Altered Spermidine/SpermineN 1-Acetyltransferase Activity as a Mechanism of Cellular Resistance to Bis(ethyl)polyamine Analogues
2000; Elsevier BV; Volume: 275; Issue: 37 Linguagem: Inglês
10.1074/jbc.m004120200
ISSN1083-351X
AutoresDiane E. McCloskey, Anthony E. Pegg,
Tópico(s)Cannabis and Cannabinoid Research
ResumoTo 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 anN 1,N 12-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 toN 1,N 12-bis(ethyl)spermine andN 1,N 11-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/spermineN 1-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. 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 anN 1,N 12-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 toN 1,N 12-bis(ethyl)spermine andN 1,N 11-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/spermineN 1-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. N 1,N 12-bis(ethyl)spermine N 1,N 11-bis(ethyl)norspermine spermidine/spermine N 1-acetyltransferase methylglyoxal bis(guanylhydrazone) N 1-ethyl-N 11-((cycloheptyl)methyl)-4,8-diazaundecane 1,19-di-(ethylamino)-5,10,15-triazanonadecane 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ornithine decarboxylase cytomegalovirus reverse transcription-polymerase chain reaction 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 (1Casero R.A. Pegg A.E. FASEB J. 1993; 7: 653-661Crossref PubMed Scopus (396) Google Scholar, 2Pegg A.E. Biochem. J. 1986; 234: 249-262Crossref PubMed Scopus (1454) Google Scholar, 3Pegg A.E. Cancer Res. 1988; 48: 759-774PubMed Google Scholar, 4Gabrielson E.W. Pegg A.E. Casero R.A. Clin. Cancer Res. 1999; 55: 1638-1641Google Scholar). These agents were designed to mimic the self-regulatory functions of the natural polyamines but not substitute functionally for cellular polyamine requirements (5Porter C.W. Bergeron R.J. Adv. Enzyme Regul. 1988; 27: 57-82Crossref PubMed Scopus (66) Google Scholar, 6Bergeron R.J. Neims A.H. McManis J.S. Hawthorne T.R. Vinson J.R.T. Bortell R. Ingeno M.J. J. Med. Chem. 1988; 31: 1183-1190Crossref PubMed Scopus (186) Google Scholar, 7Casero R.A. Ervin S.J. Celano P. Baylin S.B. Bergeron R.J. Cancer Res. 1989; 49: 639-643PubMed Google Scholar, 8Edwards M.L. Prakash N.J. Stemerick D.M. Sunkara S.P. Bitonti A.J. Davis G.F. Dumont J.A. J. Med. Chem. 1990; 33: 1369-1375Crossref PubMed Scopus (74) Google Scholar, 9Basu H.S. Pellarin M. Feuerstein B.G. Deen D.F. Bergeron R.J. Marton L.J. Cancer Res. 1990; 50: 3137-3140PubMed Google Scholar). 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 (1Casero R.A. Pegg A.E. FASEB J. 1993; 7: 653-661Crossref PubMed Scopus (396) Google Scholar, 10Casero R.A. Celano P. Ervin S.J. Porter C.W. Bergeron R.J. Libby P. Cancer Res. 1989; 49: 3829-3833PubMed Google Scholar, 11Davidson N.E. Mank A.R. Prestigiacomo L.J. Bergeron R.J. Casero R.A. Cancer Res. 1993; 53: 2071-2075PubMed Google Scholar, 12Porter C.W. Ganis B. Libby P.R. Bergeron R.J. Cancer Res. 1991; 51: 3715-3720PubMed Google Scholar), and BE 3-3-3 is currently in phase I clinical trials (4Gabrielson E.W. Pegg A.E. Casero R.A. Clin. Cancer Res. 1999; 55: 1638-1641Google Scholar). 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 (1Casero R.A. Pegg A.E. FASEB J. 1993; 7: 653-661Crossref PubMed Scopus (396) Google Scholar, 4Gabrielson E.W. Pegg A.E. Casero R.A. Clin. Cancer Res. 1999; 55: 1638-1641Google Scholar, 10Casero R.A. Celano P. Ervin S.J. Porter C.W. Bergeron R.J. Libby P. Cancer Res. 1989; 49: 3829-3833PubMed Google Scholar, 11Davidson N.E. Mank A.R. Prestigiacomo L.J. Bergeron R.J. Casero R.A. Cancer Res. 1993; 53: 2071-2075PubMed Google Scholar, 12Porter C.W. Ganis B. Libby P.R. Bergeron R.J. Cancer Res. 1991; 51: 3715-3720PubMed Google Scholar). 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 (13Basu H.S. Pellarin M. Feuerstein B.G. Deen D.F. Marton L.J. Anticancer Res. 1993; 13: 1525-1532PubMed Google Scholar, 14Basu H.S. Pellarin M. Feuerstein B.G. Shirahata A. Samejima K. Deen D.F. Marton L.J. Cancer Res. 1993; 53: 3948-3955PubMed Google Scholar, 15Basu H.S. Marton L.J. Pellarin M. Deen D.F. McManis J.S. Liu C.Z. Bergeron R.J. Feuerstein B.G. Cancer Res. 1994; 54: 6210-6214PubMed Google Scholar, 16Saab N.H. West E.E. Bieszk N.C. Preuss C.V. Mank A.R. Casero R.A. Woster P.M. J. Med. Chem. 1993; 36: 2998-3004Crossref PubMed Scopus (94) Google Scholar). Many of these compounds have been tested as chemotherapeutic agents with several showing promising antineoplastic activity against a variety of tumor types (13Basu H.S. Pellarin M. Feuerstein B.G. Deen D.F. Marton L.J. Anticancer Res. 1993; 13: 1525-1532PubMed Google Scholar, 14Basu H.S. Pellarin M. Feuerstein B.G. Shirahata A. Samejima K. Deen D.F. Marton L.J. Cancer Res. 1993; 53: 3948-3955PubMed Google Scholar, 16Saab N.H. West E.E. Bieszk N.C. Preuss C.V. Mank A.R. Casero R.A. Woster P.M. J. Med. Chem. 1993; 36: 2998-3004Crossref PubMed Scopus (94) Google Scholar, 17Casero R.A. Mank A.R. Saab N.H. Wu R. Dyer W.J. Woster P.M. Cancer Chemother. Pharmacol. 1995; 36: 69-74Crossref PubMed Scopus (38) Google Scholar, 18McCloskey D.E. Woster P.M. Casero R.A. Davidson N.E. Clin. Cancer Res. 2000; 6: 17-23PubMed Google Scholar, 19McCloskey D.E. Yang J. Woster P.M. Davidson N.E. Casero R.A. Clin. Cancer Res. 1996; 2: 441-446PubMed Google Scholar, 20Ha H.C. Woster P.M. Yager J.D. Casero Jr., R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11557-11562Crossref PubMed Scopus (271) Google Scholar, 21Zagaja G.P. Shrivastav M. Fleig M.J. Marton L.J. Rinker-Schaeffer C.W. Dolan M.E. Cancer Chemother. Pharmacol. 1998; 41: 505-512Crossref PubMed Scopus (30) Google Scholar). 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 (18McCloskey D.E. Woster P.M. Casero R.A. Davidson N.E. Clin. Cancer Res. 2000; 6: 17-23PubMed Google Scholar, 20Ha H.C. Woster P.M. Yager J.D. Casero Jr., R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11557-11562Crossref PubMed Scopus (271) Google Scholar, 21Zagaja G.P. Shrivastav M. Fleig M.J. Marton L.J. Rinker-Schaeffer C.W. Dolan M.E. Cancer Chemother. Pharmacol. 1998; 41: 505-512Crossref PubMed Scopus (30) Google Scholar). 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 (22Pilz R.B. Steglich C. Scheffler I.E. J. Biol. Chem. 1990; 265: 8880-8886Abstract Full Text PDF PubMed Google Scholar). 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 (23Byers T.L. Pegg A.E. Am. J. Physiol. 1989; 256: C545-C553Crossref Google Scholar, 24Byers T.L. Pegg A.E. J. Cell. Physiol. 1990; 143: 460-467Crossref PubMed Scopus (50) Google Scholar, 25Kramer D.L. Miller J.T. Bergeron R.J. Khomutov R. Khomutov A. Porter C.W. J. Cell. Physiol. 1993; 155: 399-407Crossref PubMed Scopus (62) Google Scholar) 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 (1Casero R.A. Pegg A.E. FASEB J. 1993; 7: 653-661Crossref PubMed Scopus (396) Google Scholar), 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. [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). The Chinese hamster ovary cell line C55.7 (22Pilz R.B. Steglich C. Scheffler I.E. J. Biol. Chem. 1990; 265: 8880-8886Abstract Full Text PDF PubMed Google Scholar), a kind gift from Dr. Immo Scheffler (University of California at San Diego, La Jolla, CA), and its derivatives were maintained in minimum essential α-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. Exponentially growing cells were plated in triplicate at 2 × 103 cells/cm2in 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, theA 570 (a value directly proportional to the number of viable cells (26Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (48003) Google Scholar) 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. The transfection of pCMV-SSAT into C55.7 and C55.7Res cells was accomplished using LipofectAMINE, as described previously (27McCloskey D.E. Coleman C.S. Pegg A.E. J. Biol. Chem. 1999; 274: 6175-6182Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), but with the addition of 100 μm putrescine to the culture medium. Exponentially growing cells were plated in triplicate at 2–4 × 104cells/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 (28Matsui I. Wiegand L. Pegg A.E. J. Biol. Chem. 1981; 256: 2454-2459Abstract Full Text PDF PubMed Google Scholar). 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. 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 witho-phthalaldehyde as described previously (29Pegg A.E. Wechter R. Poulin R. Woster P.M. Coward J.K. Biochemistry. 1989; 28: 8446-8453Crossref PubMed Scopus (54) Google Scholar). 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 (30Casero Jr., R.A. Gabrielson E.W. Pegg A.E. Cancer Res. 1994; 54: 3955-3958PubMed Google Scholar)) 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. 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 (31Casero Jr., R.A. Celano P. Ervin S.J. Applegren N.B. Wiest L. Pegg A.E. J. Biol. Chem. 1991; 266: 810-814Abstract Full Text PDF PubMed Google Scholar). The Vistra signal amplification kit was used for signal detection, and visualization and quantitation were the same as for the Western analysis. 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. The polyamine analogue BE 3-4-3 was used to select a resistant derivative of the Chinese hamster ovary cell line C55.7 (22Pilz R.B. Steglich C. Scheffler I.E. J. Biol. Chem. 1990; 265: 8880-8886Abstract Full Text PDF PubMed Google Scholar). 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 (32Pizzorno G. Mini E. Coronello M. McGuire J.J. Moroson B.A. Cashmore A.R. Dreyer R.N. Lin J.T. Mazzei T. Periti P. Bertino J.R. Cancer Res. 1988; 48: 2149-2155PubMed Google Scholar). As the population of resistant cells increases, the recovery period becomes shorter (32Pizzorno G. Mini E. Coronello M. McGuire J.J. Moroson B.A. Cashmore A.R. Dreyer R.N. Lin J.T. Mazzei T. Periti P. Bertino J.R. Cancer Res. 1988; 48: 2149-2155PubMed Google Scholar). 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.Table IScheme of drug treatment and recovery periods used to select a BE 3-4-3-resistant cell lineCycle no.[BE 3-4-3]Exposure timeTime for recoveryμmhdays110962021014423310012022410120<5 Open table in a new tab 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. 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. 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 ofS-adenosylmethionine decarboxylase (33Seppänen P. Alhonen-Hongisto L. Jänne J. Eur. J. Biochem. 1981; 18: 571-576Crossref Scopus (38) Google Scholar, 34Seppänen P. Alhonen-Hongisto L. Jänne J. Biochim. Biophys. Acta. 1981; 674: 169-177Crossref PubMed Scopus (40) Google Scholar), adriamycin is a classic indicator of multi-drug resistance through ap-glycoprotein efflux pump (35Germann U.A. Pastan I. Gottesman M.M. Semin. Cell Biol. 1993; 4: 63-76Crossref PubMed Scopus (148) Google Scholar), and a cisplatin-resistant ovarian carcinoma cell line has been shown to be cross-resistant to BE 3-4-3 (36Marverti G. Piccinini G. Ghiaroni S. Barbieri D. Quaglino D. Moruzzi M.S. Int. J. Cancer. 1998; 78: 33-40Crossref PubMed Scopus (23) Google Scholar). 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 (TableII), 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.Table IISensitivity of C55.7 and C55.7Res cells to MGBG, adriamycin, and cisplatinDrugIC50 valueC55.7C55.7ResmMGBG1.4 × 10−51.9 × 10−5Adriamycin1.1 × 10−71.1 × 10−7Cisplatin4.0 × 10−74.0 × 10−7IC50 values were determined as described under “Experimental Procedures.” Values reported are averages from duplicate experiments. Open table in a new tab IC50 values were determined as described under “Experimental Procedures.” Values reported are averages from duplicate experiments. SSAT is the rate-limiting enzyme of polyamine catabolism, and its activity is known to be induced in many cell systems by polyamine analogues (1Casero R.A. Pegg A.E. FASEB J. 1993; 7: 653-661Crossref PubMed Scopus (396) Google Scholar, 10Casero R.A. Celano P. Ervin S.J. Porter C.W. Bergeron R.J. Libby P. Cancer Res. 1989; 49: 3829-3833PubMed Google Scholar, 11Davidson N.E. Mank A.R. Prestigiacomo L.J. Bergeron R.J. Casero R.A. Cancer Res. 1993; 53: 2071-2075PubMed Google Scholar, 12Porter C.W. Ganis B. Libby P.R. Bergeron R.J. Cancer Res. 1991; 51: 3715-3720PubMed Google Scholar). 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. 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 (TableIII) 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. N 1-Acetylspermidine and N 8-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.Table IIIIntracellular polyamine content of C55.7 and C55.7Res cellsCell lineTreatmentIntracellular polyaminesBE 3-3-3PutrescineSpermidineSpermineN 1-AcetylspermidineN 8-Acetylspermidinenmol/mg of protein0 h3-aTime 0 was 12–16 h after cells were plated. C55.7No drug23.7 ± 5.616.0 ± 3.67.8 ± 2.30.2 ± 0.10.2 ± 0.2 C55.7ResNo drug5.1 ± 1.917.2 ± 5.712.4 ± 4.7ND3-bND, not detected.ND24 h C55.7No drug9.6 ± 1.814.9 ± 1.98.5 ± 1.5NDND C55.710 μM BE 3-3-319.0 ± 3.89.1 ± 1.74.9 ± 0.80.8 ± 0.20.2 ± 0.0212.8 ± 1.3 C55.7100 μM BE 3-3-37.4 ± 0.93.6 ± 0.42.3 ± 0.30.4 ± 0.2ND21.1 ± 5.2 C55.7ResNo drug1.5 ± 0.310.6 ± 0.89.6 ± 0.2NDND C55.7Res10 μM BE 3-3-31.0 ± 0.28.6 ± 1.28.7 ± 1.2NDND5.2 ± 0.4 C55.7Res100 μM BE 3-3-30.6 ± 0.25.2 ± 0.77.2 ± 1.2NDND12.8 ± 1.048 h C55.7No drug9.2 ± 0.618.4 ± 1.610.3 ± 2.0NDND C55.710 μM BE 3-3-322.9 ± 2.47.7 ± 0.63.6 ± 0.20.4 ± 0.040.3 ± 0.0312.7 ± 1.5 C55.7100 μM BE 3-3-311.4 ± 0.61.9 ± 0.10.8 ± 0.2NDND27.7 ± 3.0 C55.7ResNo drug2.5 ± 0.512.9 ± 3.613.3 ± 3.4NDND C55.7Res10 μM BE 3-3-31.6 ± 0.110.8 ± 0.57.5 ± 0.7NDND5.7 ± 1.1 C55.7Res100 μM BE 3-3-31.3 ± 0.15.3 ± 0.34.6 ± 0.8NDND13.9 ± 1.5Cells were incubated in the absence or presence of BE 3-3-3 for the indicated times, and the intracellular polyamine content was determined as under “Experimental Procedures.”3-a Time 0 was 12–16 h after cells were plated.3-b ND, not detected. Open table in a new tab Cells were incubated in the absence or presence of BE 3-3-3 for the indicated times, and the intracellular polyamine content was determined as under “Experimental Procedures.” 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 (27McCloskey D.E. Coleman C.S. Pegg A.E. J. Biol. Chem. 1999; 274: 6175-6182Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) 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 IC50values of the C55.7Res + pCMV-SSAT clones were all lower than that
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