considerably
lower than the 650-1900 pmol/min/mg protein that we obtained
previously from 13 of the 44 CHO clones transfected with pCMV-wtSSAT
(22). Western analysis indicated that the seven C55.7Res+L156F clones
exhibiting the highest SSAT activities expressed levels of SSAT protein
ranging from 0.6-2.4 times that of wtSSAT clone 43, which has basal
SSAT activity of ~1800 pmol/min/mg protein (data not shown). These results suggest that the lack of SSAT activity was not a result of
failure of the C55.7Res+L156F cells to express significant amounts of
the L156F-SSAT protein.
Analysis of Western blots of purified SSAT protein and cell extracts
was used to determine the amount of SSAT protein in the extracts of
cells expressing wtSSAT or L156F-SSAT. That value was then used to
calculate the SSAT activity per nanogram of SSAT protein (with
spermidine as the substrate) in the same cell extracts. Table
II indicates that the L156F-SSAT activity
in the cell extracts was >17 times lower than that of the wtSSAT
activity, which agreed well with the data obtained for the purified
proteins where L156F-SSAT activity was 14-fold less than that of
wtSSAT. The cellular activities for both L156F-SSAT and wtSSAT with
spermidine substrate also correlated well with the
Vmax values determined for the purified enzymes
(1.9 and 26.0 pmol/min/ng protein, respectively).
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Table II
SSAT protein amounts and specific activities in cells transfected with
L156F-SSAT or wtSSAT
The amount of SSAT protein in cell extracts was determined by Western
blot by comparison to a standard curve of purified SSAT protein. This
value was then used to calculate the specific SSAT activity per
nanogram of SSAT protein following an assay of SSAT activity in the
same cell extract using spermidine substrate.
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Effects of Expression of L156F-SSAT Activity in C55.7Res
Cells--
It was of interest to determine whether the increased
expression of L156F-SSAT that brought basal SSAT activity to a
measurable level similar to or greater than that of the
analogue-sensitive cells could restore cellular sensitivity to BE
3-3-3. The two C55.7Res+L156F clones that exhibited the highest basal
SSAT activity were used to test the sensitivity to BE 3-3-3 by
determining the IC50 values compared with C55.7 and
C55.7Res cells. The results, shown in Fig.
1, indicated that both C55.7Res+L156F
clones were nearly as resistant to BE 3-3-3 as was the C55.7Res cell
line. Therefore, increased expression of the L156F-SSAT protein that raised basal SSAT activity to levels comparable or slightly higher than
normal basal SSAT activity from wtSSAT protein was unable to restore
cellular sensitivity to the polyamine analogue.

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Fig. 1.
BE 3-3-3 sensitivity of C55.7Res cells
expressing L156F-SSAT. C55.7, C55.7Res cells, and cells of
C55.7Res clones transfected with pCMV-L156F-SSAT as described under
"Experimental Procedures" were incubated for 144 h in the
absence or presence of 6 BE 3-3-3 concentrations, and then MTT was
added to assay for the presence of viable cells as described under
"Experimental Procedures." Values shown are
A570 values of treated cells per the
A570 values of untreated control cells and are
mean ± S.D. of triplicate samples from a representative
experiment.
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The results of the BE 3-3-3 sensitivity experiments indicate that, even
though there was measurable basal SSAT activity in the
L156F-SSAT-transfected cells, in the presence of the polyamine analogue
the activity of the mutant SSAT enzyme did not reach levels that would
result in increased toxicity, suggesting that BE 3-3-3 is unable to
induce activity of L156F-SSAT. To test this hypothesis, SSAT
activity was measured in cells of C55.7Res+L156F-SSAT clone 1 following
48 h of exposure to several concentrations of BE 3-3-3 and
compared with that of C55.7 and C55.7Res cells. The results, shown in
Fig. 2, indicated that BE 3-3-3 concentrations of up to 100 µM failed to induce
L156F-SSAT more than 3.8 times, whereas 25 µM BE 3-3-3 resulted in ~300-fold induction of the wtSSAT of the C55.7 cells.

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Fig. 2.
Effect of BE 3-3-3 on SSAT activity of
C55.7Res cells expressing L156F-SSAT. C55.7, C55.7Res, and
C55.7Res+L156-FSSAT clone 1 cells were incubated for 48 h in the
absence or presence of BE 3-3-3; cells were harvested, cell extracts
made, and SSAT activity was measured as described under "Experimental
Procedures." The BE 3-3-3 concentrations used were 0-25
µM for C55.7 and C55.7Res cells and 0-100
µM for C55.7Res+L156F cells. Values shown are mean ± S.D. of triplicate samples from a representative experiment.
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BE 3-4-3 and CPENSpm are polyamine analogues that also have been
demonstrated to cause significant induction of SSAT, whereas CHENSpm
causes only minimal increases in SSAT activity. C55.7Res cells exhibit
cross-resistance to both BE 3-4-3 and CPENSpm but are as sensitive to
CHENSpm as C55.7 cells (data not shown). Because it is likely that the
leucine to phenylalanine amino acid change of the mutant SSAT protein
causes a conformational change in this protein as compared with the
wild-type SSAT, it is possible that individual analogues might exhibit
different responses with the mutant protein. Therefore, we compared
SSAT activity of cells of C55.7Res+L156F-SSAT clone 1 and wtSSAT clone
43 in the absence or presence of the four polyamine analogues (Fig.
3). It should be noted that the basal
SSAT activity of the wtSSAT clone 43 cells is elevated to ~1800
pmol/min/mg protein as compared with the ~15 pmol/min/mg protein in
the C55.7 cells, and, therefore, even though the induced SSAT activity
reached as high as ~35,000 pmol/min/mg protein, the fold induction
over untreated at
20 is less than the ~300-fold observed for the
C55.7 cell line as shown in Fig. 2. The results indicated that, with
the analogues that highly induced wtSSAT activity (13-20-fold
induction), L156F-SSAT activity was induced, at most, 4-fold with BE
3-3-3 and <1.5-fold for BE 3-4-3 and CPENSpm. Although CHENSpm
resulted in much less induction of the wtSSAT protein (at most 3-fold),
the induction of the L156F-SSAT was still even less (at most
1.4-fold).

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Fig. 3.
Effect of other polyamine analogues on SSAT
activity of C55.7Res cells expressing L156F-SSAT. wtSSAT clone 43 (labeled wtSSAT) and C55.7Res+L156-FSSAT clone 1 (labeled
L156FSSAT) cells were incubated for 48 h in the absence
or presence of BE 3-3-3, BE 3-4-3, CPENSpm, or CHENSpm. For wtSSAT
clone 43 the analogue concentrations used were 1-25 µM
for BE 3-3-3, BE 3-4-3, and CPENSpm, and 1-100 µM for
CHENSpm. For L156FSSAT the analogue concentrations used were 1-100
µM for BE 3-3-3, BE 3-4-3, and CPENSpm, and 25-100
µM for CHENSpm. Cells were then harvested, cell extracts
made, and SSAT activity was measured as described under "Experimental
Procedures." Values shown are the fold induction of the average SSAT
activity (n = 3) in the presence of analogue over the
average activity of the untreated cells (n = 3).
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Effects of BE 3-3-3 on the Degradation of L156F-SSAT in
Vitro--
Because the L156F mutation was suppressing induction of
SSAT activity by BE 3-3-3, it was logical to investigate the effect of
the analogue on degradation of the mutant protein. Shown in Fig.
4 are the time courses of degradation
in vitro of the two SSAT proteins in the absence or presence
of 1 mM BE 3-3-3. The wtSSAT protein is rapidly degraded
over the 90 min period in the absence of BE 3-3-3; however, when the
analogue is present there is almost complete stabilization and lack of
degradation over the same time period (Fig. 4A). This is
reflected in the half-life measurements of the proteins as calculated
from regression analysis of the plots of SSAT protein amounts
versus time under each condition. The half-life of the
untreated wtSSAT protein was 22 min, whereas it was greatly extended to
~144 h when the protein was exposed to BE 3-3-3. In contrast to this
behavior, the L156F-SSAT protein was rapidly degraded regardless of
whether BE 3-3-3 was present or not (Fig. 4B). The half-life
of the untreated mutant protein, at 15 min, was similar to that of the
wtSSAT; however, the L156F-SSAT half-life was not significantly
increased (18 min) when the protein was exposed to BE 3-3-3.

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Fig. 4.
Effect of BE 3-3-3 on degradation of
L156F-SSAT in vitro. 35S-labeled
wtSSAT (A), L156F-SSAT (B), L156F/E170stop-SSAT
(C), and L156F/MATEEAA-SSAT (D) proteins were
synthesized in vitro and then used as substrates in a rabbit
reticulocyte degradation system with aliquots removed for analysis at
the indicated times (in minutes) as described under "Experimental
Procedures." Following resolution on 15% polyacrylamide gels, the
gels were dried, imaged on a PhosphorImager SI, and quantitated with
ImageQuant software.
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The degradation in vitro of L156F/E170stop-SSAT and
L156F-SSAT with two additional alanine residues added to the carboxyl terminus (L156F/MATEEAA-SSAT) was also investigated. Both of these modifications to the carboxyl-terminal end of the L156F-SSAT resulted in stabilization of the protein even in the absence of polyamine analogue (Fig. 4, C and D). The half-life
measurements for the untreated proteins, at 105 min for
L156F/E170stop-SSAT and 154 min for L156F/MATEEAA-SSAT, were
significantly increased over that of the untreated wtSSAT at 22 min.
Exposure of the double mutants to BE 3-3-3 had no additional effect.
We also investigated the ability of BE 3-3-3 to block ubiquitination of
the L156F-SSAT protein. The rabbit reticulocyte degradation system used
for the studies described above was used again, but with the
modification that ubiquitin aldehyde and MG 132 were added to the
reticulocyte lysate to prevent breakdown of polyubiquitinated SSAT
protein by inhibiting polyubiquitin chain hydrolysis and proteasomal
degradation. This allowed resolution on the polyacrylamide gel and the
subsequent visualization of ubiquitinated species of the L156F-SSAT
protein present in aliquots from the degradation assay. It can be seen
in Fig. 5, that there was no difference between the pattern of ubiquitination obtained for the untreated L156F-SSAT protein and that observed following the addition of BE 3-3-3 to the assay system. These results indicated that the polyamine
analogue failed to prevent ubiquitination of the mutant SSAT protein
in vitro. This was in contrast to results reported by
Coleman and Pegg (24) for the wtSSAT protein, where 100 µM BE 3-3-3 prevented formation of
SSAT-Ubn conjugates and caused stabilization of
the wtSSAT protein.

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Fig. 5.
Effect of BE 3-3-3 on ubiquitination of the
L156F-SSAT protein in vitro.
35S-labeled L156F-SSAT protein synthesized in
vitro was used as substrate in the rabbit reticulocyte degradation
assay to which MG132, Ubal, and Ub had been added to prevent the
breakdown of polyubiquitinated SSAT protein by inhibiting polyubiquitin
chain hydrolysis and proteasomal degradation. Aliquots were removed
from the degradation assay at the indicated times (in minutes),
processed as described under "Experimental Procedures," resolved on
a 15% polyacrylamide gel, and visualized using a PhosphorImager
SI.
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Effects of BE 3-3-3 on the Degradation of Cellular L156F-SSAT
Activity--
To determine whether the effect of BE 3-3-3 on the
mutant SSAT protein was the same in the cell system as that observed
in vitro, the C55.7Res+L156F-SSAT clone 1 was used for
half-life measurements of both cellular L156F-SSAT protein and activity (Fig. 6). Regression analysis of the
plots of L156F-SSAT band volume from the Western blot versus
time was used to calculate the half-life of the cellular protein in the
absence or presence of BE 3-3-3 (Fig. 6A). The results
indicated that there was rapid degradation of cellular L156F-SSAT
protein despite the presence of the polyamine analogue and that the
protein half-life was not increased over that of the untreated control
(24.1 and 24.9 min half-life for untreated and BE 3-3-3-treated cells,
respectively). Measurements of SSAT activity of the same cell extracts
indicated a similar lack of BE 3-3-3 prolongation of enzyme activity,
as the half-life of L156F-SSAT activity was 17.1 min in untreated cells
and 18.1 min in those exposed to BE 3-3-3 (Fig. 6B). The activity of wtSSAT expressed in C55.7Res cells was, as expected, short-lived in the absence of BE 3-3-3 (21 min half-life) and stabilized by the polyamine analogue (248 min half-life) (Fig. 6B). These results corroborated the in vitro
degradation results (Fig. 4).

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Fig. 6.
Effects of BE 3-3-3 on half-life of cellular
SSAT activity and protein. Cells of C55.7Res+L156F-SSAT clone 1 were grown for 48 h, 200 µM cycloheximide was added
to stop protein synthesis, and cells were harvested at the times shown
for Western analysis (A) and assay of SSAT activity
(B) as described under "Experimental Procedures." Values
shown in panel A are from quantitation of a
Western blot of single samples from a representative experiment. SSAT
activity values shown in panel B are mean ± S.D.
(n = 3) of a representative experiment.
|
|
 |
DISCUSSION |
The first definitive evidence that induction of SSAT activity was
an integral part of the actions of BE 3-3-3 and similar polyamine
analogues was the selection of the analogue-resistant C55.7Res cell
line that exhibited decreased basal SSAT activity and a lack of SSAT
induction upon exposure to the analogue (13). Restoration of wtSSAT
activity in that cell line also restored sensitivity to BE 3-3-3 and
provided a solid link between the altered SSAT activity and the
resistance to the analogue. A point mutation was identified in the SSAT
mRNA of the analogue-resistant C55.7Res cells that results in an
amino acid change from leucine to phenylalanine at position 156 of the
SSAT protein. Although previous studies (25-28) had examined the
effects of mutations on activity and degradation of the SSAT protein,
all of that work was carried out in vitro with the SSAT
protein to which the mutation of interest had been introduced. The fact
that the L156F-SSAT has arisen within a cell system and replaced the
wtSSAT within those cells selected to be resistant to polyamine
analogues has provided us with a unique opportunity to address several
previously unanswerable questions.
At the time of selection of the C55.7Res cell line and identification
of the mutation in the SSAT mRNA, it was uncertain whether or not
the mutant mRNA could be translated to protein within the cells.
The transfection of human L156F-SSAT cDNA into the C55.7Res cells
and the subsequent detection of L156F-SSAT protein in those cells have
now confirmed that the mutant SSAT mRNA can be translated. Levels
of expression of the mutant SSAT protein that were attained were
similar to those previously achieved when wtSSAT cDNA was transfected into CHO cells, and a comparison of the basal activity of
the two proteins demonstrated that the activity of L156F-SSAT was
significantly lower than that of the wtSSAT.
The explanation of the cause of the decreased cellular SSAT activity of
the C55.7Res cells was provided by the determination of the kinetic
parameters of the purified L156F-SSAT as compared with those of the
wtSSAT. With both of the natural substrates, spermidine and spermine,
Km values were increased,
Vmax values were decreased, and rate of turnover
of substrate to product was significantly reduced for L156F-SSAT. These
data indicate that the protein interactions with the natural substrates
are altered and that this mutation has rendered the enzyme an inferior and less efficient acetyltransferase than the wtSSAT. This suggests that the single amino acid change in the SSAT sequence has significant effects on the protein conformation that decrease the ability of the
enzyme and substrate to come together to efficiently carry out the
reaction to form the acetylated polyamines. The observed increases in
Km values and decreases in
Vmax with spermidine and spermine substrates are
consistent with the effects reported by Coleman et al. (25,
26) of different mutations induced in the same region of the SSAT
sequence. The mutation of amino acid 152 from glutamic acid to either
glutamine or lysine or the mutation of amino acid 155 from arginine to
alanine caused a reduction in activity and an increase in
Km with spermidine substrate (25, 26). These
demonstrations that a single amino acid change can have profound
effects on the ability of SSAT to interact with its natural substrates
help to explain why the region of the SSAT protein around residue 156, where the leucine to phenylalanine mutation occurred, is fully
conserved across all of the known mammalian species where the SSAT
sequence has been deduced.
It is important to note that, although the basal SSAT activity of the
L156F-SSAT in the C55.7Res cells is 17 to 28-fold lower than that of
wtSSAT in the parental cells, there is no significant difference in the
growth of the untreated cells. As the rate-limiting enzyme of polyamine
catabolism, SSAT functions to help maintain the steady state level of
total cellular polyamines and prevent high levels of spermidine and
spermine that could be toxic to cells (1, 29). SSAT can rapidly respond
to physiological changes that modify the distribution and/or total
levels of cellular polyamines, and it can aid redistribution by the
conversion of spermine to spermidine and/or spermidine to putrescine as
well as decrease total cellular polyamines, because the acetylated forms of spermidine and spermine are more readily excreted from the
cell. Under normal physiological conditions, SSAT levels in cells are
very low, suggesting that little SSAT activity is required to maintain
polyamine homeostasis, perhaps due to the numerous regulatory
mechanisms that control ornithine decarboxylase and S-adenosylmethionine decarboxylase levels. It is therefore
consistent that the 17-28-fold reduction in SSAT activity in the
C55.7Res cells does not appear to have any effect on normal cellular
growth or functioning of the untreated cells. It is also known that
SSAT activity becomes more important in response to stimuli that alter normal physiological conditions. Other than the polyamines spermine and
spermidine themselves, other stimuli that have been demonstrated to
induce SSAT activity and thus invoke its role in maintaining polyamine
homeostasis include natural hormones and growth factors such as
corticosteroids, estradiol, and catecholamines, compounds known to
cause toxicity such as carbon tetrachloride and dialkylnitrosamines, and a variety of compounds used as anticancer drugs, such as
adriamycin, 5-fluorouracil and methotrexate, as well as the polyamine
analogues (1). Therefore it is also consistent that the effects of the L156F-SSAT mutation are evident under conditions of BE 3-3-3-treatment where SSAT would be an important response factor in attempting to
restore normal polyamine homeostasis and maintain cell health. It has
recently been suggested that there may be an additional cellular system
contributing to polyamine homeostasis through the back conversion of
spermine to spermidine (30), but more data will be needed to understand
the nature of and relative importance of such a system. However, it is
clear that the SSAT response is important to the action of BE 3-3-3 and
similar polyamine analogues, because the inability to reach high levels
of enzyme activity results in resistance to the analogue.
It was unclear in the original studies whether the basal SSAT activity
in the C55.7Res cells was just so low that the enzyme was induced by
the analogue, but the levels were not high enough to result in toxicity
even following induction, or whether the L156F-SSAT could not be
induced by BE 3-3-3. Because, as shown above, it is now clear
that the activity of the mutant enzyme is reduced with the natural
polyamine substrates, this is an important issue. The current studies
have made it certain that increasing basal SSAT activity to levels
normally observed within cells without restoring the capability to
induce high levels of SSAT activity in response to BE 3-3-3 is
insufficient to restore sensitivity to the analogue. Expression of
wtSSAT protein in the C55.7Res cells also restored the ability to
induce the SSAT activity upon analogue treatment. However, the C55.7Res
+L156F-SSAT clones that exhibited basal levels of SSAT at least
equivalent to that of parental C55.7 cells were still resistant to BE
3-3-3, because SSAT activity was not increased more than 4-fold upon
challenge with the analogue. The failure to induce the activity of the
mutant enzyme was not limited to BE 3-3-3, as BE 3-4-3 and CPENSpm, two related analogues known to highly induce SSAT activity, also produced only minimal increases in the activity of L156F-SSAT. Therefore, the
L156F mutation not only affects the interaction of the natural substrates with SSAT, but also decreases the ability of the polyamine analogues to bind to the SSAT protein and cause induction of the protein and activity.
The current studies have also demonstrated that the failure of BE 3-3-3 to induce L156F-SSAT activity results from the inability of the
analogue to protect the mutant protein from rapid degradation as it
does the wild-type SSAT (22). The short half-life of the SSAT protein
is one of the characteristics that allows it to function effectively in
the regulation of polyamine homeostasis, because the rapid adjustment
of cellular SSAT activity can be achieved through synthesis or
degradation of the enzyme. Coleman and Pegg (24) have recently
demonstrated that BE 3-3-3 protects the SSAT protein by interfering
with the ubiquitination necessary to target the protein for proteasomal
degradation. The current data supports the conclusion that the
conformational change brought about by the L156F mutation of the SSAT
protein prevents the interaction between the analogue and the protein
that is necessary to prevent the ubiquitination of SSAT. There were no
differences observed in the polyubiquitination patterns of the
L156F-SSAT in the absence or presence of BE 3-3-3 in vitro,
and these data, coupled with the failure of the analogue to increase
the cellular half-life of either the protein or activity, suggest that
it is this specific lack of ability to interfere with SSAT
ubiquitination that results in resistance to the polyamine analogue.
The stabilization of L156F-SSAT by mutation of the carboxyl terminus of
the protein is consistent with the results of Coleman and Pegg for
wtSSAT (24) and also supports the conclusions stated above. If the conformational change resulting from the L156F mutation merely caused
the protein to be so altered that it was recognized as a misfolded
protein and thus rapidly degraded, it is unlikely that the mutant
protein would respond as wtSSAT does to the additional mutation of the
carboxyl-terminal end. Therefore, it seems more likely that it is the
alteration of the specific interaction of the protein with the
polyamine analogue that is resulting in the failure to shield the
protein from ubiquitination. The fact that the toxicity of the analogue
is attenuated when the half-life is not lengthened and that the
activity thus does not increase to high levels indicates that this
ability to protect the SSAT protein from degradation is one of the
properties of the analogue that is essential for its toxic action.
Resistance to chemotherapeutic agents is one of the major limitations
encountered in their clinical use. Because BE 3-3-3 is now in phase II
clinical trials, it is not premature to consider whether resistance
such as was selected in the C55.7Res cells could occur with therapeutic
use of the polyamine analogue. A recent report on a Phase I clinical
trial using BE 3-3-3 reported the peak plasma levels achieved at the
MTD to be 17 µM BE 3-3-3 (12). Therefore, the 10 µM concentration of the polyamine analogue used to select
for the C55.7Res resistance to BE 3-3-3 is a clinically relevant
concentration as is the method of short exposure to the drug followed
by a period of recovery. The fact that the observed mechanism of
resistance to BE 3-3-3 is the result of a mutation that does not alter
the normal SSAT function in the absence of analogue drastically enough
to limit its occurrence suggests that the potential exists that other
relatively innocuous mutations may be present in the SSAT gene in
tumors that are targets of polyamine analogue use. The inherent genetic
instability of transformed tissues and tumors would suggest that this
is more than a hypothetical concern. As long as such mutations do not
reduce SSAT activity to critical levels that interfere with polyamine
homeostasis under normal physiological conditions, there would be no
selection pressure against their propagation, and the effects would
only become important upon exposure to the polyamine analogue, which
would be rendered less effective.