©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stable Amplification of the S-Adenosylmethionine Decarboxylase Gene in Chinese Hamster Ovary Cells (*)

(Received for publication, August 31, 1994; and in revised form, October 25, 1994)

Debora Kramer (1) Helmut Mett (2) Amanda Evans (2) Urs Regenass (2) Paula Diegelman (1) Carl W. Porter (1)(§)

From the  (1)Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263 and the (2)Pharmaceuticals Division, Research Department, CIBA-GEIGY Limited, K-125.4.10, CH-4002 Basel, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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/10^6 cells to 1000-1500 pmol/10^6 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/10^6 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.


INTRODUCTION

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) (^1)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, alpha-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.


MATERIALS AND METHODS

Chemicals and Plasmids

CGP-48664 and CGP-39937 were synthesized as described earlier(19, 20) . DENSPM was generously provided by Dr. Raymond Bergeron(23, 24) . DFMO was provided by Marion Merrel Dow Inc. (Cincinnati, OH). MGBG was obtained from Aldrich (Milwaukee, WI). The nucleoside analog inhibitor of AdoMetDC, S-(5`-deoxy-5`-adenosylmethylthioethylhydroxylamine (AMA) was synthesized (14) and generously provided by Drs. Radii and Alex Khomutov (Russian Academy of Sciences, Moscow). MDL-73811 was synthesized with certain modifications to published procedures (17) and kindly provided by Drs. Canio Marasco and Janice Sufrin (Roswell Park Cancer Institute). Putrescine, spermidine, spermine, and hydroxyurea were obtained from Sigma. The pCM9 plasmid construct containing the coding region for the cDNA of human AdoMetDC (7) was kindly provided by Dr. Bruce Stanley (Hershey Medical Center, Hershey, PA). The human glyceraldehyde-3-phosphate dehydrogenase cDNA probe was obtained from Clontech Laboratories, Inc. (Palo Alto, CA) and used to quantitate loading differences. [P]dCTP was obtained from DuPont NEN. The oligolabeling kit was obtained from Pharmacia Biotech Inc. All restriction enzymes were acquired from Boehringer Mannheim.

Maintenance of CHO Cell Lines

The monolayer CHO cells (originating from American Type Culture Collection CCL61, CHO-K1 cells) were grown in 1:1 ratio of Ham's F-12 and Dulbecco's modified Eagle's media (Life Technologies, Inc.) containing 10% fetal calf serum (Atlanta Biologicals, Norcross, GA.), 1% non-essential amino acids (Life Technologies, Inc.), 1% glutamine (Life Technologies, Inc.), 1% sodium pyruvate (Life Technologies, Inc.), and 1 mM aminoguanidine (Sigma). It should be noted that this commercial media formulation contains 0.33 µM putrescine. For growth inhibition studies with DFMO, the CHO cells were adapted to alpha-MEM (Life Technologies, Inc.) medium, which does not contain putrescine. Cell cultures were maintained at 37 °C with 5% CO(2) in a humidified incubator. Vented T-75-cm^2 flasks (Falcon, Becton-Dickinson, Bedford, MA) were used routinely. These cells were detached by 1 mM EDTA or 1 times trypsin (Life Technologies, Inc.) with prompt removal of excess trypsin. CHO cells resistant to methylglyoxal-bis(guanylhydrazone) (CHO/MGBG) were obtained from Dr. W. Flintoff (University of Western Ontario, London, Ontario, Canada; (25) ) and routinely grown in RPMI 1640 medium containing 100 µM MGBG.

CGP-48664-resistant CHO Cells (CHO/664)

The parent CHO cell line was chronically exposed to increasing levels of CGP-48664 for several weeks starting at 3 µM with stepwise increments up to 150 µM. After 3 months a resistant colony was isolated and developed into the subline designated CHO/664(22) . These cells are routinely cultured in Ham's F-12/Dulbecco's modified Eagle's media described above containing 100 or 150 µM CGP-48664. Studies were performed with CHO/664 cells grown in the presence of the inhibitor or after removal of the inhibitor for lengths of time as indicated.

Cell Growth Studies

Exponentially growing cells were seeded for each experiment at a density of 1.5 times 10^5 cells for the parent CHO cells and 4.5 times 10^5 cells for the resistant CHO/664 cells. These different initial densities were employed to compensate for the difference in growth rates between the two cell lines (doubling times for the CHO and CHO/664 cells were 14 and 24 h, respectively). Cells were exposed to various concentrations of AdoMetDC inhibitors MGBG, CGP-48664, AMA, and MDL-73811 and an ODC inhibitor, DFMO, from 0.03 µM to 1 mM to determine the IC values. For growth inhibition studies with DFMO, the CHO cells were adapted to alpha-MEM (Life Technologies, Inc.) medium, which does not contain putrescine. CHO/664 cells grown in the absence of drug for 20 days were treated with 100 µM hydroxyurea for 72 h prior to re-exposure to CGP-48664. Unless otherwise indicated, all growth and biochemical assays were carried out for 72 h. Cell growth was determined by electronic particle counting. For growth inhibition prevention studies, exogenous spermidine (Spd) was added concurrently with CGP-48664.

Spermidine Transport

Spermidine transport was evaluated by V(max) values determined as described previously (26) . Cells were seeded into six-well plates at a density of 1 times 10^4 cells/well and allowed to grow with or without 10 µM DENSPM for 72 h prior to performing the assay.

Polyamine Enzyme Activities

Following various cell treatments, ODC and AdoMetDC activities were determined from cellular extracts prepared and measured as described by Porter et al.(27) . SSAT activity was determined as described elsewhere(28) . For isolated enzyme sensitivity studies, partially purified AdoMetDC enzyme preparations were generated by liberating the enzyme from various cells and tissues via freeze-thawing. Endogenous inhibitors were removed from cell extracts by ammonium sulfate precipitation and extensive dialysis. Extracts were then incubated for 60 min at 37 °C in the presence and absence of the inhibitors CGP-48664, MGBG, CGP-39937, and AMA to determine the concentration required to inhibit AdoMetDC activity by 50% (IC values).

Polyamine and Polyamine Analog Pools

Intracellular polyamines were extracted from cell pellets with 0.6 N perchloric acid(28) , dansylated, and measured by reverse phase HPLC as described previously (27) with the following modifications. Dansylated samples (100 µl) were injected onto a 25-cm Econosil C18 column (5-µm particle size, Alltech, Deerfield, IL) with a column temperature of 50 °C and eluted by a two-solvent gradient using a Waters 590 programmable pump (Waters Associates, Milford, MA), a Waters WISP 710B autosampler, and a low pressure gradient Autochrom M112-3 CIM generator (Autochrome, Milford, MA). Solvent A contained 55% 10 mM ammonium phosphate, 45% acetonitrile at pH 4.4. Solvent B contained 100% acetonitrile. At 1.8 ml/min, the gradient began at 100% solvent A and progressed linearly to 82% solvent B over 30 min with a 15-min hold. Compounds were detected using a McPherson FL-750 fluorescence detector (McPherson, Acton, MA) with excitation wavelength of 340 nm and emission wavelength of 520 nm. The data were collected using an interface instrument NAI 7625B (Nelson Analytical Instruments), a Hewlett Packard HP9816 computer and software XTRACHROM, version 7.2 and M320, version 1.0 (Hewlett Packard).

Media Preparation for HPLC Analysis

To 6 ml of media, 1.5 ml of 50% trichloroacetic acid was added. The samples were placed on ice for 15 min and spun at 15,000 times g for 15 min. The supernatants were collected, 12 ml of 0.01 M ammonium phosphate buffer (pH 8) was added, and the pH was readjusted to pH 8 prior to column extraction. Each solution was loaded into a Bond Elut carboxylic acid solid phase preparatory column (Analytichem International, Harbor City, CA). The column was prewashed with methanol followed by ammonium phosphate buffer. Samples were loaded, washed with buffer, and eluted with 1 ml of 0.1 N HCl in methanol. Each sample was dried under nitrogen at 37 °C, reconstituted with 150 µl of distilled water, and dansylated as described previously(29) .

AdoMet and dcAdoMet Pools

HPLC analysis of intracellular AdoMet and metabolites was performed with the same perchloric acid extracts used for polyamine pool analysis. The chromatographic conditions were adapted from Yarlett and Bacchi (30) with the following modifications. Samples (50 µl) were eluted from a C18 column (21 °C) at a flow rate of 1 ml/min with a linear gradient starting with solvent A (0.1 M NaH(2)PO(4), 8 mM octane sulfonic acid, 0.05 mM EDTA, 2% acetonitrile) at 85% and solvent B (0.15 mM NaH(2)PO(4), 8 mM octane sulfonic acid, 26% acetonitrile) at 15%. Over the course of 40 min, the gradient increased to 100% solvent B. Effluent was monitored with a Waters 481 UV detector, and data processed using instrumentation described for polyamine analysis.

Intracellular Drug Accumulation

The CHO/664 and the CHO/MGBG cell lines were grown for 3 weeks in the absence of CGP-48664 and MGBG, respectively, and prior to harvest the subconfluent cultures were treated for 24 h with 1 µM CGP-48664 or 0.2 µM MGBG. Cellular extracts were prepared and analyzed for CGP-48664 as described(21, 22) . Data presented in Table 2were generated by this method. Subsequently, it was realized that CGP-48664 could be detected as a single peak in chromatograms of AdoMet and metabolites (see above). Its identity in cell extracts was confirmed by peak spiking with authentic CGP-48664. It was quantitated by the addition of known amounts of CGP-48664 to cell lysates and by the inclusion of the inhibitor in the mixture of HPLC standards. Thus, the same HPLC methodology used to measure AdoMet and dcAdoMet (30) was employed to measure CGP-48664 or MGBG. CGP-48664 data presented in Table 5were generated by this means.





Northern Blot Analysis

Methods follow those reported in greater detail elsewhere(31, 32) . To reduce nonspecific binding of the AdoMetDC probe to total RNA, membrane hybridizaton at 65 °C was followed by longer washing times at 65 °C and a final wash at 68 °C. This more stringent washing of membranes containing total RNA resulted in banding patterns for AdoMetDC mRNA that were similar to those achieved with poly(A) RNA. Autoradiograms were scanned and bands of RNA or DNA quantitated using a Molecular Dynamics densitometer with ImageQuant Software (Molecular Dynamics, Sunnyvale, CA).

Southern Blot Analysis

Genomic DNA was isolated using the buffers for cell lysis described by Anderson et al.(33) and proteinase K, RNase, and phenol extraction procedures as described by Maniatis(34) .


RESULTS

Growth Characteristics

The IC dose of CGP-48664 was found to be 0.15 µM for the CHO cells and >1500-fold higher (250 µM) for the CHO/664 cells (Fig. 1, Table 1). At concentrations up to 300 µM CGP-48664, growth inhibition of CHO/664 cells was preventable with 2 µM exogenous spermidine, indicating that even at this high concentration, the antiproliferative effect was due to interference with polyamine biosynthesis. Additionally, exogenous spermidine did not compete with CGP-48664 for uptake since the intracellular content was 237 pmol/10^6 cells, only slightly lower than that found in cells treated with 300 µM CGP-48664 alone, which was 300 pmol/10^6 cells. Time-dependent growth characteristics in the presence and absence of CGP-48664 of the CHO parent and resistant (CHO/664) cell lines are shown in Fig. 2. Whereas the parent line was growth-inhibited with 10 µM and 100 µM CGP-48664, the CHO/664 cells continued growing in 100 µM drug. Exponentially growing CHO parental cells doubled every 14 h, CHO/664 cells grown continuously in 100 µM CGP-48664 doubled every 24 h, and CHO/664 cells removed from the drug for 20 days doubled every 18 h (Fig. 2B). Resistance was maintained in CHO/664 grown continuously in the drug or in the absence of the drug for 20 days, thereby indicating the stability of the resistant phenotype. In addition to growing more slowly than CHO cells, the variants appeared larger microscopically and were found to contain 1.65-fold greater protein per cell (not shown).


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 (bullet), 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; bullet, 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; bullet, 20 days out, no drug; , 20 days out, 100 µM CGP-48664. Standard deviations were within the size of the symbols.



Cross-resistance

Although CGP-48664 shares certain structural features with MGBG, the CHO/664 cells were much less (i.e. 5-10-fold) resistant to MGBG than to the derivative (Table 1). This finding is not unexpected, since MGBG has various other actions unrelated to AdoMetDC (36, 37, 38) and since the CHO/664 cells were found to accumulate greater amounts of MGBG (see below in Table 2). The sensitivity of CHO/664 cells to other AdoMetDC inhibitors regarded as highly specific for the enzyme was also examined. Dose-response evaluations (Table 1, Fig. 1B) after 72 h of treatment revealed that relative to the parental cells, the CHO/664 cells were >100-fold cross-resistant to the mechanism-based nucleoside analog inhibitor, MDL-73811(17) . They were also resistant to another nucleoside analog inhibitor, AMA, but because of low potency in the parent line (200 µM), the magnitude of the resistance in the CHO/664 cells could not be quantitated (Table 1). Although the CHO/664 cells exhibited increased polyamine transport (Table 2), their growth sensitivity to the polyamine analog N^1,N-bis(ethyl)norspermine (DENSPM), which utilizes the polyamine transporter to enter cells and disrupts polyamine homeostasis(35) , was similar to that of the parental line. The CHO/664 cells were only weakly resistant to the ODC inhibitor, DFMO(8) .

Transport Characteristics

The CHO/664 subline was examined to determine whether resistance involved decreased inhibitor transport and accumulation. The amount of intracellular CGP-48664 following 24 h of treatment was compared in the parent CHO and CHO/664, as well as in the subline CHO/MGBG, which is known to be deficient in polyamine transport capability(25) . As shown in Table 2, CGP-48664 accumulated to similar levels in all three cell lines, indicating that a deficiency in transport was not responsible for resistance in the CHO/664 cells and that the inhibitor was not dependent on the polyamine uptake system for entry into the CHO/MGBG cells. By contrast, MGBG, which is known to enter via the spermidine uptake system(36, 37) , accumulated to much higher levels than CGP-48664 in both the CHO and CHO/664 cells. The dependence of MGBG uptake on polyamine transport is further indicated by its reduced accumulation in CHO/MGBG cells. Interestingly, the CHO/664 cells actually accumulated 2-fold more MGBG than the parent line, suggesting that increased polyamine uptake may be a functional or incidental feature of the resistant phenotype. This was further confirmed by kinetic studies with radiolabeled spermidine (Table 2), which revealed that the V(max) was increased 3-4-fold to 7100 pmol/mg protein/h in the CHO/664 cells and the K(m) increased somewhat. As expected(25) , the V(max) for spermidine was negligible (23 pmol/mg protein/h) in the CHO/MGBG cells.

AdoMetDC Gene Copy

Genomic DNA from parental cells and CHO/664 cells grown out of CGP-48664 for 3 and 20 days was analyzed (Fig. 3). There was a marked increase in certain bands hybridizing with the AdoMetDC cDNA in the CHO/664 cells, which, by densitometric quantitation of the dot blot analysis, were found to be increased 10-fold compared to the parental line. This increase in gene copy remained constant in the variant cells grown in the absence of drug for 20 days. As shown by digestion with BamHI, one major amplified band (18 kb) and two nonamplified minor bands (presumed to be pseudogenes; see Refs. 7, 39, and 40) hybridized to the AdoMetDC probe. When only the amplified bands were compared by densitometric readings, the apparent gene copy differential was 16 ± 3.5-fold as opposed to 10-fold by dot blot analysis.


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.

AdoMetDC mRNA Expression

Northern blot analysis of CHO and CHO/664 cells (Fig. 4) revealed two transcripts (3.4 and 2.1 kb), which hybridized to the AdoMetDC cDNA probe (7) and probably represent variations in polyadenylation(7) . Treatment of parental CHO cells with 10 µM CGP-48664 for 72 h resulted in a 3.3-fold increase in AdoMetDC mRNA due presumably to decreases in polyamine pools(7) . By contrast, CHO/664 cells grown continuously in 100 µM CGP-48664 contained AdoMetDC mRNA levels 66-fold higher than untreated CHO cells. When removed from the inhibitor, AdoMetDC mRNA levels for both transcripts declined steadily with time over a period of 3-7 days and remained at levels 20 times those of CHO to at least day 35. Thus, for both CHO and CHO/664 cells, the level of AdoMetDC mRNA is about 3 times higher in the presence of inhibitor than in its absence.


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.



Sensitivity of Isolated AdoMetDC

Although not indicated by restriction enzyme digest analysis of CHO/664 genomic DNA, the possibility that the sensitivity of AdoMetDC enzyme to CGP-48664 might be altered was considered. Thus, sensitivity of enzyme extracts isolated from parent cells, CHO/664 cells and rat liver to inhibition by MGBG, CGP-48664, CGP-39937(21) , and the nucleoside analog inhibitor AMA (15) were examined (Table 3). By comparing the doses required to inhibit activity by 50% (IC values), the enzymes from all three sources were found to be similarly sensitive to each of the inhibitors, suggesting that the resistant phenotype does not involve protein structural changes.



AdoMet/dcAdoMet Pool Kinetics

CHO/664 cells were grown in the presence of 100 µM CGP-48664 and in its absence for up to 40 days in order to examine the temporal sequence for AdoMet and dcAdoMet pool fluctuations (Fig. 5, Table 4). AdoMet pools remained relatively constant under all the treatment conditions. By contrast, dcAdoMet increased from almost undetectable levels (20 pmol/10^6 cells) in the presence of inhibitor to 1200 pmol/10^6 cells at 3 days following inhibitor removal. This increase was followed by a steady decline, which stabilized at 100-200 pmol/10^6 cells at 20-40 days (Fig. 5) as compared to the steady-state levels in parent cells of <5 pmol/10^6 cells (Table 4). Thus, CHO/664 cells maintained in the absence of drug for extended periods retained significantly higher levels of dcAdoMet than CHO cells.


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 (bullet) and dcAdoMet (+). The levels shown at -3 days are those found in the parental CHO cells.





Polyamine Metabolic Effects

The key polyamine enzymes, polyamine pools, and AdoMet/dcAdoMet pools were compared in the parent cells and in CHO/664 cells grown in the presence and absence of CGP-48664 for varying times (Table 4). When CHO cells are compared with CHO/664 cells grown continuously in 100 µM inhibitor, the differences in polyamine enzyme and various pool profiles were not especially profound. AdoMetDC activity, dcAdoMet, and putrescine pools were all elevated 2-4-fold. However, when the cells were grown in the absence of inhibitor, more dramatic changes were seen. After 3 days in the absence of drug, AdoMetDC activity increased only 3-fold presumably because there was still inhibitor contained within the cells, and dcAdoMet increased to 1245 pmol/10^6 cells without appreciably affecting AdoMet levels, which remained steady at around 70-110 pmol/10^6 cells. Polyamine pools were shifted toward spermine-putrescine, and spermidine pools decreased while spermine pools increased. This latter effect would seem to reflect the availability of excess dcAdoMet and its importance in regulating polyamine pool profiles. At 20 days in the absence of inhibitor, CHO/664 cells showed a 10-fold increase in AdoMetDC activity with no decrease in ODC activity. Decarboxylated AdoMet levels remained elevated at 550 pmol/10^6 cells, and, as before, polyamine pools were shifted toward spermine accumulation. After 20 days, AdoMetDC activity became extremely variable (ranging from 4 to 40 nmol/h/mg) despite the stably elevated levels of both AdoMetDC mRNA and dcAdoMet. Possibly, this was due to regulatory fluctuations by the enzyme in response to varying intracellular spermine levels.

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/10^6 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/10^6 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.

Regulation of Polyamine Uptake

The basis for the increased uptake of DENSPM by CHO/664 cells was further investigated (Table 6). From spermidine uptake studies, it was found that relative to CHO cells, the spermidine V(max) of the variants grown in the absence of drug was about 30% higher. Perhaps more importantly, it was found that 72 h of treatment with DENSPM lowered the spermidine V(max) by 90% in the CHO cells and only 50% in the CHO/664 cells despite the fact that they accumulated enormous levels of the analog.




DISCUSSION

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/10^6 cells), but when the inhibitor was removed, they increased to greater than 1200 pmol/10^6 cells after 3 days and then declined steadily to levels of 100-200 pmol/10^6 cells after 30 days as compared to <5 pmol/10^6 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(max) 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.


FOOTNOTES

*
This work was supported in part by Grants CA-22153 (to C. W. P.) and CA-16406 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, NY 14263.

(^1)
The abbreviations and trivial names used are: ODC, ornithine decarboxylase; AdoMet, S-adenosylmethionine; AdoMetDC, S-adenosylmethionine decarboxylase (EC 4.1.1.50); AMA, S-(5`deoxy-5`adenosyl)methylthioethylhydroxylamine; CGP-48664, 4-(aminoiminomethyl)-2,3-dihydro-1H-inden-1-one-diaminomethylenehydrazone; CGP-39937, [2,2`-bipyridine]-6,6`-bis(carboximidamide); CHO cells, Chinese hamster ovary cells; CHO/664, CHO cells resistant to CGP-48664: CHO/MGBG, CHO cells resistant to MGBG; dcAdoMet, decarboxylated S-adenosylmethionine; DFMO, alpha-difluoromethylornithine; MDL-73811, 5`{[(Z)-4-amino-2-butenyl]methylamino}-5`-deoxyadenosine, also known as AbeAdo; MGBG, methylglyoxal-bis(guanylhydrazone); SSAT, spermidine/spermine N^1-acetyltransferase; DENSPM, N^1,N-bis(ethyl)norspermine; Put, putrescine; Spd, spermidine; Spm, spermine; HPLC, high pressure liquid chromatography; kb, kilobase pair(s); dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.


ACKNOWLEDGEMENTS

We gratefully acknowledge the skilled technical assistance of Laetitia Duboc, Iris Oberkirch, Zenghui Mi, John Miller, Robert Reuter, and Barbara Schacher Fritz Wenger and invaluable consultations with Dr. Mirjana Fogel-Petrovic and Dr. Jennifer Black.


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