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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.

The clinically relevant polyamine analogue N(1),N(11)-diethylnorspermine (DENSPM) inhibits cell growth by down-regulating polyamine biosynthesis, up-regulating polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase (SSAT), and depleting intracellular polyamine pools. Among human melanoma cell lines, the analogue causes rapid apoptosis in SK-MEL-28 cells and a sharp G(1) arrest in MALME-3M cells. This study reveals that DENSPM potently activates the mitogen-activated protein kinase (MAPK) pathways in melanoma cells and investigates the role of this response in determining cellular outcomes. Onset of apoptosis was preceded by an intense phosphorylation of the MAPKs, including extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase, and p38 in both SK-MEL-28 and MALME-3M cells. A panel of DENSPM analogues differing only in their ability to induce SSAT was used to show that MAPK activation was causally linked to induction of SSAT activity and related oxidative events. The latter was confirmed with the polyamine oxidase inhibitor MDL-75275 and the antioxidant N-acetyl-L-cysteine, which when used in combination with DENSPM, decreased MAPK activation and as previously shown, reduced apoptosis. The MAP/extracellular signal-regulated kinase-1 inhibitor PD 98059 reduced activation of all three kinases but failed to alter apoptosis in DENSPM-treated SK-MEL-28 cells. By contrast, the inhibitor prevented p21(waf1/cip1) induction and enhanced apoptosis in MALME-3M cells as indicated by accelerated caspase-3 activation and positive annexin V staining. The generality of this effect was demonstrated in DENSPM-treated A375 and LOX human melanoma cells. Taken together, the importance of the MAPK pathways in determining the biological response to DENSPM treatment is dependent on the genetic environment of the cell.

The human methylthioadenosine phosphorylase (MTAP) gene is located on 9p21 and is frequently homozygously deleted, along with p16(cdkN2a/ARF), in a wide variety of human tumors and human tumor-derived cell lines. The function of MTAP is to salvage methylthioadenosine, which is produced as a byproduct of polyamine metabolism. We have reintroduced MTAP into MCF-7 breast adenocarcinoma cells and have examined its effect on the tumorigenic properties of these cells. MTAP expression does not affect the growth rate of cells in standard tissue culture conditions but severely inhibits their ability to form colonies in soft agar or collagen. In addition, MTAP-expressing cells are suppressed for tumor formation when implanted into SCID mice. This suppression of anchorage-independent growth appears to be because of the enzymatic activity of MTAP, as a protein with a missense mutation in the active site does not exhibit this phenotype. MTAP expression causes a significant decrease in intracellular polyamine levels and alters the ratio of putrescine to total polyamines. Consistent with this observation, the polyamine biosynthesis inhibitor alpha-difluoromethylornithine inhibits the ability of MTAP-deficient cells to form colonies in soft agar, whereas addition of the polyamine putrescine stimulates colony formation in MTAP-expressing cells. These results indicate that MTAP has tumor suppressor activity and suggest that its effects may be mediated by altering intracellular polyamine pools.

Purpose: SAM486A is a novel inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (SAMDC). This study was performed to characterize the toxicity profile and the pharmacological behavior and to determine the maximum tolerated dose (MTD) of SAM486A administered by a 1-h i.v. infusion daily for 5 days every 3 weeks in patients with advanced cancer., Experimental Design: Twenty-three patients received 46 cycles of SAM486A at dose levels ranging from 3.6 to 202.8 mg/m(2)/day. SAM486A plasma concentrations were measured during the first cycle for pharmacokinetic and pharmacodynamic evaluations. Paired tumor biopsy specimens pre- and posttreatment were obtained in 1 patient to assess the impact of SAM486A on intratumoral enzymes and metabolites involved in the polyamine biosynthetic pathway., Results: The dose-limiting toxicity of SAM486A on this schedule was myelosuppression. Nonhematological toxicities, including nausea, vomiting, anorexia, and fatigue, were mild to moderate in severity. The MTD of SAM486A was 102.4 mg/m(2)/day. Pharmacokinetic analyses demonstrated a rapid initial decrease in plasma drug concentrations at the end of infusion, followed by a long terminal elimination phase with a mean (+/- SD) terminal elimination half-life of 65.4 +/- 55.6 h. Dose and area under the concentration-time curve correlated with the appearance of grade 4 neutropenia with correlation coefficients of 0.70 and 0.69, respectively. Analysis of paired tumor biopsy specimens taken before and after SAM486A treatment in 1 patient with metastatic melanoma revealed decreased SAMDC activity, increased ornithine decarboxylase activity, increased levels of putrescine, and depleted levels of decarboxylated S-adenosylmethionine and spermine, all of which are consistent with the proposed mode of action of SAM486A., Conclusions: SAM486A was well tolerated on this schedule of administration with the MTD established at 102.4 mg/m(2)/day. Neutropenia was dose-limiting and correlated with dose and area under the concentration-time curve. Pharmacodynamic assessment of tumoral tissues in 1 study patient demonstrated changes in the levels of polyamines and their biosynthetic enzymes consistent with SAMDC inhibition.

The cell cycle regulatory events that interface with polyamine requirements for cell growth have not yet been clearly identified. Here we use specific inhibitors of polyamine biosynthetic enzymes to investigate the effect of polyamine pool depletion on cell cycle regulation. Treatment of MALME-3M cells with either the ornithine decarboxylase inhibitor alpha-difluoromethylornithine or the S-adenosylmethionine decarboxylase inhibitor MDL-73811 lowered specific polyamine pools and slowed cell growth but did not induce cell cycle arrest. By contrast, treatment with the combination of inhibitors halted cell growth and caused a distinct G1 arrest. The latter was associated with marked reduction of all three polyamine pools, a strong increase in p21(WAF1/CIP1/SDI1) (p21), and hypophosphorylation of retinoblastoma protein. All effects were fully prevented by exogenous polyamines. p21 induction preceded p53 stabilization in MALME-3M cells and also occurred in a polyamine-depleted, p53-nonfunctional melanoma cell line, indicating that p21 is induced at least in part through p53-independent mechanisms. Conditional overexpression of p21 in a fibrosarcoma cell line was shown previously to inhibit the expression of multiple proliferation-associated genes and to induce the expression of genes associated with various aspects of cell senescence and organism aging. Polyamine depletion in MALME-3M cells was associated with inhibition of seven of seven tested p21-inhibited genes and with induction of 13 of 14 tested p21-induced genes. p21 expression is also known to induce a senescence-like phenotype, and phenotypic features of senescence were observed in polyamine-depleted MALME-3M cells. Cells increased in size, appeared more granular, and expressed senescence-associated beta-galactosidase. Cells released from the polyamine inhibition lost the ability to form colonies, failed to replicate their DNA, and approximately 25% became bi- or multinucleated. These events parallel the outcome of prolonged p21 induction in fibrosarcoma cells. The results of this study indicate that polyamine pool depletion achieved by specific biosynthetic enzyme inhibitors causes p21-mediated G1 cell cycle arrest followed by p21-mediated changes in gene expression, development of a senescence-like phenotype, and loss of cellular proliferative capacity.

N(1),N(11)-Diethylnorspermine (DENSPM) is a polyamine analogue with clinicalrelevance as an experimental anticancer agent and the ability to elicit a profound apoptotic response in certain cell types. Here, we characterize the polyamine effects and apoptotic signaling events initiated by treatment of SK-MEL-28 human melanoma with 10 microM DENSPM. Maximal induction of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine pool depletion were seen by 16 h, whereas early apoptosis was first apparent at 36 h. Intermediate events related to apoptotic signaling were sought between 16 and 36 h. A loss of mitochondrial transmembrane potential (Deltapsi(m)) beginning at 24 h was followed by the release of cytochrome c into the cytosol at 30 h. Loss of mitochondrial integrity was accompanied by caspase-3 activation and poly(ADP-ribose) polymerase digestion from 30 to 36 h. The caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone rendered cells resistant to analogue-induced caspase-3 activation and reduced the apoptotic response in a dose-dependent manner. Because polyamine reduction achieved by inhibitors of polyamine biosynthesis inhibited growth but did not cause apoptosis, we looked for alternative polyamine-related events, focusing on induction of SSAT. Three DENSPM analogues that differentially induced SSAT activity but similarly depleted polyamine pools revealed a close correlation between enzyme induction and cytochrome c release, caspase activation, and apoptosis. Dose-dependent inhibition of polyamine oxidase, an enzyme that oxidizes acetylated polyamines generated by SSAT and releases toxic by-products such as H(2)O(2) and aldehydes, prevented cytochrome c release, caspase activation, and apoptosis. Taken together, the findings indicate that DENSPM-induced apoptosis is at least partially initiated via massive induction of SSAT and related oxidative events and subsequently mediated by the mitochondrial apoptotic signaling pathway as indicated by cytochrome c release and caspase activation.

Polyamine oxidase functions in the polyamine catabolic pathway, converting N1-acetyl-spermidine and -spermine into putrescine (Put) and spermidine (Spd), respectively, thereby facilitating homeostasis of intracellular polyamine pools. Inhibition of polyamine oxidase in hematopoietic cells by a specific inhibitor, N,N'-bis(2,3-butadienyl)-1,4-butanediamine (MDL-72,527), reduces the levels of Put and Spd and induces the accumulation of N1-acetylated Spd. Although previously thought to be relatively nontoxic, we now report that this inhibitor overrides survival factors to induce cell death of several immortal and malignant murine and human hematopoietic cells, but not of primary myeloid progenitors. Cells treated with MDL-72,527 displayed biochemical changes typical of apoptosis, and cell death was associated with the down-regulation of the antiapoptotic protein Bcl-X(L). However, enforced overexpression of Bcl-X(L), or treatment with the universal caspase inhibitor zVAD-fmk, failed to block MDL-72,527-induced apoptosis in these hematopoietic cells. Despite decreases in Put and Spd pools, MDL-72,527-induced apoptosis was not blocked by cotreatment with exogenous Put or Spd, nor was it influenced by overexpression or inhibition of the polyamine biosynthetic enzyme ornithine decarboxylase. Significantly, MDL-72,527-induced apoptosis was associated with the rapid formation of numerous lysosomally derived vacuoles. Malignant leukemia cells were variably sensitive to the lysosomotropic effects of MDL-72,527, yet pretreatment with the ornithine decarboxylase inhibitor L-alpha-difluoromethylornithine sensitized all of these leukemia cells to the deleterious effects of the inhibitor by stimulating its intracellular accumulation. The lysosomotropic nature of select polyamine analogues may, thus, provide a novel chemotherapeutic strategy to selectively induce apoptosis of malignant hematopoietic cells.

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 microM N1,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 (approximately 100-fold at maximum) and a concomitant increase in the cyclin-dependent kinase inhibitor, p21WAF1/CIP1 (p21; approximately 5-fold at maximum). Another cyclin-dependent kinase inhibitor, p27KIP1, and cyclin D 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.

CGP-48664, an inhibitor of the polyamine biosynthetic enzyme S-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. Biol. 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 N1,N11-diethylnorspermine (DENSPM)-a polyamine analogue also undergoing Phase 1 clinical trials-despite accumulating approximately 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 microM CGP-48664 by chronic exposure were used to demonstrate that vacuole formation correlated with the accumulation 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 microM CGP-48664 displayed a 170-fold increase in AdoMetDC mRNA levels and formed vacuoles in response to DENSPM, whereas those resistant to 10 microM 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 N1-acetyltransferase activity in cells that formed vacuoles than in those that did not. Overall, natural polyamines failed to induce vacuoles and various analogues 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-48664-resistant 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.

On the basis of encouraging preclinical findings, polyamine analogues have emerged as a novel class of experimental antitumor agents. The spermine derivative N1,N11-diethylnorspermine (DE-333, also known as DENSPM) is currently undergoing Phase I clinical trials against solid tumors. A series of systematically modified DE-333 analogues differing in intra-amine carbon distances and in N-alkyl terminal substituents (i.e., methyl, ethyl, and propyl) were evaluated in MALME-3M human melanoma cells, a cell line known to be cytotoxically affected by DE-333 and especially responsive to analogue induction of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. Analogues accumulated to comparable intracellular concentrations and similarly affected cell growth with IC50 values in the 0.5-1.0 microM range. During prolonged incubations, diethyl and dipropyl analogues were cytotoxic, whereas two dimethyl analogues were cytostatic. Cell cycle analysis following treatment with the cytotoxic analogues revealed a prominent G1 block apparent as an accumulation of cells in G0/G1 and depletion of S-phase cells as well as a less restrictive G2 block. By contrast, cytostatic analogues incompletely arrested cells in G1, leaving a significant number of S-phase cells. Morphological and immunocytochemical analysis of detached cells revealed a far greater proportion of apoptotic cells with cytotoxic analogues than with cytostatic analogues. Although spermidine/spermine N1-acetyltransferase activity was differentially induced by the analogues, there was no obvious correlation with cell cycle effects. Overall, these data indicate a previously unrecognized combined effect of polyamine analogues on cell cycle progression and apoptosis. On the basis of structure-function relationships, these activities may be manipulated to optimize therapeutic efficacy.

The spermine analogue N1,N11-diethylnorspermine (DENSPM) has been shown to induce the polyamine-acetylating enzyme spermidine/spermine N1-acetyltransferase, disrupt polyamine pool homeostasis, and inhibit tumor growth. DENSPM is currently being developed as an anti-neoplastic agent and is about to enter Phase II clinical trials. In this report, the antitumor efficacy of DENSPM was evaluated against a human transitional cell bladder BL13 carcinoma xenograft implanted orthotopically and s.c. in nude athymic mice. DENSPM was administered via continuous s.c. infusion at 93 mg/kg/day for 5 days. Treatment with DENSPM was well tolerated and produced tumor regressions in all mice with a significant proportion (up to 50%) of apparent cures. On the basis of low toxicity and good therapeutic efficacy, there is a strong rationale for evaluation of the therapeutic efficacy of DENSPM against bladder carcinomas in Phase II clinical trials.

Certain N-alkylated analogues of the natural polyamine spermine have been found to disrupt polyamine pool homeostasis and inhibit tumor cell growth. The most effective of these analogues, N1, N11-diethylnorspermine (DENSPM), apparently depletes intracellular polyamine pools primarily by inducing the polyamine acetylating enzyme spermidine/spermine N1-acetyltransferase, which contributes to polyamine depletion via increased polyamine excretion and catabolism. In this report, the experimental therapeutic efficacy of DENSPM was further examined with the use of other human solid tumor xenografts, including A121 ovarian carcinoma, A549 lung adenocarcinoma, HT29 colon carcinoma, and SH-1 melanoma, and compared with previously obtained findings with MALME-3M and PANUT-3 human melanomas. In vitro studies indicated that the growth sensitivity of most tumor cell lines to DENSPM was similar, with characteristically flat dose-response curves and IC50s ranging between 0.1 and 1 micrometer the only exception was the HT29 colon carcinoma cell line, which had an IC50 of >100 micrometer. For in vivo studies, DENSPM was administered by i.p. injection to female nude athymic mice at 40 and/or 80 mg/kg 3 times a day (every 8 h) for 6 days or by continuous s.c. infusion with the use of Alzet pumps at 120, 240, or 360 mg/kg/day for 4 days. Treatment began after s.c. tumor xenografts had reached 100-200 mm3. The SH-1 melanoma, A549 lung adenocarcinoma, and A121 ovarian carcinoma xenografts responded well to the i.p. administration of analogue with obvious tumor regressions, long-term tumor growth suppressions, and a significant proportion (up to 40%) of apparent cures (i.e., lack of tumor regrowth). However, in similarity to in vitro findings, HT29 colon carcinoma xenografts responded poorly to DENSPM treatment. Massive induction of N1-acetyltransferase activity and extensive depletion of polyamine pools were consistent findings in most tumor types after in vivo or in vitro treatment with DENSPM. The rapidly growing human LOX melanoma xenograft, however, demonstrated poor induction of N1-acetyltransferase activity and the poorest response to DENSPM treatment. In nude athymic mice with MALME-3M melanoma xenografts, constant infusion delivery of DENSPM resulted in prolonged inhibition of tumor growth and long-term tumor regressions comparable to those produced by multiple i.p. injections. On the basis of the unique structure of DENSPM, novel target and mode of intervention, mild host toxicity, and activity against different human solid tumor xenografts, DENSPM is currently being developed as an antitumor agent in humans.

Certain N-alkylated analogues of the natural polyamine spermine, such as N1,N11-diethylnorspermine (DENSPM), rapidly deplete intracellular polyamine pools by down-regulating the biosynthetic enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, and by potently up-regulating the polyamine catabolizing enzyme, spermidine/spermine N1-acetyltransferase. On the basis of previously reported antitumor activity in human tumor xenograft model systems, DENSPM is currently undergoing Phase I clinical trials against human melanoma and other solid tumors. The antiproliferative activity of this analogue against the multidrug resistance (MDR) phenotype was examined in three MDR sublines of human melanoma RPMI-7932 cells, which were shown to be 2-to 10-fold resistant to classical MDR agents. These MDR lines had been separately derived using different selecting agents (Lemontt et al., Cancer Res., 48: 6344-6353, 1988). Subline functional resistance due to P-glycoprotein was confirmed by decreased retention of rhodamine 123 relative to parent cells as detected by flow cytometry. Although the three sublines were 2- to 10-fold less sensitive than the parent line to classical MDR-type agents, they were found in dose-response studies to be significantly more sensitive to DENSPM than the parent line. In addition, they showed a distinct cytotoxic response after a 48-h treatment with 10 microM DENSPM, which was not apparent in the parent line. Growth sensitivity of the sublines to the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine, or the S-adenosylmethionine decarboxylase inhibitor, CGP-48664, was found to be similar to parent cells. The ratio of the key biosynthetic enzyme activities for ornithine decarboxylase and S-adenosylmethionine decarboxylase was found to be 3.5- to 5-fold higher in all three sublines, due mainly to increases in the former enzyme. This imbalance produced unusually high putrescine pools. Although DENSPM down-regulation of decarboxylase activities and potent up-regulation of spermidine/spermine N1-acetyltransferase activity occurred similarly in both parent and variant lines, polyamine depletion was greater in the variant lines. Collateral sensitivity of the MDR sublines to DENSPM is partially attributable to the finding that analogue (and spermidine) uptake in the sublines was about 2-fold higher (after 2 h) than in the parent cells. The presence of disturbances in polyamine homeostasis and increased sensitivity to DENSPM in three independently selected cell line variants suggests that they may be generally associated with the MDR phenotype in human melanoma and possibly other tumor cells. The collateral sensitivity of human melanoma MDR variants to DENSPM represents a possible therapeutic indication which should be considered during the ongoing clinical evaluation of this drug.

Inhibitors of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (SAMDC), derived from methylglyoxal-bis(guanylhydrazone) (MGBG), have been shown to have significant antitumor activity in several human solid tumor systems (U. Regenass et al., Cancer Res., 52:4712-4718, 1992). From an ongoing effort to synthesize derivatives with increased enzyme specificity and potency and improved antitumor efficacy, we have now identified CGP 48664, a 4-amidinoindan-1-one 2'-amidinohydrazone (J. Stanek et al., J. Med. Chem., 36:2168-2171, 1993). The compound displays potent inhibition of SAMDC (50% inhibitory concentration, 5 nM), modest inhibition of diamine oxidase (50% inhibitory concentration, 4 microM), and no detectable inhibition of ornithine decarboxylase. CGP 48664 inhibits the growth of a panel of human and mouse tumor cell lines, including one which expresses the multidrug resistance phenotype, with 50% inhibitory concentrations ranging between 0.3 and 3 microM. CGP 48664 does not seem to utilize the polyamine transport carrier system since it competes poorly with spermidine for uptake into L1210 cells (Ki 161 microM) and inhibits the growth of polyamine transport-deficient Chinese hamster ovary cells. Relative to MGBG or previously described MGBG analogues, CGP 48664 accumulates to much lower intracellular concentrations. Treatment of the L1210 cell for 48 h with 3 microM CGP 48664 decreases SAMDC activity to < 10% of control and initiates a compensatory 3-fold rise in ornithine decarboxylase. Consistent with SAMDC inhibition, putrescine pools increase 10-fold, whereas spermidine and spermine pools fall to < 10% of control. In contrast to MGBG, CGP 48664 displays attenuated antimitochondrial activity as indicated by a lack of effect on pyruvate oxidation and mitochondrial DNA levels under treatment conditions which inhibit cell proliferation. Specificity of drug action was indicated further by prevention of L1210 cell growth inhibition by exogenous spermidine or spermine. More convincingly, Chinese hamster ovary cells made approximately 1000-fold resistant by chronic exposure to the analogue were found to selectively overexpress SAMDC mRNA due to gene amplification. The new SAMDC inhibitor showed potent antitumor activity against syngeneic tumors (B16 melanoma and Lewis lung carcinoma) and nude mouse human tumor xenografts (T-24 bladder carcinoma, SK MEL-24 melanoma, and MALME-3M melanoma). On the basis of its novel structure, its apparent specificity of action, and its potent antitumor activity, CGP 48664 is the candidate drug for further preclinical development.

Pharmacokinetics of alpha-difluoromethylornithine (DFMO) in plasma and polyamine levels in urine and erythrocytes (RBC) of subjects considered to be at a higher-than-normal risk for developing cancer and receiving DFMO in a phase I chemoprevention trial were monitored over a period of 6 months at DFMO doses ranging from 200 to 6400 mg/m2/day. DFMO pharmacokinetics was linear and attained an average peak plasma concentration of 58 micrograms/ml and an average area under the concentration x time curve from 0 to 6 h of 240 micrograms/ml.h at an administered dose of 1600 mg/m2. Transient decreases in RBC polyamine levels were observed in only 3 of 22 subjects; all of the others showed an increase in the levels at some time during DFMO administration. In contrast to these findings, 17 of 22 subjects showed a decline in urinary polyamines; 10 of 22 showed this decline by the end of the first month and the remaining subjects during subsequent administration of the drug. One subject with familial polyposis who had high RBC and urinary polyamine levels prior to DFMO treatment showed a significant decline in urinary polyamines and responded to DFMO treatment with nearly complete resolution of the polyps in the rectal stump. Our results suggest that (a) DFMO concentrations achieved in this study are adequate to modulate polyamine pools as reflected by their reduced urinary excretion; (b) the red blood cell polyamines are not reliable indicators of DFMO activity; and (c) the modulation of polyamines occurs at doses of DFMO that are tolerated by a majority of the subjects.

In in vitro systems, the spermine analogue, N1,N11-bis(ethyl)norspermine (BENSPM), suppresses the polyamine biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase (ornithine decarboxylase and S-adenosylmethionine decarboxylase, respectively), greatly induces the polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT), depletes polyamine pools, and inhibits cell growth. Against MALME-3 M human melanoma xenografts, BENSPM and related homologues demonstrate potent antitumor activity that has been found to correlate positively with their ability to induce SSAT activity in vitro. Herein, we further evaluate the antitumor activity of BENSPM and at the same time characterize the biochemical effects of BENSPM treatment on polyamine metabolism of selected normal and tumor tissues. At 40 mg/kg 3 times/day for 6 days i.p., BENSPM suppressed growth of MALME-3 M human melanoma xenografts during treatment and for 65 days afterwards. Similar antitumor activity was obtained with 120 mg/kg once daily for 6 days and 40 mg/kg once daily for 6 days, indicating that against this tumor model, the dosing schedule can be relaxed up to sixfold without compromising antitumor activity. When MALME-3 M tumor-bearing mice were retreated with BENSPM 2 weeks after the first treatment at 40 mg/kg 3 times/day for 6 days, initial tumor volumes of 85 mm3 were reduced to < 10 mm3. Analysis of melanoma, liver, and kidney tissues from mice treated with 40 mg/kg 3 times/day for 6 days revealed relatively similar accumulations of BENSPM in all tissues at levels greater than the original total content of polyamine pools. By 2 weeks following treatment, BENSPM pools in normal tissues were almost gone, whereas in tumor tissues significant amounts (40%) were still retained. The biosynthetic enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, gave no indication of enzyme suppression (or increase) by the analogue as typically occurs in vitro. By contrast, SSAT was induced from an average of < 50 pmol/min/mg in control tissues to 320 pmol/min/mg in liver, 1255 pmol/min/mg in kidney, and 13,710 pmol/min/mg in MALME-3M tumor. Two weeks later, SSAT activity was still 12 times higher in tumor than in kidney. Polyamine pools (putrescine, spermidine, and spermine) were reduced after treatment in all tissues and approached near-total depletion in the tumor. Good antitumor activity and even more potent induction of SSAT (i.e., 26,680 pmol/min/mg) was also observed in PANUT-3 human melanoma xenografts. Overall, the findings reveal meaningful antitumor activity by BENSPM against 2 human melanoma xenografts and provide in vivo evidence consistent with SSAT-induced polyamine depletion playing a determining role in at least the initial phase of the antitumor response.

Methylglyoxal bis(guanylhydrazone) (MGBG) has been studied clinically as an antitumor and antileukemic agent and is recognized as a potent but nonspecific inhibitor of the key polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (SAMDC). A series of four SAMDC inhibitors with structural features similar to MGBG have been found to have improved potency and specificity toward the target enzyme, SAMDC. Relative to MGBG, the new derivatives were much more effective in inhibiting partially purified preparations of SAMDC (50% inhibitory concentration, 10 to 100 nM), much less effective at inhibiting diamine oxidase, and inactive toward ornithine decarboxylase. The inhibitors varied relative to MGBG in their ability to compete with spermidine for uptake, with two being similar and two being less effective. Against L1210 leukemic cells and T24 bladder carcinoma cells, the compounds were slightly less effective than MGBG at inhibiting cell growth, with 50% inhibitory concentration values of 1 to 10 microM as compared with 0.5 and 1.1 microM, respectively, for MGBG. Under 50% growth-inhibitory conditions, the inhibitors decreased SAMDC activity, increased ornithine decarboxylase activity and putrescine pools, and markedly depleted spermidine and spermine pools of L1210 cells. At the same time, mitochondrial integrity as assessed by whole-cell pyruvate oxidation and mitochondrial DNA content was not affected as it was with MGBG. At doses less than one tenth that of the maximally tolerated dose, all of the new inhibitors strongly suppressed the growth of B16 melanoma in vivo with minimal weight loss or toxicity. At doses less than one sixth the maximally tolerated dose, they effectively inhibited the growth of T24 human bladder carcinoma xenografts. In these same systems, MGBG showed only marginal antitumor activity. These studies identify two potent and efficacious inhibitors of SAMDC as potential antitumor agents and reaffirm the importance of SAMDC as a target in anticancer drug discovery.

The spermine analogues, N1,N12-bis(ethyl)spermine (BESPM), N1,N11-bis(ethyl)norspermine (BENSPM), and N1,N14-bis(ethyl)-homospermine (BEHSPM) behave similarly in down-regulating the key polyamine biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase, but differ distinctly in their abilities to induce the polyamine catabolic enzyme, spermidine/spermine-N1-acetyltransferase; BENSPM is 6-fold more effective than BESPM in increasing spermidine/spermine-N1-acetyltransferase activity and BEHSPM is 10-fold less effective. Since MALME-3 human melanoma cells are extremely responsive to spermidine/spermine-N1-acetyltransferase induction (i.e., increases greater than 200-fold) and since this induction correlates with growth inhibition among melanoma cell lines, the ability of these homologues to inhibit the growth of MALME-3 xenografts was examined. Analogues were administered i.p. three times per day (i.e., every 8 h) for 6 days at the following doses per injection: BEHSPM, 1.5, 3, or 6 mg/kg; BESPM, 10, 20, or 40 mg/kg; BENSPM, 20, 40, or 80 mg/kg. At the highest tolerated doses, all of the analogues fully suppressed growth of established (100-200 mm3) MALME-3 tumor during treatment and sustained tumor growth inhibition following treatment as follows: BEHSPM, 14 days; BESPM, 27 days, and BENSPM, 37 days. The tumor delay (to reach 1000 mm3 relative to control) at the highest tolerated doses was as follows: BEHSPM, 20 days; BESPM, 34 days, and BENSPM, 63 days. The rank order of analogue host toxicity as indicated by weight loss was opposite that for antitumor activity, BEHSPM was most toxic, BESPM, intermediate, and BENSPM, least toxic. Thus, the most effective of the three homologues, BENSPM, was best tolerated, and produced an initial tumor regression, full suppression of tumor regrowth during treatment, and sustained inhibition of tumor regrowth for 37 days after treatment stopped. Owing to its potent antitumor activity, mild host toxicity, and novel apparent mechanism of action, BENSPM is being considered for further development toward clinical trial.

The polyamine analogue, N1,N12-bis(ethyl(-spermine (BESPM), is known to suppress ornithine and S-adenosylmethionine decarboxylase levels, deplete intracellular polyamine pools, and inhibit cell growth. Among human melanoma cell lines, MALME-3 cells were found to be typically sensitive to the antiproliferative activity of the BESPM, whereas LOX cells were atypically insensitive to the analogue. A comparison of polyamine-related parameters revealed that the most differentially altered activity between the 2 BESPM-treated cell lines was that of spermidine/spermine N1-acetyltransferase (SSAT), which increased from 50 pmol/min/mg to greater than 10,000 pmol/min/mg in MALME-3 cells and from 16 pmol/min/mg to only 120 pmol/min/mg in LOX cells over 48 h. The basis for the large difference seems to be related to increased enzyme synthesis in both cell lines coupled with differences in prolongation of SSAT half-life (greater than 12 h in MALME-3 cells versus 1.6 h in LOX cells) after BESPM treatment. In MALME-3 cells, SSAT accumulation was found to be differentially modulated by the BESPM homologues, N1,N11-bis-(ethyl)norspermine and N1,N14-bis-(ethyl)homospermine, which were 5-fold more and 9-fold less effective, respectively, than BESPM in increasing SSAT but similar in analogue uptake and effects on polyamine biosynthesis and cell growth inhibition. Treatment of MALME-3 cells with BESPM resulted in an accumulation of N-acetylspermidine in cells and the enhanced excretion of putrescine, spermidine, and N-acetylspermidine into the medium. The relationship between SSAT induction and growth sensitivity was deduced to be a possible function of increased excretion of acetylated polyamines leading to enhanced polyamine pool depletion. The data suggest that, in cell types in which it occurs, unusually high increases in SSAT activity may serve as a determinant of growth sensitivity to bis-ethyl spermine analogues or, alternatively, as a target for appropriately designed chemotherapeutic strategies.

The polyamines are known to be essential for cellular proliferation. Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the synthesis of these amines, and activity is elevated in colorectal tumors and polyps. Two ODC genes (designated ODC1 and ODC2) were localized by somatic cell hybridization and in situ techniques to 2p25 and 7q31-qter, respectively. Investigation of the expression of ODC in colorectal neoplasia reveals a consistent increase in mRNA expression compared with normal adjacent mucosa and control mucosa, ranging from 1.3- to 12.2-fold. No amplification of the loci was seen. Comparison of ODC mRNA expression with ODC activity from the same samples revealed no direct correlation, suggesting that regulation of ODC in this system occurs at the posttranscriptional level.

Biochemical modulation of methylation processes can be accomplished by agents which either reduce pools of S-adenosylmethionine (AdoMet), the principal methyl donor, or alternatively, which raise levels of S-adenosylhomocysteine (AdoHcy), a potent product inhibitor of methyltransferase reactions. Both strategies have apparent limitations arising from their direct interference with only one determinant of the intracellular AdoHcy/AdoMet ratio, a parameter proposed to be indicative of methylation inhibition. The biological consequences of maximally altering this ratio have been examined by the combined use of an inhibitor of AdoMet synthetase, L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB), with inhibitors of AdoHcy hydrolase, 9-(trans-2',trans-3'-di-hydroxycyclopent- 4'-enyl)adenine (DHCA) and neplanocin A. At concentrations which inhibited growth of L1210 cells by 50% at 48 h, L-cisAMB alone rapidly depleted AdoMet pools, while neplanocin A or DHCA alone led to an accumulation of AdoHcy. When L-cisAMB was combined with either neplanocin A or DHCA, AdoHcy increased and, concomitantly, AdoMet pools decreased. The resultant AdoHcy/AdoMet ratios for up to 48 h ranged from 2.2 to 3.6-a value 4-fold greater than those achieved with neplanocin A or DHCA alone. This elevation in the AdoHcy/AdoMet ratio was accompanied by marked and sustained interference with DNA and RNA methylation and with a near-total inhibition of cell growth for a period of 24 to 96 h. Thus, the combined treatment with these two types of mechanistically different methylation inhibitors resulted in significantly enhanced interference with nucleic acid methylation and cell growth, both of which correlated directly with unprecedented increases in the AdoHcy/AdoMet ratio. This approach may have therapeutic implications in antiviral and/or antitumor strategies targeting methylation.

Treatment of cultured L1210 cells with the putrescine analogue, 2,5-diamino-3-hexyne, at 0.5 nM resulted in the rapid (1-2 h) appearance of numerous cytoplasmic vacuoles which were highly visible by light microscopy. Ultrastructural examination revealed that the vacuoles contained numerous membrane vesicles and electron-dense structures resembling endosomal elements. Other cellular organelles were unaffected by the drug. The overall morphological effect by 2,5-diamino-3-hexyne was nearly identical to that produced in the same cells by the known lysosomotropic agent, chloroquine. Since the putrescine analogue, 1,4-diamino-2-butyne at 1.2 mM, also produced comparable cytoplasmic vacuolation, and putrescine itself failed to do so at concentrations as high as 5 mM, it was concluded that the apparent lysosomotropic activity of the putrescine analogues was probably due to their weaker basicity related to the presence of an internal triple bond. Although it is uncertain whether the effect of the analogues on the endosomal system is related to the natural function of polyamines, the finding points out the previously unrecognized potential for certain polyamine analogues to act in this manner.

Stable variants of the human cell line, VA2-B, have been developed which are 10- to 20-fold less sensitive to the antiproliferative effects of methylglyoxal bis(guanylhydrazone) (MGBG) than the parent cell lines and which are not drug transport deficient. The lines were characterized biochemically giving particular attention to parameters related to the two known sites of MGBG action, mitochondria and polyamine metabolism. Dose-response studies with MGBG (0 to 30 microM for 40 to 48 hr) revealed that, of the parameters related to polyamine metabolism (i.e., polyamine pools, S-adenosylmethionine, and ornithine decarboxylase activities), only spermine pool size reduction seemed to correlate with inhibition of cell growth by MGBG. By contrast, decreases in pyruvate oxidation (used here as a measure of mitochondrial function) closely paralleled growth inhibition in all cell lines. Similarly, MGBG-induced changes in mitochondrial ultrastructure were less conspicuous in the variants than in the parent cell line and also corresponded with growth inhibition. Respiration of isolated mitochondria from one of the variant lines was about 2-fold more resistant to the inhibitory effects of MGBG than mitochondria from the VA2 cells. Finally, treatment with alpha-difluoromethylornithine, a potent inhibitor of polyamine biosynthesis having no known effect on mitochondrial function, resulted in comparable inhibition of growth in variant and parent cell lines. Overall, the data suggest that a phenotypic alteration in mitochondrial function, rather than in polyamine metabolism, may represent the basis for MGBG resistance in these variant cell lines.

The effects of the enzyme-activated irreversible inhibitors of ornithine decarboxylase, alpha-difluoromethylornithine, alpha-(fluoromethyl)dehydroornithine, alpha-(fluoromethyl)dehydroornithine methyl ester, and (2R,5R)-6-heptyne-2,5-diamine (RR-MAP), on cell growth and parameters related to polyamine biosynthesis were compared in L5178Y and L1210 cells under identical culture conditions. The two lines are murine lymphocytic leukemia cells which differ in their ability to metabolize 5'-methylthioadenosine, the by-product of polyamine biosynthesis: L5178Y cells contain a specific 5'-methylthioadenosine phosphorylase; L1210 cells do not. In L1210 cells, the 50% inhibitory concentrations (lC50S) of the various analogues were 3.0 mM for alpha-difluoromethylornithine, 0.2 mM for alpha-(fluoromethyl)dehydroornithine, 0.1 mM for alpha-(fluoromethyl)dehydroornithine methyl ester, and 0.01 mM for RR-MAP. L5178Y cells were somewhat more sensitive to the inhibitors with lC50 values of 0.5 mM for alpha-difluoromethylornithine, 0.06 mM for alpha-(fluoromethyl)dehydroornithine, 0.03 mM for alpha-(fluoromethyl)dehydroornithine methyl ester, and 0.002 mM for RR-MAP. In all cases, growth inhibition was fully prevented by exogenous putrescine. The effects of the inhibitors on parameters related to polyamine metabolism were compared at drug concentrations approximating the average of lC50 values for the two cell lines. Under these treatment conditions, polyamine pools were similarly affected by the various inhibitors. Typically, putrescine and spermidine were depleted, but effects on spermine pools differed according to the cell line, increasing slightly in L1210 cells and decreasing by about 50% in L5178Y cells. Spermine pools in L1210 cells could be reduced by RR-MAP at concentrations higher than the lC50 (i.e., 0.1 mM). Clonogenicity in soft agar was decreased about 50% by putrescine and spermidine depletion and was not further affected by spermine depletion. The inhibitors elevated S-adenosylmethionine decarboxylase activity in both cell lines with a 2-fold greater increase in L5178Y cells than in L1210 cells. Finally, the inhibitors decreased S-adenosylmethionine pools in L1210 cells by about 50% but had little effect on these pools in L5178Y cells with the exception of RR-MAP, which decreased S-adenosylmethionine pools by about 40%. Whether the different polyamine responses of the two cell lines are related to their ability to metabolize 5'-methylthioadenosine is uncertain. It is apparent, however, that the presence or absence of methylthioadenosine phosphorylase does not substantially modulate the antiproliferative activity of ornithine decarboxylase inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)

The distribution of isotopically labeled hematoporphyrin derivative (HPD) has been studied in mice bearing the spontaneous mammary tumor (fast growing). In stomach, liver, spleen, and pancreas, 3 hr after i.p. injection of [3H]HPD, grains were uniformly distributed over the tissue sections. After 24 hr, the grain density overlying parenchymous areas of these tissues was lower than that over the stromal or reticuloendothelial areas. In the spontaneous mammary tumor (fast growing), higher grain densities were seen over pseudocapsule, stromal septa, and necrotic areas at 3, 6, 12, 24, and 48 hr after injection. At 168 hr postinjection, only isolated stomal cells, presumably macrophages, showed high grain densities. From the temporal changes observed in the distributions of HPD in normal tissues and the relative stability of the distribution seen in the spontaneous mammary tumor (fast growing), we speculate that tissue factors such as vascular permeability, lack of an adequate lymphatic drainage, and nonspecific binding of serum proteins to stromal elements may be responsible for or contribute to the preferential uptake and/or retention of HPD observed in both human and animal tumors.

The distribution of the carcinogen-metabolizing enzyme system, aryl hydrocarbon hydroxylase (AHH), was biochemically determined in the intestinal epithelium of the rat. A method of epithelial cell isolation in which fractions of cells are sequentially collected as a villus tip-to-crypt gradient was used. AHH activity was highest in the midvillus region, 40% lower at the villus tip, and practically nonexistent in the crypt region where active cell proliferation takes place. This distribution differed from those of sucrase and alkaline phosphatase (used here as markers for cellular differentiation), which were characteristically lowest in activity at the crypts and increased continuously to the villus tips. Conceivably, the midvillus peak of AHH activity may serve to protect or enhance the susceptibility of cells undergoing cell division in the nearby crypt regions, depending on whether the predominant function of AHH in the intestinal epithelium involves detoxication or activation of polyaromatic carcinogens.

The ability of methylglyoxal-bis(guanylhydrazone) (MGBG) and 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) to interact with the hypoglycemic agent, phenethylbiguanide (DBI), in affecting the bioenergetic functions of isolated rat liver mitochondria was studied. DBI was found to increase markedly the inhibitory effect of either 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) or MGBG on respiration of isolated rat liver mitochondria. Conversely, these bis(guanylhydrazones) enhanced the inhibitory potency of DBI and increased the apparent affinity of mitochondria for the drug. As with MGBG and 4,4'-diacetyldiphenylurea-bis(guanylhydrazone), the potassium cationophore, valinomycin, increased the sensitivity of mitochondrial respiration to DBI. It is suggested that the enhancement of bis(guanylhydrazone) inhibition of mitochondrial respiration by DBI involves inhibition of proton fluxes across the inner mitochondrial membrane and the subsequent alkalinization of the mitochondrial matrix. This drug interaction was extended to the level of antiproliferative activity in which DBI was found to potentiate the growth-inhibitory effects of MGBG on murine L1210 leukemia in vivo.

Eleven novel spermidine (SPD) derivatives were synthesized as potential anticancer agents and evaluated for their ability to compete with [3H]SPD for cellular uptake, to inhibit cell growth, to affect polyamine biosynthesis, to suppress enzyme activity, and to substitute for SPD in supporting growth of cultured L1210 leukemia cells. The compounds included a series of N4-SPD derivatives (N4-methyl-SPD, N4-ethyl-SPD, N4-acetyl-SPD, N4-hexyl-SPD, N4-hexanoyl-SPD, N4-benzyl-SPD, and N4-benzoyl-SPD) and a series of N1,N8-SPD derivatives [N1,N8-bis(ethyl)-SPD, N1,N8-bis(acetyl)-SPD, N1,N8-bis(propyl)-SPD, and N1,N8-bis(propionyl)-SPD]. Uptake studies revealed N4-alkyl derivatives to be the most effective competitive inhibitors of [3H]SPD uptake (Ki, 26 to 43 microM) followed by N1,N8-alkyl derivatives (Ki, 71 to 115 microM), then N4-acyl derivatives (Ki, 115 to greater than 500 microM), and N1,N8-acyl derivatives (Ki, greater than 500 microM). The data indicate the relative importance of the terminal amines and of charge as determinants of cellular uptake. Of the 11 derivatives, only N4-hexyl-SPD, N1,N8-bis(ethyl)-SPD, and N1,N8-bis(propyl)-SPD demonstrated antiproliferative activity at 0.1 mM with 50%-inhibitory concentration values at 48 h of 30, 40, and 50 microM, respectively. In the case of the N1,N8-SPD derivatives, recovery from growth inhibition was enhanced considerably by exogenous SPD, suggesting involvement of polyamine depletion. At 10 to 30 microM, both N1,N8-bis(ethyl)-SPD and N1,N8-bis(propyl)-SPD (but not N4-hexyl-SPD) inhibited polyamine biosynthesis as indicated by significant reductions in polyamine pools and in biosynthetic enzyme activities. The more effective of the two, N1,N8-bis(ethyl)-SPD, depleted intracellular putrescine and SPD and reduced spermine by approximately 50% at 96 h and decreased ornithine and S-adenosylmethionine decarboxylase activities by 98 and 62%, respectively. Since neither derivative (at 5 mM) directly inhibited these enzymes from untreated cell extracts by significantly more than SPD itself, it is suspected that they act by regulating enzyme levels. As a measure of regulatory potential of the derivatives, ornithine decarboxylase was assayed in cells treated for 24 h and compared to the effects of 10 microM SPD which reduced the enzyme activity by 80%. None of the N4-SPD derivatives affected ornithine decarboxylase activity, while N1,N8-bis(ethyl)- and (propyl)-SPD were nearly as effective as SPD. Apparently, the central amine of the molecule is critical for regulatory function.(ABSTRACT TRUNCATED AT 400 WORDS)

The use of methylglyoxal-bis(guanylhydrazone) (MGBG) in the clinical treatment of myeloid and lymphoid disorders has been limited by severe host toxicity to renewing tissues, particularly the intestinal mucosa. Since the drug is a potent inhibitor of spermidine biosynthesis, the distributions of ornithine and S-adenosylmethionine decarboxylases and polyamine pools have been characterized in the rat intestinal mucosa in an attempt to discern the basis for MGBG toxicity. A method of epithelial cell isolation in which fractions of cells are sequentially collected in a villus tip-to-crypt gradient was used. Ornithine decarboxylase activity was highest in the villus tip region and unexpectedly lowest in the crypts, while S-adenosylmethionine decarboxylase activity showed the opposite pattern. Intracellular polyamine pools were uniform along the gradient corresponding to the villus length and increased appreciably in the crypt region. The relative concentrations of the individual polyamines were highest in the crypts, with spermidine and spermine being nearly equivalent in all regions. Twenty-four hr after a single i.p. injection of MGBG (50 mg/kg), S-adenosylmethionine decarboxylase activity increased markedly, especially in the crypt region (approximately 50-fold), while ornithine decarboxylase activity also increased but to a lesser extent. Putrescine pools were most affected by MGBG and were elevated 5- to 6-fold, especially in the crypt region. The results are consistent with an alteration of polyamine biosynthesis by MGBG being involved in the antiproliferative toxicity of the drug.

The proliferation kinetics of a fast-growing spontaneous mouse mammary tumor subline (SMT-F) and a slow-growing spontaneous mouse mammary tumor subline (SMT-S) tumor have been determined autoradiographically at 2 different stages of tumor growth. The length of the cell cycle and the growth fraction for SMT-F were 11.2 hr and 0.85, respectively, on Day 14 and 12.1 hr and 0.78 on Day 28. For SMT-S these same parameters were 15.6 hr and 0.50 on Day 14 and 16. 1 hr and 0.45 on Day 28. On Days 14 and 28 the mitotic indices were 1.3 and 1.0%, respectively, for SMT-S, compared to 2.2 and 1.9% for SMT-F. The cell loss rate, cell loss factor, and cell loss were significantly higher for SMT-F than for SMT-S. The difference in the growth rates for these 2 tumor lines was attributable to a slight prolongation of the length of the cell cycle and a reduction in the growth fraction of SMT-S.

The cellular effects of alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), and N1,N8-bis(ethyl)spermidine (BES), an apparent regulator of the enzyme were compared in cultured L1210 cells. Unlike DFMO, BES has no direct inhibitory effect on ODC activity. Rather the polyamine analogue is believed, from previous studies, to behave similarly to exogenous spermidine in its ability to suppress intracellular ODC activity but not in its ability to perform functions required for cell growth. The kinetics and extent of growth inhibition by 30 microM or 100 microM BES and 1 mM DFMO were nearly identical as were their effects on macromolecular precursor incorporation with leucine being the first and most significantly affected. By flow cytometry, neither BES nor DFMO induced obvious perturbations in the cell cycle. Both compounds effectively eliminated ODC activity in treated cells and depleted putrescine and spermidine pools with very similar kinetics of decline. These close similarities in drug effects between BES and DFMO, an established polyamine inhibitor, support previous indications that BES induces growth inhibition by depletion of cellular polyamines. BES differed distinctly from the ODC inhibitor by decreasing spermine pools, and by not increasing S-adenosyl-methionine decarboxylase activity, S-adenosylmethionine pools, or stimulating cellular uptake of polyamines. The data suggest that enzyme regulation by polyamine analogues such as BES represents a viable alternative to enzyme inhibition as an antiproliferative strategy directed at polyamine biosynthesis.

Four variants (VA2/MGBG) of the human cell line, VA2, have been isolated which are 10- to 20-fold more resistant than the parent line to the antiproliferative effects of the anticancer agent, methylglyoxal bis(guanylhydrazone) (MGBG). Drug resistance was not cytoplasmically transmitted by cytoplast cell fusion for any of the four sublines, suggesting that the genes responsible for resistance may be of nuclear rather than mitochondrial origin. Uptake properties were characterized in the VA2 cells and two of the four variant lines. Uptake of [14C]MGBG during long-term (0.5 to 28 hr) incubations was 3 to 4 times greater in the VA2 cells than in the VA2/MGBG sublines. However, during short-term (2 to 60 min) incubations, the uptake of [14C]MGBG or [3H]spermidine (which competes for MGBG uptake) was similar for all cell lines. This was further supported by kinetic data which indicated that, for [14C]MGBG uptake at 4 min, the apparent Km for all cell lines was 5.8 to 13.4 microM, and the Vmax, 44 to 53 pmol/mg/min. For [3H]spermidine uptake, the apparent Km values were approximately 1 microM and Vmax, 54 to 69 pmol/mg/min. Efflux studies performed on cells incubated for 30 min in 10 microM [14C]MGBG revealed that the two VA2/MGBG sublines released drug much more rapidly than did VA2 cells over an 8-hr period. Thus, while the variants may transport MGBG at a rate similar to VA2 cells, the drug is not accumulated to the same extent during long-term incubations, probably because of altered intracellular binding sites for MGBG. The identification of these sites may provide insight into the basis for antiproliferative action of MGBG.

A series of diamine homologues of putrescine and triamine homologues of spermidine was used to determine the structural specificity of the polyamine transport system in ascites L1210 leukemia cells by measuring their ability to compete with [3H]-putrescine, [3H]spermidine, or [3H]spermine for uptake. Transport specificity among the diamines (as indicated by K1 constants) was greatest for those having chain lengths similar to that of spermidine and least for those similar to putrescine. Among the triamines, transport specificity was greatest for those having an overall chain length similar to those of spermidine and spermine. The homologue competition profiles were relatively the same for [3H]putrescine, [3H]spermidine, or [3H]spermine, suggesting that all three polyamines utilize the same transport system. This was further substantiated by uptake kinetic plots which showed that the three polyamines were competitive inhibitors of one another. In terms of receptor specificity, the ranking order among the polyamines was as follows: spermine (apparent Km, 1.6 microM) greater than spermidine (apparent Km, 2.2 microM) greater than putrescine (apparent Km, 8.5 microM). This information should prove useful in designing anticancer agents which are intended to utilize this transport system.

A number of studies have demonstrated that pretreatment of tumor-bearing animals with the inhibitor of polyamine biosynthesis, alpha-difluoromethylornithine (DFMO), potentiates the antitumor activity of methylglyoxal bis(guanylhydrazone) (MGBG). The present study examines whether this phenomenon is related to a DFMO-mediated increase in the selectivity of MGBG for tumor tissue. Specifically, the effect of DFMO pretreatment on the tissue distribution and content of MGBG was investigated in mice bearing ascites L1210 leukemia. At 3 and 18 h following a single i.v. injection of [14C]MGBG (50 mg/kg), L1210 cells and seven tissues from nonpretreated (control) and DFMO-pretreated (3% by drinking water for 3 days) animals were compared for their [14C]MGBG content. In control mice, the greatest amount of drug was found in L1210 cells, small intestine, and kidney (in decreasing order of magnitude) at both 3 and 18 h. This distribution was not altered following DFMO pretreatment, but the relative MGBG content of other tissues was shifted. On an average, DFMO pretreatment increased the accumulation of MGBG by 30% in normal tissues and 32% in tumor tissues at 3 h and 56% and 69%, respectively, at 18 h. Thus, pretreatment of leukemic mice with DFMO fails to improve the selectivity of MGBG for L1210 cells. It is possible that other tumor systems might demonstrate sufficient DFMO-mediated increases in MGBG uptake to enhance drug selectivity but not without significantly increasing MGBG uptake (and hence toxicity) in normal tissues.

Ethylglyoxal bis(guanylhydrazone) (EGBG) was compared as an inhibitor of polyamine biosynthesis with methylglyoxal bis(guanylhydrazone) (MGBG) in bovine small lymphocytes stimulated by concanavalin A. EGBG brought about a decrease in spermidine and spermine levels equal to that found with MGBG, but at a 5-fold lower intracellular drug concentration. Despite identical polyamine levels, the degree of inhibition of DNA and protein synthesis by EGBG was smaller than that observed with MGBG, in either the presence or absence of the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. It was found that in vitro protein synthesis and in vivo mitochondrial function were inhibited by concentrations of MGBG necessary to inhibit polyamine synthesis in cells (1 to 3 mM), but not by efficacious levels of EGBG (0.2 to 0.6 mM). These results suggest that EGBG is more suitable as a specific inhibitor of polyamine biosynthesis and that use of this drug, rather than MGBG, in combination with alpha-difluoromethylornithine may be useful for studying the physiological functions of polyamines in animal cells.

Methylglyoxal-bis(guanylhydrazone) (MGBG) is a polycationic drug which is useful in the chemotherapy of lymphoid and myeloid proliferative disorders. The drug has recently been shown to produce selective ultrastructural damage to the mitochondria of proliferating cell populations. It is important to understand the molecular basis for this action, since it may be related to the known ability of MGBG to block polyamine biosynthesis. Accordingly, the effect of MGBG treatment on the incorporation of [3H]thymidine into both mitochondrial and nuclear DNA has been examined. Exponentially growing L1210 leukemia cells were prelabeled with [14C]thymidine, treated with MGBG for 1.5 to 16 hr, and then pulse labeled with [3H]-thymidine. Incorporation of [3H]thymidine into mitochondrial DNA was selectively inhibited at 5 hr with concentrations of 1 to 10 microM MGBG. Nuclear DNA, however, was not similarly affected until 8 to 11 hr of drug treatment. Dye-CsCl gradients of mitochondrial DNA indicated that the inhibition of synthesis occurred in replicative forms of circular DNA. Uptake studies excluded the possibility of drug interference with cellular uptake of thymidine. Ultrastructural studies revealed a very close correlation between the dose-response curve for mitochondrial damage and that for MGBG inhibition of mitochondrial DNA synthesis. This correlation suggests a direct cause-and-effect relationship between inhibition of mitochondrial DNA synthesis and ultrastructural damage, but the possibility of both phenomena being related to another action by the drug, such as inhibition of polyamine biosynthesis, or a drug effect on mitochondrial function, must also be considered.

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.

Polyamine depletion by pretreatment with alpha-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase, potentiates the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in L1210 leukemia cells grown in a modified soft agar system. The dose enhancement ratio was 1.97 at a control colony formation level of 5%. The basis for this enhancement was investigated at the level of DNA damage using a modified fluorometric assay to quantitate the production of alkaline-labile strand breaks per relative DNA molecular mass. Pretreatment of cultured L1210 cells for 48 hr with 5 mM DFMO depleted intracellular putrescine and spermidine (but not spermine) pools and resulted in a 2.3-fold increase in BCNU-induced (10 micrograms/ml, 2 hr) DNA strand breaks per relative DNA molecular mass. The inclusion of 10 microM spermidine during the DFMO pretreatment fully prevented growth inhibition and enhancement of BCNU-induced DNA damage while maintaining cellular spermidine pools at control levels. The inclusion of 2 microM putrescine or spermidine also prevented growth inhibition and enhancement of DNA damage while maintaining spermidine pools at only 25 to 35% of control. Thus, the portion of spermidine essential for cell growth appears to be associated with DNA. BCNU itself was found to reduce cellular polyamine levels by causing their leakage from cells. In addition, BCNU was found to react directly with spermidine in a cell-free system, resulting in a major reaction product detectable by high-performance liquid chromatography. While decreased interaction of BCNU with polyamines could account, in part, for enhancement effects of DFMO, it is more probable that alterations in DNA structure secondary to polyamine depletion are responsible for these effects.

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.

alpha-Difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, has been shown to be growth inhibitory in a wide variety of normal and tumor cell systems. Since cells of the host defense system are among the most rapidly proliferating cells in the body, DFMO may inhibit certain components of this system. In order to assess this possibility, four randomized groups of C57BL/6 mice were maintained either on water throughout (controls) or on 2% DFMO in drinking water for various periods of time. Mice were given DFMO from Days -4 to 0, Days 0 to +4, or Day 0 to day of assay. On Day 0 randomly selected mice from each group received P815 tumor allografts. Daily from Days +3 to +14, pools of spleen cells from three mice per group were assessed for allospecific cytolytic T-lymphocyte, antibody formation, natural killer cell, and phagocytic cell activities. While natural killer cell and phagocytic cell activities remained essentially unchanged under all conditions, both cytotoxic T-lymphocyte and antibody responses were modified. Somewhat similar effects were seen with both responses and involved to varying degrees: (a) a delay of the initiation of rapid increase in the response but not in the onset of first detectable response; (b) delay in the time of peak response; (c) increased level of maximal response; (d) two peaks of maximal response. The data indicate that DFMO treatment of whole animals, dependent upon schedule of administration and time of assay, induces very selective effects on both cytotoxic T-lymphocyte and antibody responses, without apparent modification of nonspecific host defense mechanisms, with the overall effect being a prolongation of the period of specific response.

Exposure of cultured leukemia L1210 cells to 0.1 micron methylglyoxal-bis(guanylhydrazone) resulted in a concentration-dependent inhibition of cellular proliferation, beginning after about 1 to 2 generation times (12 to 24 hr). Ultrastructural examination of the cells treated at and above 1.0 micron concentrations of drug for 24 hr revealed unifrom damage to mitochondria. The basic lesion involved extensive swelling of the mitochondrion, a deterioration and eventual loss of cristae, and a decrease in the matrix density. This damage preceded growth inhibition by about 12 hr and did not immediately affect cell viability as detected by trypan blue dye exclusion. Whether these findings are related to the known actions of the drug on polyamine metabolism is not clear at present.

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.

Ultrastructural studies of rats or mice treated for 24 hr with a toxic dose (100 mg/kg) of methylglyoxal-bis(guanylhydrazone) revealed the presence of damaged mitochondria in the crypt cells of the intestinal epithelium. Mitochondria were severely swollen and electron lucent, and appeared to be similar to those observed previously in a variety of cell types treated in vitro and in vivo with methylglyoxal-bis(guanylhydrazone). Since thymidine incorporation into the intestine was not found to be decreased until after 24 hr, it is concluded that the mitochondrial damage of methylglyoxal-bis(guanylhydrazone) could be responsible for the antiproliferative toxicities of the drug.

Microbial siderophores represent a class of iron chelators characterized by their high affinity (i.e., formation constants, greater than 10(40) M) for ferric iron. Previously, we demonstrated that the bacterial siderophores, N-[3-(2,3-dihydroxybenzamido)propyl]-N-[4-(2, 3-dihydroxybenzamino)butryl]-2-(2-hydroxyphenyl) trans-5-methyloxazoline-4-carboxamide (Parabactin) and N1,N8-bis(2,3-dihydroxybenzoyl)spermidine (Compound II), inhibit the growth of L1210 cells and the replication of DNA (but not RNA) viruses at low micromolar concentrations (Biochem. Biophys. Res. Commun., 121: 848-854, 1984). The basis for this antiproliferative effect on L1210 cells has now been investigated further. Onset of growth inhibition induced by 5 microM Parabactin occurs much earlier than with an equimolar concentration of Compound II but, once established by either chelator, inhibition appears to be irreversible. Growth inhibition was fully preventable with exogenous FeCl3 when given at the same time as the chelators. Flow cytometric analysis revealed a G1-S cycle block following treatment for 4 h with either 5 microM Parabactin or 30 microM Compound II. The block was readily reversed with exogenous FeCl3, allowing cells to progress to mid-S phase by 3 h and to G1 again by 9 h. Thereafter, cells accumulated at a second block located at S phase. The treatment conditions required for the initial cell cycle block (at 4 h) were adapted for subsequent studies. Clonogenicity of L1210 cells in soft agar following a 4-h exposure was reduced to 22% of control by 5 microM Parabactin and to 16% by 30 microM Compound II. Neither growth inhibition in suspension culture nor decreased clonogenicity in soft agar could be reversed with exogenous iron, following treatment with the chelators. Both chelators caused an early and significant decrease in [14C]thymidine incorporation over the 4-h period (50% inhibitory concentration at 4 h, 0.4 microM for Parabactin and 6.0 microM for Compound II). [3H]Uridine incorporation was inhibited later than [14C]thymidine and to a much lesser extent, while [3H]leucine incorporation was not significantly affected. Treatment of cells with 5 microM Parabactin or Compound II for 4 h decreased deoxy-adenosine triphosphate pools by 38 and 70%, respectively, and increased deoxythymidine triphosphate pools by 67 and 36%, respectively, suggesting interference with ribonucleotide reductase. Indeed, extracts of cells treated for 4 h with either 5 microM Parabactin or 30 microM Compound II exhibit a 97 to 98% decrease in cytidine-5'-diphosphate reductase activity compared to control, whereas DNA polymerase was elevated slightly.(ABSTRACT TRUNCATED AT 400 WORDS)