Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
[CANCER RESEARCH 58, 3883-3890. September I, 1998] Lysosomal Sequestration of Polyamine Analogues in Chinese Hamster Ovary Cells Resistant to the S-Adenosylmethionine Decarboxylase Inhibitor, CGP-486641 Debora L. Kramer, Jennifer D. Black, Helmut Mett, Raymond J. Bergeron, and Carl W. Porter ice Cancer Drug Center, Roswett Park Cancer Institute, Buffalo, New York 14263 ID. L. K., J. D. B., C. W. P.}; Research Lttboratories. Grac Novartis Pharmaceuticals Pharmaceittic, '-4002. Basel. Switzerland [H. M.¡;and Department of Medicinal Chemistry. University of Florida. J. Hillis Miller Health Center, Gainesville. Florida 326/0 [K. J. BJ CH-4002 CGP-48664 ABSTRACT CGP-48664, an inhibitor of the polyamine biosynthetic enzyme \- adenosylmethionine decarboxylase (AdoMetDC), is presently undergoing Phase 1 clinical trials as an experimental anticancer agent. We have shown previously (D. L. Kramer et al, J. Bici. Chem., 270: 2124-2132, 1995) that Chinese hamster ovary (CHO) cells that are made resistant to the growth inhibitory effects of the drug overexpress AdoMetDC because of a stable gene amplification. Unexpectedly, these same cells (CHO/644) were found to be insensitive to the growth inhibitory effects of Ai'^Vll-diethylnorspermine (DENSPM)—a polyamine analogue also undergoing Phase 1 clinical trials—despite accumulating ~5 times more analogue than parental cells. We now report that treatment of CHO/664 cells with DENSPM results in the formation of numerous large cytoplasmic vacuoles, which on the basis of electron microscopy and cytochemical staining seem to be lysosomal in origin. A series of newly established CHO cell lines made differentially resistant to 1, 3,10, 30, and 100 /IM CGP-4S664 by chronic exposure were used to demonstrate that vacuole formation correlated with the accumu lation of extremely high levels of DENSPM without increasing growth inhibition. These same cells were used to show that AdoMetDC gene overexpression as indicated by mRNA levels was unrelated to vacuole formation; cells resistant to 100 /¿MCGP-48664 displayed a 170-fold increase in AdoMetDC mRNA levels and formed vacuoles in response to DENSPM, whereas those resistant to 10 JIM CGP-48664 displayed a 120-fold increase in AdoMetDC mRNA levels and failed to form vacuoles. Despite accumulating to high intracellular levels, DENSPM was much less effective than spermine at down-regulating ornithine decarboxylase and polyamine transport activities in highly resistant cells. Similarly, DENSPM was less able to induce spermidine/spermine W'-acetyltransferase activity in cells that formed vacuoles than in those that did not. Overall, natural polyamines failed to induce vacuoles and various ana logues of DENSPM were used to probe the structural specificity of the effect. The data are consistent with the probability that DENSPM is sequestered to high concentrations in lysosomal vacuoles of CGP-48664resistant cells and is, therefore, not available to interact with polyamine regulatory sites or to cytotoxically affect cell growth. In addition to implicating the lysosome as a potential new site of CGP-48664 drug action that could be involved in antitumor activity and/or host toxicities, the findings also suggest a potential mechanism of cell resistance to analogues such as DENSPM. INTRODUCTION As an alternative to anticancer and chemopreventive strategies targeting the polyamine biosynthetic enzyme ornithine decarboxylase (1, 2), investigators at Novartis Pharmaceuticals (Basel, Switzerland) have developed analogues of MGBG,3 also known as methylGAG, as potential inhibitors of the other key biosynthetic enzyme, AdoMetDC. AG., (Fig. 1) was selected from a relatively large series of MGBG analogues because of its potent and selective enzyme inhib itory properties, its metabolic stability, and, most importantly, its minimized antimitochondrial activity (3-5). The specificity of the inhibitor in cell culture conditions is based on observed alterations in polyamine pool perturbations that are characteristic of AdoMetDC inhibition and on the finding that CHO cells that are made resistant (CHO/664) to the growth inhibitory effects of CGP-48664 overex press AdoMetDC (5, 6). On the basis of broad spectrum in vitro antiproliferative activity and promising antitumor activity against a variety of human tumor xenografts, CGP-48664 is presently under going Phase 1 clinical trials (4, 5). Although subsequent studies (7-9) failed to correlate antitumor activity with apparent polyamine meta bolic effects, we have recently confirmed that, in a melanoma biopsy, CGP-48664 acts as an inhibitor of AdoMetDC under conditions of clinical use (10). The CHO/664 cells mentioned above were derived by chronic exposure to increasing concentrations of CGP-48664. They represent the first cell line in which the AdoMetDC gene is overexpressed because of gene amplification as indicated by genomic Southern blot analysis and increased expression of AdoMetDC mRNA and enzyme activity (6). The initial characterization of this cell line led to the observation that these same cells were only cytostatically affected by the polyamine analogue DENSPM (Fig. 1) despite accumulating at least 5 times more analogue than parental cells. This observation was unexpected inasmuch as DENSPM is known to have multiple sites of action within cells in addition to the down-regulation of AdoMetDC. These include suppression of ODC and polyamine transport activities and the potent up-regulation of the polyamine catabolic enzyme SSAT (11-13). In fact, recent studies with fibroblasts derived from transgenie animals that overexpress SSAT (14, 15) suggest strongly that the induction of SSAT may be the most critically important of these various regulatory responses in terms of depleting polyamine pools and affecting cell growth (16). It has also been observed that DENSPM treatment of CHO/664 cells leads to the appearance of numerous cytoplasmic vacuoles, and the possibility has been suggested that the analogue is contained within them (6). Herein, we provide morphological evidence to indi cate that the vacuoles are lysosomal in origin, that intracellular ana logue is sequestered in these vacuoles, and that their appearance in response to DENSPM is not linked to overexpression of the AdoMetDC gene. Because DENSPM exhibits promising antitumor activity in several model systems (17-19) and is also undergoing Phase 1 clinical trials, these studies may provide new insight into the cellular effects of both DENSPM and CGP-48664, insight that could Received 4/1/98; accepted 7/2/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by National Cancer Institute Grant ROI CA22153 prove to be clinically relevant to antitumor activity, tissue toxicity, or drug resistance. Roswell Park Cancer Institute Elm and Carlton Streets, Buffalo. NY 14263. Phone: (716) 845-3002: Fax: (716) 845-8857: E-mail: [email protected]. 3 The abbreviations used are: MGBG, methylglyoxal-bis(guanylhydrazone); AdoMetDC, S-adenosylmethionine decarboxylase: dcAdoMet, decarboxylated 5-adenosylmethionine; CGP-48664, 4-amidinoindan-l-one 2'-amidinohydrazone; CHO. Chinese CHO/3, Chinese hamster ovary cells resistant to 3 LIMCGP-48664; CHO/10. Chinese hamster ovary cells resistant to 10 LIMCGP-48664; CHO/30, Chinese hamster ovary cells resistant to 30 LIMCGP-48664: CHO/IOO, Chinese hamster ovary cells resistant to 100 LIM CGP-48664; CHO/664. Chinese hamster ovary cells resistant to 100 LIMCGP-48664 derived previously (6): DENSPM. /V'.N1 '-diethylnorspermine [also known as BENSPM. /V'./V1'-bis(ethyl)norspermine]; ODC, ornithine decarboxyla.se: SSAT, spermidine/sperm ine /V'-acetyltransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCA, hamster ovary cells: CHO/1. Chinese hamster ovary cells resistant to 1 /AMCGP-48664: perchloric acid. (to C. W. P.) and Roswell Park Cancer Institute Core Grant CA16056 (to J. B.). 2 To whom requests for reprints should be addressed, at Grace Cancer Drug Center, 3883 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1998 American Association for Cancer Research. LYSOSOMAL SEQUESTRATION OF POLYAMINE ANALOGUES Cell Growth Studies. Exponentially growing resistant cells were placed in the absence of CGP-48664 for at least 72 h before treatment with DENSPM and related analogues. The doubling time for the parental CHO cells and each resistant CHO/664 cell line was similar (—20h). Dose-response studies with DENSPM DENSPM were performed at concentrations from 0.01 to 100 JU.Mand the cell lines were seeded at 1 X IO5 cells into T-25 flasks. Unless otherwise indicated, all of the growth and biochemical assays were performed after analogue treatment for 72 h. Cell growth was determined by electronic particle counting. Light and Electron Microscopy. Cells were washed, fixed in situ in 3% phosphate-buffered glutaraldehyde (pH 7.4), postfixed in 1% osmium tetroxide. embedded in Epon-Araldite plastic resin as described previously (23) and sectioned at -500 nm for viewing by light microscopy after being stained with toluidine blue and at —¿100nm for viewing in a Siemens 101 electron CGP-48664 Fig. 1. Slniclures of the polyaminc analogue DENSPM and the AdoMelDC inhibitor CGP-48664. microscope after being stained with uranyl acetate and lead citrate. For detection of lysosomal structures. CHO/100 cells were cultured for 3 days in the absence of CGP-48664 before being plated on coverslips in the absence or Table I Relame effects of DENSPM on growth and mornhotogv of CHO and CHO/664 cells presence of 10 /UMDENSPM and incubated for the time periods indicated. After DENSPM treatment for 24 h, certain coverslips were exposed to l ¡J.M of an acidotropic dye (LysoSensor Green DND-153, Molecular Probes, Inc.) for linesCHOCHO/664''Treatment Cell h)noneDENSPM (10 /¿M,72 noneDENSPM% control growth10052 analogue" (pmol/106 cells)none6.000 vacuolesnononoyesIntracellular 10078Cytoplasmic none35,000 " Values represent the average of at least six separate determinations with SD < 20%. * Grown in the absence of CGP-48664 for al least 72 h before treatment with DENSPM. MATERIALS Materials. 1 h to detect the intracellular localization of this fluorescent dye in relation to vacuole formation. Cells were observed and photographed using a Zeiss epifluorescence microscope equipped with appropriate optics and filter mod ules. Cells were also photographed using a Leica phase contrast microscope (Leitz, Model DMIL) with optics as indicated. Polyamine and Polyamine Analogue Pools. Inlracellular polyamines were extracted from cell pellets with 0.6 N PCA with a ratio of 2 X IO7 cells AND METHODS CGP-48664 was synthesi/.ed as described previously (3). The polyamine analogue DENSPM was a gift from Warner Lambert Parke Davis (Ann Arbor. MI). The polyamines spermidine and spermine were purchased from Sigma (St. Louis. MO); norspermine was purchased from Aldrich (Mil waukee. WI). All of the other spermidine and spermine analogues were synthesi/.ed as described previously (20, 21). Tritiated spermidine and [32P]dCTP was obtained from DuPont NEN (Boston, MA). The pCM9 plasmid construct containing the coding region for the cDNA of human AdoMetDC (22) was kindly provided by Dr. Bruce Stanley (Hershey Medical Center, Hershey. PA). The human GAPDH cDNA probe, used to quantitate Northern blot loading differences, was obtained from Clontech Laboratories. Inc. (Palo Alto. CA). The oligolabeling kit was obtained from Pharmacia LKB Biotech nology (Piscataway, NJ). LysoSensor Green DND-153 was purchased from Molecular Probes, Inc. (Eugene, OR) for detection of lysosomal acidic or- per ml PCA, dansylated, and measured by reverse phase high-pressure liquid chromatography as described previously with minor modifications (6). Polyamine Enzyme Activities. After various cell treatments, ODC, AdoMetDC, and SSAT activities were determined from cellular extracts pre pared and measured as described by Porter et al. (13). AdoMet, dcAdoMet, and CGP-48664 Pools. High-pressure liquid Chro matographie analysis of intracellular AdoMet and metabolites was performed with the same PCA extracts used for polyamine pool analysis. The Chromato graphie conditions were adapted from Yarlett and Bacchi (24) with modifica tions (6). Spermidine Transport. Tritiated spermidine transport was evaluated by Vmaxvalues determined as described previously (25). Cells were seeded into six well plates at a density of 1 x IO4 cells per well and allowed to grow in the presence or absence of 10 /J.MDENSPM for 72 h before performing the assay. ganelles. Maintenance of CHO Cell Lines. The monolayer CHO cells, obtained from American Type Culture Collection (CHO-K1 cells), were grown in 100 DMEM/F12 medium (Life Technologies. Grand Island. NY) containing 10% PCS (Atlanta Biologicals. Norcross, GA.), 1% nonessential amino acids (Life Technologies), I % sodium pyruvate (Life Technologies), and I mM aminoguanidine (Sigma). Cell cultures were maintained at 37°Cwith 5% CO2 in a humidified incubator. Vented T-75 cm2 flasks (Falcon, Becton Dickinson, Vacuoles Bedford. MA) were used routinely for cell culture. CHO cells were detached by trypsin (Life Technologies) with prompt removal of excess trypsin by washing. Derivation of CGP-48664-Resistant CHO Cells. The parent CHO cell line was chronically exposed to increasing levels of CGP-48664 for at least o u eight passages beginning at 0.1 /J.Mdrug as described previously (6). Cell lines were serially exposed to increasing concentrations until a panel of sublines was obtained resistant to 1 (CHO/1). 3 (CHO/3), 10 (CHO/10), 30 (CHO/30), and 100 (CHO/100) P.M CGP-48664 for comparative studies, which initially fo cused on AdoMetDC mRNA as an indication of gene overexpression and presumably gene amplification (6). In terms of biochemical and molecular characteristics, the CHO/100 cells, newly developed for these studies, were found to be nearly identical to the original resistant line designated CHO/664 cells (5. 6).The cells were routinely cultured in DMEM/F12 described above containing the appropriate amount of CGP-48664. Characterization studies were performed with cells grown in the presence of the inhibitor or at various times after removal of the inhibitor as indicated (typically, 72 h). CHO -•- CHO/664(+) 25 -n- 0.01 CHO/664(-) 0.1 1 10 100 DENSPM Treatment Fig. 2. Comparison of growth sensitivity of CHO and CHO/664 lo increasing concen trations of DENSPM during a 72 h incubation in the presence ( + ) and absence (-) of CGP-48664. Noie thai in the absence of CGP-48664, CHO/664 cells form vacuoles and are less sensitive than cells in the presence of CGP-48664 before and during DENSPM treatment that do not form vacuoles. 3884 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1998 American Association for Cancer Research. LYSOSOMAL SEQUESTRATION OF POLYAMINE ANALOGUES Fig. 3. Light microscopy of the CHO parental cells treated with 10 ¿IM DENSPM for 72 h (A), and newly derived panels of CHO/10 (ß),CHO/30 (O. and CHO/100 (O) cells grown out of CGP-48664 for 72 h and treated with 10 /IM DENSPM for 72 h. The differences between these cell lines in growth re sponse (as shown in Fig. 2) are not reflected here because regions of similar cell density were chosen for photography. Treated CHO/100 cells (D) were washed, placed in drug-free medium, and allowed to recover for 24 h (E) and 48 h (F). Notice the gradual disappearance of vacuoles. Vacuoles were noi ob served in CHO/100 cells grown in the presence of CGP-48664 before and during 10 /¿M DENSPM treat ment (C) nor in CHO/100 cells removed from CGP48664 (as above) and treated with 10 /IM spermine for 72 h (H). XI200. Northern Blot Analysis. Methods follow those reported previously in greater detail (26). Total RNA was extracted with guanidine isothiocyanate and purified by a CsCI gradient centrifugation from the parental CHO cells and each of the sublines CHO/1, CHO/3. CHO/10, CHO/30, CHO/100 grown continuously in the presence of CGP-48664. Each sample (10 /ig) was electrophoresed onto a 1.5% agarose gel, transferred to Duralon membrane and probed with the Sacl/Xba fragment (~1(KX) bp) cut from the pCM9 plasmid containing the human AdoMetDC cDNA coding region (23). A GAPDH cDNA probe was used to adjust quantitation for loading differences. Modifi cations specific for probing with AdoMetDC cDNA were noted previously (6). Autoradiograms were scanned and bands of RNA quantitated using a Molec ular Dynamics densitometer with Image Quant Software (Molecular Dynam ics, Sunnyvale. CA). RESULTS CHO/664 cells containing an amplified AdoMetDC gene were derived originally (6) by chronic exposure to increasing concentra tions of CGP-48664 up to 100 ¡JLM. When those cells were grown in the absence of CGP-48664 for up to 3 weeks, the gene amplification was found to be stable, they were cross-resistant to other known AdoMetDC inhibitors, and they were found to be less sensitive to DENSPM despite accumulating ~5-fold more of the analogue than the parental line (6). In this subsequent detailed characterization. CHO/664 (also designated CHO/100) cells were found again to ac cumulate extremely high intracellular levels of DENSPM (Table 1), unexpectedly accompanied by much less of an effect on cell growth (Fig. 2). A prominent feature of the treated cells was that they contained numerous cytoplasmic vacuoles. Interestingly, when the CHO/664 cells were grown in the presence of CGP-48664 up to and during treatment with DENSPM, vacuole formation was not observed, and these cells were growth-inhibited and contained levels of DENSPM similar to the parental CHO cells (Fig. 2). Possible reasons for this differential response to DENSPM in the absence and presence of CGP-48664 will be discussed later. One goal of the present study was to determine the origin of the 3885 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1998 American Association for Cancer Research. LYSOSOMAL SEQUESTRATION OF POLYAMINE ANALOGUES Fig. 4. CHO/HX) cells cultured on coverslips in the absence (A) and Ihe presence (Bl of 100 U.Mchloroquine for 3 h or (O with K) /IM DENSPM for 24 h, and then siained for l h wiih l /IM lysosensor lluorescent acidotropic dye (Lysosensor Green DND-153. Molec ular Probes. Inc.). Observations using a fluorescence microscope showed thai the same cells in B and C containing numerous large vacuoles (left pam-I) were siained more intensely with the lysosome-specific dye (right panel) compared with untreated cells. In panel D. lysosensor dye, added I h before DENSPM treat ment, prevented the formation of large vacuoles, bar (jiitnt't A), 10 /im. cytoplasmic vacuoles that appeared in DENSPM-treated CHO/664 cells. To gain insight into the nature of the vacuoles, CHO/664 (hereafter designated as CHO/100) cells were removed from CGP- exaggerated form (not shown). Examination of CHO/100 cells treated with 10 JUMDENSPM indicated that cytoplasmic vacuolation was well established by 24 h (Fig. 4C) and, when treated CHO/100 cells that had contained vacuoles for 48 h were placed in drug-free medium, 48664 for 3 days, treated for 72 h with 10 JUMDENSPM. and the vacuoles shrank (Fig. 3, £and F) and cell growth resumed. examined by light and electron microscopy. By light microscopic As further confirmation of the apparent lysosomal nature of the preview, 95% of the cells were observed to contain a large number of cytoplasmic vacuoles (Figs. 3D and 4C) that were not apparent in analogue-induced vacuoles. CHO/100 cells were treated with 10 /XM analogue-treated CHO parental cells (Fig. 3/4). A nearly identical DENSPM for 24 h and then stained with 1 p.M lysosensor green (a morphological effect was obtained with chloroquine (Fig. 4ß),a fluorescent dye specific for lysosomes; Ref. 30), 1 h before exami well-known lysosomotropic agent (27-29). Ultrastructural studies re nation by fluorescence microscopy (Fig. 4). The cytoplasm of DENSPM-treated CHO/100 was seen to contain large, brightly fluo vealed that virtually every cell contained swollen cytoplasmic vacu oles with electron-dense material typical of secondary lysosomes as rescent bodies that corresponded to the swollen vacuoles observed in well as numerous tiny vesicles presumed to be endosomal in origin the same field of cells with phase optics (Fig. 4C). The response of these cells to 100 /MMchloroquine for 3 h showed similar swollen and (Fig. 5). In many cases, the vacuoles appeared to be fusing with one another. All of the other organdÃ-es including mitochondria, endoplasfluorescent vacuoles (Fig. 4B). The cytoplasm of untreated CHO/100 mic reticulum, and nuclear membrane appeared normal and unswolcells (Fig. 4/4) contained much smaller fluorescent bodies, which were len. Ultrastructural studies confirmed light microscopic indications identical to those seen in untreated CHO cells (not shown) and that vacuoles did not form in the parental CHO cells even in a less presumed to be lysosomes. Interestingly, exposure to the fluorescent 3886 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1998 American Association for Cancer Research. LYSOSOMAL SEQUESTRATION OF POLYAMINE ANALOGUES B Fig. 5. Electron micrograph of untreated CHO/ 100cells (A) and those Ireated with 10 fiM DENSPM for 72 h (B and O showing the presenceof numerous cytoplasmic vacuoles containing electron dense ma terial and numerous small vesicles. Note that mito chondria and other cellular organdÃ-esare unaffected by analogue treatment, bar. I firn. dye for only l h before DENSPM treatment fully prevented vacuole formation (Fig. 4D). Taken together, the light and electron micro scopic data indicated that the vacuoles were most likely lysosomal in origin and suggested that DENSPM may possess lysosomotropic activity in CHO/100 cells similar to that reported for certain other polyamine analogues in LI210 cells (23). Because the lysosomal vacuolation only appeared in DENSPMtreated CHO/100 cells, two additional goals of the study were: (a) to determine whether it was a consequence of AdoMetDC gene overexpression; and (b) whether there exists a possible relationship between vacuole formation and DENSPM accumulation. For these purposes, a panel of CHO cells that were differentially resistant to CGP-48664 was derived by chronically treating cells with the drug beginning at concentrations of 0.1 JU.Mand increasing to a maximum of 100 /AM over the course of several months. Cells were grown for a minimum of at least eight passages before increasing the drug concentration. Adapted cells were removed from the common culture at 1,3, 10, 30, and 100 JAMCGP-48664 and maintained separately at those concen trations. The cells were then subjected to biochemical and molecular characterization. The CHO/100 cells were found to be remarkably similar to the originally derived CHO/664 cells, which were also resistant to 100 /J.M CGP-48664. AdoMetDC mRNA levels were measured by Northern blot analysis of resistant cells growing in the presence of CGP-48664 (Fig. 6). Quantitation of AdoMetDC mRNA levels relative to untreated CHO parental cells and normalized to GAPDH message, indicated an 11- to 15-fold increase for cells adapted to 1 and 3 JU.MCGP-48664, respectively, and an increase of 120- to 170-fold in cells adapted to 10 /J.Mand 100 /U.MCGP-48664 (Fig. 6; Table 2). These same cells were then compared for vacuole formation and analogue accumulation after treatment for 72 h with DENSPM (Fig. 3; Table 2). Two findings emerged: (a) vacuole formation seemed unrelated to gene overexpression inasmuch as it occurred in CHO/100 cells and to some degree in CHO/30 cells but not in CHO/10 cells, which, according to AdoMetDC transcript level, displayed a similar level of AdoMetDC gene overexpression. This suggests that, at sufficiently high drug levels, CGP-48664 apparently alters lysosomal membranes and/or contents. In this regard, it is relevant that vacuole formation did occur in CHO/100 cells when they were not removed from CGP-48664 before treatment with DENSPM (Fig. 3G), which suggests that, as with lysosensor green (Fig. 4D), there are direct effects on the lysosome; and (b) major increases in intracellular DENSPM levels correlated with the appearance of vacu oles (Table 2) suggesting that the analogue is sequestered within the vacuoles. mRNA _kb_ 3.4 AdoMetDC 2.1 GAPDH x11 x15 x120 x125 x170 x1 Fold Increase of AdoMetDC / GAPDH mRNA Fig. 6. Northern hint analysis of AdoMetDC mRNA from CHO cells for comparison with the CHO cell panel designated CHO/1. CHOß. CHO/10. CHO/30. CHO/100 described in "Materials and Methods." Note the presence of two delectable AdoMetDC transcripts of 3.4- and 2. l-kb lengths. The fold-increase values are based on the ratio of the 3.4-kb AdoMetDC transcript to the GAPDH mRNA to adjust for loading. 3887 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1998 American Association for Cancer Research. LYSOSOMAL SEQUESTRATION OF POLYAMINE ANALOGUES Table 2 Comparison of CHO cells that are differentially resistant to CGP-48664 were unable to induce the formation of vacuoles in the CHO/100 cells and did not accumulate to higher levels in CHO/IOO cells than in the h)fcCclls/CGP-48664resistant parental CHO cells (Table 5). On the other hand, all of the mono- and di-alkylated spermidine and spermine analogues behaved similarly to i-lliil.uCGP-48664"(pmol/IO6 AdoMettnRNA"1IIIS120125170Vacuoleformation(% DENSPM in their ability to induce vacuoles except for DMSPM and cells)<53060IÃŽ32211350Relative cells)6,5458,6507,6256,21513,69532,200 cells)000n4896IntraccllularDENSPM'(pmol/106 (/IM)CHO A/'-MESPM. In every case in which vacuoles were observed, ana logue levels were 3- to 5-fold higher (range, 13-31 nmol/106 cells) parentalCHO/ICHO/3CHO/ 10 ;ÃŒM DENSPM-trealed(72 than levels of analogue that failed to produce vacuoles. Unlike DM SPM, the two other dimethyl-analogues, A'1,Ai1'-dimethylnorspermine and Ar',/V'4-dimethylhomospermine, did induce vacuoles, which indi II)CHO/30CHO/IOOliin.i. cated that intra-amine carbon distances may play a role in lysosomal recognition. Thus, alkylation of the terminal amines seemed to be important, but not the only, structural determinant for triggering the lysosomotropic response. " Values determined on cells maintained in the presence of CGP-48664. * Cells grown in the absence of CGP-48664 for 72 h before DENSPM treatment. ' Values represent averages of six separate determinations with SD < 20%. The relative metabolic effects of differential amplification of the AdoMetDC gene are compared in Table 3. Because AdoMetDC activity is inhibited in the presence of CGP-48664, we measured enzyme activity, dcAdoMet pools, and polyamine pools on cells grown in the absence of drug for 72 h. We observed that enzyme activity did not seem to correlate closely with mRNA, in part because of compensatory responses to drug withdrawal and also because of the well-known posttranscriptional regulation of the enzyme activity (31). Nonetheless, enzyme activity was clearly increased in cells resistant to 10 JAMCGP-48664 and higher. These increases were paralleled by rises in dcAdoMet pools from <10 pmol/106 cells in CHO, CHO/1, and CHO/3 cells up to >6000 pmol/106 cells in CHO/IOO cells. In the CHO/IOO cells. AdoMetDC activity did not closely parallel the rise in pools. One possible explanation for this is that, although undetectable, residual CGP-48664 in the cell extract partially inhibits the enzyme under in vitro assay conditions. AdoMet pools were unaffected (data not shown). Shifts in polyamine pools favored spermine synthesis for the CHO/10, CHO/30, and CHO/IOO cells in which dcAdoMet pools were accumulated to levels that were no longer rate-limiting. Thus, despite the dramatic shift in the polyamine pool profile including an 80% decrease in spermidine pools, the cells remained viable with a similar growth rate. The similar polyamine profile in the CHO/10 and CHO/IOO cells would argue against vacuole formation being depend ent on the shifts in the pools created with CGP-48664 withdrawal. As further indication that DENSPM was contained within the vacuoles, we compared how effective the analogue was at mediating the various regulatory responses involving ODC, SSAT, and transport activities between the CHO. CHO/10, and CHO/IOO cells. The cells were removed from CGP-48664 for 72 h and cultured for an addi tional 72 h in the presence or absence of 10 /XMDENSPM. In CHO and CHO/10 cells, ODC activity was reduced by 98% whereas in CHO/IOO it decreased by only -40% (Table 4). A similar trend was seen with spermidine transport activity as indicated by DENSPMmediated decreases of >80% in Vm.lxin CHO and CHO/10 but not CHO/IOO cells. Notice that both ODC and transport were downregulated similarly in all of the three lines treated with spermine. The lack of regulatory response was most evident with SSAT activity where the analogue caused ~120- and 200-fold induction in enzyme activity in CHO and CHO/10 cells, respectively, but only a —¿6-fold DISCUSSION The present findings extend our earlier characterization of the CHO cell line resistant to CGP-48664, the only cell line known to contain an amplified AdoMetDC gene (6). Grown in the absence of CGP48664, in addition to being cross-resistant to other AdoMetDC inhib itors, these cells were unexpectedly found to be insensitive to the antiproliferative activity of DENSPM. It seemed relevant that treat ment with the analogue resulted in the formation of numerous cytoplasmic vacuoles. We now report that these vacuoles are part of the lysosomal system and that they contribute to the massive accumula tion of DENSPM and account for the growth insensitivity of these cells by compartmentalizing the analogue. Morphological evidence strongly suggests that the vacuoles are lysosomal in origin. By electron microscopy, the swollen vacuoles were seen to contain electron-dense material and endosomes typical of secondary lysosomes. In addition, the vacuoles were selectively stained by the supravital dye lysosensor green, which is known to accumulate specifically in that organelle (30). Finally, pretreatment of the cells with the dye interfered with vacuole formation by the analogue. In terms of light microscopy and dye staining, the vacuoles were found to be nearly identical to those produced by chloroquine, a well-known lysosomotropic agent (27-29). Other organdÃ-es includ ing the endoplasmic reticulum, the Golgi apparatus, the mitochondria, and the nuclear membrane were unaffected by the drugs, which indicates that the effect was not due to some generalized osmotic or toxic effect. It is interesting that CGP-48664 resistance should be associated with a lysosomal effect inasmuch as the compound from which it was conceptually derived, MGBG, is well known for its selective effect on mitochondria leading to a cytoplasmic vacuolation that at the light microscopic level appears to be nearly identical to the one observed here (32). Thus far, the profound lysosomal vacuolation seen with DENSPM only occurs in CHO cells resistant to CGP-48664. Because such vacuoles do not form in parental cells, they must represent an adap tation to treatment with CGP-48664 during derivation of the resistant increase in the CHO/IOO cells. Importantly, DENSPM accumulated to much higher levels in CHO/IOO cells than in either CHO or CHO/10 cells. Overall, the data are consistent with the interpretation that in CHO/30 and CHO/IOO cells, DENSPM is sequestered inside the vacuoles and is unavailable to interact at various polyamine regulatory sites located elsewhere in the cell. Finally, we examined whether natural polyamines and certain an alogues of DENSPM could induce vacuoles in CHO/100 cells. Data in Fig. 3 indicate that the natural polyamines, as well as norspermine. Table 3 Characterization of metabolic shifts in CHO cells that are differentially resistant to CGP-48664 Polyamine pools* (pmol/106 cells) Cells/CGP-48664 AdoMetDC'' resistance" (JÕM) (nmol/h/mg) dcAdoMet'' (pmol/lO6 cells) Pulrescine Spermidine Spermine CHO parental <IO<10140020406100270 0.700.35353522<10 241520652305001602510 3605352533253430 CHO/1CHO/3CHO/10CHO/30CHO/IOO0.75 115854560752340 " These cells were grown in the absence of CGP-48664 for 72 h. '' Values represent at least three separate determinaiions with SD < 20%. 3888 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1998 American Association for Cancer Research. LYSOSOMAL SEQUESTRATIONOF POLYAMINE ANALOGUES Table 4 DENSPM regulation of enzyme and transpon activities in CHO ceux that are differentially rexisttwt to CGP-48664 DENSPM" Cell/CGP-48664 cells)6,5457.62532.200average (pmol/106 (nmol/h/mg)0.850.020.03 (JIM)CHOCHO/IO''CHO/100''" resistance (10 h)NoneDENSPMSPMNoneDENSPMSPMNoneDENSPMSPMIntracellular JIM. 72 1.150.060.01 1.100.500.03with Values represent the of at least two separate determinations performed in duplicate ' Resistant cells wereTreatment grown in the absence of CGP-48664 for 72 h before treatment.ODCactivity" line. The possibility was examined that vacuole formation might somehow be related to overexpression of the AdoMetDC gene as has been shown to be the case in CHO/664 cells (6). It is possible, for example, that cells that overexpress AdoMetDC have metabolic per turbations, such as overproduction of spermine, that lead to adaptive responses intended to minimize resulting toxic effects. We examined this possibility by deriving and characterizing a panel of CHO cells that were differentially resistant to CGP-48664. Although on the basis of transcript level the AdoMetDC gene was differentially overexpressed in all of the CHO cell variants chronically exposed to CGP48664. DENSPM-induced vacuole formation only occurred in the CHO/100 cells and to a lesser degree in CHO/30 cells. Because vacuolation did not occur in CHO/10 cells with a polyamine-pool profile similar to that in the CHO/30 and CHO/100 cells, related metabolic effects such as a possible rapid accumulation of spermine could not account for the lysosomal response to DENSPM. On the other hand, we did not encounter cells that formed vacuoles but did not overexpress AdoMetDC. Thus, although AdoMetDC overexpression alone can account for low level CGP-48664 resist ance, it is not clear whether lysosomal changes are required for high-level resistance or whether they are simply a consequence of the higher levels of drug exposure. Because CGP-48664 is a basic com pound, it is possible that high levels of drug exposure result in the interaction of the drug with the lysosomal system without producing the swelling seen with the analogue. When the CHO/100 and CHO/30 cells are continuously grown in CGP-48664 before and during transport" (pmol/min/mg)3,700415150 2.63020095 3.1601.39090SSAT activity" (pmol/min/mg)784510 71.43022 95010 SD < 10%.Spermidine DENSPM treatment, vacuole formation is not observed and DENSPM is only accumulated to levels similar to those seen in CHO and CHO/10 cells. This suggests that CGP-48664 is bound at intracellular sites, thus preventing analogue accumulation into the lysosome. The fact that the level of DENSPM accumulation in cells that form vacuoles (CHO/100) is ~5 times higher than in cells that do not form vacuoles (CHO/10) suggests that analogue may actually be contained in the swollen organelles. Further evidence for this is provided by a comparison of growth inhibitory and gene regulatory responses in CHO/100 cells treated with either the analogue or the natural poly amine spermine. Despite accumulating to extremely high levels, DENSPM is not particularly more growth inhibitory and is much less effective than spermine at down-regulating ODC and transport. The two are known to behave almost identically in this regard (13, 25), including the CHO cells shown here (Table 4). Likewise, although DENSPM accumulated to higher levels in CHO/100 cells than in CHO/10 cells, induction of SSAT by the analogue was less apparent in the former cells. Both induction of SSAT and down-regulation of ODC are regarded as largely posttranscriptional events and thus should occur in the cytosol (33, 34). Thus, the overall data are consistent with the likelihood that most intracellular DENSPM is sequestered in the vacuoles and unable to interact at various regula tory sites represented elsewhere within the cell. There is a biological precedent for storage of polyamines in vacu oles. Davis and colleagues have shown (35, 36) that under normal cell culture conditions, as much as 25% of the total spermidine of Neu rospora cells resides specifically within the cytoplasmic vacuole (often compared with the animal lysosome) of these organisms where Table 5 Comparison of polyamine and polyamine analogue acctinndiiiion and induction of cytoplasmic vacuoles in CHO and CHO/100 cells" it complexes with polyphosphates. In addition, we have shown pre viously (23) that certain polyamine analogues that are structurally cells'*VacuolesnononoyesyesyesyesyesyesyesnoyesnoAccumulation'(nmol/106 cellsTreatment(10 CHO dissimilar and many that are structurally similar to DENSPM (Table 5) can also behave as lysosomotropic agents. Depending on the net 'nononononononononononononoAccumulation*(nmol/106 cells)5427435444432CHO/100 cells)345311821262727137154 h)SpermidineSpermineNorspermineDESPDN'-MESPDN8-MESPDDENSPMDESPMDEHSPMDMNSPMDMSPMDMHSPMN'-MESPMVacuoles JIM. 72 pK.t of a molecule, compounds such as chloroquine (27-29) enter lysosomes and become irreversibly trapped in the organelle by pro- "DESPD. W'A^-diethylspermidine: W'-MESPD. W'-monoethylspermidine; MESPD, /V^-monoethylspermidine; DESPM, /v'./Vl2-diethylspermine: DEHSPM, Af',JVl4-dietnylhomospermine; DMNSPM, Nl V-dimethylnorspermine: DMSPM, ¿V'JVl2-dimethylspermine: DMHSPM. AT1 JV14-dimelhylhomosperniinc: /v'-MESPM. /V1monoethylspermine. * Vacuoles were visualized under a phase contrast microscope using L40/0.50 Phaco 1a optics. ' Values represent the average of three separate determinations with SD < 20%. Cells were grown in the absence of CGP-48664 for 72 h before analogue treatment. tonation. The continued accumulation of the drug leads to lysosomal swelling and the eventual formation of visible vacuoles identical to those we have described here. Because in the case of DENSPM, vacuole formation has not been observed to occur in other cell types, we presume that the lysosomal properties of CHO/KX) cells have been altered by CGP-48664 in such a way that the analogue can either enter the lysosome more readily or is more effectively protonated once inside. In this regard, drug accumulation into lysosomes has been attributed to alterations in the intralysosomal milieu (37). Changes in lysosomal pH or membrane permeability brought about by previous exposure to CGP-48664 represent two possibilities. As noted above, these vacuoles do not seem to form in cells treated with spermine and, therefore, probably do not represent a drug-mediated exaggeration of a biological mechanism for processing or storing naturally occurring polyamines. Although the pK, of the molecule may be one character- 3889 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1998 American Association for Cancer Research. LYSOSOMAL SEQUESTRATION istic responsible for the vacuole formation in the CHO/100 cells, this does not seem to be the case inasmuch as Bergeron et al. (38) reported that the pK., values for the four charged nitrogens of spermine (pA"a values = 7.95, 8.82, 10.05 and 10.86) are similar to those of DENSPM (pA"a values = 7.68, 8.51, 9.91 and 10.83). Studies with DENSPM analogues (Table 5) indicated that vacuolation may be a function of terminal alkylation as well as intra-amine carbon dis tances. Thus, the overall mechanism remains to be defined. It would seem that vacuole formation represents a potential means by which cells can become resistant to the growth inhibitory effects of DENSPM. As shown here, cells that form vacuoles (i.e., CHO/100 cells) are less sensitive to growth inhibition by the analogue than those that do not (i.e., CHO/10 and parental cells). The unusual feature of this form of resistance is that the cells actually accumulate more of the analogue rather than less, unlike the more classical form of drug resistance involving defects in polyamine transport (39). The findings also have obvious therapeutic relevance for CGP-48664 inasmuch as they seem to indicate another site of action of the drug that could have greater relevance under in vivo conditions. At least two groups have reported (7-9) that the magnitude of the inhibitor effects on tumor polyamine pools does not seem to correlate with the substantial antitumor activity seen in certain in vivo systems. The present finding provides an additional or alternative possible mechanism for in vivo antitumor activity and/or host toxicities. ACKNOWLEDGMENTS The authors gratefully acknowledge the technical assistance Diegelmun. John Miller. Deborah Ogden, and Edward Hurley. of Paula REFERENCES 1. Pegg. A. E. Polyamine metabolism and its importance in neoplastic growth and as a target for chemotherapy. Cancer Res.. -Hi: 759-774, 1988. 2. Marlon. L. J.. and Pegg. A. E. Polyamines as targets for therapeutic intervention. Annu. Rev. Pharmacol. Toxico].. 35: 55-91. 1995. 3. Stanek. J.. Cravatti. G.. Frei. J.. Furet. P.. Melt. H.. Schneider. P.. and Regenass. U. 4-Amidinoindan-l-one 2'-amidinohydrazone: a new potent selective inhibitor of S-adenosylmclhionine decarhoxylase. J. Med. Chem. 36: 2168-2171. 1993. 4. Regenass. U.. Caravatti. G.. Melt. H.. Slanek. J.. Schneider. P.. Muller. M.. Malier. A.. Vertino. P.. and Porter. C. W. New S-adenosylmethionine decarboxylase inhibi tors with potent antilumor activity. Cancer Res.. 52: 4712-4718, 1992. 5. Regenass. U.. Me». H.. Slanek, J.. Muller. M.. Kramer, D., and Porter, C. W. CGP-48664. a new S-adenosylmelhionine decarboxylase inhibitor with broad specIruin antiprolifcralive and antitumor activity. Cancer Res.. 54: 3210-3217, 1994. 6. Kramer. D. L.. Melt. H.. Regenass, U., Diegelman. P.. and Porter. C. W. Stable amplificalion of ihe .C-adenosylmelhionine decarboxylase gene in Chinese hamster ovary cells. J. Biol. Chem.. 270: 2124-2132, 1995. 7. Delworth. M. G., Nishioka. K.. Pellaway. C.. Gulman. M.. Killion. J. J., von Eschenbach, A. C., and Fidler, I. J. Syslcmic adminislralion of 4-amidinoindanon-l-(2'-amidino)hydrazone. a new inhibitor of .S'-adcnosylmethionine decarboxylase. produces cylostasis of human prostale cancer in athymic nude mice. Ini. J. Oncol.. 6: 293-299, 1995. 8. Dorhoul. B.. le Velde. R. J.. Ferwada. H.. Kingma. A. W.. and Muskiet. F. A. In vivo effecls of 4-amidinoindane-l-one 2'amidinohydra7.one (CGP-48664) and adifluoromelhylomilhine (DFMO) on LI2IO cell growlh. cell cycle phase dislribulion and polyamine contents. Int. J. Cancer. 62: 738-742. 1995. 9. Dorhout, B., Odink. M. F. G., deHoog, E.. Kigma. A. W.. van der Veer, E., and Muskiet. F. A. J. 4-Amidinoindan-l-one-2'amidinohydrazone (CGP-48664) exerts in vitro growth inhibitory effecls lhal are noi only relaled to 5-adenosylmelhionine decarboxylase (SAMdc) inhibilion. Biochim. Biophys. Acia, 1335: 144-152, 1997. 10. Porter, C. W.. Sui. L.. Kramer, D. L., Meli. H.. Barbet. N.. Linnarts. R., and Eckhardl. S. G. Pharmacodynamic confirmation of CGP-48664 as an inhibitor of the polyamine biosynthetic en/.yme .S-adenosylmelhionine decarboxylase (SAMDC) in an advanced melanoma patient. Tenth NCI.EORTC Symposium on New Drugs in Cancer Therapy, p. 128. 1998. 11. Casero. R. A.. Celano. P., Ervin. S. J., Porter, C. W.. Bergeron. R. J., and Libby, P. R. Differential induction of spermidine/spermine N'-acetyllransferase in human lung cancer cells by Ihe bislelhyllpolyamine analogues. Cancer Res., 49: 3829-3833. 1989. 12. Pegg, A. E.. Wechter. R.. Pakala. R.. and Bergeron. R. J. Effect of N1. N12his(elhyl)spermine and relaled compounds on growlh and polyamine acelylalion, content, and excretion in human colon lumor cells. J. Biol. Chem.. 264: 1174411749. 1989. OF POLYAMINE ANALOGUES 13. Porter, C. W.. Ganis. B.. Libby. P. R., and Bergeron. R. J. Correlations between polyamine analog-induced increases in spermidine/spermine N'-acetyltransferase aclivily and growlh inhibilion in human melanoma cell lines. Cancer Res., 51: 37153720, 1991. 14. Pielilä.M., Alhonen, L.. Halmekylö, M., Kanler. P.. Janne. J.. and Porter, C. W. Aclivalion of polyamine calabolism profoundly allers lissue polyamine pools and affecls hair growlh and female fertility in Iransgenic mice overexpression spermidine/ spermine ¿V'-acelyllransferase. J. Biol. Chem.. 272: I8746-I875I, 1997. 15. Alhonen. L.. Karppinen, A.. Uusi-Oukari, M.. Vujcic, S., Korhonen. V-P., Halmekylö. M., Kramer, D. L., Hiñes.R., Jänne,J., and Porter. C. W. Correlalion of polyamine and growlh responses lo A/",W"-dielhylnorspermine in primary felal fibroblasts derived from transgenic mice overexpressing spermidine/spermine N'acetyllransferase. J. Biol. Chem.. 273: 1964-1969, 1998. 16. Porter. C. W., Regenass. U., and Bergeron. R. J. Polyamine inhibitors and analogs as potenlial anticancer agents. In: R. H. Dowling. U. R. Folsch. and C. Loser (eds.), Falk Symposium on Polyamines in Ihe Gaslroinleslinal Tract, pp. 301-322. Dordrecht. Germany: Kluwer Academic Publishers, 1992. 17. Bernacki. R. J.. Bergeron. R. J.. and Porter. C. W. Antitumor activity of W'JV12bis(elhyl)spermine homologs againsl human MALME-3 melanoma xenografls. Can cer Res.. 52: 2424-2430. 1992. 18. Porter. C. W.. Bemacki. R. J.. Miller. J.. and Bergeron. R. J. Antitumor activity of Arl.W"-bis(ethyl)-norspermine against human melanoma xenografts and possible biochemical correlales of drug action. Cancer Res., 53: 581-586. 1993. 19. Bernacki. R. J.. Oberman. E. J.. Bergeron. R. J.. and Porter. C. W. Broad prcclinical antitumor efficacy with the polyamine analog, AflJV"-dielhylnorspermine. Clin. Cancer Res.. /: 847-857. 1995. 20. Bergeron. R. J., McManis. J. S.. Liu. C. Z.. Feng, Y.. Weimer. W. R.. Luchetia, G. R., Wu. Q.. Ortiz-Ocasio. J.. Vinson. J. R. T.. Kramer. D.. and Porter, C. W. Antiproliferative properties of polyamine analogues: a struclure-aclivily study. J. Med. Chem.. 37: 3464-3476. 1994. 21. Bergeron, R. J.. Neims, A. H.. McManis. J. S.. Hawthorne. T. R.. Vinson, J. R. T.. Bortell. R.. and Ingeno. M. J.. Synthetic polyamine analogues as antineoplaslics. J. Med. Chem.. 31: 1183-1190. 1988. 22. Pajunen. A., Crozal. K. A.. Janne. O., Ihalainen. R., Lailinen, P., Slanley, B., Madhubala. R., and Pegg. A. E. Structure and regulation of mammalian 5-adenosylmethionine decarboxylase. J. Biol. Chem., 263: 17040-17049, 1988. 23. Porter. C. W., Stanek. J.. Black. J.. and Pleshkewych, A. Morphologic evidence for lysosomolropic aclivily by unsaturaled pulrescine analogs. Cancer Res.. 50: 1929-1935, 1990. 24. Yarletl. N.. and Bacchi. C. J. Effect of DL-a-difluoromethylornilhine on methionine cycle inlermediales in Trypantmima hmcei brucei. Mol. Biochem. Parásito!., 27: 1-10. 1988. 25. Kramer. D. L., Miller. J. T.. Khomulov. R.. Khomulov. A.. Bergeron, R. J., and Porter. C. W. Regulalion of polyamine Iransport by polyamines and polyamine analogs. J. Cell Physiol., 115: 399-407. 1993. 26. Fogel-Pelrovic. M.. Shappell. N. W.. Bergeron. R. J.. and Porter, C. W. Polyamine and polyamine analog regulalion of spermidine/spermine N ' -acelyltransferase mRNA accumulation in MALME-3M human melanoma cells. J. Biol. Chem. 268: 191 1819125. 1993. 27. Fedorko. M. E.. Hirsch. J. G.. and Cohn. Z. A. Aulophagic vacuoles produced in vitro. I. Sludies on cullurcd macrophages exposed to chloroquine. J. Cell. Biol.. 38: 377-391, 1968. 28. Fedorko. M. E.. Hirsch. J. G.. and Cohn, Z. A. Autophagic vacuoles produced in vilro. II. Studies on the mechanism of formation of autophagic vacuoles produced by chloroquine. J. Cell Biol., 38: 392-402, 1968. 29. DeDuve, C.. De Barsey. T.. Poole. B.. Trauet. A.. Tulkens. P., and Van Hoof, F. Lysosomotropic agents. Biochem. Pharmacol.. 23: 2495-2531. 1974. 30. Haugland. R. Cell-permeant probes for lysosomes and other acidic organdÃ-es. In: M. Spence (ed.). Handbook of Fluorescent Probes and Research Chemicals, 6lh ed., p. 275. Eugene. OR: Molecular Probes. Inc.. 19%. 31. Pegg. A. E., and McCann. P. P. 5-adenosylmelhionine decarboxylase as an enzyme largel for Iherapy. Pharmacol. Therap.. 56: 359-377, 1992. 32. Mikles-Robertson, F., Feuerslein, B.. Dave. C., and Porter. C. W. The generality of methylglyoxal-bis(guanylhydrazone) (MGBG)-induced milochondrial damage and the dependence of this effect on proliferaiâ„¢ activity. Cancer Res., 39: 1919-1926. 1979. 33. Pegg, A. E., Shantz, L. M., and Coleman, C. S. Ornithine decarboxylase structure, function and translational regulalion. Biochem. Soc. Trans.. 22: 846-852. 1994. 34. Fogel-Petrovic, M., Vujcic. S.. Miller, J., and Porter. C. W. Differential poslIranscriplional control of omithine decarboxylase and spermidine/spermine N1acelyllransferase by polyamines. FEES Lett.. 391: 89-94, 1996. 35. Paulus. T. J.. Cramer. C. L.. and Davis. R. H. Compartmenlalion of spermidine in Neurospora crossa. 1. Biol. Chem.. 255: 8608-8612. 1983. 36. Cramer. C. L., and Davis. R. H. Polyphosphate-cation inleraclion in Ihe amino acid containing vacuole oÃ-Neurospora crossa. L Biol. Chem., 259: 5152-5157. 1984. 37. Ohkuma. S.. and Poole. B. Cyloplasmic vacuolation of mouse peritoneal macro phages and Ihe uplake into lysosomes of weakly basic subslances. J. Cell. Biol.. 90: 656-664. 1981. 38. Bergeron. R. J.. Weimer. W. R.. Wu, Q.. Feng. Y.. and McManis, J. S. Polyamine analogue regulalion of NMDA MK-801 binding: a slruclure-aclivily sludy. J. Med. Chem.. 39: 5257-5266. 1996. 39. Shao, D.. Xiao. L., Hyo-Chol. H.. and Casero. R. A. Isolalion of a polyamine Iransport deficient cell line from the human non-small cell lung carcinoma line NCI HI57. J. Cell. Physiol., 166: 43-46. 1996. 3890 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1998 American Association for Cancer Research. Lysosomal Sequestration of Polyamine Analogues in Chinese Hamster Ovary Cells Resistant to the S-Adenosylmethionine Decarboxylase Inhibitor, CGP-48664 Debora L. Kramer, Jennifer D. Black, Helmut Mett, et al. Cancer Res 1998;58:3883-3890. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/58/17/3883 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1998 American Association for Cancer Research.