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.

Spermidine requirement for cell proliferation in eukaryotic cells: structural specificity and quantitation.

Six structural homologs of spermidine and five of its precursor, putrescine, were studied for their ability to prevent cytostasis of cultured L1210 leukemia cells induced by alpha-difluoromethylornithine (DFMO), a specific inhibitor of putrescine biosynthesis. High-performance liquid chromatography and competition studies with spermidine indicated that the homologs, which vary in the length of the carbon chain separating the amines, penetrated the cells. The structural specificity of the spermidine carrier was defined. Three of the six spermidine homologs supported cell growth during a 48-hour incubation in the presence of DFMO, indicating that a two-carbon extension of spermidine structure was tolerated for biological function. Two of the five putrescine homologs supported growth after being converted by the cells to their respective spermidine homologs. The central nitrogen of spermidine appears to be essential for function since diamines of chain length comparable to that of spermidine did not prevent DFMO cytostasis. No more than 15 percent of the spermidine normally present in L1210 cells was required for cell proliferation in the presence of DFMO.

Generation of a mouse model for arginase II deficiency by targeted disruption of the arginase II gene.

Mammals express two isoforms of arginase, designated types I and II. Arginase I is a component of the urea cycle, and inherited defects in arginase I have deleterious consequences in humans. In contrast, the physiologic role of arginase II has not been defined, and no deficiencies in arginase II have been identified in humans. Mice with a disruption in the arginase II gene were created to investigate the role of this enzyme. Homozygous arginase II-deficient mice were viable and apparently indistinguishable from wild-type mice, except for an elevated plasma arginine level which indicates that arginase II plays an important role in arginine homeostasis.

Biochemical localization of aryl hydrocarbon hydroxylase in the intestinal epithelium of the rat.

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.

A selective effect of methylglyoxal-bis(guanylhydrazone) on the synthesis of mitochondrial DNA of cultured L1210 leukemia 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.

Autoradiographic distribution of hematoporphyrin derivative in normal and tumor tissue of the mouse.

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.

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.

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.

Ultrastructural changes in the mitochondria of intestinal epithelium of rodents treated with methylglyoxal-bis(guanylhydrazone).

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.

Morphological evidence for an antimitochondrial action by methylglyoxal-bis(guanylhydrazone).

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.

Potentiation of the antimitochondrial and antiproliferative effects of bis(guanylhydrazones) by phenethylbiguanide.

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.

Antitumor activity of N,N'-bis(ethyl)spermine homologues against human MALME-3 melanoma xenografts.

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.

Aliphatic chain length specificity of the polyamine transport system in ascites L1210 leukemia cells.

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.

Comparison of specificity of inhibition of polyamine synthesis in bovine lymphocytes by ethylglyoxal bis(guanylhydrazone) and methylglyoxal bis(guanylhydrazone).

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.

Enhancement of 1,3-bis(2-chloroethyl)-1-nitrosourea-induced cytotoxicity and DNA damage by alpha-difluoromethylornithine in L1210 leukemia cells.

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.

Antitumor activity of N1,N11-bis(ethyl)norspermine against human melanoma xenografts and possible biochemical correlates of drug action.

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.

Polyamines and biosynthetic enzymes in the rat intestinal mucosa and the influence of methylglyoxal-bis(guanylhydrazone).

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.

Selective modulation by alpha-difluoromethylornithine of T-lymphocyte and antibody-mediated cytotoxic responses to mouse tumor allografts.

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.

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)

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.