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[Cancer Research 59, 1278-1286, March 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 1278-1286, March 15, 1999]
© 1999 American Association for Cancer Research

Experimental Therapeutics

Polyamine Analogue Induction of the p53-p21WAF1/CIP1-Rb Pathway and G1 Arrest in Human Melanoma Cells1

Debora L. Kramer, Slavoljub Vujcic, Paula Diegelman, James Alderfer, John T. Miller, Jennifer D. Black, Raymond J. Bergeron and Carl W. Porter2

Grace Cancer Drug Center [D. L. K., S. V., P. D., J. T. M., J. D. B., C. W. P.] and Molecular and Cellular Biophysics [J. A.], Roswell Park Cancer Institute, Buffalo, New York 14263; and Department of Medicinal Chemistry, University of Florida J. Hillis Health Center, Gainesville, Florida 32610 [R. J. B.]

ABSTRACT

Although polyamines are well recognized for their critical involvement in cell growth, the cell cycle specificity of this requirement has not yet been characterized with respect to the newly delineated regulatory pathways. We recently reported that polyamine analogues having close structural and functional similarities to the natural polyamines produce a distinct G1 and G2-M cell cycle arrest in MALME-3M human melanoma cells. To determine a molecular basis for this observation, we examined the effects of N1,N11-diethylnorspermine on cell cycle regulatory proteins associated with G1 arrest. The analogue is known to deplete polyamine pools by suppressing biosynthetic enzymes and potently inducing the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. Treatment of MALME-3M cells with 10 µMN1,N11-diethylnorspermine caused an increase in hypophosphorylated Rb, which correlated temporally with the onset of G1 arrest at 16–24 h. Rb hypophosphorylation was preceded by an increase in wild-type p53 (~10-fold at maximum) and a concomitant increase in the cyclin-dependent kinase inhibitor, p21WAF1/CIP1 (p21; ~5-fold at maximum). Another cyclin-dependent kinase inhibitor, p27KIP1, and cyclin D1 increased slightly, whereas proliferating cell nuclear antigen and p130 remained unchanged. Induction of p21 protein was accompanied by an increase in p21 mRNA, whereas induction of p53 protein was not, suggesting transcriptional activation of the former and posttranscriptional regulation of the latter. SK-MEL-28 human melanoma cells, which contain a mutated p53, failed to induce p53 or p21 and did not arrest in G1. Rather, these cells rapidly underwent programmed cell death within 48 h. Overall, these findings provide the first indication of the cell cycle regulatory pathways by which polyamine antagonists such as analogues might inhibit growth in cells containing wild-type p53 and further suggest a mechanistic basis for differential cellular responses to these agents.

INTRODUCTION

Polyamines are organic cations that are synthesized by all cells and are likely to be involved in multiple functions required for cell growth. Evidence from clinical studies (1, 2, 3) strongly suggests that dependence on polyamines for growth may be greater in neoplastic cells because polyamine pools and enzyme activities of tumors are significantly higher than those of the surrounding normal tissues, even in rapidly growing systems, such as the gastrointestinal tract epithelium. The requirement for polyamines in cell cycle progression has been frequently investigated but variably defined. Some reports implicate their involvement in the G1-S transition (4, 5, 6, 7, 8, 9) , and others suggest involvement the S and/or G2 phases of the cell cycle (10, 11, 12) ; still others suggest a general lengthening of all phases of the cycle (13) . This lack of literature consensus is probably attributable to differences in the cell type being studied, the enzyme being inhibited, and/or the specificity of the inhibitors being used. In addition, there are certain inherent technical difficulties imposed by the length of time required to deplete natural polyamines with inhibitors. It is relevant that dramatic increases in the activity of the sentinel polyamine biosynthetic enzyme ODC3 are associated with the G1-S transition of virtually all cell lines (14, 15, 16, 17, 18, 19, 20, 21) . A molecular basis for this is derived from the finding that ODC is among the few genes known to be transactivated by the cell cycle-regulated transcription factors, c-myc and N-myc (17, 18, 19) , a finding that clearly implicates polyamine participation in the G1-S transition.

Three scientific developments provide the opportunity to more precisely elucidate the polyamine requirement in cell cycle progression and, at the same time, to gain insight into their function: (a) the availability of polyamine analogues having close structural and functional similarities to the natural polyamines; (b) the discovery of well-characterized inhibitors that are highly specific for key polyamine biosynthetic enzymes; and (c) more significantly, the elucidation of complex regulatory pathways that control the cell cycle. Thus far, these advantages have not been extended to mapping the polyamine requirement in cell growth, and only a few previous reports describe the effects of polyamine depletion on a cell cycle regulatory protein. Thomas and Thomas (20) found that treatment of MCF-7 cells with an ODC inhibitor increased cyclin B1 mRNA levels 2–3-fold, and Casero and coworkers (21 , 22) reported that lowered c-myc expression precedes growth inhibition in polyamine-depleted cells.

Here, we have chosen to examine the effects of a well-characterized polyamine analogue on cell cycle regulatory pathways. The analogue, DENSPM (Fig. 1Citation ; also known as DE-333), is known to inhibit cell growth by what appears to be disruption of intracellular polyamine pool homeostasis (23, 24, 25) . More specifically, it down-regulates the polyamine biosynthetic enzymes ODC and S-adenosylmethionine decarboxylase and potently up-regulates the catabolic enzyme SSAT. The net result is a rapid and near total depletion of intracellular polyamine pools. The analogue is currently undergoing Phase II clinical evaluation as an anticancer agent. We recently reported that DENSPM and a series of related analogues cause G1 and G2-M arrests, followed by a delayed apoptotic response in MALME-3M human melanoma cells (26) . Our present findings indicate that the G1 arrest is associated with a sharp and early induction of the p53-p21-Rb pathway and that this response is not seen in melanoma cells containing mt-p53. This is a unique and obvious example of a positive gene response to polyamine analogues (or polyamines) that is not related to polyamine metabolism or transport. In combination with newly available inhibitors and analogues, the system provides a sensitive and early end point for defining the role of polyamines in cell proliferation, beginning with the upstream effectors involved in the p53 response. Portions of this work have been published in an abbreviated form as a meeting presentation (27) .



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  		Fig. 1. Structures of the natural polyamines and polyamine analogues DENSPM (or DE-333), 3,7,12,17-tetra-azanonadecane[N1,N13-diethyl(aminopropyl)homospermidine] (DE-443), N1,N12-diethylspermine (DE-343), and N1,N14-diethylhomospermine (DE-444). Note that these tetra-amine homologues differ only in the intra-amine carbon distances.

 
MATERIALS AND METHODS

Materials.
The polyamine analogue DENSPM (Fig. 1)Citation was provided by Parke Davis (Ann Arbor, MI). Three additional DENSPM analogues (Fig. 1)Citation were synthesized as published recently (28) and provided by Dr. Raymond Bergeron (University of Florida, Gainesville, FL). The inhibitor of polyamine oxidase, MDL-72,527 [N1-methyl-N2-(2,3-butadienyl)butane-1,4-diamine; Ref. 29 ], was generously provided by Hoechst Marion Roussel (Bridgewater, NJ). MALME-3M human melanoma cells containing wt-p53 (30) and SK-MEL-28 cells containing mt-p53 (30) were both purchased from the American Tissue Type Culture Collection (Manassas, VA).

Cell Culture.
MALME-3M and SK-MEL-28 human melanoma cells were maintained as monolayer cultures growing in RPMI 1640 containing 10% Nu-Serum (Collaborative Research Products, Bedford, MA), 1 mM aminoguanidine, penicillin (50 units/ml), and streptomycin (50 µg/ml). Under these conditions, both MALME-3M and SK-MEL-28 cell lines were found to have a doubling time of 40–45 h. Cells were seeded 24 h prior to treatment, and growth inhibition was assessed by dose-response curves at 96 h and by time course growth studies at 10 µM concentrations unless otherwise indicated. Other assays were determined on cells treated with 10 µM analogue for variable time periods. Cell number was determined electronically using a Model ZM Coulter Counter (Coulter Electronics, Hialeah, FL). Dose-response data were expressed as a percentage of the initial seeding density to define IC50s as well as the CTD. The latter was identified as the treatment dose at which cell density fell below that of the seeding density.

Assay Time Lines.
Biochemical parameters, including polyamine metabolic enzyme activities, polyamine pool levels, and analogue accumulation, were determined following 24 h of treatment with 10 µM analogue. Cell cycle analysis was determined on the attached cells after treatment periods of 48 h for MALME-3M cells and 24 h for SK-MEL-28 cells. Analysis of apoptosis on pooled attached and detached cell populations with 10 µM DENSPM was carried out following 24-, 48-, 72-, and 96-h incubations of both cell lines. Extended growth curves evaluating cytotoxicity in MALME-3M cells were carried out by continuous exposure to 10 µM analogue for 6 days.

Polyamine Enzymes and Pools.
All basic biochemical assays, including cell culture, enzyme assays, polyamine pool, and analogue determinations, were carried out as described recently (26 , 31) . The polyamine pool assay is based on high-performance liquid chromatography separation and detection. The system also detects polyamine analogues, thus allowing for normalization of biological effects according to intracellular drug levels.

Flow Cytometry.
Following treatment of MALME-3M cells for 48 h and SK-MEL-28 cells for 24 h, cell samples were stained with propidium iodide using a procedure described by Krishan (32) and subjected to cell flow analysis using a FACScan flow cytometry unit (Becton Dickinson, San Jose, CA), available at the Institute Core facility under the direction of Dr. Carleton Stewart. Modifications of these methodologies have recently been successfully applied in the analysis of analogue-treated cells by Kramer et al. (26) .

Apoptosis.
Loss in cell number, as detected by growth analysis, was taken as an initial indication of programmed cell death. One quantitative method used to assess apoptosis involved proton NMR spectroscopy for detection of membrane phospholipid spectral intensity changes (33) , which have been shown to correlate very closely with the surface expression of phosphatidylserine, an early marker of apoptosis, as determined by fluorescein-annexin V detection using flow cytometry (34) . To prepare cells for NMR analysis, ~5 x 107 cells were washed twice in PBS made with deuterium water (D2O) obtained from Cambridge Isotope Laboratories (Andover, MA), resuspended in 500 µl of PBS-D2O, and place on ice until data acquisition as described previously (33 , 34) . The areas included in the methylene (CH2) and methyl (CH3) peaks at 1.3 and 0.9 ppm, respectively, were determined, and CH2:CH3 ratios were calculated. SK-MEL-28 cells were stained for annexin V, as indicated in a commercial apoptosis detection kit (R&D Systems, Minneapolis, MN). Further confirmation of apoptosis was carried out by quantitative morphological analysis using detached cell populations stained with H&E (26) and by fluorescence-activated cell sorting analysis, in which apoptotic cells are typically apparent as a sub-G1 population, as described previously (26) .

Northern and Western Blot Assays.
Northern and Western blots were used for mRNA and protein detection, respectively, as reported in Fogel-Petrovic et al. (35 , 36) . Antibodies used for detecting specific cell cycle regulatory proteins by Western blot were obtained commercially as follows. Rabbit polyclonal p130 (C-20), mouse monoclonal p53 (DO-1), and goat polyclonal p16 (C-20) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal PCNA (Ab-1), rat monoclonal cyclin D1, and mouse monoclonal cyclin E were purchased from Oncogene Research Products (Cambridge, MA). Mouse monoclonal Rb and p21 was purchased from PharMingen (San Diego, CA); mouse monoclonal p27 was from Transduction Labs (Lexington, KY); and mouse monoclonal ß-actin, which was used to as a lane-loading control, was from Sigma Chemical Co. (St. Louis, MO). cDNA probes used in Northern blot hybridization of p21 and p53 mRNA were obtained from Dr. Bert Vogelstein (Ref. 37 ; Johns Hopkins Oncology Center, Baltimore, MD) and from Dr. Stephen Friend (Ref. 38 ; Fred Hutchinson Cancer Research Center, Seattle, WA), respectively.

To ensure that control cells gave reproducible baseline protein levels, we carefully maintained cell cultures in a constant semiconfluent and logarithmically growing state throughout each experiment. Multiple controls seeded at varying densities yielded similar protein signals (data not shown). For time course experiments, treatments were staggered to allow simultaneous processing of the untreated and treated samples.

RESULTS

The time course kinetics were determined in MALME-3M cells treated with 10 µM DENSPM for 0–96 h by flow cytometry. The onset of G1 arrest began at ~16 h and progressed as indicated by a steady loss in S-phase cells and increase in G1 cells (Fig. 2)Citation . The accumulation of cells in G1 was complete by 48 h, at which time the proportion of cells in S phase had declined from an original level of 20% to <2%. Despite this marked loss in S-phase cells, the proportion of cells in G2-M phase remained ~10%, suggesting the coexistence of a G2-M block. In contrast, the natural polyamine spermine at 10 µM had no similar effect on either cell growth or cell cycle progression (data not shown). The DENSPM-associated G2-M block was more obvious in synchronized MALME-3M cells, as shown in Fig. 3Citation . Following serum deprivation for 120 h, the majority of cells were accumulated in G1 (>95%). When released with serum, a large proportion moved into S phase in the absence or presence of 10 µM DENSPM. However, by 48 h, cells released in the presence of DENSPM were partitioned in the G1 (70%) and G2-M (30%) phases of the cycle, with no detectable S-phase cells. The continuation of this cell cycle profile at 60 h suggests that the cells were arrested at both checkpoints, although the possibility that the G2-M cells were actually 4 N cells arrested in G1 (39) could not be excluded.



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  		Fig. 2. Time-dependent effects of 10 µM DENSPM on asynchronous MALME-3M human melanoma cell growth (A) and cell cycle distribution (B). Note in A that cells continue to grow steadily for the first 24 h and then plateau and begin to decline in number from 48 h onward. Cell cycle data in B represent relative percentage of total cells in G1 and G2-M (left axis) or S (right axis) phases of the cell cycle. Note that G1 arrest begins at 16 h, as indicated by a concomitant increase of cells in G1 and a decrease of those in S. Data points, means based on determinations from three separate experiments; bars, SD. Only the attached (viable by trypan blue exclusion) cell populations were used for these studies.

 


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  		Fig. 3. Evidence for a DENSPM-induced G1 and G2-M cell cycle block in MALME-3M cells synchronized in G0/G1 for 120 h by serum starvation (designated as -S). Following serum addition (designated as +S), untreated cells (left) resumed an asynchronous cycling profile by 48 h, whereas DENSPM-treated cells (right) accumulated in G1 and G2-M (arrow). The data are representative of two separate experiments.

 
We next treated asynchronously growing cells with 10 µM DENSPM from 0 to 48 h to determine whether changes in G1-associated regulatory proteins had occurred, focusing on the retinoblastoma protein Rb. The analysis revealed a shift of Rb from a hyperphosphorylated (ppRb) to hypophosphorylated (pRb) state, which began with the appearance of the pRb species at ~16 h and a concomitant decrease in the ppRb species (Fig. 4)Citation . This shift in Rb status correlated closely with the onset of G1 arrest (Fig. 2)Citation . There was a 2-fold increase in cyclin D1 at 20 h (Fig. 4)Citation and a ~50% decrease in cyclin E over the 48-h time course. This transient 2-fold increase at 20 h of cyclin D1 may represent a compensatory cellular attempt to reestablish cycling or, more likely, stabilization of the cdk/cyclinD1 complex due to p21 binding. We note that it was only seen at the 20 h time point. Cyclin-cdk kinase activities were not measured.



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  		Fig. 4. Western blot analysis of cell cycle regulatory proteins p53, p21, p27, Rb, cylcin D1, cyclin E, and PCNA in asynchronously growing MALME-3M cells treated for 0–48 h with 10 µM DENSPM. See Fig. 7Citation for quantitative determinations of protein level changes. These data are representative of at least three separate determinations. Bands for Rb are designated as ppRb, hyperphosphorylated protein (top band), and pRb, hypophosphorylated protein (bottom band). ß-actin was used as an indicator for equality of lane loading.

 
We next explored the possible basis for Rb hypophosphorylation, focusing on p53 and the cdk inhibitors p21, p27, and p16. As shown in Fig. 4Citation , the shift in Rb phosphorylation status at 16 h was preceded slightly by an increase in wt-p53, which began ~12 h and reached a ~10-fold maximum at 30 h before declining. This, in turn, was accompanied by an increase in the cdk inhibitor p21 that began at ~12 h and also peaked with a 5-fold maximum at 30 h. The level of induction of p27 was much less, reaching a maximum of ~1.5-fold at 24–30 h. Despite the increase in p21, levels of PCNA, which is known to interact with p21 in the cyclin-cdk complex (40) , were unchanged. The temporal relationship between the increases in p53 and p21 and the shift in Rb phosphorylation status is shown in Fig. 6Citation . The two species of Rb are depicted as a ratio (pRb:ppRb), which began at 0.16 ± 0.05 (n = 8) for logarithmically growing cells. At 16 h, the ratio rose to 1.15 ± 0.2 and increased steadily in close association with the increased in p53 and p21 proteins; by 30 h, pRb was the predominant species.



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  		Fig. 6. Relative changes in cell cycle regulatory protein expression (Fig. 4)Citation and p21 mRNA levels (Fig. 5)Citation during treatment of MALME-3M cells with 10 µM DENSPM. Proteins and mRNA were quantitated densitometrically and normalized relative to ß-actin protein and glyceraldehyde-3-dehydrogenase mRNA levels, respectively. Fold changes in protein and mRNA were calculated relative to 0-h treatment time (left axis). Data points, ratios of hypophosphorylated Rb (pRb) to hyperphosphorylated Rb (ppRb) protein was quantitated from three separate experiments (right axis).

 
By Northern blot analysis (Fig. 5)Citation , p53 mRNA remained constant during analogue treatment, indicating that the rise in p53 protein was probably due to posttranscriptional mechanisms. Because p21 is among the genes known to be transactivated by p53 (41 , 42) , we also examined whether p21 mRNA was affected. As shown in Figs. 5Citation and 6Citation , p21 mRNA began to increase at 12 h and continued to rise to ~10-fold at 48 h, which is well beyond the p21 protein peak at 30 h. Thus, the p21 mRNA response coincided with the rise in p53 protein and the initial increase in p21 protein (Fig. 6)Citation . The findings are consistent with the probability that induction of p21 was mediated by p53.



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  		Fig. 5. Northern blot analysis of p53, p21, and glyceraldehyde-3-dehydrogenase (GAPDH) mRNA levels in MALME-3M cells treated for 0–48 h with 10 µM DENSPM as in Fig. 4Citation . Numbers at the bottom of each lane indicate the relative increase in p21 mRNA following densitometric quantitation and normalization to the GAPDH signal to account for lane loading differences.

 
DENSPM effects on growth, cell cycle, and cell cycle proteins were also examined in SK-MEL-28 human melanoma cells, which differ from the wt-p53-containing MALME-3M cells by expressing a mt-p53 protein (30) . More specifically, it contains a cysteine-to-valine substitution at amino acid position 145 located in the DNA binding region of the molecule. It should be noted that following normalization of autoradiograms, the level of p53 in SK-MEL-28 cells is ~2-fold higher than in MALME-3M cells. As shown in Fig. 7Citation , SK-MEL-28 cells differed remarkably from MALME-3M cells in their response to DENSPM. As noted above, the growth of MALME-3M cells was relatively unchanged during the first 24 h and cytostatically affected up to 48 h. By 72 h and beyond, there was evidence for programmed cell death, as indicated by a modest but steady decrease in cell number. In contrast, SK-MEL-28 cultures stopped growing after only 24 h of analogue treatment and displayed massive cell loss by 48 h. NMR spectral changes were used to confirm that the loss in cell number seen in MALME-3M cells after 72 h and in SK-MEL-28 cells after only 24 h is directly attributable to apoptosis (Fig. 8)Citation . The technique records the methylene (CH2) and methyl (CH3) content of the lipid bilayer in whole cells as separate peaks (33 , 34) . Thus, CH2:CH3 spectral ratios of 0.5–1 are typical of untreated cells. Programmed cell death increases the CH2 signal without effecting the CH3 peak, whereas cell death by necrosis has no effect on the ratio. We have observed that, in DENSPM-treated SK-MEL-28 cells at 48 h, the ratio of 2.6 (Fig. 8)Citation corresponds to >90% apoptotic cells, as indicated by annexin V-positive and propidium iodide-negative staining (data not shown). Further confirmation of apoptosis included standard flow analysis of ethanol-fixed detached cells, which eluted as a sub-G1 peak and morphological analysis of H&E-stained cells (data not shown).



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  		Fig. 7. Comparison of effects of 10 µM DENSPM on cell growth (A) and cell cycle regulatory proteins (B) in MALME-3M and SK-MEL-28 cells. Note that SK-MEL-28 cells differ from MALME-3M cells by undergoing a profound and rapid cytotoxic response and by failing to induce p53 or p21 protein. Dose-response curves to DENSPM at 96 h (C) demonstrate that the CTD (i.e., the dose required to reduce cell density below seeding density) was 100-fold lower for SK-MEL-28 compared to the MALME-3M cells. Data are representative of at least two separate experiments.

 


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  		Fig. 8. Comparison of the effects of 10 µM DENSPM on apoptosis-related NMR spectral intensity changes in the membrane lipids of MALME-3M and SK-MEL-28 cells. A, representative NMR spectral tracings for control and DENSPM-treated SK-MEL-28 cells. The concomitant changes in the methylene (CH2) and methyl (CH3) peaks reflect cell surface expression of apoptotic lipid changes. B, time-dependent increase in CH2:CH3 ratio for the two cell types. When the ratio was maximal at 48 h in SK-MEL-28 cells, morphological assessment and annexin V staining showed cells were >90% apoptotic. Data points, mean ratios; bars, SD.

 
SK-MEL-28 cells achieved only a 50% decrease in S-phase cells at 24 h before undergoing the rapid cell death due to apoptosis. Consistent with this finding, there was no induction of p53 at 24 h and no increase in p21 over the barely detectable levels seen under basal conditions (Fig. 7)Citation . Similarly, the cdk inhibitors p16 and p27 were also unchanged by DENSPM. As reported by others (43) , p16 was undetectable in MALME-3M (Fig. 7B)Citation . Thus, SK-MEL-28 cells differ from MALME-3M cells by rapidly proceeding to a near-total programmed cell death response in the absence of p53 induction and/or G1 arrest. We also examined the possibility that the difference in cellular responses between the two melanoma lines might be dose-related. The dose at which cytotoxicity became apparent as defined by cell loss (CTD at 96 h) for SK-MEL-28 cells (~1 µM) was 100-fold lower than that for MALME-3M cells (~100 µM; Fig. 7CCitation ). Treating MALME-3M cells with concentrations of DENSPM from 10 to 100 µM accelerated the onset of apoptosis from 72 to 48 h but still caused a G1 arrest. Similarly, treatment of SK-MEL-28 cells with DENSPM concentrations as low as 1 µM delayed the onset of apoptosis from 24 to 48 h and still did not cause a G1 arrest prior to cell death.

Despite different responses to DENSPM, both cell lines underwent similar early and near-total reductions in polyamine pools. As shown in Table 1Citation , intracellular polyamine pools were depleted after only 24 h treatment, presumably due to down-regulation of the polyamine biosynthetic enzymes ODC and S-adenosylmethionine decarboxylase and a very potent induction of the polyamine catabolic enzyme SSAT. In both MALME-3M and SK-MEL-28 cells, SSAT activity increased from ~40 to >25,000 pmol/min/mg. DENSPM accumulated to similar levels in both cell lines (6010 pmol/106 MALME-3M cells and 7520 pmol/106 SK-MEL-28 cells). Thus, there was no obvious polyamine-related drug effect that could be responsible for the differential cellular responses.


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  		Table 1 DENSPM effects on polyamine metabolism in MALME-3M and SK-MEL-28 human melanoma cellsa

 
We have previously demonstrated a correlation between SSAT induction by analogues and the delayed apoptotic response seen in MALME-3M cells (26) . Given the distinctive apoptotic response in SK-MEL-28 cells, we sought to examine whether a more obvious correlation could be established in this cell line. The relative effects of DENSPM and a series of three DENSPM analogues (Fig. 1)Citation that differentially induce SSAT but similarly deplete intracellular polyamine pools (24, 25, 26) were compared. As shown in Fig. 9Citation , N1,N14-diethylhomospermine (also known as DE-444) increased SSAT activity to 930 pmol/min/mg and produced a cytostatic response, whereas DENSPM induced SSAT to 31,920 pmol/min/mg and resulted in a rapid apoptotic response. The intermediate responses for the two remaining analogues support a correlation between SSAT induction and apoptosis. As shown in Table 2Citation , all four analogues accumulated to similar intracellular concentrations and similarly depleted polyamine pools, indicating that SSAT induction was more closely associated with apoptosis than polyamine pool depletion. Another more subtle possibility could involve residual spermine pools, which, in turn, are depleted in accordance with levels of SSAT induction (24 , 25) . For example, DENSPM (DE-333) produced a 95% decrease in spermine pools, DE-444 produced a 65% decrease, and N1,N12-diethylspermine (also known as DE-343) and 3,7,12,17-tetra-azanonadecane[N1,N13-diethyl(aminopropyl)homospermidine] (also known as DE-443) produced intermediate effects. Also included in the experiment was the demonstration that inhibition of polyamine oxidase with 100 µM MDL-72,527 (29) had only a minimal effect on DENSPM-induced apoptosis. Accumulation of acetylated spermine indicated the metabolic effectiveness of these treatment conditions (Table 2)Citation . The results indicate that production of hydrogen peroxide as a down-stream metabolic consequence of SSAT induction was probably not involved in the apoptotic response of the SK-MEL-28 cells.



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  		Fig. 9. Differential effects of DENSPM (or DE-333) and three homologues, DE-343, DE-443, and DE-444 (see structures in Fig. 1Citation ), on cell growth in SK-MEL-28 cells. All four analogues accumulate to comparable intracellular concentrations and similarly down-regulate ODC but differ in their ability to induce SSAT activity (see Table 2Citation ). · · · · ·, cotreatment of cells with 10 µM DENSPM plus 100 µM MDL-72,527, a specific inhibitor of polyamine oxidase (29) . Treatment with 250 µM MDL-72,527 as used by Ha et al. (57) was found to be toxic to SK-MEL-28 cells. {triangleup}, control; {blacktriangleup}, DE-444; {blacktriangledown}, DE-443; {blacksquare}, DE-343; {square}, DE-333; {diamond}, DE-333+72,527.

 

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  		Table 2 Relative DENSPM analogue effects on polyamine metabolism in SK-MEL-28 cellsa

 
DISCUSSION

The polyamine analogue DENSPM induces a distinct G1 arrest in MALME-3M cells, which is accompanied by a less obvious G2-M arrest and followed by a modest apoptotic response. One goal of this study was to define the cell cycle regulatory pathway responsible for the G1 arrest. Progression through the cell cycle is elegantly controlled by the combined effects of kinases, phosphatases, and inhibitory proteins, mediated by protein partnering and positive- and negative-acting phosphorylation. One of the best defined systems is the cyclin D1-cdk4-pRb pathway, which is activated in G1 and initiates progression toward S phase (41) ; the cyclin E-cdk2 complex sustains that progression. A critical step in the pathway is the G1 cyclin-cdk complex phosphorylation of Rb and the related proteins p107 and p130, leading to their inability to sequester the transcription factor E2F. Unbound E2F transactivates a number of key genes required for DNA synthesis and S-phase progression. These findings indicate that the G1 arrest produced by DENSPM is the end result of events preventing phosphorylation of Rb. The close temporal relationship between the shift in Rb phosphorylation status beginning at 16 h and the onset of G1 arrest supports this contention and further confirms the specificity of DENSPM as a G1 blocking agent. As discussed below, induction of certain known upstream effectors of this pathway, including p53 and p21, lends further credence to this likelihood.

Cell cycle progression is characterized by checkpoints at which the cell hesitates to determine whether previous steps have been successfully completed before moving forward. The p53 network (42 , 44) serves as the key molecular sensor for the G1 checkpoint and monitors DNA damage, nucleotide pool levels, mitotic spindle status, and genotoxic stress. In coordination with these sensory functions, the p53 network also regulates effector functions, which include cell cycle progression, programmed cell death, replicative senescence, and, possibly, differentiation. In perhaps the best defined of these responses, the DNA damage pathway (45) , p53 increases in amount via protein stabilization and transcriptionally activates several genes including the potent cdk inhibitor, p21. Increases in p53 can either promote apoptosis or G1 arrest, depending on the cell type and the nature of the cellular insult. Three lines of evidence suggest that DENSPM-induced G1 arrest in MALME-3M cells occurs via the p53 response pathway: (a) the G1 arrest and concomitant shift of Rb to a hypophosphorylated state was preceded by induction of p53 and p21 (Figs. 2Citation , 4Citation , and 6Citation ) in a manner consistent with other systems; (b) induction of p21 is accompanied by increases in p21 mRNA (Figs. 5Citation and 6Citation ), as would be expected during transactivation by p53; and (c) the p53-nonresponsive cdk-inhibitory protein p27 was not significantly increased. It is also probable that induction of p53 and p21 is involved in the G2-M block because they have been shown to contribute to this activity in other systems (44, 45, 46, 47) , but this remains to be demonstrated.

The upstream events leading to induction of p21 by DENSPM are of obvious interest to us. Although a variety of agents (48, 49, 50) induce p21 via p53-independent mechanisms, the evidence presented here is more consistent with DENSPM activation of a p53-dependent pathway. In particular, induction of p53 protein precedes G1 arrest and Rb hypophosphorylation and coincides with increases in p21. In addition, the increase in p21 protein is accompanied by a sharp rise in p21 mRNA, which is consistent with transactivation by the p53 protein. In keeping with what is known about the p53 network (41 , 42) , the following DENSPM effects could serve to activate the appropriate p53-related sensors: (a) reduction of polyamine pools, similar to the depletion of ribonucleotide triphosphate pools (51) ; (b) analogue interactions with DNA and/or chromatin (52, 53, 54, 55) ; (c) oxidative stress (56) due to massive analogue induction of SSAT (57) ; (d) generalized cellular stress; and/or (e) other pathways.

Although the SK-MEL-28 cells were similar to MALME-3M cells in their ability to induce SSAT in response to DENSPM, they differed by expressing mt-p53. Thus, we observed that SK-MEL-28 cells failed by induce p53 or p21 and were unable to sustain a G1 arrest in response to DENSPM. Rather, they underwent profound and rapid apoptosis. The finding is consistent with the emerging concept that induced or forced expression of p21 causes a G1 arrest, which delays or prevents apoptosis (58, 59, 60, 61) . In fact, Gorospe et al. (62) have reported that overexpression of p21 protects against p53-mediated apoptosis in human melanoma cells. We emphasize that this limited observation between the cellular response to DENSPM and p53 status needs to be validated in additional cell lines; however, this is complicated by the fact that other tumor types (63) and even other melanomas (24 , 64) vary remarkably in their ability to induce SSAT in response to analogues. Thus, more definitive studies will require transfecting MALME-3M cells with dominant-negative p53 plasmids and the SK-MEL-28 cells with functional p53. However, if these findings are shown to be generalities among other melanoma lines, they could have therapeutic implications for DENSPM because the majority of human solid tumors contain a mt-p53 or deleted p53 gene and are inherently resistant to commonly used DNA-damaging agents (45) .

The intensity of the apoptotic response in SK-MEL-28 cells provided a useful opportunity to examine whether analogue induction of SSAT might be responsible for this effect. For this purpose, we used a unique series of DENSPM analogues, which differentially induced SSAT but similarly depleted intracellular polyamine pools. Although additional sites of analogue action could also be involved, the extent of apoptosis by the analogues correlated closely with the level SSAT induction. The possibility of selective analogue depletion of spermine pools could not be excluded; however, this, too, would be expected to be mediated by SSAT. A previously observed correlation between analogue-induced apoptosis and SSAT induction in MALME-3M cells (24 , 26) was not nearly as striking as that seen in the SK-MEL-28 cells, presumably because wt-p53 or some other sensor gene system exerts a dampening effect. Although the ultimate cytotoxic response to DENSPM was similar in both cell lines, the time required to achieve this end point was much shorter in the SK-MEL-28 cells (Fig. 8)Citation .

SSAT induction by analogues is a highly heterogeneous response among tumor cell lines, and, as a generality, melanoma cell lines are more likely to hyperinduce the enzyme than most other tumor cell lines (24 , 36 , 64) , although the basis for this has not yet been defined. The potential for SSAT to participate in cytotoxicity was first noted in correlative studies with lung carcinoma cell lines that differentially induce the enzyme (63) and then extended to melanoma cell lines (24 , 64) . It has recently been reported by the Casero and coworkers (57) that the mechanistic linkage between potent SSAT induction and analogue-induced programmed cell death in certain lung carcinoma cells seems to involve the release of hydrogen peroxide by the polyamine oxidase reaction which follows polyamine acetylation by SSAT in polyamine catabolism. Hydrogen peroxide is known to induce single-strand breaks in DNA (65) and apoptotic nuclear fragmentation (66) in tumor cells, whereas a senescence-like growth arrest was induced by brief exposures in normal fibroblasts (67) . Although attractive, the possibility does not seem applicable to the SK-MEL-28 cells because we found that cotreatment with DENSPM and a specific inhibitor of polyamine oxidase failed to reduce apoptosis (Fig. 9)Citation . Thus, other conceivable linkages between SSAT induction, cell cycle effects and/or apoptosis might include: (a) selective depletion of spermine pools (Table 2)Citation ; (b) depletion of intracellular acetyl CoA pools; (c) interference with histone (68) or p53 acetylation (69) ; and/or (d) accumulation of acetylated polyamines (24) .

Taken together, these findings illustrate for the first time that a polyamine antagonist, in this case, the polyamine analogue DENSPM, is capable of activating the p53-p21-pRb pathway and inducing G1 arrest in certain melanoma cells. To our knowledge, induction of p21 and p53 by the analogue represents a unique example of positive regulation of gene expression involving systems that are not obviously polyamine related. With well-characterized analogues and highly specific polyamine inhibitors and p53 as a molecular end point, it should now be possible to identify upstream sensor systems and, from there, to more readily investigate the role of polyamines in cell proliferation.

ACKNOWLEDGMENTS

This manuscript is dedicated to the memory of John Miller, a beloved and productive member of the Porter laboratory for more than 24 years. We also gratefully acknowledge the technical assistance of Michael Rajecki and the encouraging discussions and materials provided by Dr. Ron Merriman (Parke Davis, Ann Arbor, MI).

FOOTNOTES

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.

1 This work was supported in part by National Cancer Institute Grant RO1 CA-22153 (to C. W. P.), the Roswell Park Alliance Foundation, and Institute Core Grant CA-16056, which funded the Flow Cytometry, Cell Analysis, and NMR Facilities. Back

2 To whom requests for reprints should be addressed, at Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: (716) 845-3002; Fax: (716) 845-8857; E-mail: Porter{at}sc3101.med.buffalo.edu Back

3 The abbreviations used are: ODC, ornithine decarboxylase; DENSPM, N1,N11-diethylnorspermine; SSAT, spermidine/spermine N1-acetyltransferase; p21, p21WAF1/CIP1; mt-p53, mutated p53; wt-p53, wild-type p53 protein; CTD, cytotoxic dose; NMR, nuclear magnetic resonance; PCNA, proliferating cell nuclear antigen; p27, p27KIP1; cdk, cyclin-dependent kinase; p16, p16INK4. Back

Received 9/28/98. Accepted 1/21/99.

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Y. Chen, D. L. Kramer, J. Jell, S. Vujcic, and C. W. Porter
Small Interfering RNA Suppression of Polyamine Analog-Induced Spermidine/Spermine N1-Acetyltransferase
Mol. Pharmacol., November 1, 2003; 64(5): 1153 - 1159.
[Abstract] [Full Text] [PDF]