(Received for publication, August 23, 1995; and in revised form, November 15, 1995)
From the
The plant amino acid, mimosine, is an extremely effective
inhibitor of DNA replication in mammalian cells (Mosca, P. J., Dijkwel,
P. A., and Hamlin, J. L.(1992) Mol. Cell. Biol. 12,
4375-4383). Mimosine appears to prevent the formation of
replication forks at early-firing origins when delivered to mammalian
cells approaching the G/S boundary, and blocks DNA
replication when added to S phase cells after a lag of
2.5 h. We
have shown previously that [
H]mimosine can be
specifically photo-cross-linked both in vivo and in vitro to a 50-kDa polypeptide (p50) in Chinese hamster ovary (CHO)
cells. In the present study, six tryptic peptides (58 residues total)
from p50 were sequenced by tandem mass spectrometry and their sequences
were found to be at least 77.5% identical and 96.5% similar to
sequences in rabbit mitochondrial serine hydroxymethyltransferase
(mSHMT). This assignment was verified by precipitating the
[
H]mimosine-p50 complex with a polyclonal
antibody to rabbit cSHMT. The 50-kDa cross-linked product was almost
undetectable in a mimosine-resistant CHO cell line and in a CHO gly
cell line that lacks mitochondrial, but
not cytosolic, SHMT activity. The gly
cell
line is still sensitive to mimosine, suggesting that the drug may
inhibit both the mitochondrial and the cytosolic forms. SHMT is
involved in the penultimate step of thymidylate biosynthesis in
mammalian cells and, as such, is a potential target for chemotherapy in
the treatment of cancer.
Our laboratory's interest is the regulation of DNA
synthesis in mammalian cells and, in particular, the nature of origins
of replication. Although it is known that mammalian DNA is replicated
from bidirectional origins spaced 100 kilobase pairs
apart(1) , the molecular mechanisms of this process remain
elusive (see (2) , for review).
In the absence of a viable
assay for identifying the genetic elements (replicators) that control
initiation in mammalian cells, attention has been focussed on
determining the positions at which replication initiates, which should
lie close to replicators. This approach requires methods for obtaining
cell populations in which initiation at a given origin is occurring at
the same time. In a commonly used synchronization protocol, cells are
first arrested in the G (non-proliferating) compartment by
nutritional or serum starvation, followed by release into an inhibitor
of DNA synthesis (e.g.(3, 4, 5) ).
The drug treatment is enforced for a time long enough to allow all
cells in the population to arrive at the beginning of the S period (a
time when at least some origins are sure to be firing); the drug is
then removed, allowing cells to enter S in a semi-synchronous wave.
Unfortunately, this protocol is not entirely satisfactory for examining
initiation events at the beginning of S, because even the most
efficacious replication inhibitors do not inhibit initiation per
se; rather, they slow the rate of replication fork movement by
affecting DNA polymerases (e.g. aphidicolin (6) ) or
by lowering deoxyribonucleotide pools (e.g. hydroxyurea (7) and 5`-fluorodeoxyuridine(8) ).
About five years
ago it was reported that the plant amino acid, mimosine, arrests
mammalian cells at a specific point in the late G phase of
the cell cycle(9, 10) . Therefore, mimosine could be a
superior agent for synchronizing cells prior to initiation at
early-firing origins. However, when we examined the effects of mimosine
on cell cycle progression, specific G
arrest was not
observed(11) . Instead, we showed that the drug inhibits
replication per se, but in a manner different from any known
chain elongation inhibitor. For example, mimosine completely prevents
the uptake of [
H]thymidine into DNA when added to
CHO (
)cells that have already entered the S period, but only
after
2.5 h, whereas aphidicolin and hydroxyurea inhibit
replication almost immediately (e.g.(12) ). After the
2.5-h lag, mimosine effectively prevents S phase cells from progressing
any further in the cell cycle for at least 48 h, as assessed by
fluorescence-activated cell sorter analysis; this contrasts with
aphidicolin and hydroxyurea, which are relatively leaky even at high
concentrations and allow cells to slowly traverse the S period (11, 13) . When added to cells as they attempt to
cross the G
/S boundary, mimosine completely prevents the
formation of replication forks in the dihydrofolate reductase origin in
CHO cells, while aphidicolin and hydroxyurea do
not(12, 14) . Finally, the initial rate of DNA
synthesis is zero regardless of how long cells are maintained in
mimosine after release from a G
block, suggesting again
that mimosine prevents the formation of replication forks; in contrast,
with aphidicolin or hydroxyurea, the initial rate of
[
H]thymidine incorporation increases with the
duration of the block, arguing that both initiation and a significant
amount of chain elongation occur in their presence(14) .
Thus, the possibility arose that mimosine could inhibit initiation
itself, either by interfering with an initiator protein or by somehow
preventing the formation of replication forks. However, it is also
known that mimosine chelates iron, which is required by ribonucleotide
reductase(15) . Indeed, at relatively high concentrations,
mimosine has been reported to lower deoxynucleotide pools in mammalian
cells(16, 17) . In addition, it has no apparent effect
on DNA synthesis either in frog embryos (18) or in in vitro replication extracts prepared from mammalian
cells(16, 17) , ()both of which contain
large deoxynucleotide pools. Furthermore, the inhibitory effects of
mimosine on DNA replication in CHO cells can be overcome by adding iron
to the culture medium(19) . It has therefore been suggested
that mimosine functions solely by inhibiting ribonucleotide
reductase(16, 17) .
It is difficult to prove or
disprove the latter assertion (see arguments in (19) ).
However, a simple lowering of deoxyribonucleotide pool levels by
inhibiting ribonucleotide reductase does not explain why mimosine is
such an efficacious inhibitor and why it appears to prevent initiation
of nascent DNA chains. We recently obtained evidence for a different
(or additional) intracellular target. We showed that
[H]mimosine can be specifically cross-linked to a
50-kDa polypeptide (p50) both in vivo and in vitro at
concentrations equal to the minimum effective dose in
vivo(19, 20) . Furthermore, we demonstrated that
the p50 mimosine binding activity is almost absent in a CHO cell line
selected for resistance to 1 mM mimosine (
10 times the
lethal dose; 19, 20). p50 partitions largely with the soluble
cytoplasmic and nuclear fractions, and the ability to bind mimosine
does not fluctuate demonstrably during the cell cycle(19) .
In the present study, we have partially purified the major 50-kDa mimosine binding activity and have sequenced six of its tryptic peptides by tandem mass spectrometry(21) . The sequences of all six peptides (58 residues total) are consistent with 96.5% similarity and at least 77.5% identity to sequences in rabbit mitochondrial serine hydroxymethyltransferases (mSHMT), and all are closely related to rabbit cytosolic SHMT (cSHMT). Both of these enzymes are involved in the biogenesis of thymidine (among other activities; (22) and (23) ). Evidence is presented that mSHMT and cSHMT are, indeed, the major mimosine-binding species in vivo, as well as bona fide targets for the drug.
Given the role of SHMT in deoxyribonucleotide metabolism, mimosine would be expected to have an effect only on chain elongation, even though its overall effect on replication differs greatly from other drugs that inhibit replication by lowering nucleotide pools (e.g. hydroxyurea, methotrexate, and 5`-fluorodeoxyuridine). Possible reasons for this dichotomy are discussed.
Each sample was then transferred to a 1.5-ml tube and
centrifuged at 8,000 rpm for 5 min in a microcentrifuge. The
supernatants were brought to 40%
(NH)
SO
, incubated on ice for 10
min, and centrifuged at 10,000 rpm for 20 min at 4 °C in an HB-4
rotor (DuPont/Sorvall). Pellets were resuspended in lysis buffer
without Triton X-100 and were subjected to gel filtration on a 1.2
100-cm Sepharose CL-6B column (Sigma). Fractions (1.2 ml) were
collected and 100-µl aliquots were analyzed on a 10% sodium dodecyl
sulfate-polyacrylamide gel (PAGE)(25) . The gel was impregnated
with ENTENSIFY (DuPont Fluorofor kit) following the
manufacturer's instructions, and radioactive spots were
identified fluorographically, using Kodak X-Omat AR film. The
H-labeled p50 protein fractions thus identified were
combined and subjected to DE52 anion exchange chromatography (1.5
10 cm, Whatman): after washing the column with 40 mM KCl in lysis buffer lacking Triton X-100, fractions were eluted
sequentially with 100, 200, 500, and 1,000 mM KCl in lysis
buffer without Triton X-100. The p50-containing fraction (the 100
mM KCl wash) was then separated by PAGE and blotted onto a
nitrocellulose membrane. The membrane was stained with Ponceau S, and
the p50 spot or band, as well as several surrounding ones, were
individually excised. Approximately 5% of each was used for
scintillation counting to confirm the identity of
H-labeled
p50, and the remainder was subjected to tryptic digestion (see below).
Enrichment and recovery values at each purification step were
determined by assessing protein content with the Bradford assay (26) and radioactivity by trichloroacetic acid precipitation.
Although a large percentage of the [H]mimosine
label in the initial cell lysate is cross-linked to proteins other than
p50(19, 20) , the majority is associated exclusively
with p50 after the 8,000 rpm centrifugation step (e.g. see Fig. 2).
Figure 2:
Progress of the purification of p50.
Extract from 6
10
CHO-K1 cells was mixed with
[
H]mimosine, irradiated, centrifuged to remove
aggregated material, and adjusted to 40% saturated ammonium sulfate.
The resulting pellet was redissolved and fractionated on Sepharose
CL-6B, followed by DE52. Approximately equal amounts of protein
(
20 µg) from the pooled fractions resulting from each step
were separated on a 10% denaturing polyacrylamide gel and stained with
Coomassie Brilliant Blue. The polypeptides were then subjected to
fluorography as described in the legend to Fig. 1. Panel
A, stained gel. Panel B,
fluorogram.
Figure 1:
A
50-kDa [H]mimosine-binding polypeptide is
detected in a wide variety of mammalian cell lines. Cell extracts were
prepared and irradiated in the presence of 100 µM [
H]mimosine, as described under
``Materials and Methods.'' Samples (
100 µg of
protein) were run on a 10% denaturing polyacrylamide gel, which was
stained and photographed, and then treated with ENTENSIFY and dried
onto filter paper. Panel A, Coomassie Brilliant Blue-stained
polyacrylamide gel. Panel B, fluorograph of the gel shown in panel A, showing tritium-labeled polypeptides (7 day
exposure).
To address this
question, extracts from eight different cell lines of human, monkey,
mouse, and hamster origin were incubated with
[H]mimosine and illuminated with a xenon lamp.
The extracts were separated on a polyacrylamide gel, and the
corresponding radioactive products were detected by fluorography. The
Coomassie Brilliant Blue-stained gel is shown in Fig. 1A and the fluorogram in Fig. 1B. Seven of the eight
cell lines contain a prominent
[
H]mimosine-binding species that migrates at
50 kDa. Although the CV-1 extract displays only a faint band in
this experiment, p50 mimosine binding activity has been observed in
other preparations. (
)Also note that monkey COS cells, which
were derived from CV-1 cells, display a prominent radioactive band at
50 kDa. Thus, a mimosine binding activity appears to be conserved
among four different mammalian species. Furthermore, although some
minor differences in molecular weight are detected (e.g. compare human 293 and monkey COS cell extracts, Fig. 1B), the binding proteins all migrate in the size
range 50-53 kDa.
Additional, less prominent H-labeled bands are also detected, as well as significant
amounts of labeled high molecular weight material at the top of the
gel. Since the spectrum and intensity of the faint bands are not
reproducible from experiment to experiment, and since additional
experiments indicated that they are probably not relevant (see below),
we have not studied any of these bands further. The high molecular
weight material is not detected in non-irradiated
extracts(19) , (
)and we therefore assume that it
represents aggregates of p50, either with itself or with other
polypeptides. We have also examined extracts prepared from Saccharomyces cerevisiae and Escherichia coli, but
did not reproducibly observe specific [
H]mimosine
binding activity at any position in the gels.
This is
consistent with the observation that mimosine does not inhibit DNA
replication in either of these species at concentrations as high as 1
mM. (
)
Exponentially growing CHO-K1 cells in tissue culture dishes were
harvested, and a soluble extract was prepared by lysing with Triton
X-100 and removing nuclei by centrifugation. The resulting extract was
cross-linked with [H]mimosine and aggregated
material was removed by low speed centrifugation. The bulk of the p50
was then precipitated in a 40% saturated ammonium sulfate solution, and
the resulting pellet was redissolved and fractionated by size on
Sepharose CL-6B. The p50-enriched fractions were pooled and subjected
to chromatography on DE52, and those fractions containing the
[
H]mimosine binding activity were finally
subjected to preparative polyacrylamide gel electrophoresis.
The
approximate degrees of purification and recovery at each step in a
typical experiment are outlined in Table 1, and the progress of
the purification is shown in Fig. 2. Note that equal amounts of
protein were loaded into each well of the polyacrylamide gel in this
experiment. As can be seen in the stained gel in Fig. 2A and the corresponding fluorogram in Fig. 2B, DE52
chromatography and polyacrylamide gel electrophoresis represented the
most effective purification steps. The arrow in Fig. 2A indicates the stained band that co-migrates
with the [H]mimosine-binding band in Fig. 2B. This stained band must represent several
50-kDa polypeptide species in the early stages of the purification,
since its intensity relative to total protein does not markedly
increase in the first two steps, while the
[
H]mimosine binding activity at the corresponding
position does increase. This raises the question whether the 50-kDa
band in the final polyacrylamide gel represents a single species. In
fact, on a two-dimensional gel, the 50-kDa band separates into one
major and one very minor radioactive spot that differ only slightly in
isoelectric point, and each of these corresponds to a stained
spot.
These data suggest that the bulk of the material in
the 50-kDa band on the one-dimensional polyacrylamide gel binds to
[
H]mimosine.
In a typical purification
protocol, 6
10
cells yielded 1-2 µg
of p50 (estimated by Ponceau S staining intensities on nitrocellulose
blots relative to standards). This represented an average purification
of 6,000-fold and an average recovery of
57% from the starting
extract (Table 1).
Figure 3: Six tryptic peptides from CHO p50 are highly homologous to rabbit mSHMT. A tryptic digest of p50 was separated by high performance liquid chromatography and subjected to analysis by the tandem mass spectrometer. The sequence of six different peptides (54 residues total) are compared to the sequences of cSHMT and mSHMT from humans and rabbits (GenBank). Note that the quadrupole instrument cannot discriminate between leucine and isoleucine, resulting in ambiguity at these residues; in the CHO peptides shown, we have used the residues that are common to four other mammalian SHMT sequences. The period denotes probable identity with the lysine or arginine in the amino-terminal positions of the other comparison peptides, although these were not actually sequenced in the hamster peptide.
The primary sequences of
both cSHMT and mSHMT are highly conserved among mammals(30) .
We therefore asked whether a polyclonal antibody against rabbit cSHMT
would cross-react with the mimosine-binding p50 polypeptide. An aliquot
of the starting cell lysate was cross-linked to radiolabeled mimosine
and was then incubated with Protein A beads, either alone or coupled to
the polyclonal antibody preparation. As shown in Fig. 4B, the majority of H-labeled p50
partitioned with the antibody-coated Protein A beads (compare +Ab
pellet to +Ab supe), while no detectable
H-labeled p50
partitioned with control beads (-Ab pellet).
Figure 4:
p50 can be immunoprecipitated by antibody
against rabbit SHMT. Extracts from CHO-K1 cells were mixed with
[H]mimosine, irradiated, and centrifuged to
remove aggregated material. The clarified extracts (7 µl,
20
µg of protein) were incubated with 10 µl of antiserum raised
against rabbit cSHMT as described under ``Materials and
Methods.'' The immunoreactive material remaining with the pellet
was then eluted and run on a 10% denaturing polyacrylamide gel and
transferred to nitrocellulose. Panel A, stained gels. Panel B, fluorogram.
Figure 5:
SHMT is involved in both glycine and
thymidylate biosynthesis. SHMT transfers a methylene group from serine
to tetrahydrofolate (THF) to form glycine and N,N
-methylene-THF. The
latter intermediate then donates a methylene group to dUMP (with a
reduction to a methyl group by dTMP synthase) to form dTMP. Note that
the two other enzymes in the thymidylate biosynthetic pathway are
dihydrofolate reductase (DHFR) and thymidylate synthase, which
are targeted by the chemotherapeutic agents, methotrexate and
5-fluorodeoxyuridine (or 5-fluorouracil), respectively. Modified from (51) with permission.
In an independent test of whether mSHMT represents the major mimosine binding activity, we analyzed the effects of mimosine on a variant CHO cell line (CHO/51-11) that lacks mSHMT activity, probably as the result of a point mutation(32, 34) . This cell line was isolated as a glycine-requiring mutant, and was subsequently shown to have greatly reduced levels of mSHMT enzyme activity but near-normal levels of cSHMT activity(32) .
As shown in Fig. 6B, extracts prepared from the gly CHO/51-11 mutant lack the predominant
50-kDa [
H]mimosine binding activity that
characterizes wild-type CHO-K1 cells. Furthermore, p50 could not be
detected in extracts of CHO/51-11 cells regardless of whether they were
propagated in minimal essential medium supplemented with non-essential
amino acids (our standard culture medium) or in F12 (the medium in
which this cell line is usually maintained; (32) ). Thus, a
single mutation has affected both catalysis and mimosine binding to
mSHMT in the CHO/51-11 cell line, suggesting that mimosine may bind
directly to the active site.
Figure 6:
A glycine-requiring CHO variant that is
deficient in mSHMT activity does not contain demonstrable p50 mimosine
binding activity. Extracts from wild-type CHO-K1 and gly CHO-K1 cells were cross-linked to
[
H]mimosine and separated on a denaturing
polyacrylamide gel. Cells were either grown in our standard minimal
essential medium or in F12, in which this cell line is usually
propagated(32) . Panel A, stained gel. Panel
B, fluorogram.
In a different approach,
pyridoxal phosphate was covalently linked to the enzyme active site
with sodium borohydride(38) , and the binding of mimosine to
this modified preparation was determined (Fig. 7). Since sodium
borohydride is a reducing agent, it was first necessary to show that
sodium borohydride-treated mimosine (mimo) is still
capable of binding to SHMT. A preparation of mimosine and sodium
borohydride was stored at room temperature overnight and was employed
as the source of mimosine in the cross-linking experiments. The
fluorogram in Fig. 7B shows that 1 mM sodium
borohydride has little effect on mimosine binding (compare mimo
to mimo
).
Figure 7:
Reduction of Schiff bases in CHO-K1
extracts abolishes the cross-linking of mimosine (Mimo) to
p50. Mimosine was pretreated with sodium borohydride as described under
``Materials and Methods'' and was then incubated with
extracts that had been treated with the indicated concentrations of
sodium borohydride, dithiothreitol (DTT), or
-mercaptoethanol (BME) as described under
``Materials and Methods.'' After irradiation, the extracts
were separated on a 10% denaturing polyacrylamide gel. Panel
A, stained gel. Panel B,
fluorograph.
Cell extracts were then treated with
sodium borohydride and were immediately incubated for 30 min with 100
µM [H]mimosine
. The
extracts were then illuminated and the radioactive products were
separated on a gel and analyzed by fluorography. As shown in Fig. 7B, extracts treated with concentrations as low as
0.25 mM borohydride were no longer capable of binding to
[
H]mimosine. Therefore, when pyridoxal phosphate
is coupled to the enzyme through a reduced Schiff base (probably in the
active site), access to mimosine appears to be prevented. Note that,
with the exception of the material at the origin of the gel, none of
the labeled bands other than p50 were reduced by prior cross-linking to
pyridoxal phosphate. The reduction of [
H]mimosine
binding to the high molecular weight material at the origin is
consistent with the suggestion that this material represents p50
aggregates cross-linked to itself or to other proteins. As additional
controls for changes in pH and reducing conditions, extracts were
treated with 1 mM dithiothreitol, 1 mM
-mercaptoethanol, or NaOH concentrations ranging from 0.25 to
1 mM. None of these treatments significantly affected the
binding of [
H]mimosine to mSHMT (Fig. 7B and data not shown).
When we
tested the effects of the drug on SHMT activity in CHO cell extracts
(containing predominantly mSHMT(39) ), significant inhibition
was not observed. The enzyme assay measures the transfer of a methylene
group from serine to tetrahydrofolate. As shown in Fig. 8, the
rates of catalysis in the presence and absence of 200 µM mimosine are indistinguishable, regardless of whether 200
µM pyridoxal phosphate is included in the reaction
mixtures. We have obtained the same results at serine substrate
concentrations ranging from 20 µM to 20 mM. Preincubation of cells with 200 µM mimosine for 3.5
h prior to lysate preparation was equally ineffective at inhibiting
SHMT activity in vitro.
In addition, mimosine had
no significant effect on the enzyme activity of purified rabbit cSHMT
(the major form in this species; 31).
We have not attempted
to cross-link mimosine to SHMT prior to enzyme assays in cell extracts,
since the cross-linking procedure is performed on ice, which reduces
mimosine binding; furthermore, we do not know the degree of saturation
of the intracellular target achieved in our cross-linking procedure,
which would render any inhibitory effects difficult to interpret.
Figure 8: Mimosine (MIMO) does not inhibit SHMT enzyme activity in a standard in vitro assay. CHO cell lysates were assayed for SHMT activity in the absence (closed symbols) or presence (open symbols) of 200 µM mimosine. The effect of the addition of 200 µM pyridoxal phosphate (triangles) were compared to extracts without pyridoxal phosphate (squares).
Second, DNA replication in the gly CHO/51-11 cell line (which lacks mSHMT but retains cSHMT) is
still as sensitive to mimosine inhibition as in wild-type CHO-K1 cells. (
)This finding suggests that while mSHMT represents the
major [
H]mimosine binding species in CHO cells,
cSHMT may also be an important target.
The basis for mimosine action has been studied for years,
ever since it was shown to be the agent responsible for hair loss in
the cattle feed, Leucaena pudica. We became interested in this
compound as a potential synchronizing agent when it was reported to
arrest cells in the late G period(9, 10) .
However, we have shown that mimosine is actually an extremely effective
inhibitor of DNA replication(11, 12, 14) . In
fact, several of its properties suggested that mimosine might act
during initiation at origins, either at the strand separation stage or
when the replication machinery is loaded into the melted region to form
the replication forks(11, 12, 14) .
Mimosine is also known to chelate iron. Thus, it has been suggested that mimosine inhibits DNA replication by depriving ribonucleotide reductase of required iron(16, 17) . Three lines of evidence were advanced to support this proposal: 1) mimosine reduces deoxynucleotide pool levels and inhibits DNA replication, both of which effects can be reversed with added iron(17, 19) ; 2) mimosine has no effect on DNA replication in frog embryos or in vitro, in which deoxyribonucleotide precursors are present in excess(18) ; and 3) mimosine inhibits ribonucleotide reductase activity in vitro, and inhibition can be overcome by the addition of excess iron(17) .
However, we have argued that
these experiments are not conclusive, since the inhibition of many
different metal-requiring enzymes could ultimately result in imbalanced
deoxyribonucleotide pools through secondary effects(19) ;
furthermore, excess iron could inactivate mimosine itself instead of
resupplying iron to an iron-deprived ribonucleotide
reductase(19) . We have also been able to demonstrate that cell
lysates prepared from mimosine-treated cells have a reduced capacity
for supporting SV40 replication in vitro. ()In
addition, CHO cell lines selected for resistance to 300 µM mimosine are still sensitive to the iron chelator, deferoxamine,
at a concentration that chelates the same amount of iron (100
µM; 38, 40).
Finally, mimosine is much more
efficacious and has markedly different properties than does
hydroxyurea, which inhibits DNA replication by inactivating
ribonucleotide reductase directly(7, 15) ; in
particular, dATP and dGTP pool levels are maximally reduced only after
a 4-h exposure to mimosine(16) , whereas hydroxyurea requires
only a few minutes to maximally affect deoxynucleotide pool levels (7) .
It has also been suggested that mimosine may affect histone kinase activity(41) , although, again, it is difficult to separate primary from downstream effects on this activity.
We
have taken a direct approach to identifying possible intracellular
targets for mimosine. A photochemical cross-linking strategy allowed us
to detect a 50-kDa [H]mimosine-binding species in
CHO cell extracts(19, 20) . Importantly, this 50-kDa
mimosine binding activity appears to be absent in CHO cells made
resistant to 1 mM mimosine, strongly arguing that p50 is a
biologically relevant target of mimosine(19, 20) . We
also show in this report that the 50-kDa mimosine binding activity is
present in all of the wild-type mammalian cell lines examined (Fig. 1); therefore, p50 and its ability to bind to mimosine
have been conserved in mammals.
In the present report, we also
describe a simple fractionation scheme in which extracts from a modest
amount of cells (6
10
) yields a band on a
polyacrylamide gel of sufficient purity and quantity (
20 pmol) to
analyze by tandem mass spectrometric analysis. Six tryptic peptides
from this fraction were sequenced, all of which show a high degree of
similarity with rabbit mitochondrial SHMT: four of the six are
identical to rabbit mSHMT and are nearly identical to human mSHMT (Fig. 3). Although we have not yet been able to purify the
mimosine-binding peptide itself for sequence analysis,
[
H]mimosine-labeled p50 was quantitatively
precipitated by an antibody to rabbit cSHMT (Fig. 4).
Furthermore, when a gly
CHO cell line that
lacks mSHMT activity (32) was analyzed for the presence of the
mimosine binding activity, the major radioactive band at 50 kDa was
greatly reduced (Fig. 6).
Several additional lines of
evidence suggest that p50 corresponds to SHMT. For example, all known
eukaryotic SHMT enzymes (whether mitochondrial or cytosolic) migrate
between 50 and 53 kDa on denaturing SDS-polyacrylamide gels and, like
p50(20) , most have isoelectric points near
7.0(20, 42) . Not surprisingly, each of these enzymes
is purified by separation methods very similar to those employed
here(33, 43, 44, 45) . Furthermore,
[H]mimosine-labeled p50 elutes from a
nondenaturing gel filtration column as a multimer with an estimated
size of 200-250 kDa,
as does purified rabbit
mSHMT(33, 43, 44, 45) . Thus, there
is little doubt that mimosine-labeled p50 corresponds to SHMT.
The
two forms of SHMT (cytosolic and mitochondrial) from several mammalian
species are readily distinguishable by kinetic parameters and by heat
lability(32) . They have been shown to co-purify through
biochemical separation techniques very similar to those employed in the
present study (e.g.(31) ), and the two forms in
rabbit co-migrate at 53 kDa on one-dimensional SDS-PAGE
gels(31) . The amino acid sequences of each form are highly
conserved across species, but within a species the two forms are more
divergent. For example, 97% of the residues are identical in rabbit and
human mSHMT(30) , while human cSHMT and mSHMT are 63%
similar to one another(30) .
The question whether mimosine
inhibits the cytosolic or the mitochondrial form of SHMT, or both, is a
difficult one to answer. Although the proteins have distinguishing
turnover numbers and heat sensitivities(31) , the mitochondrial
form is in such great excess over the cytosolic form in the Chinese
hamster (32) that we were not able to study cSHMT in isolation.
It is therefore not surprising that mSHMT is the form from which we
obtained sequence information, regardless of whether one or both forms
bind to mimosine. Furthermore, the polyclonal antibodies prepared
against rabbit cSHMT and used here to precipitate the
[H]mimosine-p50 complex recognize both forms in
the rabbit approximately equally. (
)
The gly cell line, which lacks mSHMT activity
but retains cSHMT activity(32) , displays only a very faint
50-kDa mimosine-binding species in autoradiograms that may represent
cSHMT (Fig. 6). Since this cell line retains sensitivity to
mimosine,
it is probable that both forms are inhibited by
the enzyme. This suggestion is consistent with the presence of a
prominent [
H]mimosine-labeled 50-kDa polypeptide
in monkey and rabbit cells, in which cSHMT is the predominant enzyme
activity(43) .
It is paradoxical, therefore, that mimosine
had no obvious effect on SHMT activity when added directly to CHO cell
extracts (mostly mSHMT) or to purified cSHMT isolated from rabbits. One
possible explanation is that mimosine binding activity (but not SHMT
enzyme activity) is quite labile, especially at low temperature: when
the CHO lysate was incubated with [H]mimosine on
ice for as little as 8 min prior to photo-cross-linking, the bulk of
the mimosine binding activity was lost.
Obviously, the
rabbit cSHMT enzyme preparation encountered these conditions during
purification. The CHO cell extracts were not subjected to this
treatment, and still retain both enzyme and mimosine binding activity.
However, cell extracts prepared for enzyme activity measurements are
10- 20-fold more dilute than those used in cross-linking studies,
and we have observed that the more dilute the extracts are, the more
difficult it is to obtain reproducible cross-linking. (
)In
addition, the assay is performed in the presence of 12 mM dithiothreitol to maintain the tetrahydrofolate in a reduced
state; this high concentration of reducing agent could affect the
mimosine-mSHMT interaction significantly (although 1 mM dithiothreitol had only a marginal effect; Fig. 7). It is
also possible that the high levels of tetrahydrofolate utilized in this
assay could affect the binding of mimosine to SHMT.
The observation
that the gly CHO cell line that lacks mSHMT
activity has also lost most of its mimosine binding ability suggests
that mimosine either binds to the active site of the enzyme or to a
site critical for enzyme function in vivo. In addition, when
the Schiff base that binds pyridoxal phosphate to SHMT was reduced,
cross-linking to mimosine was specifically prevented (Fig. 7).
This result suggests that mimosine binds to SHMT either through a free
Schiff base or a free pyridoxal phosphate in or near the active site.
Many enzymes, particularly those involved in amino acid metabolism,
require pyridoxal phosphate as a cofactor for activity(46) . It
is interesting to note that an antibody against pyridoxal phosphate
detects low levels of many different proteins in both normal and tumor
cell extracts, but the signal from a 50-kDa polypeptide with a pI
of
7.0 is greatly amplified in tumor cells(38) .
Similarly, a large spectrum of faint [
H]mimosine
binding bands is detected in extracts from normal rat tissues,
while in extracts from cell lines, all of which are transformed
to some degree, the 50-kDa species predominates ( Fig. 1and Fig. 6). If SHMT is, indeed, overexpressed in transformed cell
lines, and if its overexpression is required to maintain the
tumorigenic state, SHMT could represent an important target for
chemotherapeutic protocols.
The actual effects of mimosine on cycling mammalian cells are undoubtedly complex and almost certainly depend upon drug concentration. We believe that the primary in vivo effect at the minimal effective concentration (50-100 µM for CHO cells) is probably to inhibit DNA replication by interacting with SHMT in an as yet undefined way. This raises the question why mimosine affects only dATP and dGTP pools without affecting dTTP pools(16) , since methylenetetrahydrofolate is directly involved in the thymidylate synthase reaction. As discussed below, all of our cross-linking studies were performed at 100 µM mimosine; in contrast, the deoxynucleotide pools measurements were performed in the presence of 400 µM(16) , which may, indeed, be high enough to inhibit ribonucleotide reductase. In this regard, it is interesting to note that hydroxyurea would be expected to lower pool levels of all four deoxyribonucleotides; in fact, hydroxyurea lowers only dATP and dGTP pools, while dCTP and dTTP are actually increased(7) .
Since we have not yet been able to demonstrate a direct effect on
enzyme activity in vitro, the interesting possibility arises
that mimosine might disrupt an allosteric interaction within a proposed
multienzyme replication complex(47) . This could lead to an
inhibition of replication fork formation when added to cells entering
the S period, as well as to the loss of existing forks when added to S
phase cells, both of which we have previously
observed(12, 14) . An alternative and intriguing
possibility is that mimosine affects the function of a proposed
purine-synthesizing multienzyme complex, for which there is both in
vitro and genetic evidence in various eukaryotic systems (see (23) for review). This would explain why dATP and dGTP pools
are selectively affected by mimosine, but only after exposure to
mimosine for at least 4 h(16) . Inhibition of complex formation
(or slow disruption of the complex) could also explain why it takes 4 h
for mimosine to reduce purine deoxyribonucleotide pool levels to zero,
while hydroxyurea exerts its effects within minutes(7) . It
would have to be additionally supposed that a subset of these complexes
are dedicated to providing (compartmentalizing) precursors for DNA
synthesis, however, since we do not observe a significant effect of
mimosine on RNA synthesis for at least 4 h after mimosine addition to
cultured cells.
At mimosine concentrations below 100
µM, some chelation of metal ions (e.g. iron and
copper) may occur, but chelation is probably not the critical factor
preventing DNA replication at low concentrations by the following
argument. Although we were able to isolate cell lines that were
marginally resistant to 1 mM mimosine (presumably because of a
point mutation that destroys binding ability), no resistant variants
could be isolated at higher drug concentrations. This result would be
expected if low levels of mimosine inhibit SHMT and can be overridden
by a point mutation that affects binding affinity, but higher levels
act as a general chelating agent. In fact, in the cell line selected
for resistance to 1 mM mimosine, labeled p50 was almost
undetectable in extracts exposed to 100 µM [H]mimosine, while DNA replication could
still be inhibited by 100 µM deferoxamine, a specific iron
chelator(48) .
Furthermore, in wild-type cells, the
effect of 100 µM mimosine on
[
H]thymidine incorporation into DNA could be
completely overridden with the substrate, serine, or the end product,
glycine, both of which are capable of forming a Schiff base with SHMT,
while inhibition by 100 µM deferoxamine was not affected
by these amino acids.
However, at concentrations above 100
µM mimosine, glycine or serine could not restore DNA
replication to control values. These findings may indicate that
mimosine does, in fact, inhibit ribonucleotide reductase and/or other
enzymes by metal ion chelation, and could explain why others have
observed changes in deoxynucleotide pools in CHO cells treated with 400
µM mimosine(16) .
Our initial interest in
mimosine stemmed from its utility as an efficacious and economical
synchronizing agent which, when added to cells approaching the S
period, seems to prevent the formation of replication forks at
early-firing origins(12, 14) . However, we believe
that mimosine also deserves attention as a potential anti-cancer agent.
Not only is the drug capable of completely preventing replication fork
progression for extended intervals(11) , but we were unable to
isolate cell lines displaying more than 5-10-fold higher
resistance to mimosine than the starting cell line, and then only after
a selection regimen lasting more than 18 months (20) . ()Although there are a few older reports describing
mimosine's effects on tumors in rat
models(49, 50) , perhaps this interesting drug should
be revisited in light of its possible effects on SHMT.
It is interesting to note that inhibitors of dihydrofolate reductase (methotrexate) and thymidylate synthase (5-fluorodeoxyuridine) have been available for years and are used routinely in chemotherapeutic mixtures. However, no compounds other than mimosine have been shown to target SHMT, which is the third enzyme in the deoxythymidylate biosynthetic pathway (see Fig. 5). Although we do not understand the mechanism by which mimosine disrupts the function of SHMT in vivo, it will be important to investigate its potential as a chemotherapeutic agent for cancer, alone or in combination with methotrexate and/or 5-fluorouracil.