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J. Biol. Chem., Vol. 275, Issue 37, 28708-28714, September 15, 2000

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Altered Spermidine/Spermine N¹-Acetyltransferase Activity as a Mechanism of Cellular Resistance to Bis(ethyl)polyamine Analogues^*

Diane E. McCloskey and Anthony E. Pegg

From the Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

Received for publication, May 15, 2000, and in revised form, June 28, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To develop a model system to investigate mechanisms of antiproliferative action of bis(ethyl)polyamine analogues, intermittent analogue treatments followed by recovery periods in drug-free medium were used to select an N¹,N¹²-bis(ethyl)spermine-resistant derivative of the Chinese hamster ovary cell line C55.7. The resulting C55.7Res line was at least 10-fold resistant to N¹,N¹²-bis(ethyl)spermine and N¹,N¹¹-bis(ethyl)norspermine. The stability of the resistance in the absence of selection pressure was 9 months, indicating that a heritable genotypic change was responsible for the resistance phenotype. Polyamine transport alterations and multi-drug resistance were eliminated as causes of the resistance. Spermidine/spermine N¹-acetyltransferase (SSAT) activity and regulation were altered in C55.7Res cells as basal activity was decreased, and no activity induction resulted from exposure to analogue concentrations, which caused 300-fold enzyme induction in parental cells. SSAT mRNA levels in the absence and presence of analogue were unchanged, but no SSAT protein was detected in C55.7Res cells. A point mutation, which results in the change leucine156 (a fully conserved residue) to phenylalanine, was identified in the C55.7Res SSAT cDNA. Expression of wtSSAT activity in C55.7Res cells restored sensitivity to bis(ethyl)polyamines. These results provided definitive evidence that SSAT activity is a critical target of the cytotoxic action of these analogues.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

There is an ever-present need for better chemotherapeutic agents for the clinical treatment of cancer, because most of the best drugs available are far less than 100% effective and must be used at doses that cause significant undesirable side effects. The rational design of better antineoplastic agents depends on understanding cellular mechanisms involved in cytotoxicity and/or cellular escape from cytotoxicity. One class of compounds, which has recently shown clinical promise, is structural analogues of the natural polyamines that are essential for cellular growth and differentiation (1-4). These agents were designed to mimic the self-regulatory functions of the natural polyamines but not substitute functionally for cellular polyamine requirements (5-9). Two bis(ethyl) analogues of spermine, BE 3-4-3 and BE 3-3-3¹ have been shown to have cytotoxic or cytostatic effects in model systems for non-small cell lung cancer, melanoma, pancreatic cancer, breast cancer, and prostate cancer (1, 10-12), and BE 3-3-3 is currently in phase I clinical trials (4). These analogues have been shown to down-regulate polyamine synthesis, deplete intracellular polyamine pools, inhibit cell growth, and activate programmed cell death pathways. Additionally, an apparent correlation has been demonstrated between the cytotoxic effects of BE 3-3-3 and BE 3-4-3 and their ability to induce the rate-limiting enzyme of polyamine catabolism, SSAT (1, 4, 10-12). A number of other structural polyamine analogues have also been developed, including some that are unsymmetrically alkylated and some bis(ethyl) analogues in which the numbers of carbon atoms in the internal part of the polyamine structure have been modified (13-16). Many of these compounds have been tested as chemotherapeutic agents with several showing promising antineoplastic activity against a variety of tumor types (13, 14, 16-21). Unfortunately, however, rather than clarifying the mechanisms of action, these studies have demonstrated that it is difficult to delineate the specific mechanisms of action that are integral to the cytotoxic action of the structural polyamine analogues. For example, for two analogues that show significant cytotoxic activity, CHENSpm and BE 4-4-4-4, it has been demonstrated that both analogues are poor inducers of SSAT. In addition, CHENSpm treatment does not deplete natural polyamine pools, and BE 4-4-4-4 does not activate programmed cell death pathways in prostate cancer cells, which are activities that have been linked to the effective action of the bis(ethyl) polyamine analogues BE 3-4-3 and BE 3-3-3 (18, 20, 21).

One approach to understanding cellular mechanisms of cytotoxicity is to develop a model cell system that is resistant to the chemotherapeutic agent of interest and to utilize that system to determine the changes that are responsible for cellular escape from cytotoxicity. The current studies were carried out using this approach to develop a model system of resistance to the bis(ethyl)polyamine analogues. We have used the analogue BE 3-4-3 to select a resistant derivative of the Chinese hamster ovary cell line C55.7 (22). This cell line was chosen to help ensure that mutants displaying transport alterations would not be selected. C55.7 cells lack ODC activity because of a point mutation in the ODC gene, which results in production of an inactive mutant ODC protein. Therefore, C55.7 cells are unable to synthesize the polyamine putrescine and must rely on salvage of putrescine from the external environment. This auxotrophy for putrescine requires that the polyamine transport system (23-25) of these cells be functional in order for the cells to survive. Because the structural polyamine analogues of interest also enter the cell through the specific polyamine transport system (1), use of the C55.7 cell line should favor selection of resistant mutants that do not have alterations in polyamine transport but rather exhibit mechanisms of resistance related to intracellular mechanisms of cytotoxicity. Following the selection, the cell line was characterized with respect to mechanisms of the observed resistance. These studies have demonstrated that altered SSAT regulation is a cause of the resistance and have provided definitive evidence that SSAT activity is a requisite part of the cytotoxic action of the tested bis(ethyl)polyamine analogues.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [1-¹⁴C]Acetyl-CoA (63 Ci/mol) was obtained from ICN Biochemicals (Costa Mesa, CA). LipofectAMINE and Geneticin were purchased from Life Technologies, Inc. (Rockville, MD). BE 3-4-3 and BE 3-3-3 were kindly provided by Dr. Raymond Bergeron (University of Florida, Gainesville, FL). Putrescine, MTT, MGBG, and cisplatin were purchased from Sigma (St. Louis, MO).

Cell Culture-- The Chinese hamster ovary cell line C55.7 (22), a kind gift from Dr. Immo Scheffler (University of California at San Diego, La Jolla, CA), and its derivatives were maintained in minimum essential -medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Atlanta Biological, Norcross, GA), 100 µM putrescine, 100 units/ml penicillin, and 100 units/ml streptomycin. Cultures were incubated at 37 °C in a humidified 5% CO₂ atmosphere and were passaged every 5-7 days to maintain exponential growth. For all experiments, concentrated solutions of BE 3-3-3 and BE 3-4-3 (10 mM and 1 mM, respectively, in water, stored at 20 °C) were diluted with medium to the desired concentrations.

Growth Inhibition Assay-- Exponentially growing cells were plated in triplicate at 2 × 10³ cells/cm² in 100 µl of medium/well in 96-well plates. After a 12- to 18-h period for the cells to attach, 200-µl medium containing 1.5× the desired final drug concentration was added. Cells were incubated in the absence or presence of at least six drug concentrations for 144 h, at which time, the medium was aspirated and 100 µl of 5 mg/ml MTT in Optimem (Life Technologies, Inc.) was added. The cells were incubated an additional 4-6 h at 37 °C, after which 100 µl of 50% EtOH in Me₂SO was added to each well. After 20 min, the A₅₇₀ (a value directly proportional to the number of viable cells (26) was determined using a Bio-Rad plate reader. IC₅₀ values were determined from plots of percentage of untreated control cell number versus the logarithm of the drug concentration.

Transfection of pCMV-SSAT-- The transfection of pCMV-SSAT into C55.7 and C55.7Res cells was accomplished using LipofectAMINE, as described previously (27), but with the addition of 100 µM putrescine to the culture medium.

Analysis of SSAT Activity-- Exponentially growing cells were plated in triplicate at 2-4 × 10⁴ cells/cm². Following attachment, the medium was changed and cells were incubated for the desired time. SSAT activity was determined in cell extracts by an assay that measures the incorporation of radioactivity from [1-¹⁴C]acetyl-CoA into [1-¹⁴C]acetylspermidine in 10 min at 30 °C as described previously (28). A standard assay mixture contained 50 mM Tris-HCl (pH 7.8), 3 mM spermidine, and 12.7 µM (63 mCi/mmol) [1-¹⁴C]acetyl-CoA in a total volume of 100 µl.

Analysis of Intracellular Polyamine Content-- Cells were plated as described for SSAT activity determination and then harvested and extracted with 10% (w/v) trichloroacetic acid. Aliquots were assayed for polyamine content using ion-paired, reversed phase high performance liquid chromatography and post-derivatization with o-phthalaldehyde as described previously (29).

Western Analysis-- Proteins present in cell extracts were resolved by SDS-polyacrylamide gel electrophoresis using a 15% gel. Electrotransfer to polyvinylidene difluoride membrane (Micron Separations Inc., Waterborough, MA) was followed by hybridization with a polyclonal anti-SSAT antibody (prepared as described previously (30)) and detection using the Vistra Western blot detection kit (Amersham Pharmacia Biotech). A Molecular Dynamics FluorImager model 595 and ImageQuaNT application software were used for visualization and quantitation.

Northern Analysis-- Total cellular RNA was prepared using the Totally RNA kit from Ambion (Austin, TX). Northern analysis was carried out using a Hybond N⁺ membrane according to the manufacturer's instructions (Amersham Pharmacia Biotech). Ethidium bromide staining was used as a loading control. A fluorescein-labeled full-length SSAT probe, to which membranes were hybridized, was prepared by transcription from the T3 promoter of the pSAT9.3 plasmid containing the SSAT cDNA in the Bluescript vector (31). The Vistra signal amplification kit was used for signal detection, and visualization and quantitation were the same as for the Western analysis.

RT-PCR-- Reverse transcription of total cellular RNA from C55.7 and C55.7Res cells was carried out using SuperScript II RNase H reverse transcriptase (Life Technologies, Inc.). PCR to enrich for SSAT cDNA was accomplished according to the manufacturer's instructions using the Expand High Fidelity PCR system (Roche Biochemicals, Indianapolis, IN) with sense primer 5'-GGGAAGAAAAGCAAAAGACG and antisense primer 5'-AATGGAGGTTGTCATCTACAGC. Triplicate RT and PCR reactions were carried out for each cell line followed by sequencing of the products by the Pennsylvania State University College of Medicine Macromolecular Core Facility.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Selection of a Cell Line Resistant to Bis(ethyl)polyamine Analogues-- The polyamine analogue BE 3-4-3 was used to select a resistant derivative of the Chinese hamster ovary cell line C55.7 (22). The method used for selection was a defined period of exposure to the drug (96-144 h) followed by a recovery period in drug-free medium until the cells regained exponential growth (32). As the population of resistant cells increases, the recovery period becomes shorter (32). Selection of a BE 3-4-3-resistant derivative of the C55.7 cell line was accomplished through four cycles of drug treatment and recovery as shown in Table I. The short recovery period following the fourth treatment suggested that this cell population was resistant to BE 3-4-3. To confirm resistance to the similar polyamine analogue BE 3-3-3, the cells were exposed to 100 µM BE 3-3-3 for an additional 120-h period during which their growth was observed to be only slightly slower than that of the untreated parental C55.7 cells. The resistant cell line was then designated C55.7Res and was routinely maintained in drug-free medium.

Level and Stability of the C55.7Res Resistance-- The level of resistance was assessed using 144-h growth inhibition assays. The IC₅₀ values for BE 3-3-3 were determined to be 7 and 70 µM, respectively, for the C55.7 and C55.7Res cell lines, indicating 10-fold resistance of the C55.7Res cell line to the polyamine analogue. Another indication of the resistance of the C55.7Res cell line is that these cells can be continuously passaged in 100 µM BE 3-3-3 while 10% of the parental C55.7 cells survive 96-h exposure to 10 µM BE 3-3-3. To determine whether the observed resistance phenotype was a stable trait, the C55.7Res cells were maintained in drug-free medium and the IC₅₀ values relative to the parental C55.7 cells were monitored over time. The average IC₅₀ values of 6.2 ± 1.3 µM for C55.7 (n = 8) and 63.0 ± 18 µM C55.7Res cells (n = 8), determined over a 9-month period and 40 cell passages in the absence of selective pressure, indicated that the 10-fold resistance of the C55.7Res cells to BE 3-3-3 was stable and was the result of a heritable genotypic change in the C55.7Res cell line.

Uptake of BE-3-3-3 into C55.7 and C55.7Res Cells-- To verify that the C55.7Res resistance to bis(ethyl)polyamine analogues did not result from an altered ability of the cells to transport the analogue, the intracellular level of BE 3-3-3 was measured for C55.7 and C55.7Res cells following 15- and 30-min exposure to 10 µM BE 3-3-3. The results (Fig. 1) indicated that the uptake of BE 3-3-3 by C55.7Res cells did not differ significantly from that of the parental C55.7 cells over the 30-min period. Additionally, the reduced uptake of the analogue in the presence of exogenous putrescine confirmed that the analogue was competing with putrescine for uptake by the polyamine transport system.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Uptake of BE 3-3-3 by C55.7 and C55.7Res cells. C55.7 and C55.7Res cells were incubated in the presence of 10 µM BE 3-3-3 for 15 or 30 min, in the absence or presence of 100 µM putrescine. After the incubation period, cells were harvested and the intracellular BE 3-3-3 content was determined as described in "Experimental Procedures." Values reported are from triplicate cell samples and are ±S.E.

Sensitivity to Other Agents-- As an initial investigation of possible mechanisms of resistance, the C55.7Res cells were assessed for cross-resistance to MGBG, adriamycin, and cisplatin. MGBG targets the polyamine synthetic pathways through inhibition of S-adenosylmethionine decarboxylase (33, 34), adriamycin is a classic indicator of multi-drug resistance through a p-glycoprotein efflux pump (35), and a cisplatin-resistant ovarian carcinoma cell line has been shown to be cross-resistant to BE 3-4-3 (36). If the pathways or enzymes that are targets of these agents were altered in the C55.7Res cells and thus responsible for the resistance, it would be expected that the C55.7Res cells would also exhibit cross-resistance to these agents. However, the C55.7Res cells were as sensitive to these drugs as the parental C55.7cells (Table II), suggesting that neither multi-drug resistance nor the targets of the polyamine analogue MGBG are factors contributing to the bis(ethyl)polyamine analogue resistance of the C55.7Res cells and that there is no obligatory link between resistance to cisplatin and resistance to these analogues.

SSAT Activity of C55.7 and C55.7Res Cells-- SSAT is the rate-limiting enzyme of polyamine catabolism, and its activity is known to be induced in many cell systems by polyamine analogues (1, 10-12). Therefore, because the observed resistance could result from changes in polyamine catabolism, the activity of SSAT was assessed in C55.7 and C55.7Res cells. The results (Fig. 2) indicated that SSAT activity was low, but measurable, in untreated parental C55.7 cells at both time points. However, the SSAT activity of the untreated C55.7Res cells was at or below the limit of detection of the activity assay, indicating that it was reduced in comparison to the C55.7 cells. Exposure to BE 3-3-3 resulted in a time- and concentration-dependent increase of SSAT activity in the C55.7 cells, with induction ranging from 3-fold in response to 1 µM BE 3-3-3 to ~300-fold resulting from 25 µM BE 3-3-3. In contrast, SSAT activity of the C55.7Res cells exposed to BE 3-3-3, for all conditions tested, remained at or below the limit of detection of the assay with no detectable induction of SSAT activity resulting even after 48-h exposure to 25 µM BE 3-3-3. This difference between the C55.7Res cell line and the parental cells in response to BE 3-3-3 suggested that the reduced SSAT activity and altered SSAT regulation of the C55.7Res cells was contributing to the observed resistance to polyamine analogues.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   SSAT activity levels and induction by BE 3-3-3 for the C55.7 and C55.7Res cell lines. Cells were incubated in the absence or presence of BE 3-3-3 for the indicated times, and SSAT activity was determined as under "Experimental Procedures." Values shown represent the average ± S.D. of triplicate samples from a representative experiment.

Effects of Altered SSAT Regulation on Intracellular Polyamine Levels-- The intracellular polyamine pools were assessed, following exposure to 10 or 100 µM BE 3-3-3 for up to 48 h, to examine the effects of the altered SSAT regulation in the C55.7Res cells. The polyamine profile of the parental C55.7 cells (Table III) was consistent with a normal cellular response to BE 3-3-3 in that the spermidine and spermine concentrations decreased and the putrescine concentration increased as a consequence of SSAT induction, whereas BE 3-3-3 accumulated. The response was concentration-dependent as exposure to the higher BE 3-3-3 concentration resulted in a greater decrease in spermidine and spermine (~90% depletion by 48 h) as well as a larger intracellular accumulation of the analogue. In the C55.7Res cells, the spermidine and spermine concentrations decreased to a lesser extent than in the C55.7 cells and the putrescine concentration decreased rather than increasing. The effects in the C55.7Res cells were also concentration-dependent, as lower spermidine and spermine concentrations and greater analogue accumulation resulted from exposure to 100 µM BE 3-3-3. However, the maximum decreases in the spermidine and spermine concentrations of the C55.7Res cells (59% and 65%, respectively) were still significantly less than those of the parental cells. N¹-Acetylspermidine and N⁸-acetylspermidine, products that can accumulate as a result of active polyamine catabolism, were detected only in the C55.7 cells. Intracellular BE 3-3-3 concentrations of 12.8 ± 1.3 and 21.1 ± 5.2 nmol/mg of protein for C55.7 and 5.2 ± 0.4 and 12.8 ± 1.0 for C55.7Res after 24-h exposure to 10 and 100 µM BE 3-3-3, respectively, indicated that the analogue accumulated to significant levels in a concentration-dependent manner in both cell lines. The lack of depletion of the natural polyamine pools of the C55.7Res cells in response to the polyamine analogue, which was consistent with the reduced cytotoxicity and the inability of the analogue to induce SSAT in these cells, also suggested that the altered SSAT regulation was related to the resistance phenotype of the C55.7Res cells.

Effects of Expression of SSAT Activity in C55.7Res Cells-- To determine whether restoration of SSAT activity and analogue induction in the C55.7Res cells would restore sensitivity to BE 3-3-3, the human SSAT cDNA under control of the CMV promoter (27) was expressed in C55.7 and C55.7Res cells. Three clones that exhibited detectable basal SSAT activity as well as induction of this activity in response to polyamine analogues were chosen for each cell line to test the sensitivity to BE 3-3-3. Table IV shows the SSAT activities of the selected clones in the absence and presence of 10 µM BE 3-3-3. The sensitivity of the clones transfected with pCMV-SSAT to BE 3-3-3 was assessed by determination of IC₅₀ values, and the results are shown in Fig. 3. The average BE 3-3-3 IC₅₀ values of the C55.7Res + pCMV-SSAT clones were all lower than that for the untransfected C55.7Res cell line or C55.7Res transfected with empty vector, indicating an increased sensitivity to the polyamine analogue. Using the Student t test to assess the statistical significance of the results, the average BE 3-3-3 IC₅₀ values for all three C55.7Res + pCMV-SSAT clones were significantly (p < 0.02) lower than that of the C55.7Res cell line. Additionally, the average BE 3-3-3 IC₅₀ values for C55.7Res clones 2 and 3 did not differ significantly (p = 0.06 and 0.17, respectively) from that of the C55.7 cell line. These results indicated that restoration of SSAT activity to the C55.7Res cell line also restored sensitivity to the polyamine analogue.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   BE 3-3-3 sensitivity of C55.7 and C55.7Res cells expressing SSAT. Cells of C55.7 and C55.7Res clones transfected with pCMV-SSAT as described under "Experimental Procedures" were incubated for 144 h in the absence or presence of 6 BE 3-3-3 concentrations, and IC50 values were determined. Values shown represent averages ± S.D. from three experiments.

Level of SSAT Transcription and Effect of BE 3-3-3-- Alterations of SSAT activity could result from changes at one or more levels of regulation of the enzyme. Northern analysis, using a full-length SSAT probe (27), was carried out to assess the steady-state level of SSAT mRNA (Fig. 4). Detection of SSAT mRNA at comparable levels in untreated C55.7 and C55.7Res cells indicated transcription of the SSAT gene in C55.7Res cells and also established that the SSAT gene had not been deleted in these cells. Additionally, SSAT mRNA levels increased similarly in both cell lines following 24-h exposure to 10 µM BE 3-3-3 (Fig. 4) indicating that the cellular response to the polyamine analogue had not been altered at the transcriptional level in the resistant cells.

View larger version (64K): [in this window] [in a new window]   Fig. 4.   Level of SSAT mRNA in C55.7 and C55.7Res cells. Cells were incubated in the absence or presence of 10 µM BE 3-3-3 for 24 h; total RNA was extracted and Northern analysis performed using a full-length SSAT probe as described under "Experimental Procedures."

SSAT Protein Levels and Induction by BE 3-3-3-- Western analysis was used to determine whether SSAT protein could be detected in the C55.7Res cell line. For the parental cell line, SSAT protein was undetectable in untreated cells, but was readily apparent on a Western blot following 24-h exposure to 25 µM BE 3-3-3 (Fig. 5). In contrast, no SSAT protein could be detected in the C55.7Res cells (Fig. 5) even in response to 25 µM BE 3-3-3, the same conditions where it was easily detected in the parental cell line. These results suggested that either translation of the SSAT mRNA was not occurring or the mRNA was translated but the polyamine analogue was unable to induce the SSAT enzyme of this cell line to a level that could be detected by Western analysis.

View larger version (43K): [in this window] [in a new window]   Fig. 5.   Western analysis of SSAT protein in C55.7 and C55.7Res cells. Cells were incubated in the absence or presence of 25 µM BE 3-3-3 for 24 h, and then Western analysis was carried out using a polyclonal SSAT antibody as described under "Experimental Procedures." Cell extract containing 50 µg of protein was loaded into each lane.

SSAT cDNA Sequence-- RT-PCR followed by sequencing was used to determine the sequence of Chinese hamster SSAT mRNA from the parental C55.7 cells (Fig. 6) and to compare that with the sequence of SSAT from the C55.7Res cells. The Chinese hamster SSAT cDNA base sequence was >92% homologous to the human SSAT sequence, and there were differences in only 7 of 171 amino acids (Fig. 6). Comparison of the C55.7 and C55.7Res SSAT sequences identified a point mutation of C  T at base 466 of the open reading frame, which would result in an amino acid change from leucine to phenylalanine at position 156 of the SSAT protein.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Amino acid sequence of Chinese hamster SSAT cDNA. Positions of difference between the Chinese hamster and human SSAT sequence are indicated in boldface type, with the corresponding human residues as follows: Val-5, Ala-12, Glu-32, Ile-35, Val-56, Arg-119, and Thr-170. The underlined residue at position 156 indicates the location of the point mutation identified in the C55.7Res SSAT cDNA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although there is substantial evidence that structural polyamine analogues can effectively cause cellular cytotoxicity (1, 7, 11, 18, 19, 37, 38), the precise mechanisms by which this occurs are less certain. A number of effects have been attributed to a variety of both symmetrically and unsymmetrically alkylated polyamine analogues through several studies in a wide range of cell types. However, it has been difficult to delineate which, if any, of the identified effects are integral to the cytotoxic action of the analogues. Cells that can escape the cytotoxic effects of a drug provide an opportunity to study mechanisms that are vital to drug action through determination of cellular alterations, which allow cell survival in the presence of the drug. This approach has been used successfully with a number of antineoplastic agents (32, 39-42) but has not been reported previously with polyamine analogues.

One problem inherent in the selection of a resistant cell line that will be used to examine intracellular mechanisms of drug action is that the resistant cells must not simply exclude entry of the drug into the cell. This was a particular concern with regard to polyamine analogues, because these agents are transported by the polyamine-specific transport system used by the natural polyamines that has been demonstrated to undergo mutation, which can result in exclusion of the drug from cells (43, 44). The approach combining the use of a parental cell line auxotrophic for putrescine with a selection method of intermittent drug exposure that limited the exposure to selection pressure was effective in overcoming that problem, because the C55.7Res cell line exhibits polyamine transport characteristics similar to the parental cell line. Because the cells must maintain the resistance in the absence of selection pressure during the drug-free recovery period of the selection process, this method also favors the selection of cells that have stable, heritable mutations that reflect permanent changes in the cell genotype and not just transient phenotypic changes. The stability of the 10-fold polyamine analogue resistance of the C55.7Res cells over a 9-month period and 40 cell passages in the absence of selection pressure demonstrated that a heritable genotypic change from that of the parental cells was present in this cell line.

The successful selection of the polyamine analogue-resistant cell line C55.7Res that exhibits normal polyamine and polyamine analogue transport has now allowed study of cellular mechanisms of escape from the cytotoxic effects of the bis(ethyl)polyamine analogues and thus insight into the actions integral to the cytotoxicity of these agents. The restoration of sensitivity of the C55.7Res cell line to BE 3-3-3 as a result of expression of SSAT in these cells provides definitive evidence that the diminished activity and lack of regulation of SSAT by the polyamine analogue is responsible for the observed resistance. This correlation between SSAT induction and cytotoxic response to the bis(ethyl)polyamine analogues is consistent with studies in a variety of cell lines where SSAT induction has ranged from low to extremely high levels (7, 10-12, 45-47). These results are also consistent with those of Maverti et al. who reported cross-resistance to BE 3-4-3 in a human ovarian carcinoma cell line selected for resistance to cisplatin (36). In the resistant cell line, they observed lower, but measurable SSAT induction along with slightly less depletion of the natural polyamines than in the sensitive cell line and suggested that these factors could be among several contributing to the BE 3-4-3 resistance. The current data provide strong evidence that the SSAT response is a requisite part of the action of these analogues and not an effect that merely supplements other actions.

Other effects that have been attributed to the bis(ethyl)polyamine analogues are depletion of intracellular polyamine pools and down-regulation of polyamine synthesis. It is clear that the observed effects on polyamine homeostasis, both in the untreated cells and in response to BE 3-3-3, result from the altered SSAT regulation and decreased SSAT activity and not from altered regulation of polyamine synthetic pathways. The C55.7 cell line has a point mutation that renders ODC inactive and exogenous putrescine is supplied to meet the cellular requirement for this polyamine. S-adenosylmethionine decarboxylase activities did not differ significantly between the two cell lines either in the absence or presence of the analogue (data not shown). The reduced putrescine levels in the C55.7Res cells at all times and conditions reflect the fact that SSAT is not functioning to recycle spermidine and spermine back to putrescine. The failure of the analogue to activate SSAT activity in the C55.7Res cells results in preservation of the spermidine and spermine pools that become depleted in the parental cell line in response to the analogue. The amount of polyamine depletion that can be tolerated before cytotoxicity results is not known, and perhaps future studies with the C55.7Res cell line will provide insight into this issue.

The lower concentrations of analogue measured in the C55.7Res cells as compared with those in the C55.7 cells can also be attributed to the lack of induction of SSAT in the C55.7Res cells. Cellular levels of the higher polyamines are normally strictly controlled, and it can be seen in both cell lines that the total of the spermidine, spermine, and BE 3-3-3 levels changes little over the 48 h of the experiment. It is only the relative distribution of the three that changes. In the parental C55.7 cells, as SSAT activity increases, the levels of spermidine and spermine, both substrates for the enzyme, are reduced and replaced by the polyamine analogue, which is not a substrate for the enzyme. However, in the C55.7Res cells, the natural polyamines are not metabolized by SSAT and the analogue does not accumulate to a greater extent to replace them. The lower accumulation of BE 3-3-3 in the C55.7Res cells, however, does not explain the lack of SSAT induction in this cell line, because the intracellular level of 12.8 nmol/mg of protein after 24-h exposure to 100 µM BE 3-3-3 is equivalent to the level that produces 100-fold induction of SSAT (after 24-h exposure to 10 µM BE 3-3-3) in the parental cells.

The current data support the conclusion that the genotypic change that is present in the C55.7Res cells causes altered regulation of SSAT at the protein level. The SSAT gene has not been deleted, SSAT transcription appears similar in the parental and the resistant cells, and the response to BE 3-3-3 at the transcriptional level appears unchanged. However, SSAT protein cannot be detected in the polyamine analogue-resistant cells even under conditions where it is readily detected in the parental cell line and basal activity of the enzyme is reduced in these cells.

The point mutation that has been identified in the C55.7Res SSAT gene would cause an amino acid change from leucine to phenylalanine at amino acid 156 of the SSAT protein. This is the first mutant SSAT cDNA identified from a cell system. SSAT is highly conserved among mammalian species, with the largest difference observed to date that of 8 of 171 amino acids between the human SSAT and that of the Syrian hamster (48). The wild type Chinese hamster SSAT sequence differs by one amino acid from that of the Syrian hamster and has seven amino acid changes from the human SSAT sequence. The region around residue 156, where the point mutation in C55.7Res results in a leucine to phenylalanine change, is fully conserved across all the mammalian species in which the SSAT sequence is currently known. Evidence that this region is important to SSAT function comes from Coleman et al. who demonstrated that mutation of amino acid 155 of the human SSAT protein from arginine to alanine causes a reduction in the enzyme activity and an increase in the K_m for spermidine (49) and that mutation of amino acid 152 from glutamic acid to lysine results in a decreased ability of BE 3-3-3 to protect the enzyme from trypsin digestion (50). This suggests not only that the region near the mutation now identified in the C55.7Res cell line is important to SSAT activity and induction by the analogue but also further suggests that the mutation may be responsible for the C55.7Res resistance.

This mutation of the SSAT gene could result in a SSAT mRNA that cannot be translated or one that is translated but produces an inactive or less active protein. Although the current data does not rule out the possibility that the SSAT mRNA is not translated, it suggests that any SSAT protein that is produced is altered in its interaction with the polyamine analogue. It is known that one mechanism by which the polyamine analogues induce SSAT activity is through interaction with the protein to lengthen the SSAT half-life and prevent protein degradation (27). Because SSAT is a short-lived protein (~30-min half-life), any change in protein/analogue interaction could decrease the ability of the analogue to protect the SSAT protein from degradation and result in decreased enzyme activity even in the presence of the analogue. It would be expected that a mutation in the SSAT protein that rendered the protein inactive, but did not effect regulation by the analogue, would result in accumulation of the inactive protein under conditions that normally result in SSAT induction. However, the Western analysis of C55.7Res cells indicated a lack of SSAT protein even in the presence of the analogue, a condition where SSAT is readily apparent in the parental cell line. Although we cannot currently eliminate the possibility that the antibody used for Western analysis does not bind to a mutant SSAT protein, use of a polyclonal SSAT antibody diminishes this possibility. Therefore, these results support the conclusion that SSAT regulation by the polyamine analogue has been altered in the C55.7Res cell line either exclusively or in addition to alterations in the ability of the protein to interact with the polyamine substrates. There is also evidence that translational regulation is involved in control of SSAT activity and response to the polyamine analogues (27) and the current results could be explained by effects on such regulation. Further studies will be needed to determine which, if any, of the above possibilities are responsible for the altered SSAT regulation and activity that result in the polyamine analogue resistance of the C55.7Res cells.

Another area of interest with this cell line will be to examine the response to other related polyamine analogues that have been demonstrated to have a variety of effects on SSAT. There are unsymmetrically alkylated polyamine analogues and other structural polyamine analogues with modifications of the carbon chain lengths that have little or no effect on SSAT (16, 17, 38, 51, 52) yet still have significant cellular cytotoxicity. Investigation of the sensitivity of the C55.7Res cell line to such analogues may aid understanding of the level of SSAT activity necessary to exert an effect on cytotoxicity of polyamine analogues. Also, investigation of these analogues will be useful in determining whether any mechanisms unrelated to SSAT are functional in the C55.7Res cell line and contributing to the resistance to polyamine analogues. It is also possible that the analogues that induce SSAT and those that do not induce SSAT share a common mechanism that is still functional in the C55.7Res cells, because these cells are not completely resistant to BE 3-3-3. Induction of SSAT and subsequent polyamine depletion may be essential for BE 3-3-3 to bind to a target, whereas other analogues may bind effectively without needing SSAT induction. The use of a similar approach to develop a cell line resistant to an analogue that does not alter SSAT may be useful in elucidating such mechanisms.

	FOOTNOTES

^* This work was supported by Grant GM-26290 from the National Institutes of Health and by a grant from the Pennsylvania State University Cancer Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF281149.

To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033. Tel.: 717-531-6987; Fax: 717-531-5157; E-mail: dem15@psu.edu.

Published, JBC Papers in Press, July 7, 2000, DOI 10.1074/jbc.M004120200

	ABBREVIATIONS

The abbreviations used are: BE 3-4-3, N¹,N¹²-bis(ethyl)spermine; Be 3-3-3, N¹,N¹¹-bis(ethyl)norspermine; SSAT, spermidine/spermine N¹-acetyltransferase; MGBG, methylglyoxal bis(guanylhydrazone); CHENSpm, N¹-ethyl-N¹¹-((cycloheptyl)methyl)-4,8-diazaundecane; BE 4-4-4-4, 1,19-di-(ethylamino)-5,10,15-triazanonadecane; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ODC, ornithine decarboxylase; CMV, cytomegalovirus; RT-PCR, reverse transcription-polymerase chain reaction.

	REFERENCES

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