Major increases in spermidine/spermine-N1-acetyltransferase activity by spermine analogues and their relationship to polyamine depletion and growth inhibition in L1210 cells

As an antiproliferative strategy, we are using bis(ethyl) derivatives of spermine to suppress polyamine biosynthetic enzyme activity and, thereby, deplete intracellular polyamine pools. Since certain of these analogues have recently been shown to potently increase spermidine/spermine-N1-acetyltransferase activity, we have investigated the relationship of this effect to growth inhibition and polyamine depletion. The cellular effects of N1,N12-bis(ethyl)spermine (BESPM) and two of its homologues, N1,N11-bis(ethyl)norspermine (BENSPM) and N1,N14-bis(ethyl)homospermine (BEHSPM), were compared in L1210 cells following treatments at equimolar concentrations (2 microM) and at concentrations (0.5 microM BEHSPM; 2 microM BESPM, and 20 microM BENSPM) producing comparable intracellular concentrations (2600-3000 pmol/10(6) cells) of the analogues. At 2 microM, BENSPM increased total polyamine N-acetyltransferase activity by 15-fold, BESPM, by 7-fold, and BEHSPM, by only 1.5-fold. These differences were much more exaggerated at comparable intracellular concentrations, where BENSPM increased enzyme activity 31-fold, BESPM, 7-fold, and BEHSPM had no effect. This rank order in effectiveness sharply contrasted effects on cell growth and interference with polyamine biosynthesis, which correlated more with intracellular accumulation of the analogues. At 2 microM, BEHSPM was most effective in suppressing ornithine and S-adenosylmethionine decarboxylases, depleting polyamine pools, and inhibiting cell growth, followed by BESPM and then by BENSPM. Thus, the data indicate that, in L1210 cells, the large increases in spermidine/spermine-N1-acetyltransferase activity produced by the analogues do not appear to contribute significantly to polyamine depletion or to be causally related to inhibition of cell growth. These studies also identify BENSPM as the most potent modulator of spermidine/spermine-N1-acetyltransferase activity thus far studied in cell culture systems. To a large extent, its greater effectiveness over BESPM seems to be attributable to a major increase in prolongation of enzyme half-life (3.9 versus 1.3 h), presumably due to enzyme stabilization caused by differential binding of the analogues at the enzyme active site.     

Induction of spermidine/spermine N1-acetyltransferase in breast cancer tissues treated with the polyamine analogue N1, N11-diethylnorspermine

Purpose The polyamine analogue, N¹,N¹¹-diethylnorspermine (DENSpm), is currently being evaluated in clinical trials for the treatment of solid tumors. The response of solid tumors to this drug has been associated with superinduction of the polyamine catabolic enzyme, spermine/spermidine N¹-acetyltransferase (SSAT). Therefore, to estimate the response of breast cancers to DENSpm, we measured induction of SSAT in breast cancer explants treated in vitro with this polyamine analogue.Experimental design Expression of SSAT protein was evaluated by immunohistochemistry in tissue explants from 38 invasive breast cancer tumors incubated in vitro in the presence (or absence) of DENSpm. In addition, SSAT enzymatic activity was measured in tissue explants from four tumors with high cellularity.Results SSAT expression was significantly increased in 30 of 38 tumor samples treated with DENSpm compared to untreated controls. This induction of SSAT protein expression was found specifically in neoplastic cells of the treated samples, and was seen in all histologic patterns (ductal, lobular, and mucinous) of breast cancer examined. In tumor samples evaluated for changes in SSAT enzymatic activity, these changes correlated closely with changes in protein expression.Conclusions Immunohistochemical staining for induction of SSAT correlates with measures of enzymatic activity in a small sample where measurements were possible and suggests that immunohistochemistry may be used for predicting response of breast cancers to DENSpm. A high proportion of breast cancers induced SSAT in response to DENSpm, supporting the continued consideration of this class of agents for treatment of breast cancer.Abbreviations 5-FU 5-fluorouracil - DENSpm N¹,N¹¹-diethylnorspermine - SSAT spermidine/spermine N¹-acetyltransferase     

Targeted expression of spermidine/spermine N1-acetyltransferase increases susceptibility to chemically induced skin carcinogenesis

The bovine keratin 6 gene promoter was used to target expression of spermidine/spermine N1-acetyltransferase (SSAT) to epidermal keratinocytes in the hair follicle of transgenic mice. K6-SSAT transgenic mice appeared to be phenotypically normal and were indistinguishable from normal littermates until subjected to a two-stage tumorigenesis protocol. For such tumorigenesis studies, mice were bred for six generations onto a tumor promoter resistant C57BL/6 background strain. K6-SSAT transgenic mice showed a 10-fold increase in the number of epidermal tumors that developed in response to a single application of 400 nmol of the tumor initiator 7,12-dimethylbenz[a]anthracene followed by twice weekly applications of 17 nmol of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate for 19 weeks. Tumor samples from transgenic animals showed marked elevations in SSAT enzyme activity and SSAT protein levels compared with tumors from non-transgenic littermates, and the accompanying changes in putrescine and N1-acetylspermidine pools indicated activation of SSAT-mediated polyamine catabolism in transgenic animals. An unusually high number of tumors were shown both grossly and histologically to have progressed to carcinomas in this model and these occurred with an early latency and only in mice carrying the K6-SSAT transgene. These results suggest that activation of polyamine catabolism leading to increases in putrescine and N1-acetylspermidine may play a key role in chemically induced mouse skin neoplasia.     

Studies of the mechanism by which increased spermidine/spermine N1-acetyltransferase activity increases susceptibility to skin carcinogenesis

Previous studies have shown that keratin 6 (K6)-spermidine/spermine N1-acetyltransferase (SSAT) transgenic mice, which modestly over-express SSAT in the skin, are more sensitive to tumor induction by a two-stage tumorigenesis protocol using initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). To evaluate the role of altered levels of polyamines and oxidative stress in this increase, studies were carried out with pharmacologic and genetic manipulation of K6-SSAT mice subjected to DMBA/TPA carcinogenesis. The increased tumor incidence was partially prevented by treatment with 1,4-bis-[N-(buta-2,3-dienyl)amino]butane, an inhibitor of acetylpolyamine oxidase which prevented degradation of the acetylated polyamines. This result suggests that toxic products such as reactive oxygen species and aldehydes liberated by the action of polyamine oxidase on the acetylated polyamines formed by SSAT may enhance tumor development. Breeding of the K6-SSAT mice with K6-antizyme (AZ) mice [which express AZ, a negative regulator of ornithine decarboxylase (ODC)] blocked the development of tumors. In addition, treatment of tumor-bearing K6-SSAT mice with the ODC inhibitor, alpha-difluoromethylornithine, resulted in the complete regression of established tumors. In contrast, treatment with N1,N11-bis(ethyl)norspermidine which increased SSAT activity in the tumors did not enhance regression. These results indicate that the tumor progression in K6-SSAT mice is dependent on elevated ODC activity and increased putrescine levels and may be further enhanced by oxidative stress. They support the use of strategies to modulate polyamine levels through the inhibition of ODC activity or polyamine uptake, but not via increased SSAT expression, for cancer chemoprevention in individuals at high risk for skin tumor development.     

Effect of hyperthermia on spermidine/spermine N1-acetyltransferase activity in Ehrlich ascites cells

When Ehrlich ascites tumor cells were incubated at 42° C, the activity of spermidine/spermine N¹-acetyltransferase, a rate-limiting enzyme of polyamine biodegradation, decreased to 20% of the control level within 1 hr. Protein synthesis, judged from the incorporation of [³H]leucine into the acid-insoluble fraction, was less affected by heat exposure. The decrease in the enzyme activity caused by heat treatment could be reversed by switching the incubation temperature back to 37° C. The reversion of enzyme activity was suppressed by cycloheximide but not by actinomycin D.     

Differential induction of spermidine/spermine N1-acetyltransferase in human lung cancer cells by the bis(ethyl)polyamine analogues

We have investigated the induction of an important polyamine metabolic enzyme, spermidine/spermine N1-acetyltransferase, in two human lung cancer cell lines which respond differently to treatment with the bis(ethyl)polyamine analogues. The human small cell lung carcinoma line NCI H82 has previously been shown to be minimally affected by treatment with these analogues, whereas the large cell undifferentiated lung carcinoma line, NCI H157, responds in a rapid cytotoxic manner (R.A. Casero, Jr., S. J. Ervin, P. Celano, S. B. Baylin, and R. J. Bergeron, Cancer Res., 49:639-643, 1989). The mechanisms underlying the differential response are unknown. In the responsive NCI H157 cells, the bis(ethyl)polyamines were found to induce spermidine/spermine N1-acetyltransferase in a time- and dose-dependent manner to maximum levels greater than 1700-fold over baseline. By contrast, the unresponsive NCI H82 cells exhibit minimal induction of spermidine/spermine N1-acetyltransferase to less than 7-fold increase after bis(ethyl)polyamine treatment, regardless of time or concentration examined. The results of the current study suggest that the differential induction of this key enzyme, which is rate limiting in the back conversion pathway of polyamine metabolism, may play a role in determining cell specific to the bis(ethyl)polyamine analogues.     

Potent modulation of intestinal tumorigenesis in Apcmin/+ mice by the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase

Intracellular polyamine pools are homeostatically maintained by processes involving biosynthesis, catabolism, and transport. Although most polyamine-based anticancer strategies target biosynthesis, we recently showed that activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase-1 (SSAT) suppresses tumor outgrowth in a mouse prostate cancer model. Herein, we examined the effects of differential SSAT expression on intestinal tumorigenesis in the Apc(Min/+) (MIN) mouse. When MIN mice were crossed with SSAT-overproducing transgenic mice, they developed 3- and 6-fold more adenomas in the small intestine and colon, respectively, than normal MIN mice. Despite accumulation of the SSAT product, N(1)-acetylspermidine, spermidine and spermine pools were only slightly decreased due to a huge compensatory increase in polyamine biosynthetic enzyme activities that gave rise to enhanced metabolic flux. When MIN mice were crossed with SSAT knock-out mice, they developed 75% fewer adenomas in the small intestine, suggesting that under basal conditions, SSAT contributes significantly to the MIN phenotype. Despite the loss in catabolic capability, tumor spermidine and spermine pools failed to increase significantly due to a compensatory decrease in biosynthetic enzyme activity giving rise to a reduced metabolic flux. Loss of heterozygosity at the Apc locus was observed in tumors from both SSAT-transgenic and -deficient MIN mice, indicating that loss of heterozygosity remained the predominant oncogenic mechanism. Based on these data, we propose a model in which SSAT expression alters flux through the polyamine pathway giving rise to metabolic events that promote tumorigenesis. The finding that deletion of SSAT reduces tumorigenesis suggests that small-molecule inhibition of the enzyme may represent a nontoxic prevention and/or treatment strategy for gastrointestinal cancers.     

Adenovirus-mediated expression of spermidine/spermine N1-acetyltransferase gene induces S-phase arrest in human colorectal cancer cells

Spermidine/spermine N1-acetyltransferase (SSAT) is a key enzyme of polyamine catabolism. In a previous study, we constructed a recombinant adenovirus, Ad-SSAT, which can express human SSAT. In the present study, we investigated the effect of Ad-SSAT on the growth and cell cycle of colorectal cancer cells. We found that Ad-SSAT increased the expression of SSAT and inhibited the growth of HT-29 and Lovo cells. The growth inhibition was caused by cell cycle arrest in the S phase. Furthermore, Ad-SSAT was shown to suppress the expression of cyclin A and nuclear factor E2F-1 in HT-29 and Lovo cells. The inhibitory effect of Ad-SSAT on cyclin A promoter activity was also observed in a reporter gene assay. Our results suggest that the expression of SSAT mediated by Ad-SSAT infection inhibits the growth of colorectal cancer cells and induces cell cycle arrest at the S phase, through a mechanism involving the suppression of cyclin A and E2F-1 expression.     

Differential effects of the spermine analog, N1, N12-bis(ethyl)-spermine, on polyamine metabolism and cell growth in human melanoma cell lines and melanocytes.

We have previously found that in one of two human melanoma cell lines, potent increase in the polyamine catabolizing enzyme, spermidine/spermine N1-acetyltransferase (SSAT), correlate with growth sensitivity to the spermine analog, N1, N12-bis(ethyl) spermine (BESPM). Herein, we examine the generality of this SSAT response among seven human melanoma cell lines (LOX, SH-1, STO-1, HO, PANUT-3, MALME-3 and Ebey) and normal melanocytes and further evaluate its possible correlation with growth sensitivity. Following treatment with 10 microM BESPM for 48 hr, SSAT activity among the various cell lines increased from basal levels of 20-90 pmol/min/mg to levels ranging from 170 to 30,470 pmol/min/mg. Five of the seven cell lines and melanocytes induced SSAT activity to levels to greater than 2,500 pmol/min/mg and three of these, to levels greater than 10,000 pmol/min/mg. When ranked according to SSAT responsiveness (LOX less than SH-1 less than STO-1 less than HO less than PANUT-3 less than MALME3 less than Ebey), there was a general correlation among the cell lines with growth sensitivity. Antiproliferative effects ranged from slowing of cell growth in the less SSAT responsive lines (LOX, SH-1) to total cessation of cell growth or overt cytotoxicity in the more potently SSAT responsive lines (MALME-3, Ebey). The polyamine biosynthetic enzyme activities, ornithine and S-adenosylmethionine decarboxylase, were similarly suppressed in all cell lines, presumably via analog activation of inherent regulatory mechanisms. Polyamine pool reduction occurred to a greater extent than predicted in cell lines where SSAT was increased to greater than 2500 pmol/min/mg suggesting a possible role for the enzyme in enhancing polyamine excretion and/or catabolism. The occurrence of potent SSAT induction among several human melanoma cell lines and the growth sensitivity of these same lines to BESPM suggests that the enzyme response may represent a determinant of drug action in this particular malignancy.     

Combination effects of platinum drugs and N 1, N 11 diethylnorspermine on spermidine/spermine N 1-acetyltransferase, polyamines and growth inhibition in A2780 human ovarian carcinoma cells and their oxaliplatin and cisplatin-resistant variants

Purpose

To understand the mechanisms behind platinum drug/DENSPM-induced inhibition of cancer cell growth, we compared the effects of oxaliplatin and cisplatin when combined with DENSPM on the induction of SSAT mRNA, activity, polyamines and cell growth in A2780 human ovarian carcinoma cells and their oxaliplatin- and cisplatin-resistant variants A2780/C10B and A2780/CP, respectively.

Methods

Parental and Pt-resistant cells were treated with platinum agent alone, DENSPM alone or combination (10???M each, 20?h). QRT?CPCR, radioactive product measurement and HPLC were used for mRNA, activity and polyamine pools, respectively; drug interaction on cell growth was by SRB and isobologram analysis.

Results

Both platinum agents induced SSAT mRNA in parental A2780 cells, but not in resistant cells. Platinum drug/DENSPM combinations produced high levels of SSAT activity in parental cells with significant depletion of spermine and spermidine, but not in resistant cells. Co-treatment with platinum agents increased the levels of DENSPM in all cell lines. Oxaliplatin/DENSPM combination was superior to cisplatin/DENSPM in the inhibition of cell growth in parental cells. No synergy was observed in the resistant cells.

Conclusions

Increased DENSPM levels following co-treatment with Pt agents enhances the translation and stability of SSAT protein leading to polyamine pool depletion, facilitating more Pt?CDNA adduct formation in parental cells. Oxaliplatin/DENSPM combination is superior to cisplatin/DENSPM in cell growth inhibition as DACH-Pt DNA adducts are cytotoxic even at relatively fewer numbers. Reduced platinum uptake in Pt-resistant cells contributes to reduced SSAT mRNA induction and absence of synergy when combined with DENSPM.     

Inactivation of IkappaB contributes to transcriptional activation of spermidine/spermine N(1)-acetyltransferase

Spermidine/spermine N(1)-acetyltransferase (SSAT) is a key enzyme in polyamine catabolism. We recently reported that the combination of N(1), N(11)-diethylnorspermine (DENSPM) and 5-fluorouracil (5-FU) synergistically induces SSAT expression, depletes polyamine levels and causes apoptosis in colon cancer cells. To determine whether new RNA and protein synthesis is required for SSAT induction, we examined the effect of actinomycin D (ActD) and cycloheximide (CHX). ActD alone blocked the induction of SSAT expression; however, the combination of CHX and DENSPM markedly induced SSAT expression and caused mitochondrial damage, suggesting that an inhibitory labile protein is involved in SSAT transactivation. SSAT promoter analysis identified two putative Rel/Nuclear Factor kappaB (NFkappaB) binding sites. Thus, we hypothesized that IkappaB is the labile inhibitory protein and that its removal contributes to the activation of NFkappaB. CHX quickly eliminated the IkappaB protein in the cells and increased the levels of the two subunits of NFkappaB, p65 and p50, in the nucleus. Luciferase reporter gene assay showed that SSAT promoter constructs containing the two putative NFkappaB binding elements responded to CHX as well as TNFalpha, whereas the promoter without the two sites did not. Chromatin immunoprecipitation (ChIP) assay showed that NFkappaB was indeed bound to the SSAT promoter after CHX treatment. Further, dominant negative IkappaB attenuated the CHX and DENSPM-induced SSAT expression and mitochondria damage. These results taken together suggest that the inhibition of IkappaB and activation of NFkappaB activate SSAT.     

The induction of spermidine/spermine N1-acetyltransferase (SSAT) is a common event in the response of human primary non-small cell lung carcinomas to exposure to the new antitumor polyamine analogue N1,N11-bis(ethyl)norspermine.

Several new polyamine analogues have been developed for the treatment of human solid tumors. The phenotype-specific activity of some of these analogues has been associated with the superinduction of the rate-limiting enzyme in polyamine catabolism spermidine/spermine N1-acetyltransferase (SSAT). Using immunohistochemistry, we found a majority (64%) of human primary lung cancer explants to exhibit high expression of SSAT after treatment with 10 microM N1,N11-bis(ethyl)norspermine, an agent currently undergoing Phase II clinical trials against several important human solid tumors. The staining of SSAT was found specifically in the tumor tissue and not in the neighboring normal lung tissue. These results demonstrate the ability to detect induction of SSAT in clinical specimens and suggest the potential of this test as a prognostic indicator of drug response.     

1,2-Dimethylhydrazine-induced alterations in N1-acetylspermidine levels and spermidine N1-acetyltransferase activity in rat colonic mucosa

To determine whether alterations in the "reverse" or "conversion" pathway for putrescine formation were involved in the induction of colonic tumors by 1,2-dimethylhydrazine, male albino rats of the Sherman strain were given injections s.c. of this agent (20 mg/kg body weight/week) or diluent for 5, 10, 15, and/or 26 weeks. Animals were sacrificed at each of these time periods and polyamine levels, including N1- and N8-acetylspermidine, as well as the activities of ornithine decarboxylase, spermidine N1-acetyltransferase and polyamine oxidase were measured and compared in rat proximal and distal colonic mucosa of each group. The results of these studies demonstrated that: (a) N1- and N8-acetylspermidine levels were similar in the control and treated proximal colonic segments at all time periods examined; (b) N1- and N8-acetylspermidine levels were also similar in the control and treated distal colonic segments at 5 and 10 weeks; (c) at 15 weeks the level of N1-acetylspermidine, but not N8-acetylspermidine, however, was increased in the treated distal colonic segment secondary to increases in the activity of spermidine N1-acetyltransferase; and (d) at 26 weeks, the level of N1-acetylspermidine remained higher in treated distal "uninvolved" colonic tissue and were markedly elevated in colonic tumors in both segments. Based on these findings, it would appear that the reverse pathway for putrescine formation may be involved in the 1,2-dimethylhydrazine-induced malignant transformation process of the rat colon.     

Spermidine/spermine N1-acetyltransferase transient overexpression restores sensitivity of resistant human ovarian cancer cells to N1,N12-bis(ethyl)spermine and to cisplatin

The limited induction of spermidine/spermine N1-acetyltransferase (SSAT) activity has been implicated as an important determinant of the reduced response to the spermine analogue N1,N12-bis(ethyl)spermine (BESpm) by the cisplatin or cis-diamminedichloroplatinum(II) (cDDP)-resistant human ovarian carcinoma cell line (C13*). We checked whether or not under conditions of SSAT overexpression, enzyme induction and cell sensitivity to both, BESpm and cDDP, were restored to levels comparable with those of more responsive cDDP-sensitive 2008 cells. We transiently transfected the SSAT repressed C13* cells with two expression vectors driving human SSAT overexpression by diverse promoters. We then analysed their responses in the absence and in the presence of BESpm. SSAT activity was promptly, but briefly, expressed by transfection with both pOP/SSAT and pCMV-SSAT plasmids. However, only in the presence of BESpm, did SSAT activity reach the highest levels of induction for longer duration, with different time-courses for the two vectors, that paralleled the effect on cell growth. Under these conditions, growth sensitivity to BESpm of the less-responsive C13* cells was 25% reverted to cell growth inhibition displayed by 2008 cells. More interestingly, the sensitivity to cDDP cytotoxicity also increased in parallel to SSAT overexpression. BESpm induction of pCMV-SSAT-transfected cells caused a further 20-30% reduction of cell survival induced by cDDP, almost recovering the sensitivity of 2008 cells. The enhanced effectiveness of cDDP was also confirmed by the comet assay, showing an increase in the number and length of tails of damaged DNA. These findings confirm that SSAT overexpression inhibits cell growth and enhances growth sensitivity to BESpm in C13* cells, showing for the first time that restoring high inducibility of SSAT activity subverts the reduced sensitivity to cDDP of SSAT-deficient cells, making them almost indistinguishable from the responsive parental 2008 cells.     

Role of p53/p21(Waf1/Cip1) in the regulation of polyamine analogue-induced growth inhibition and cell death in human breast cancer cells

Intracellular polyamines are absolutely required for cell proliferation and many tumors have abnormal requirements for polyamines. Therefore, the polyamine metabolic pathway represents a rational target for antineoplastic intervention. A number of polyamine analogues act as potent modulators of cellular polyamine metabolism and exhibit encouraging effects against tumor growth in both cell culture and animal studies. In this study we demonstrate that specific polyamine analogues exhibit differential inhibitory action against growth of human breast cancer MCF-7 cells. Treatment of MCF-7 cells with oligoamine analogues and the symmetrically substituted bis(alkyl)-substituted analogue, BENSpm, produced a G1 cell cycle arrest, while the unsymmetrically substituted bis(alkyl)-substituted analogue, CHENSpm, induced a G2/M cell cycle arrest. All four compounds significantly upregulated p53 and p21 expression in MCF-7 cells. Stable transfection of small interfering RNA (siRNA) targeting p53 blocked the expression of p21 induced by the polyamine analogues and significantly reduced polyamine analogue-induced growth inhibition and apoptosis, suggesting that polyamine analogue-induced p21 expression occurs through p53-dependent mechanisms. The effects of analogue exposure on cyclins and cyclin dependent kinases varied with the specific agent used. Expression of p53 siRNA reversed only BENSpm-modulated the cell cycle arrest, suggesting that regulation of cell cycle arrest by p53/p21 induced by polyamine analogues occurs through agent-specific mechanisms. Understanding the mechanism of p53-mediated cellular responses to polyamine analogue may help to improve the therapeutic efficacy of polyamine analogues in human breast cancer.     

Spermidine/spermine N1-acetyltransferase, a new biochemical marker for epithelial proliferation in rat bladder.

We examined the activity of spermidine/spermine N1-acetyltransferase (SAT), a rate-limiting enzyme of the biodegradation of polyamines, in N-butyl-N-(4-hydroxybutyl)nitrosamine-induced transitional cell carcinoma (TCC) and melamine-induced papillomatosis of rat bladder, and compared the activity to that of ornithine decarboxylase (ODC). Both activities were higher in both lesions than in control rats. The difference between SAT and ODC activities in cancerous tissue and papillomatosis was not significant. Cells stained for proliferating cell nuclear antigen (PCNA) were abundant in papillomatosis. TCC had areas with much PCNA. The results indicated that an elevation of SAT activity occurs in both reversible and irreversible proliferation of bladder epithelium and could be important in bladder carcinogenesis.     

Comparisons between sensitive and resistant human tumor cell lines regarding effects of polyamine depletion on chloroethylnitrosourea efficacy

We have reported that 2-difluoromethylornithine (DFMO)-induced polyamine (PA) depletion sensitized five chloroethylnitrosourea (CENU)-resistant, O6-alkylguanine repair-proficient (Mer+) human tumor cell lines to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), but failed to alter BCNU efficacy in a single CENU-sensitive, repair-deficient (Mer-) line. Further, alkaline elution assays of DNA interstrand cross-links (ISC) found no BCNU-induced ISC in either PA-depleted or control Mer+ cells, suggesting that targets other than ISC may be involved in the DFMO/BCNU drug interaction. To verify that DFMO-induced enhancement of BCNU action segregates with Mer phenotype, we tested three additional Mer- lines for effects of DFMO pretreatment on BCNU efficacy. We found no potentiation of BCNU by PA depletion in any of our human Mer- lines. We also used streptozotocin (STZ) to deplete the repair capacity of Mer+ cell lines, thus converting their BCNU sensitivity to near that of Mer- cells. Combined pretreatment with DFMO then STZ did enhance BCNU cell kill relative to STZ pretreatment alone. Exogenous putrescine restored BCNU sensitivity of (DFMO plus STZ)-pretreated cells to that of cells pretreated with STZ alone. Measurements of O6-alkylguanine DNA alkyltransferase activity verified that in at least one of the Mer+ lines (HT-29), STZ did deplete repair capacity to below detectable limits. These results suggest that in HT-29 cells, STZ and DFMO probably act via differing mechanisms to potentiate BCNU. Our observations also imply that targets for CENUs may differ between Mer+ and Mer- cells, with importance of ISC possibly limited to Mer- cells. Our data further suggest that PA depletion may potentiate CENUs only at targets critical in Mer+ cells. We also noted that 48-h treatments with DFMO markedly reduced clonogenicity of Mer- cells. Exogenous putrescine restored Mer- cell survival after DFMO to near that of controls. In contrast, Mer+ cells showed little, if any, effect of DFMO treatment on plating efficiency. These results suggest that PA depletion may be cytocidal to some Mer- cells.     

N1,N12-bis(ethyl)spermine effect on growth of cis-diamminedichloroplatinum(II)-sensitive and -resistant human ovarian-carcinoma cell lines

The results presented here demonstrate that a cis-diamminedichloroplatinum(II) (DDP)-resistant human ovarian-carcinoma cell line is also cross-resistant to the spermine analogue N¹,N¹²-bis(ethyl)spermine (BESPM). We report that C13* cells, which are approximately 20-fold resistant to DDP, similarly showed 7-fold resistance to BESPM by colony-forming assay with an IC₅₀ value of 24.6 ± 2 μM vs. 3.4 ± 0.8 μM of 2008 cells. Resistance appears to be the result of many effects, such as different morphological and functional modifications of mitochondria. Furthermore, although BESPM accumulation was almost identical in sensitive and resistant cells, the intracellular polyamine pool of the 2 cell lines was differentially affected by this polyamine analogue. In fact, when spermidine (SPD) was still detectable in C13* cells, in 2008 cells it was not, and the spermine (SPM) content was always more markedly reduced in sensitive cells than in the resistant variant. The lower polyamine content of 2008 cells could be related to a higher degree of induction of spermidine/spermine N¹-acetyltransferase (SSAT) activity by BESPM in sensitive cells than in their resistant counterpart. Despite the observed cross-resistance, the combination of the 2 drugs resulted in supra-additive and synergistic effects in both cell lines, depending on concentration, as assessed by median-effect analysis of the survival data. The effectiveness of this combination was also confirmed by the increased accumulation of cells in the G₂/M phase of the cell cycle in both cell lines. Taken together, these data suggest that BESPM effect on cell growth of DDP-sensitive and DDP-resistant cells involves multiple mechanisms that are differently modulated by the DDP-resistant phenotype. Int. J. Cancer 78:33–40, 1998.© 1998 Wiley-Liss, Inc.     

Relative abilities of bis(ethyl) derivatives of putrescine, spermidine, and spermine to regulate polyamine biosynthesis and inhibit L1210 leukemia cell growth

It has been shown previously (Porter et al., Cancer Res., 45: 2050-2057, 1985) that the N1,N8-bis(ethyl) derivative of spermidine has significant antiproliferative activity which appears to derive from its regulatory effects on the polyamine biosynthetic pathway, particularly on ornithine decarboxylase activity. In the present study, N1,N4-bis(ethyl)putrescine (BEP) and N1,N12-bis(ethyl)spermine (BESm) were compared with N1,N8-bis(ethyl)spermidine (BES) in their ability to inhibit cell growth and regulate polyamine biosynthesis. With cultured L1210 murine leukemia cells, the IC50 values at 48 h were approximately 2 mM for BEP, 30 microM for BES, and 1 microM for BESm making the latter the most effective polyamine inhibitor or analogue thus far identified. At concentrations which approximated IC50 values and yielded similar intracellular concentrations at 48 h (1500-2000 pmol/10(6) cells), the effects of the analogues on polyamine biosynthesis generally correlated with their antiproliferative activity. BEP, at 1 mM, exerted relatively minor effects on polyamine biosynthesis. By contrast, 100 microM BES totally eliminated ornithine decarboxylase activity, depleted putrescine and spermidine pools, and decreased spermine pools by 40%. AdoMet decarboxylase activity was lowered slightly. The most impressive effects were obtained with 10 microM BESm which decreased ornithine and AdoMet decarboxylase activities by 99 and 84%, respectively; depleted putrescine and spermidine pools; and decreased spermine pools by 73%. None of the analogues, at 1 or 3 mM, had significant direct inhibitory effects on the decarboxylase activities from untreated cells with the exception of BESm which inhibited ornithine but not AdoMet decarboxylase activity. Thus, the effects of the analogues on these enzymes in treated cells are presumed to be mainly mediated by regulatory mechanisms. In this regard, BESm was superior to BES since both ornithine and AdoMet decarboxylase activities were suppressed. Given its unique activities, BESm would seem to have potential as both an antiproliferative agent and also as an experimental probe for studying regulation of the polyamine pathway, particularly AdoMet decarboxylase.