(Received for publication, August 31, 1994; and in revised form, October 25, 1994)
From the
A Chinese hamster ovary cell subline (CHO/664) >1000-fold
resistant to the S-adenosylmethionine decarboxylase (AdoMetDC)
inhibitor, CGP-48664
(4-(aminoiminomethyl)-2,3-dihydro-1H-inden-1-one-diaminomethylenehydrazone),
has been developed and characterized. The cells were also
cross-resistant to the highly specific nucleoside analog inhibitor of
AdoMetDC, MDL-73811. These unique cells stably overexpress AdoMetDC due
to a 10-16-fold amplification of the AdoMetDC gene, which
resulted in a similar increase in AdoMetDC transcript levels. In the
presence of 100 µM CGP-48664, the CHO/664 cells displayed
AdoMetDC activities similar to the parental line. Following removal of
the inhibitor, AdoMetDC activity increased steadily over 20 days to
10-12 times that found in parental CHO cells. Decarboxylated (dc)
AdoMet pools accumulated rapidly from <5 pmol/10 cells
to
1000-1500 pmol/10
cells at 3 days due to
diffusion away of intracellular inhibitor and to the depletion of
putrescine and spermidine as aminopropyl acceptors in dcAdoMet-mediated
synthase reactions. Polyamine pools shifted as putrescine, and
spermidine pools were processed forward to spermine. During the period
from 3 days to 20 days, dcAdoMet pools fell steadily and eventually
stabilized at 100-200 pmol/10
cells. Providing excess
putrescine at this time as an aminopropyl acceptor rapidly lowered
dcAdoMet pools and led to a near normalization of polyamine pools,
indicating that both dcAdoMet and putrescine are essential in
maintaining steady-state polyamine pool profiles. As with cell line
variants that overproduce ornithine decarboxylase, polyamine transport
was found to be increased in CHO/664 cells due to an apparent inability
of the system to down-regulate polyamine transport in response to
polyamine excess. Given the unique metabolic disturbances seen in these
cells, we anticipate that in addition to providing a useful system for
evaluating the specificity of newly developed AdoMetDC inhibitors, they
will undoubtedly prove valuable for investigating the various
regulatory interrelationships involved in polyamine homeostasis and
possibly other aspects of purine metabolism.
The polyamines putrescine, spermidine, and spermine are known to
be critically involved in cell growth and have been implicated recently
in the process of cell transformation(1, 2) . In most
mammalian cells, polyamine biosynthesis seems to be simultaneously
regulated by ornithine decarboxylase (ODC) ()and S-adenosylmethionine decarboxylase (AdoMetDC) in response to
growth stimuli as well as to changes in intracellular polyamine pools
(reviewed in (3) and (4) ). Although intensely
studied, the regulatory relationships between these enzymes, polyamine
pools, and other effectors of polyamine homeostasis require further
definition if the role of these enzymes in cell growth is to be more
clearly understood. One approach has been to develop cell lines that
display exaggerated expression of either enzyme. In addition to
confirming the specificity of the relationship between enzyme
inhibition and cell growth, cell line variants resistant to specific
inhibitors of either enzyme offer the opportunity to study (a)
regulation of that enzyme and its relationship to intracellular
polyamine pools, (b) the effects of increased enzyme
expression on other effectors of polyamine pool homeostasis, (c) the consequences of increased enzyme expression on
cellular behavior, and (d) the specificity of other inhibitors
to that enzyme. While these same issues can also be addressed with
gene-transfected cells, only ODC has been stably overexpressed in
cells(5, 6) ; AdoMetDC has only been transiently
transfected in cells(7) . It has been speculated that because
AdoMetDC may be more critical than ODC in regulating the synthesis of
spermidine and spermine, its overexpression may constitute a toxic
event to cells.
Using the specific inhibitor of ODC,
-difluoromethylornithine (DFMO; (8) ), investigators have
developed at least eight resistant cell lines that overexpress ODC due
to gene amplification (reviewed in (9) ). One such line
produced the enzyme by an amount equivalent to about 15% of its total
protein(10) . Because ODC is typically found in relatively low
amounts in most cells, amplified variants have facilitated cloning of
the enzyme (11) and provided useful systems for studying its
regulation(12, 13) . Although specific nucleoside
analog inhibitors of AdoMetDC have been available for some
time(14, 15, 16, 17, 18) ,
resistant cell lines that overexpress the enzyme have not yet been
derived. From a synthetic effort focusing on derivatives of the
AdoMetDC inhibitor, methylglyoxal-bis(guanylhydrazone) (MGBG; (19, 20, 21, 22) ) as potential
anticancer agents, we have recently identified CGP-48664, a cyclic
analog with increased selectivity and potency toward the target enzyme
(IC
, 5 nM; Refs. 20 and 22). In contrast to MGBG,
the cyclic analog displays attenuated antimitochondrial activity, as
indicated by a lack of effect on pyruvate oxidation and mitochondrial
DNA levels under treatment conditions that inhibit cell growth. Using
this inhibitor, we recently developed a CHO cell line variant (CHO/664)
that is >1000-fold resistant to CGP-48664(22) . In this
study, we determined that a critical component of the resistant
phenotype is overproduction of AdoMetDC activity due to gene
amplification.
To our knowledge, the CHO/664 cells are the first variants to be described that stably overexpress the AdoMetDC gene. With this exaggerated phenotype, the CHO/664 cells offer a unique opportunity to examine various regulatory interrelationships involved in polyamine homeostasis and/or purine metabolism. Toward this end, we have characterized aspects of AdoMet metabolism related to polyamine biosynthesis and uptake.
Figure 1:
Dose-response curves of CHO and CHO/664
cells to CGP-48664 (A) and the nucleoside analog inhibitor of
AdoMetDC, MDL-73811 (B). The curves represent the CHO
parental line (+), CHO/664 cells grown in 100
µM CGP-48664 prior to retreatment (), CHO/664 cells
grown in the absence of drug for 20 days prior to retreatment (
),
and CHO/664 cells treated with CGP-48664 in the presence of 2
µM spermidine (
). These data represent the average
of at least two separate determinations performed in
duplicate.
Figure 2:
Time dependent growth inhibition by
CGP-48664 in CHO (A) and CHO/664 cells (B). A, CHO cells were treated with 10 and 100 µM CGP-48664 and cell growth was monitored for 5 days. +,
control; , 10 µM CHO/664; +, 100 µM CHO/664. B, the CHO/664 cells were grown in the presence
or absence of 100 µM CGP-48664 for 20 days prior to
retreatment with 100 µM drug. +, 0 days out, 100
µM CGP-48664;
, 20 days out, no drug;
, 20
days out, 100 µM CGP-48664. Standard deviations were
within the size of the symbols.
Figure 3:
Southern blot of genomic DNA isolated from
parental CHO cells and the resistant CHO/664 cells grown either in the
presence or absence of CGP-48664 for 3 and 20 days. A, genomic
DNA (10 µg/lane) was digested with restriction enzymes (BamHI or EcoRI). B, the dot blot represents
0.5, 0.25, and 0.1 µg of DNA (legends are as in the lanes from A). Blots were probed with a P-labeled fragment
of the human AdoMetDC cDNA, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used to adjust for variations in
loading.
Comparative cell line analysis of the banding patterns for EcoRI and BamHI in the genomic Southern blots
indicated that there was no change in gene structure or organization in
the CHO/664 cells. Similar banding patterns between the two cell lines
were also observed following digestion with Pst, HindIII, SacI, Sma, PvuII, HpaII, and MspI (data not shown). In an attempt to
further characterize the nature of the amplified sequences, CHO/664
cells were treated for 72 h with 100 µM hydroxyurea, which
is known to destabilize amplified sequences associated with
extrachromosomal elements(41) . Following such treatment, the
antiproliferative IC value for the CHO/664 cells was found
to be 60 µM as compared to 100 µM for
untreated CHO/664 cells, suggesting that the amplified copies were not
extrachromosomal.
Figure 4: Northern blot analysis of AdoMetDC poly(A) mRNA from CHO and CHO/664 cells showing amplification of AdoMetDC sequences in the resistant cells. Total RNA samples (10 µg) were loaded and probed with either AdoMetDC or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as described under ``Materials and Methods.'' Lane 1, CHO parent cells; lane 2, CHO cells treated with 10 µM CGP-48664 for 72 h. Each lane thereafter contains RNA from CHO/664 cells removed from drug for 0, 3, 7, 15, 20, 28, and 35 days. Fold increase values were determined relative to untreated CHO cells for the 3.4-kb AdoMetDC transcript and normalized for loading using the glyceraldehyde-3-phosphate dehydrogenase transcript. Under exposure conditions, the 2.1-kb transcript was not visible in untreated CHO cells but is known from previous studies (22) to be present.
Figure 5:
Changes in intracellular AdoMet and
dcAdoMet pools in CHO/664 cells following removal of CGP-48664. The
variant line was routinely maintained in 100 µM CGP-48664
at which point (0 days) the drug was removed and cells were sampled
every 3 or 4 days to determine by HPLC the intracellular levels of
AdoMet () and dcAdoMet (+). The levels shown at -3
days are those found in the parental CHO cells.
The effects of various treatments on
both CHO and CHO/664 cells are shown in Table 5. Whereas the
AdoMetDC activity of CHO cells treated with CGP-48664 was completely
inhibited, the AdoMetDC activity of CHO/664 cells grown in the absence
of drug for 20 days and then retreated was much less affected and
polyamine pools were less depleted. Cells were also treated with 100
µM putrescine to determine the metabolic consequences of
providing excess acceptor for aminopropyl transfer to cells with excess
dcAdoMet. In CHO/664 cells grown for 20 days in the absence of
CGP-48664, the addition of putrescine for 72 h caused a near-total
normalization of dcAdoMet pools (from 548 to 15 pmol/10 cells) and a net incorporation of >1550 pmol of aminopropyl
units into spermidine and spermine. This rapid production of polyamines
was not toxic, nor did it provide a growth advantage to cells. It did,
however, result in the down-regulation of both ODC and AdoMetDC, which
probably helped to limit the amount of polyamines produced. During this
same treatment, at similar cell culture densities the media levels of
spermidine and spermine were found to be 21.2 and 1.14 pmol/culture,
respectively, for CHO/664 cells as opposed to 12.5 pmol of
spermidine/culture for the CHO cells; no spermine or acetylated
polyamines were detected. Exogenous spermidine treatment yielded
regulatory responses similar to those of putrescine in the absence of
apparent growth advantage or cytotoxicity.
The above noted
regulatory response of ODC and AdoMetDC was further investigated by
treatment with the spermine analog, DENSPM. Both enzymes were potently
down-regulated in CHO/664 cells after growth for 20 days in the absence
of CGP-48664, and dcAdoMet pools fell by about 50%. Consistent with the
earlier observation that CHO/664 cells had an activated polyamine
transport system, DENSPM accumulated to extraordinarily high levels in
these cells (27,015 pmol/10 cells). Cell growth in these
cells was no more affected at 72 h than in CHO cells that had
accumulated only one-fifth the amount of the analog. Induction of SSAT
also failed to correlate with DENSPM accumulation in CHO and CHO/664
cells. It should be noted that the CHO/664 cells treated with DENSPM
contained numerous cytoplasmic vacuoles, which by electron microscopy
(not shown) were found to be lysosomal elements.
A major incentive in deriving the CHO/664 cells was to determine whether the antiproliferative activity of CGP-48664 is due to inhibition of AdoMetDC activity. The CHO/664 cells clearly indicate that overexpression of that enzyme and resistance to CGP-48664 growth inhibition are causally linked. The findings that CHO/664 cells are cross-resistant to the mechanism-based irreversible inhibitor of AdoMetDC, MDL-73811 (17) and that growth inhibition by CGP-48664 at concentrations as high as 300 µM can be prevented by exogenous spermidine further reinforce this conclusion. Thus, overexpression of AdoMetDC seems to have evolved as an adaptive response to the antiproliferative pressure of sustained exposure to CGP-48664. The possibility cannot be excluded, however, that during the course of that selection, other undefined sites of drug action have also changed.
A number of cell line variants which overproduce the polyamine biosynthetic enzyme ODC have been described (reviewed in (9) ). By contrast, derivation of sublines that similarly overexpress AdoMetDC has not been as readily achieved despite the long-time availability of highly specific and irreversible inhibitors of the enzyme. Although Pajunen et al.(7) reported temporary increases in AdoMetDC activity by transiently transfecting CHO cells with AdoMetDC cDNA, sustained overproduction of the enzyme has not been achieved. Suzuki et al.(42) described variants of mouse FM3A cells resistant to the AdoMetDC inhibitor ethylglyoxal-bis(guanylhydrazone)(38) , which overexpressed AdoMetDC activity by about 5-fold. Although AdoMetDC mRNA was increased, the gene itself did not seem to be affected. The stability of the resistance phenotype in the absence of drug was not described.
Our success in deriving an AdoMetDC-overproducing cell line may reside in the selecting agent used. CGP-48664 is a derivative of MGBG, a potent but nonspecific inhibitor of AdoMetDC(36, 37) . Although a relatively large number of MGBG-resistant sublines have been reported (25, 43, 44, 45, 46, 47) , they were all found to be at least partially deficient in MGBG transport and failed to show increased AdoMetDC expression or function. Unlike MGBG, CGP-48664 is taken up independently of the polyamine transport apparatus(22) , and by design, it is a more specific and more potent inhibitor of the enzyme(21, 22) . It may also be relevant that the CHO cells were selected in Ham's F-12 medium, which contains low levels (0.33 µM) of putrescine, thus allowing AdoMetDC to overexpress in the presence of sufficient precursor to ensure that spermidine and spermine could be synthesized at the levels required for cell growth. Under these circumstances, ODC would not become rate-limiting to polyamine biosynthesis and overexpression of AdoMetDC could then be of direct benefit to the cell.
Genomic DNA analysis revealed that the AdoMetDC gene is amplified by 10-16-fold depending on whether it was quantitated by dot blot analysis or genomic Southern blot analysis, respectively. Such amplified sequences typically localize to two types of abnormal chromosomal structures, expanded chromosomal regions referred to as homogeneously staining regions and double-minute chromosomes, which are extrachromosomal. Since treatment with hydroxyurea, which is known to greatly accelerate the loss of extrachromosomal units(41) , had no effect on the sensitivity of the variants to CGP-48664, we tentatively conclude that the amplified gene copies are probably chromosomally located although this needs to be examined more directly. By genomic Southern analysis, there was no evidence for gene rearrangement or change in organization. Similarly, biochemical analysis indicated that the sensitivity of the CHO/664 enzyme to the inhibitor was unchanged.
In the presence of maintenance levels of CGP-48664 (100 µM), the 10-16-fold amplification of the AdoMetDC gene results in levels of AdoMetDC activity that are only about twice that of the parental line: an amount apparently sufficient to maintain polyamine pools and support cell growth. However, when the inhibitor is removed for long enough to allow for removal of intracellular CGP-48664, the enzyme level eventually increases to about 10-12-fold that of the parent line. The fact that, during this same time, AdoMetDC mRNA levels actually decrease to a level comparable to the gene copy number probably reflects the loss of inhibitor pressure. Once steady-state conditions are achieved, gene copy number, mRNA level, and enzyme activity all seem to be stoichiometrically related.
Even though CHO/664 cells grown in the absence of drug have high levels of AdoMetDC activity, their ODC activity was not decreased relative to control cells as might be expected. Typically, increases in spermine pools such as are apparent in CHO/664 cells grown out of drug for 20 days result in down-regulation of the enzyme(48, 49) . The enzyme, however, has not lost its potential to be down-regulated by polyamines since both ODC and the overproduced AdoMetDC were clearly suppressed by the spermine analog DENSPM, spermidine, or even by putrescine treatment once it is converted to higher polyamines. In addition, the CHO/664 cells seem to have adapted a means to minimize the accumulation of excess spermine. In this regard, it is interesting that even though CHO/664 cells overproduce a key polyamine biosynthetic enzyme, their total polyamine pool is actually lower than that of parent cells due mainly to the apparently rapid conversion of putrescine and spermidine to spermine in the presence of excess dcAdoMet. It should be noted that a small but persistent spermidine pool is always maintained, presumably because small quantities of this particular polyamine are essential for cell growth(15) . In the presence of exogenously provided putrescine as an acceptor for aminopropyl transfer from dcAdoMet, spermine fails to accumulate to cytoxic levels, suggesting that it may be exported out of the cell. Indeed, some indication for this was obtained in media samples.
Perhaps the most impressive finding with
the CHO/664 cells is the massive amounts of dcAdoMet that accumulated
as a result of AdoMetDC overproduction. In the presence of CGP-48664,
dcAdoMet pools were normal (i.e. <5 pmol/10 cells), but when the inhibitor was removed, they increased to
greater than 1200 pmol/10
cells after 3 days and then
declined steadily to levels of 100-200 pmol/10
cells
after 30 days as compared to <5 pmol/10
cells in the
parent line. The reason for this decline is not immediately apparent
since, paradoxically, AdoMetDC activity was seen to increase during
this time. One possibility is that there may be increased availability
of putrescine and spermidine to serve as aminopropyl acceptors. Unlike
putrescine, dcAdoMet is ordinarily found in extremely low levels in
cells and is generally regarded as rate-limiting to polyamine
biosynthesis. Thus, in the presence of excess dcAdoMet, such as in
CHO/664 cells grown in drug-free medium for >20 days, putrescine
becomes rate-limiting in polyamine biosynthesis. Since providing the
CHO/664 cells with exogenous putrescine tends to normalize polyamine
pool profiles, it would appear that under steady-state conditions, the
polyamine pool distribution in normal cells is probably maintained by
the combined regulation of both dcAdoMet and putrescine. Transient
increases in dcAdoMet similar in magnitude to those seen here have also
been reported during treatment with DFMO(50, 51) . In
this case, the elevated metabolite is due to a compensatory increase in
AdoMetDC activity related to ODC inhibition and to the absence of
putrescine and spermidine to serve as aminopropyl acceptors.
It is interesting that despite the overexpression of AdoMetDC and the accumulation of massive amounts of dcAdoMet, AdoMet pools remain relatively unchanged. This may be partially explained by the depletion of acceptor molecules such as putrescine and spermidine. However, when cells were provided with sufficient acceptor (putrescine) to rapidly deplete dcAdoMet pools, AdoMet pools were still not decreased, suggesting that their levels are strictly controlled in the cell. Although AdoMet synthase is generally regarded as a stable enzyme, these findings suggest that it may be regulated under certain circumstances.
Finally, we have observed that like many
ODC-overproducing variants, the CHO/664 cells seem to have an enhanced
or deregulated ability to transport polyamines as indicated by their
high accumulation of the polyamine analog DENSPM and to increased V values for spermidine uptake. The finding is
unexpected since cells that overexpress polyamine biosynthetic enzymes
would not seem to need polyamines from extracellular sources. In the
case of ODC overproducers, one possible explanation for increased
transport is based on the recent suggestion (52) that both ODC
and the polyamine transporter are regulated by the same unstable
protein, antizyme(13) . Thus, in the presence of excess ODC,
antizyme levels are depleted so that none is available to down-regulate
transport. While this remains a possibility, the present finding that
AdoMetDC overproducers display a similar perturbation in transport
argues against it since there is no known indication that antizyme is
involved in the regulation of this enzyme. It would appear from the
uptake studies presented in Table 6that the increased transport
is due to an insensitivity of the transporter to down-regulation by
polyamines and their analogs. Although this could be due to changes in
the transport system, it could, in the case of DENSPM, also involve
intracelllular sequestration of the analog.