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INTRODUCTION |
Spermidine/spermine N1-acetyltransferase
(SSAT)1 is the catabolic
enzyme primarily responsible for regulation of intracellular polyamine
concentrations in mammalian cells (1, 2). SSAT converts spermine and
spermidine to their N1-acetyl derivatives, which
can be excreted from the cell or further metabolized, by polyamine
oxidase, ultimately to putrescine. SSAT activity is highly regulated
and is induced in response to high intracellular levels of natural
polyamines (3, 4). Additionally, SSAT activity can be induced by a
number of other stimuli, including hormones, physiological stimuli,
drugs, and toxic agents (1, 2). Cellular levels of SSAT are usually
very low, and therefore, it has been difficult to study the regulation
of this enzyme under cellular conditions where it is not induced.
Studies of SSAT under conditions where the enzyme has been induced have
provided evidence of SSAT regulation at the levels of transcription (5,
6), mRNA stabilization (6, 7), mRNA translation (8), and protein degradation (8-11).
Structural polyamine analogues of the bis(ethyl)polyamine type, which
are currently of interest as cancer chemotherapeutic agents because of
antineoplastic effects against a variety of tumor types, are the most
potent inducers of SSAT activity identified to date (1, 12-14). It
should be noted that although these analogues are quite similar in
structure to the natural polyamines and can induce SSAT activity, they
are not themselves substrates of the acetyltransferase because of their
N terminal substituents. The bis(ethyl)polyamine analogues accumulate
intracellularly, induce SSAT activity, and deplete intracellular
polyamine pools. Correlations have been demonstrated between the
cytotoxicity of these analogues and the SSAT induction, which in some
cell types can reach greater than 1000-fold. However, it is not yet
clear whether SSAT induction is integral to analogue cytotoxicity. How
the various levels of SSAT regulation combine to produce the rapid and
high induction of enzyme activity, which results from exposure to these
analogues is not yet fully understood.
The current studies were therefore carried out to obtain a greater
understanding of the natural cellular regulation of the SSAT enzyme in
its noninduced state and to examine SSAT regulation during induction of
the enzyme by the polyamine analogue
N1,N12-bis(ethyl)spermine
(BE 3-4-3). We now report the establishment of a CHO cell line stably
transfected with human SSAT and which expresses levels of SSAT
activity which have allowed investigation of cellular properties and
regulation of the enzyme in its noninduced state. Induction of SSAT by
BE 3-4-3 was used to provide a more detailed understanding of SSAT
regulation by contrasting the properties of SSAT in the presence
of the analogue to those in the noninduced state.
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EXPERIMENTAL PROCEDURES |
Materials--
[1-14C]acetyl-CoA (50 or 63 Ci/mol)
was obtained from ICN Biochemicals (Costa Mesa, CA). The proteasome
inhibitor MG 132 was purchased from Calbiochem. Cycloheximide was
obtained from Sigma. LipofectAMINE and Geneticin were purchased from
Life Technologies, Inc. BE 3-4-3 was kindly provided by Dr. Raymond
Bergeron (University of Florida, Gainesville, FL).
Cell Culture--
CHO cells were maintained in minimum essential
-medium (Life Technologies, Inc.) supplemented with 10% fetal
bovine serum (Atlanta Biological, Norcross, GA), 100 units/ml
penicillin, and 100 units/ml streptomycin (CHO medium). Cultures were
incubated at 37 °C in a humidified 5% CO2
atmosphere and were passaged every 5-7 days to maintain exponential
growth. To assess cell growth over time, cells were plated at 2 × 10 3 cells/cm2 in 24-well plates in 2 ml of
medium and incubated at 37 °C in a humidified 5% CO2
atmosphere. Cells from triplicate wells were harvested at
appropriate times and counted using a model ZF Coulter Counter (Coulter Electronics, Hialeah, FL).
Assay of Colony Forming Ability--
To assess the colony
forming ability of the transfected cell lines, single cell suspensions
of each cell line were plated in triplicate in 60-mm culture dishes, at
densities of 25, 50, 100, and 150 cells/dish, in 6 ml of CHO medium
supplemented with 0.5 mg/ml Geneticin (selective medium). Cells were
grown for 5 or 10 days and then stained with crystal violet (5% (w/v)
in 95% ethanol) to visualize colonies. Colonies of
50 cells were counted.
Construction of pCMV-SSAT--
The plasmid pSAT9.3 containing
the SSAT cDNA in the Bluescript vector (15) was used as a template
for polymerase chain reaction to introduce a BamHI
restriction site upstream of the initiation codon and to change the
existing BamHI site downstream of the stop codon to a unique
NheI site. The resulting product contained the entire SSAT
coding region flanked by 31 and 123 base pairs of 5' and 3'-noncoding
sequences, respectively. The BamHI/NheI fragment
was sequenced to confirm the absence of polymerase chain reaction-induced mutations and ligated into the same sites of a
pCMV-Neo-Bam vector generously provided by Dr. B. Vogelstein (The Johns
Hopkins University, Baltimore MD) in which the multiple cloning site
had been expanded to include unique AflII and
NheI sites. The resulting plasmid was termed pCMV-SSAT.
Transfection of pCMV-SSAT into CHO Cells--
Exponentially
growing CHO cells were plated at a density of 1 × 104
cells/cm2 and 12-18 h allowed for adherence to the culture
plate. For transfection, cells were incubated with LipofectAMINE (20 µg/plate) and 2 µg of plasmid DNA for 5 h at 37 °C in a
humidified 5% CO2 atmosphere. After 5 h, the medium
was aspirated, fresh medium (minimum essential
-medium supplemented
with 10% fetal bovine serum) was added, and cultures were incubated
for an additional 43 h. Cells were then replated at low density in
selective medium and incubated for 12-16 days, at which time
individual colonies were selected and transferred to individual wells
of a 24-well plate.
Analysis of SSAT Activity and Determination of SSAT
Half-life--
Exponentially growing cells were plated in triplicate
at 5-6 × 104 cells/cm2. Following
attachment, the medium was changed, and the cells were incubated for
48 h, unless otherwise indicated. SSAT activity was determined in
cellular extracts by an assay which measures the amount of
incorporation of radioactivity from [1-14C]acetyl-CoA
into [1-14C]acetylspermidine in 10 min at 30 °C as
described previously (16). A standard assay mixture contained 50 mM Tris-HCl (pH 7.8), 3 mM spermidine, and 16 µM (50 mCi/mmol) or 12.7 µM (63 mCi/mmol)
[1-14C]acetyl-CoA in a total volume of 100 µl. For
assessment of SSAT half-life, at the end of the 48-h incubation period,
200 µM cycloheximide was added in the absence or presence
of any other agent. Cells were harvested at the time of cycloheximide
addition and at no fewer than four subsequent times, and SSAT activity
was measured. SSAT activity half-life was determined from an
exponential curve fit applied to plots of SSAT activity
versus time. Values reported were derived from several
individual experiments, each of which contained triplicate cultures at
each time point and are the mean ± S.D. where n = the number of individual samples assayed. To adjust for variation
between complete experiments, all values were normalized to the mean
SSAT activity at time 0.
Analysis of Intracellular Polyamine Content--
Cells were
plated as described for SSAT activity determination, harvested, and
extracted with 10% (w/v) trichloroacetic acid. Aliquots were then
assayed for polyamine content using an ion-pair reversed phase high
performance liquid chromatography separation method and
postderivatization with o-phthalaldehyde as described previously (17).
Western Blot Analysis--
Proteins present in cellular 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 (18)) and
detection using the Vistra Western blot detection kit (Amersham
Pharmacia Biotech). Visualization and quantitation were accomplished
using a Molecular Dynamics Fluorimager model 595 and ImageQuant
application software.
Expression and Degradation of SSAT in Vitro--
The
35S-labeled wtSSAT protein was synthesized in the
TNT-coupled transcription/translation system (Promega, Madison, WI)
from the T7 promoter of the plasmid pSAT9.3 containing the SSAT
cDNA in the Bluescript vector (15) as described previously (19). To
follow the rate of SSAT degradation, 30-µl aliquots were removed from
the crude reticulocyte lysate assay at the desired times, mixed with
SDS sample buffer, boiled for 10 min, and resolved by SDS-PAGE as
described previously.
Northern Analysis--
Total cellular RNA was isolated using
Qiashredder columns and the RNeasy RNA purification kit from Qiagen
(Chatsworth, CA). Northern analysis was carried out using Hybond
N+ membrane according to the manufacturer's instructions
(Amersham Pharmacia Biotech). Ethidium bromide staining was used as a
loading control. A fluorescein-labeled full-length SSAT probe, to which membranes were hybridized, was prepared by transcription from the T3
promoter of the pSAT9.3 plasmid containing the SSAT cDNA in the
Bluescript vector (15). Signal detection utilized the Vistra signal
amplification kit (Amersham Pharmacia Biotech), and visualization and
quantitation were the same as for Western blot analysis.
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RESULTS |
Expression of wtSSAT in CHO Cells--
In order to establish a
cell line that would express increased levels of SSAT activity, CHO
cells were stably transfected with pCMV-SSAT, a plasmid coding for
human wtSSAT under control of the CMV promoter. Forty-four clones were
selected and screened for SSAT activity levels in the absence and
presence of BE 3-4-3, which was used to determine whether the SSAT
activity of the clone was inducible by a polyamine analogue. Of the 44 clones selected, 13 exhibited significantly greater basal SSAT activity
(650-1900 pmol/min/mg protein) than control cells transfected with
empty vector (24 pmol/min/mg protein) (Fig.
1). SSAT activity of the same 13 clones
was also induced, after 48 h in the presence of 0.5 or 1 µM BE 3-4-3, to levels significantly higher than those induced in control cells under the same conditions (Fig. 1).

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Fig. 1.
SSAT activity of CHO clones stably
transfected with human wtSSAT. CHO cells were stably transfected
with pCMV SSAT and assayed for SSAT activity as described under
"Experimental Procedures." Shown is the SSAT activity following
48-h incubation in the absence of drug (black) or in the
presence of 0.5 µM BE 3-4-3 (light gray) or 1 µM BE 3-4-3 (dark gray). V
indicates control cells transfected with empty vector. Values represent
averages of triplicate determinations.
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The clones were numbered sequentially at the time that each was
transferred from its initial culture well when confluence had been
reached. Therefore, the numbering reflects the relative initial growth
rates of the individual clones with clone 1 the fastest and clone 44 the slowest. Twelve of the 13 clones that expressed increased basal
levels of SSAT were among the clones numbered 22-44, suggesting that
the increased SSAT levels may have had an effect on the initial growth
of these clones and slowed the establishment of thriving colonies. The
wtSSAT clone 43, which exhibited increased basal SSAT expression both
in the absence and presence of the inducing agent, was selected for
further studies and is the clonal cell line referred to unless
otherwise stated.
Growth Characteristics of the wtSSAT Clone--
Since one
objective of these studies was to determine whether increased SSAT
activity has an effect on cell growth and viability, the colony forming
ability and population growth characteristics of the wtSSAT clone were
compared with those of control cells containing empty vector.
Assessment of the ability to form colonies from single cells revealed
that the size of the colonies resulting from growth of the wtSSAT
clone, either 5 days (Fig. 2) or 10 days
following plating, was considerably smaller than that of colonies
formed from control cells. Visual inspection of cell size and cell
number per colony indicated that this difference in colony size was a
result of cell number per colony and cannot be attributed to
differences in the sizes of individual cells between the wtSSAT clone
and the control. This indicates a difference in the rate of growth
between the two cell lines with growth of the wtSSAT cells slower than
the vector control under these clonal conditions. The percent of
colonies formed, however, was quite similar for the wtSSAT clone
(46 ± 3 at day 5 and 51 ± 8 at day 10) and the vector
control (50 ± 8 at day 5 and 55 ± 10 at day 10).

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Fig. 2.
Colony forming ability of control and wtSSAT
cells. To assess colony forming ability, cells were plated in
single cell suspensions, incubated for 5 days, and stained to visualize
colonies as described under "Experimental Procedures." Shown is a
photograph of representative plates of colonies resulting from growth
of control cells transfected with empty vector (left) and
wtSSAT cells (right).
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Despite the slower rate of growth of the wtSSAT cells observed in the
colony forming experiments described above, there was no detrimental
effect of SSAT expression on the growth of exponentially growing
cultures of the wtSSAT cells. Populations of untreated wtSSAT clone
cells and vector control cells grew similarly, with doubling times of
19.2 ± 1.6 and 17.5 ± 0.6 h, respectively and a
plateau density for each of 4-5 × 105
cell/cm2.
The presence of higher levels of SSAT also had little effect on cell
growth in the presence of 1 µM BE 3-4-3, with cessation of exponential growth between 48 and 72 h and a final density 8-10 times the original cell number for the vector control and 14-17
times the original cell number for the wtSSAT clone. IC50 values determined following 120-h incubation with BE 3-4-3 were 2 × 10
8 M and 9 × 10
9
M for the wtSSAT clone and vector control, respectively,
indicating a slight decrease in the sensitivity of the wtSSAT clone to
this polyamine analogue.
Two other wtSSAT clones (clones 29 and 41) that had increased levels of
basal SSAT activity similar to clone 43 were also tested for colony
forming ability, population growth, and sensitivity to BE 3-4-3. Results were similar to those reported for wtSSAT clone 43 (data not
shown). These results suggest that an increase in basal SSAT activity
from 24 to ~1600 pmol/min/mg protein may have an effect on initial
cell growth rates from a single cell but does not adversely affect the
growth of the exponentially growing cell populations. In fact, the
results suggest that the increased SSAT activity may even render these
cells marginally less sensitive to the growth inhibitory effects of the
polyamine analogue BE 3-4-3.
Effects of Increased SSAT Expression on Polyamine-related Enzyme
Activities and Intracellular Polyamine Levels--
To assess the
effects of the increased basal SSAT levels on polyamine homeostasis in
the wtSSAT clone, intracellular polyamine pools, SSAT activity, and
activity of ornithine decarboxylase (ODC) and
S-adenosylmethionine decarboxylase (AdoMetDC), two of the
polyamine synthetic enzymes, were measured over time (Table I). The SSAT activity of the wtSSAT clone
was initially higher than control and rose throughout the 48-h period
of the measurements, while that of the control cells remained
comparatively low at all time points. ODC activity increased in both
the control and wtSSAT clone over time, but was higher in the wtSSAT
clone at all times. AdoMetDC activity was initially higher in the
wtSSAT clone and increased to a greater extent over time than that of the control. Distinct polyamine pool perturbations were observed at all
time points in wtSSAT cells, where putrescine levels were highly
increased, spermidine levels were moderately decreased, and
N1-acetylspermidine and
N8-acetylspermidine were present in measurable
quantities.
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Table I
Polyamine pools and SSAT, ODC, and AdoMetDC activities in wtSSAT and
vector control cells
Intracellular polyamine levels, and the activity of SSAT, ODC, and
AdoMetDC, were determined as described under "Experimental
Procedures," using cell extracts of exponentially growing control or
wtSSAT clone cells harvested at 0, 24, and 48 h. Values are
averages ± S.D. of measurements from triplicate cultures of a
representative experiment.
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SSAT Half-life and Degradation--
The level of SSAT activity
measured in the wtSSAT clone under normal growth conditions was
sufficiently high to allow detailed investigation of the degradation of
the SSAT protein and its intracellular half-life. Cells were incubated
for 48 h, treated with cycloheximide to prevent new protein
synthesis, and then harvested for SSAT activity measurements and
Western blot analysis to quantitate the amount of SSAT protein. From
the assay results (Fig. 3), the SSAT
activity half-life was determined to be 29 min and the SSAT protein
half-life was 26 min. These data indicate that, in the noninduced
state, the SSAT protein is short-lived and that decreases in protein
are responsible for and directly proportional to reductions in enzyme
activity.

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Fig. 3.
SSAT activity and SSAT protein levels in the
absence of new protein synthesis. Cells of the wtSSAT clone were
grown for 48 h, 200 µM cycloheximide was added to
stop protein synthesis, and cells were harvested at the times shown for
assay of SSAT activity (A) and Western analysis
(B) as described under "Experimental Procedures." In
A, the circles represent SSAT activity, and the
squares represent relative amounts of SSAT protein as
determined from the Western analysis. SSAT activity values represent
mean ± S.D. (n = 15). For the Western analysis,
150 µg of protein were loaded in each lane.
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Preliminary indications that SSAT may be degraded via the
ubiquitin/proteasomal pathway have been published based on in
vitro studies of the breakdown of SSAT protein synthesized in a
coupled transcription/translation system in reticulocyte lysates (19). We have now found that a specific inhibitor of the 26 S proteasome, MG
132 (20), has a profound effect on SSAT degradation in this system
(Fig. 4). In the absence of the
proteasomal inhibitor, SSAT was rapidly degraded and higher molecular
weight bands of labeled protein, consistent with ubiquitination of
SSAT, appeared at 15 min and remained present through 1 h. In the
presence of 50 µM MG 132, the rate of degradation of SSAT
was reduced and the SSAT-ubiquitin bands were visible throughout
the 3-h period of the assay (Fig. 4).

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Fig. 4.
Effects of the proteasomal inhibitor MG 132 on the degradation of SSAT in vitro. The
35S-labeled wtSSAT protein was synthesized in the
TNT-coupled transcription/translation system and used as a substrate
for degradation as described under "Experimental Procedures."
Protein was incubated in the degradation assay at 37 °C in the
absence or presence of 50 µM MG 132. Aliquots of 30 µl
were removed at the times indicated and analyzed by SDS-PAGE.
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To confirm that intracellular degradation of SSAT occurs through the
proteasome complex as suggested by the in vitro data, two
types of experiments were carried out using the transfected CHO cells.
First, SSAT activity and SSAT protein amounts were assessed in the
presence of 25 µM MG 132 added concurrently with cycloheximide so that the effect of the proteasomal inhibitor on the
intracellular half-life of SSAT could be determined (Fig. 5). In the presence of MG 132, the SSAT
activity and protein amounts were directly proportional (Fig. 5), as
they were in the absence of the inhibitor (Fig. 3). The SSAT activity
and SSAT protein half-lives were each extended significantly from 29 min to >300 min in the presence of the proteasomal inhibitor.

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Fig. 5.
Intracellular degradation of SSAT in the
presence of the proteasomal inhibitor MG 132. Cells of the wtSSAT
clone were grown for 48 h, 200 µM cycloheximide and
25 µM MG 132 were added concurrently, and cells were
harvested at the times shown for assay of SSAT activity (A)
and Western analysis (B) as described under "Experimental
Procedures." In A, the circles represent SSAT
activity, and the squares represent relative amounts of SSAT
protein as determined from the Western analysis. SSAT activity values
are mean ± S.D. (n = 3). For the Western
analysis, 75 µg of protein were loaded in each lane.
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Second, the cellular SSAT activity was assessed at the beginning and
end of a 4 h incubation in the absence or presence of 10, 25, and
50 µM MG 132. With no drug present, SSAT activity increased only slightly from 1338 ± 140 to 1587 ± 140 pmol/min/mg protein after 4 h. In contrast, SSAT activity was
increased significantly to 4805 ± 383, 4668 ± 113, and
4182 ± 430 pmol/min/mg protein, following 4-h exposure to 10, 25, or 50 µM MG 132, respectively. These results correlate
with the reduced protein degradation in the presence of MG 132 observed
with the SSAT half-life determination. Together, these data indicate
that cellular SSAT degradation is occurring through proteasomal action
with loss of protein and enzyme activity occurring simultaneously.
Effects of BE 3-4-3 on SSAT Expression and Activity--
Having
established SSAT parameters under noninducing conditions, it was
possible to investigate changes in SSAT regulation during induction by
the polyamine analogue BE 3-4-3, one of the most potent SSAT-inducing
agents known. SSAT activity and protein expression were assessed in
crude extracts of the wtSSAT clone and vector control following 48-h
incubation with BE 3-4-3 concentrations ranging from 0 to 25 µM. Cells of the wtSSAT clone exhibited basal SSAT
activity of ~700 pmol/min/mg protein, a 2-fold increase in SSAT
activity was noted at 0.1 µM BE 3-4-3, the lowest
concentration tested, and there was a steep
concentration-dependent increase of activity to a maximum
of ~60,000 pmol/min/mg protein reached after exposure to 5 µM BE 3-4-3 (Fig.
6A). Western blot analysis indicated a concentration-dependent increase of SSAT
protein, which correlated directly with the observed SSAT activity
increase (Fig. 6B). Although the SSAT activity was much
lower in the control CHO cells (basal SSAT activity of ~25
pmol/min/mg protein), similar effects of BE 3-4-3 were observed. There
were SSAT activity increases of 1.5-, 2-, and 7-fold following exposure
to 0.1, 0.25, and 0.5 µM BE 3-4-3, respectively. The SSAT
activity increase was much greater after exposure to 1 µM
BE 3-4-3, and the maximum activity of ~10,000 pmol/min/mg protein was
reached at 2 µM BE 3-4-3 (Fig. 6A). The SSAT
protein of the control was detectable only in extracts from cells
exposed to
1 µM BE 3-4-3 where a
concentration-dependent increase of SSAT protein directly
correlating with the SSAT activity increase was observed (Fig.
6B). These data indicate that the effects of BE 3-4-3 on
SSAT regulation are similar for the control and the wtSSAT cells.

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Fig. 6.
Effect of BE 3-4-3 concentration on SSAT
activity and SSAT protein levels. Cells were incubated in the
absence or presence of BE 3-4-3 for 48 h and then harvested for
assay of SSAT activity and Western analysis as described under
"Experimental Procedures." A shows SSAT activity of the
vector control (circles) and the wtSSAT clone
(squares), and B shows the Western blot of the
SSAT protein of each cell line. SSAT activity values are mean ± S.D. (n 6). SSAT activity values of the wtSSAT clone
represent total SSAT activity at each concentration minus the SSAT
activity of the vector control at the same concentration. For the
Western analysis, 125 and 25 µg of protein were loaded in each lane
for the vector control and the wtSSAT clone, respectively.
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Effect of BE 3-4-3 on SSAT Half-life and mRNA Levels--
The
rapid and large increase of SSAT protein and activity in the presence
of BE 3-4-3 may result from the effects of the analogue at one or more
levels of SSAT regulation. To investigate whether this polyamine
analogue was exerting an effect on the longevity of the SSAT protein,
the half-life of SSAT in the presence of BE 3-4-3 was determined using
two different protocols. First, cells of the wtSSAT clone were
incubated in the presence of 0.5 µM BE 3-4-3 for 48 h, at which time cycloheximide was added and cells were harvested at
several time points for SSAT activity measurements and half-life
determination. Second, cells were incubated in drug-free conditions for
48 h, at which time 0.5 µM BE 3-4-3 was added
concurrently with cycloheximide, and cells were harvested at several
times for determination of SSAT activity and half-life. The presence of
BE 3-4-3 for 48 h prior to the addition of cycloheximide resulted
in significant lengthening of the SSAT half-life to >200 min as
compared with 29 min in the absence of the polyamine analogue (Fig.
7). Concurrent addition of the analogue
with the inhibitor of new protein synthesis also resulted in
significant lengthening of the SSAT half-life from 29 to 85 min (Fig.
7). These data indicate that interaction between BE 3-4-3 and existing
protein prevents degradation of the enzyme and prolongs its period of
activity.

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Fig. 7.
Effect of BE 3-4-3 on SSAT degradation.
Cells of the wtSSAT clone were grown for 48 h, 200 µM cycloheximide was added, and cells were harvested at
the times shown for assay of SSAT activity as described under
"Experimental Procedures." SSAT activity is shown for cells
incubated in the absence of drug for 48 h followed by addition of
cycloheximide alone (circles), or cycloheximide plus 0.5 µM BE 3-4-3 (squares), and cells incubated in
the presence of 0.5 µM BE 3-4-3 for 48 h followed by
the addition of cycloheximide (triangles). Values represent
mean ± S.D. (n = 15 for untreated,
n = 6 for concurrent addition of BE 3-4-3 and
cycloheximide, and n = 9 for 48 h exposure to BE
3-4-3).
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To investigate the regulation of SSAT at the level of SSAT message,
steady state SSAT mRNA levels were determined in control and wtSSAT
cultures which had been exposed either to no drug or varying
concentrations of BE 3-4-3 for 48 h. In the absence of the
inducing agent, the SSAT message level of the wtSSAT clone was 5.7 ± 2.8 (n = 9) times greater than that of the control
cells, indicating increased SSAT transcription with the CMV promoter. Thus, in the noninduced state an ~6-fold increase in SSAT mRNA (described above) resulted in a ~28-fold SSAT activity increase (Fig.
6). The effect of BE 3-4-3 on the SSAT message was similar in the two
cell lines with SSAT mRNA increased only slightly over that in each
cell line in the absence of the polyamine analogue. At BE 3-4-3 concentrations of 1, 5, and 10 µM, in a representative experiment, the SSAT message increases were 1.6-, 2.9-, and 2.4-fold, respectively, for the control, and 1.2-, 2.1-, and 2.0-fold,
respectively, for the wtSSAT clone. Therefore these data indicate that
the
3-fold increases in steady state levels of SSAT mRNA in the
presence of BE 3-4-3 are far too small to fully account for the
90-fold concentration-dependent induction of SSAT
activity mediated by BE 3-4-3 in either the control or the wtSSAT clone.
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DISCUSSION |
Although there is abundant evidence that SSAT plays a very
important role in polyamine metabolism in mammalian cells (1, 7, 21),
details of the mechanisms of SSAT regulation are not well understood.
One of the difficulties inherent to the study of SSAT has been that the
very low cellular levels of this enzyme have prevented accurate
assessment of its properties under normal cellular conditions.
Information related to SSAT properties and regulation has come largely
from studies of the enzyme following exposure to agents that induce its
activity, often quite dramatically, and that may alter its regulation.
Additionally, effects on cell growth and survival often result from the
use of inducing agents, and it has been difficult to assess which
effects are directly attributable to the increased SSAT activity. The
SSAT protein has been expressed at high levels in bacterial systems
(22); however, stable overexpression in mammalian cells has not
previously been achieved. The successful stable transfection and
overexpression of the SSAT protein in CHO cells reported here has now
enabled study of the properties and regulation of SSAT without the use of an inducing agent. Furthermore, the SSAT activity of all of the
clones exhibiting increased basal levels of SSAT was highly inducible
by BE 3-4-3, a characteristic that provided the basis for assessing
changes in SSAT regulation during its induction.
The basal level of SSAT activity achieved in the clones transfected
with SSAT cDNA, while substantially greater than the endogenous level in CHO cells, was still modest compared with SSAT activity levels
observed in many cell lines in the presence of an inducing agent.
However, it should be noted that many of the potent SSAT inducers are
either substrates or inhibitors of the enzyme. Therefore, the extent to
which such induction alters SSAT activity toward endogenous polyamines
is not clear.
The increased basal level of SSAT in the transfected CHO cells (from 24 to ~1600 pmol/min/mg protein) clearly caused altered polyamine
homeostasis with perturbations in the polyamine distribution that were
consistent with increased flux through the catabolic pathways.
Putrescine levels were highly increased, spermidine was decreased, and
N1-acetylspermidine accumulated. The polyamine
synthetic enzymes ODC and AdoMetDC are responsible for maintaining
intracellular spermidine and spermine levels, and the observed
increases in the activities of these enzymes would function to restore
the polyamine pools depleted by SSAT mediated polyamine catabolism. These compensatory actions prevent depletion of the polyamine pools and
may partially explain why there is little effect on growth of the
wtSSAT clone. Polyamine depletion has been implicated in cytostasis and
cytotoxicity resulting from polyamine analogues, even when SSAT
activity is only moderately induced (14, 23, 24). However, in those
instances, ODC was also down-regulated by the analogue and therefore no
increase in polyamine synthesis occurred to replenish the pools. There
was also an increase in the total intracellular polyamine content in
the wtSSAT clones, a cellular parameter that is usually tightly
controlled by the actions of the polyamine metabolic enzymes. The
production of detectable levels of
N8-acetylspermidine, which is unlikely to be
produced directly by SSAT since SSAT is highly specific for the
formation of N1-acetylspermidine (25), may be a
cellular response to limit the total polyamine level since the only
known fate of N8-acetylspermidine is
de-acetylation or excretion from the cell.
The profound effects on cell growth brought about by exposure to
polyamine analogues that are powerful inducers of SSAT has raised the
question of whether increased SSAT activity is in itself detrimental to
cell growth and survival. The current data suggest that increased SSAT
activity may at least retard cell growth from the single cell stage,
since the majority of the clones that exhibited increased SSAT activity
were among the slowest growing of all the clones selected, no clones
exhibiting extremely high levels of SSAT activity were isolated, and
the wtSSAT clone 43 grew more slowly under clonal conditions. The fact
that the population growth characteristics of the clones expressing
increased SSAT activity were similar to the control cells suggests that
the observed increase of SSAT activity does not adversely affect the
growth of exponentially growing cell populations. This latter
observation is consistent with the recent findings of Alhonen et
al. (21) that primary fetal fibroblasts, derived from transgenic
mice overexpressing SSAT and which express a 20-fold increase in SSAT
activity, grew similarly to primary fibroblasts derived from
nontransgenic counterparts. The current finding that the wtSSAT clone
was actually marginally less sensitive to growth inhibition by BE
3-4-3 than the control cells, however, differs from the observation in
that study where the SSAT overexpressing fetal fibroblasts displayed an
increased sensitivity to a polyamine analogue.
The increase in SSAT expression in the transfected CHO cells was
clearly due to an increase in mRNA content. There was an approximately 6-fold increase in mRNA levels in the uninduced state. This increase is probably due to the greater strength of the CMV
promoter compared with the endogenous SSAT promoter, but an increased
gene copy number in the transfected cells may also contribute. The
observation that the magnitude of the SSAT activity increase is greater
than the SSAT mRNA increase suggests translational regulation of
SSAT. Regulation at the level of translation has been demonstrated for
both of the polyamine synthetic enzymes ODC and AdoMetDC (26), and this
may be a common feature of the polyamine-related enzymes. Since the
SSAT derived from the transfected plasmid construct does not contain
the complete 5'- and 3'-UTR regions of the endogenous gene, yet the
data suggest translational regulation of SSAT, we can conclude that
these regions are not factors in regulation of SSAT translation in the
noninduced state. Parry et al. (8) have previously reported
that in COS-7 cells, neither the 5'- nor 3'-UTR of SSAT mRNA was
important for translational regulation of SSAT by BE 3-4-3.
We have now provided data that substantiate previous estimates (8-10)
that, under conditions where the enzyme is not induced, SSAT is a
short-lived protein. Our results indicate that under normal cellular
growth conditions the SSAT protein has a half-life of 26-29 min and
SSAT degradation occurs through the proteasomal complex. SSAT activity
is directly proportional to the amount of SSAT protein.
When BE 3-4-3 is present, the degradation of SSAT is inhibited, and
this stabilization contributes to the increases in SSAT protein but is
far less than is needed to account for the total increase observed. The
lengthened SSAT half-life in the presence of the analogue, even in the
absence of new protein synthesis, suggests that a direct stabilizing
interaction between BE 3-4-3 and the SSAT protein may be one factor
contributing to the increased SSAT activity. The results indicate that
the SSAT half-life in the presence of BE 3-4-3 is increased from 29 min
to greater than 200 min.
The presence of BE 3-4-3 also slightly increases the amount of SSAT
mRNA in a concentration-dependent manner, but this
increase is also far too small to account for the massive increase in
SSAT activity. Therefore, we can conclude that transcriptional
regulation is not a major factor in the observed SSAT induction. The
small increase in SSAT message observed may result from stabilization of the SSAT mRNA and a lengthening of the SSAT mRNA half-life. Such an effect has been observed by Fogel-Petrovic et al.
(6), where they reported an increase of SSAT message half-life from 17 to 64 h in the presence of BE 3-4-3. The similarity of the SSAT
message increase for both the control and the wtSSAT clone would
indicate that if such stabilization is occurring, then it does not
involve the portions of the 5'- and 3'-UTRs, which are not present in
the wtSSAT clone.