Polyamine biosynthesis impacts cellular folate requirements necessary to maintain S-adenosylmethionine and nucleotide pools.

Folate (vitamin B9) is utilized for synthesis of both S-adenosylmethionine (AdoMet) and deoxythymidine monophosphate (dTMP), which are required for methylation reactions and DNA synthesis, respectively. Folate depletion leads to an imbalance in both AdoMet and nucleotide pools, causing epigenetic and genetic damage capable of initiating tumorigenesis. Polyamine biosynthesis also utilizes AdoMet, but polyamine pools are not reduced under a regimen of folate depletion. We hypothesized that high polyamine biosynthesis, due to the high demand on AdoMet pools, might be a factor in determining sensitivity to folate depletion. We found a significant correlation (P<0.001) between polyamine biosynthesis and the amount of folate required to sustain cell line proliferation. We manipulated polyamine biosynthesis by genetic and pharmacological intervention and mechanistically demonstrated that we could thereby alter AdoMet pools and increase or decrease demand on folate availability needed to sustain cellular proliferation. Furthermore, growing a panel of cell lines with 100 nM folate led to imbalanced nucleotide and AdoMet pools only in cells with endogenously high polyamine biosynthesis. These data demonstrate that polyamine biosynthesis is a critical factor in determining sensitivity to folate depletion and may be particularly important in the prostate, where biosynthesis of polyamines is characteristically high due to its secretory function.

Is sperm cheap? Limited male fertility and female choice in the lemon tetra (pisces, characidae).

In the laboratory, fertilization rates achieved by male lemon tetras decline with spawning frequency. Even when the number of females is not limited, males can produce only four times as many offspring as females. Females show a preference for males that have not recently spawned as opposed to those that have. The cost of producing sufficient sperm to maximize fertilization rates may therefore reduce the intensity of sexual selection in this polygamous fish species.

Isolation and uptake characteristics of human cell variants resistant to the antiproliferative effects of methylglyoxal bis(guanylhydrazone).

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.

Effect of pretreatment with alpha-difluoromethylornithine on the selectivity of methylglyoxal bis(guanylhydrazone) for tumor tissue in L1210 leukemic mice.

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.

Biochemical and ultrastructural characterization of human cell variants resistant to the antiproliferative effects of methylglyoxal bis(guanylhydrazone).

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.

Combined modulation of S-adenosylmethionine biosynthesis and S-adenosylhomocysteine metabolism enhances inhibition of nucleic acid methylation and L1210 cell growth.

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.

Apoptotic signaling in polyamine analogue-treated SK-MEL-28 human melanoma cells.

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.

Comparison of the biological effects of four irreversible inhibitors of ornithine decarboxylase in two murine lymphocytic leukemia 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)

Collateral sensitivity of human melanoma multidrug-resistant variants to the polyamine analogue, N1,N11-diethylnorspermine.

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.

Comparison and characterization of growth inhibition in L1210 cells by alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, and N1,N8-bis(ethyl)spermidine, an apparent regulator of the enzyme.

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.

The polyamine oxidase inhibitor MDL-72,527 selectively induces apoptosis of transformed hematopoietic cells through lysosomotropic effects.

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.

Lysosomal sequestration of polyamine analogues in Chinese hamster ovary cells resistant to the S-adenosylmethionine decarboxylase inhibitor, CGP-48664.

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.

Effects of novel spermine analogues on cell cycle progression and apoptosis in MALME-3M human melanoma cells.

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.

Polyamine analogue induction of the p53-p21WAF1/CIP1-Rb pathway and G1 arrest in human melanoma 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.

Polyamine depletion in human melanoma cells leads to G1 arrest associated with induction of p21WAF1/CIP1/SDI1, changes in the expression of p21-regulated genes, and a senescence-like phenotype.

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.

Cloning and sequence analysis of the gene and cDNA encoding mouse spermidine/spermine N1-acetyltransferase--a gene uniquely regulated by polyamines and their analogs.

The polyamine catabolizing enzyme, spermidine/spermine N1-acetyltransferase (SSAT), has been implicated as a critical determinant of polyamine pool maintenance. SSAT has recently been shown to be positively regulated in human cell lines by polyamines and their analogs at the level of mRNA accumulation. Mouse LA-4 lung adenoma cells treated with either spermine or the spermine analog, N1,N12-bis(ethyl)spermine, produced a 2.3 and 6.5-fold increase, respectively, in SSAT mRNA. Prior evidence for transcriptional control of the enzyme prompted investigation of SSAT gene structure and its regulatory elements. The mouse SSAT gene was isolated as a 3650 bp EcoRI fragment from a lambda-J1 Mus saxicola genomic library by hybridization with human SSAT cDNA. An additional 431 bp downstream from the 3' EcoRI site were sequenced from a BamHI fragment (total gene sequence, 4066 bp). The gene contains six exons and five introns. Sequence analysis of the 774 bp of the 5' non-coding region revealed the absence of TATAA or CCAAT sequence motifs and the presence of a number of binding motifs in the 5' region of the gene with consensus binding sequences for transcription factors SP1, AP1, E2F, AP2, PEA-3 and others. The deduced amino acid sequence of the coding region differs from that of the human SSAT cDNA by five amino acids. The 527 bp of the 3' non-coding region contains four possible polyadenylation signal sites of which only one displays a typical consensus sequence. A 940 bp SSAT cDNA was isolated from Mus domesticus (BALB-C) liver lambda gt11 cDNA library. It contains a 5' untranslated region 89 bp in length and a 3' untranslated region 376 bp in length. The amino acid sequence deduced from Mus domesticus differs from that of Mus saxicola by one amino acid, from the hamster cDNA, by four amino acids and from the human cDNA by six amino acids. Further elucidation of the structural features of the SSAT gene may reveal how it is positively regulated by polyamines and their analogs.

Serine/threonine phosphorylation of IRS-1 triggers its degradation: possible regulation by tyrosine phosphorylation.

Insulin receptor substrate (IRS)-1 protein expression is markedly reduced in many insulin-resistant states, although the mechanism for this downregulation is unclear. In this study, we have investigated the early events in the insulin pathway that trigger the degradation of IRS-1. Incubation of the adipocytes with insulin induced a fast electrophoretic mobility shift of IRS-1 and a subsequent degradation of the protein. Wortmannin and rapamycin blocked this mobility shift of IRS-1, maintained the insulin-induced tyrosine phosphorylation of IRS-1, and blocked its degradation. In contrast, a glycogen synthase kinase 3 inhibitor, a mitogen-activated protein kinase/extracellular-regulated kinase inhibitor, and various protein kinase C inhibitors had no effect. Incubation with okadaic acid increased the serine/threonine phosphorylation of IRS-1 and its degradation, mimicking insulin, and its effect was prevented by the proteasome inhibitor lactacystin, as well as by rapamycin. Treatment of the cells with the tyrosine phosphatase inhibitor orthovanadate in the presence of insulin or okadaic acid partially inhibited the degradation of IRS-1. We propose that a rapamycin-dependent pathway participates as a negative regulator of IRS-1, increasing its serine/threonine phosphorylation, which triggers degradation. Thus, regulation of serine/threonine versus tyrosine phosphorylation may modulate IRS-1 degradation, affecting insulin sensitivity.

Molecular analysis of the region of distal 1p commonly deleted in neuroblastoma.

Cellular, cytogenetic, and molecular evidence indicates that chromosome band 1p36 is often deleted in neuroblastoma cell lines and tumours, suggesting the presence of one or more tumour suppressor genes in this region. We used a multifaceted approach to analyse the commonly deleted region, 28 distal 1p-specific polymorphic loci were used to detect loss of heterozygosity (LOH) in a panel of primary neuroblastoma tumours. Thirty-two of 122 tumours (26%) demonstrated LOH at three or more loci. In addition, a patient with a constitutional deletion of 1p36.2-.3 and two neuroblastoma cell lines with 1p36 abnormalities were characterised by FISH. When combined with the LOH data, a single consensus region of deletion was defined proximally by PLOD and distally by D1S80, a region spanning approximately five megabases. Several proposed candidate tumour suppressor genes, including ID3, CDC2L1, DAN, PAX7, E2F2, TNFR2 and TCEB3, map outside of this region; however, the transcription factor HKR3 cannot be excluded. LOH for 1p is correlated with adverse clinical and biological features and a poor prognosis, but 1p LOH is not an independent predictor of overall survival. To identify additional candidate genes, an integrated physical map of 1p35-36 is being constructed. The current map includes 445 polymerase chain reaction (PCR)-formatted markers and 608 YACs. This map will help identify region-specific transcripts by direct selection and sequencing.

Synthesis and antiproliferative effects of novel 5'-fluorinated analogues of 5'-deoxy-5'-(methylthio)adenosine.

5'-Deoxy-5'-[(monofluoromethyl)thio]adenosine (9) and 5'-deoxy-5'-fluoro-5'-(methylthio)adenosine (10), two novel analogues of 5'-deoxy-5'-(methylthio)adenosine (MTA), have been synthesized and evaluated for their substrate and inhibitory activities toward MTA phosphorylase and for their biological effects in L1210 (MTA phosphorylase deficient) and L5178Y (MTA phosphorylase containing) murine leukemia cell lines. Compound 9 was a potent competitive inhibitor of MTA phosphorylase with a Ki value of 3.3 microM and was also a substrate, with activity approximately 53% that of MTA. Compound 10 was significantly less inhibitory toward the phosphorylase with a Ki value of 141 microM; its lack of substrate activity was attributed to rapid nonenzymatic degradation. The 50% growth inhibitory concentrations (48 h) of 9 were 300 and 200 microM in L1210 and L5178Y cells, respectively; for 10, these respective values were 2 and 0.7 microM. The initial characterization of 9 in these systems reveals that it differs from MTA by not acting as a product regulator of the polyamine biosynthetic pathway.

Targeting 5'-deoxy-5'-(methylthio)adenosine phosphorylase by 5'-haloalkyl analogues of 5'-deoxy-5'-(methylthio)adenosine.

A series of 5'-haloalkyl-modified analogues of 5'-deoxy-5'-(methylthio)adenosine (MTA), a nucleoside byproduct of polyamine biosynthesis, has been synthesized: 5'-deoxy-5'-[(2-monofluoroethyl)thio]adenosine (10), 5'-deoxy-5'-[(2-chloroethyl)thio]adenosine (4), 5'-deoxy-5'-[(2-bromoethyl)thio] adenosine (5), and 5'-deoxy-5'-[(3-monofluoropropyl)thio]adenosine (13). On the basis of their abilities to serve as substrates of MTA phosphorylase prepared from mouse liver, several of these analogues were characterized for their growth inhibitory effects in MTA phosphorylase-containing (murine L5178Y and human MOLT-4) and MTA phosphorylase-deficient (murine L1210 and human CCRF-CEM) leukemia cell lines. The MTA phosphorylase-containing tumor cell lines, especially of human origin, were found to be more sensitive to treatment by these analogues. Of the analogue series, 10 was the most potent inhibitor of growth in each of the cell lines tested. The analogues, especially compound 10, displayed a reduced capacity to alter polyamine pools relative to MTA, mechanistically indicating a decreased potential for interactions at sites other than MTA phosphorylase. The results indicate that of the analogues tested, compound 10 displayed the best inhibitor/substrate interaction with MTA phosphorylase, which, in turn, correlated with more potent growth inhibition in tumor cell lines containing MTA phosphorylase. Overall, this supports the concept that MTA phosphorylase plays a role in the activation of such analogues.