Growth regulatory effects of cyclic AMP and polyamine depletion are dissociable in cultured mouse lymphoma cells.

Treatment of mouse lymphoma S49 cells with D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, depleted cellular polyamine levels and stopped cell growth. The cells were arrested predominantly in G1. Thus, polyamine depletion may lead to a regulatory growth arrest in S49 cells. We tested two hypotheses regarding the relationship of growth arrest mediated by polyamine limitation to that mediated by cyclic AMP (cAMP). The hypothesis that cAMP-induced arrest results from polyamine depletion is not tenable, because the arrest could not be reversed by addition of exogenous polyamines, and because cellular polyamine levels do not drop in dibuturyl cyclic AMP (Bt2cAMP)-arrested cells. The hypothesis that polyamine-mediated growth arrest is effected via modulation of cAMP levels or cAMP-dependent protein kinase activity was also shown to be incorrect, because a S49 variant deficient in cAMP-dependent protein kinase was arrested by DFMO. The activities of the polyamine-synthesizing enzymes ornithine decarboxylase (ODC) and S-adenosyl methionine decarboxylase (SAMD) are both reduced in Bt2cAMP-treated cells to about 10% of that in control populations, as shown previously. DFMO diminishes ODC activity and augments SAMD activity in both untreated and Bt2cAMP-treated cells, leading to polyamine depletion in both cases.

Ornithine decarboxylase is important in intestinal mucosal maturation and recovery from injury in rats.

A transient increase in ornithine decarboxylase activity and polyamine biosynthesis occurs in the intestinal mucosa of the newborn rat in the third week after birth. During this period, there is a rapid conversion of the mucosa from a fetal to a mature adult status. A similar increase in ornithine decarboxylase activity also accompanies the rapid recovery of the mucosa 1 week after an injury is induced by chemotherapy in adult rats. In vivo, alpha-difluoromethyl ornithine, a highly selective, enzyme-activated, irreversible inhibitor, suppresses these increases in mucosal ornithine decarboxylase and delays both intestinal mucosal maturation and recovery from injury. Thus increased ornithine decarboxylase activity, with the resultant increase in polyamine content, may play an essential role in intestinal mucosal maturation and regeneration in the rat.

alpha-Difluoromethylornithine-induced polyamine depletion of 9L tumor cells modifies drug-induced DNA cross-link formation.

Depletion of intracellular levels of polyamines, which are believed to have a role in the intranuclear stabilization of DNA, alters the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea and cis-diamminedichloroplatinum II in 9L rat brain tumor cells. Alkaline elution techniques were used to show that polyamine depletion alters the number of DNA cross-links formed by these cytotoxic agents.

Polyamine depletion influences drug-induced chromosomal damage.

Polyamines have been implicated in the intracellular stabilization of DNA. Depletion of intracellular polyamines influences the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea and cis-diamminedichloroplatinum II. By means of the sister chromatid exchange assay, it was found that intracellular polyamine depletion can also alter the induction of chromosomal damage by these cytotoxic agents.

Depletion of intracellular polyamines may alter DNA conformation in 9L rat brain tumor cells.

Depletion of polyamines in 9L rat brain tumor cells by treatment with alpha-difluoromethylornithine dramatically altered DNA conformation as measured by viscoelastometry. The reduction of intracellular putrescine and spermidine concentrations to less than 5 percent of their concentrations in control cells decreased the sensitivity of 9L cell DNA to x-irradiation and increased the maximum viscoelastic retardation time of the DNA. Both of these phenomena were reversed by addition of exogenous putrescine.

Antizyme and antizyme inhibitor activities influence cellular responses to polyamine analogs.

Close structural analogs of spermidine and spermine, polyamine mimetics, are potential chemotheraputic agents as they depress cellular polyamines required for tumor growth. Specific mimetic analogs stimulate synthesis of the regulatory protein antizyme (AZ), which not only inactivates the initial enzyme in polyamine biosynthesis but also inhibits cellular uptake of polyamines. The role of AZ induction in influencing cellular uptake of representative analogs was investigated using three analogs produced by Cellgate Inc., CGC-11047, CGC-11102, and CGC-11144, which exhibit markedly distinct AZ-inducing potential. An inverse correlation was noted between the AZ-inducing activity of a compound and the steady-state levels accumulated in cells. As some tumor cells over express AZI as a means of enhancing the polyamines required for aggressive growth, analog sensitivity was examined in transgenic CHO cells expressing exogenous antizyme inhibitor protein (AZI). Although AZI over expression increased cell sensitivity to analogs, the degree of this affect varied with the analog used.

Biochemical markers of central nervous system tumors measured in cerebrospinal fluid and their potential use in diagnosis and patient management: a review.

The concept of tumor markers was reviewed, and the potential uses of markers of central nervous system (CNS) tumors and methods for their evaluation were discussed. Markers examined included lactate dehydrogenase, aspartate aminotransferase, fructose-bisphosphate aldolase, the polyamines, desmosterol, and several other enzymatic, nonenzymatic, and immunologic markers. Data collated from the clinical studies surveyed showed isocitrate dehydrogenase, desmosterol, and the polyamines to have the greatest potential utility in the diagnosis of CNS tumors.

Effects of DL-alpha-methylornithine on proliferation and polyamine content of 9L rat brain tumor cells.

Treatment of 9L rat brain tumor cells with 10 mM DL-alpha-methylornithine (alpha MO) resulted in cytostasis when cells were plated in monolayer culture at an initial cell density of 5 x 10(5)/flask but not 1 x 10(6). Fifty mM caused cytostasis at both initial densities but more effectively at the lower one. Cytocidal effects measured by a colony-forming efficiency assay were observed at 100 mM but not at 75 mM or less. Both concentrations at both initial cell densities depleted intracellular putrescine content to less than 5% of control by 12 hr and spermidine content to less than 20% of control by 48 hr posttreatment. Intracellular spermine content increased between 1.5- and 2-fold with either concentration of alpha MO and at both densities. Flow cytometry revealed no differences in cell cycle distribution between controls and cells treated with 10 mM alpha MO. The cytostatic effect of 10 mM alpha MO on 9L cultures of lower initial density appeared to be a specific result of polyamine depletion since it was reversible by exogenous putrescine added 24 hr after treatment. The effect of 50 mM alpha MO was not reversible by exogenous putrescine or pyridoxal added simultaneously or 24 hr after treatment. Treatment of 9L cells with 50 mM DL-or L-ornithine caused growth inhibition equal to that produced by 50 mM alpha MO. The effect of 50 mM alpha MO is thus not attributable to polyamine depletion.

Effects of dicyclohexylamine sulfate, a spermidine synthase inhibitor, in 9L rat brain tumor cells.

Growth characteristics, polyamine levels, and distribution of cells in the cell cycle were determined for 9L rat brain tumor cells treated for various periods with 1 mM dicyclohexylamine sulfate (DCHA). Continuous treatment of cells with DCHA caused growth inhibition at 2 days of treatment. After 2 days of treatment the growth rate of cells increased to approximately the same rate as control cells, even though treatment was continuous. Levels of spermidine were depleted to less than 10% of control levels, spermine levels were essentially unchanged, and putrescine levels were elevated to more than 350% of control levels after 9L cells were treated with DCHA for 2 days. In contrast to results found for the polyamine biosynthesis inhibitor alpha-difluoromethylornithine, treatment of 9L cells with DCHA did not potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea. To mimic the effects on polyamine levels caused by treatment with DCHA, 9L cells were treated with 5 mM putrescine alone or with 5 mM putrescine and 1 mM DCHA after treatment with 1 mM alpha-difluoromethylornithine. Results of these experiments suggest that treatment with DCHA alone does not potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea because elevated levels of putrescine caused by treatment counteract the effects of decreased spermidine levels.

Modification of uptake and antiproliferative effect of methylglyoxal bis(guanylhydrazone) by treatment with alpha-difluoromethylornithine in rodent cell lines with different sensitivities to methylglyoxal bis(guanylhydrazone).

Uptake characteristics and growth-inhibitory effects of methylglyoxal bis(guanylhydrazone) (MGBG), a competitive inhibitor of S-adenosylmethionine decarboxylase, were investigated in 9L rat brain tumor cells and in V79 hamster lung cells. Proliferation of 9L cells was only slightly inhibited by treatment with 40 microM MGBG alone, but when used in combination with 0.5 mM alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, proliferation was much more effectively inhibited. The intracellular concentration of MGBG was approximately 2-fold higher 4 days after cells were treated with both DFMO and MGBG, either simultaneously or when MGBG was added after a 48-hr DFMO pretreatment, than that in cells treated with MGBG alone. Polyamine levels in DFMO- and MGBG-treated cells correlated with the antiproliferative effects of the drugs. Used either alone or in combination with 1 mM DFMO, 0.5 microM MGBG inhibited the growth of and eventually killed V79 cells. Simultaneous or sequential treatment with DFMO and MGBG increased intracellular concentrations of MGBG at 4 days by 2- and 3-fold, respectively, compared to treatment with MGBG alone. Intracellular polyamine levels did not correlate with the antiproliferative effect of the two drugs in V79 cells. In both cell lines, polyamines and MGBG share a common transport system. The net transport of polyamines and MGBG was more temperature dependent and up to 10-fold more active in V79 cells than in 9L cells. The Km and Vmax values for spermidine and MGBG measured 10 sec after addition (initial permeation) were not affected by DFMO pretreatment in either cell line. However, 1 hr after administration, the Vmax values for spermidine and MGBG uptake were doubled in V79 cells pretreated for 48 hr with DFMO; no significant change occurred in 9L cells. Mitochondrial function, assessed by pyruvate oxidation, was substantially impaired by MGBG in V79 cells but not in 9L cells when the intracellular concentrations of MGBG were equal in each cell line. Pretreatment with DFMO did not increase MGBG-induced inhibition of pyruvate oxidation in V79 cells. These results show that, compared with V79 cells, the decreased sensitivity of 9L cells to MGBG may be related to decreased intracellular MGBG accumulation but not to cellular permeation such as carrier transport. Results of measurements of both polyamine levels and mitochondrial function indicate that V79 cells may be more susceptible to nonpolyamine-dependent effects of MGBG than are 9L cells.

Influence of polyamine depletion caused by alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on alkylation- and carbamoylation-induced cytotoxicity in 9L rat brain tumor cells in vitro.

Using a colony-forming efficiency assay, we studied the effect of polyamine depletion on the cytotoxicity of four nitrosoureas with different capacities to alkylate and/or carbamoylate biomolecules. 9L rat brain tumor cells were treated with 10 mM alpha-difluoromethylornithine for 48 hr before a 1-hr treatment with nitrosoureas. The cytotoxicity of 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose, which alkylates and subsequently cross-links DNA but does not carbamoylate, was significantly increased by depletion of intracellular polyamines; the dose enhancement ratio of 1.3 is identical to that found for 1,3-bis(2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-trans-4-methylcyclohexyl-1-nitrosourea in previous studies. Addition of exogenous putrescine to polyamine-depleted 9L cells 24 hr before treatment prevented this phenomenon. In contrast, the cytotoxicity of 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea, which carbamoylates only, was significantly decreased in polyamine-depleted cells. This compound alone reduced intracellular polyamine levels. Polyamine depletion did not affect the cytotoxicity of the monoalkylating nitrosoureas N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea. Thus, polyamine depletion apparently potentiates the cytotoxicity only of chloroethylnitrosoureas that alkylate and cross-link.

Effect of difluoromethylornithine, an inhibitor of polyamine biosynthesis, on the topoisomerase II-mediated DNA scission produced by 4'-(9-acridinylamino)methanesulfon-m-anisidide in L1210 murine leukemia cells.

Treatment of mouse leukemia L1210 cells with the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) increased the magnitude of the DNA scission produced by the DNA intercalator 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). This enhanced DNA scission was protein concealed and protein associated, as was the m-AMSA-induced scission in cells unexposed to DFMO. The effect of DFMO required more than 6 hr to develop and was greater at 48 hr than at 24 hr of exposure to DFMO. Exogenously added putrescine partially reversed the effects of DFMO, while exerting no effect on m-AMSA-induced DNA scission in cells unexposed to DFMO. The cellular uptake of [14C]-m-AMSA was the same in DFMO-treated or untreated cells. The DNA scission and DNA-protein cross-linking produced by m-AMSA appear to represent the stabilization of an intermediate in the normal cycle of topoisomerase II function (Nelson, E.M., Tewey, K.M., and Liu, L.F., Proc. Natl. Acad. Sci. USA, 81: 1361-1365, 1984). Since polyamine depletion appears to affect the magnitude of this effect in cells, and since polyamines can alter topoisomerase II function in vitro, polyamines may be involved in topoisomerase function in vivo either directly or through secondary effects, such as alterations of the conformation of chromatin, the intracellular site at which topoisomerase acts.

Decreased cytotoxicity of cis-diamminedichloroplatinum(II) by alpha-difluoromethylornithine depletion of polyamines in 9L rat brain tumor cells in vitro.

Before treatment with the cytotoxic drug cis-dichlorodiammineplatinum(II) (cis-platinum), 9L rat brain gliosarcoma cells grown in vitro were depleted of intracellular polyamines by alpha-difluoromethylornithine (DFMO; 10 mM, 48 hr), an enzyme-activated, irreversible inhibitor of the polyamine-synthetic enzyme ornithine decarboxylase. In contrast to studies that showed that the cytotoxicity of 1,3-bis (2-chloroethyl)-1-nitrosourea as measured by colony-forming efficiency is enhanced by DFMO depletion of intracellular polyamines, the cytotoxicity of cis-platinum was significantly decreased in polyamine-depleted 9L cells. At 10, 1, and 0.1% survival levels, the dose modification factors were 2.0 to 2.1. Addition of exogenous putrescine to polyamine-depleted 9L cells 24 hr before treatment with cis-platinum partially reversed this phenomenon. When 9L cells were treated either with DFMO and cis-platinum or with DFMO, putrescine, and cis-platinum for 1 hr, a time period that is too short for DFMO to affect intracellular polyamine levels, the cytotoxicity of cis-platinum was decreased, but to a significantly lesser extent than by the 48-hr DFMO pretreatment. Putrescine alone did not alter the cytotoxic effect of cis-platinum when administered either 24 or 1 hr before treatment. DFMO appears to affect cis-platinum cytotoxicity by two different mechanisms, the first mediated through polyamine depletion and the second through a direct interaction with cis-platinum.

Changes in the glutathione content of rat 9L cells induced by treatment with the ornithine decarboxylase inhibitor alpha-difluoromethylornithine.

Treatment of 9L cells with the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) depletes cells of putrescine and spermidine and sensitizes cells to the cytotoxic effects of the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea. Because depletion of intracellular levels of the sulfhydryl compound glutathione also increases the cytotoxicity of alkylating agents, the effect of DFMO on intracellular glutathione content was investigated to determine whether DFMO-induced sensitization could be explained by a sulfhydryl-related mechanism. Treatment of 9L cells with 1 mM DFMO caused no change in the glutathione concentration within 48 h; sensitization of cells to 1,3-bis(2-chloroethyl)-1-nitrosourea is generally studied after 48 h of treatment with DFMO. Incubation of cells with DFMO for longer periods caused an increase in glutathione that is related to the depletion of polyamines and not to a decrease in growth rate or a cell cycle effect. Increased glutathione content is not due to a decrease in glutathione catabolism, but rather to an apparent increase in the rate of synthesis of the tripeptide.

Decreased cytotoxicity of aziridinylbenzoquinone caused by polyamine depletion in 9L rat brain tumor cells in vitro.

The in vitro cytotoxicity of aziridinylbenzoquinone (AZQ) used either alone or after induced intracellular polyamine depletion in 9L rat brain tumor cells was studied using a colony-forming efficiency assay. Used alone, AZQ was cytotoxic to 9L cells; however, depletion of intracellular putrescine and spermidine levels by treatment with 1 mM alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, for 72 hr decreased significantly the cytotoxicity of AZQ. Dose modification factors were 1.9 and 1.8 at 10 and 1% survival levels, respectively. Decreased cytotoxicity could be almost completely prevented by addition of putrescine to polyamine-depleted cells 24 hr before AZQ treatment. Although AZQ alone was cytotoxic against 9L cells, metabolic activation by the S-9 rat liver microsomal fraction increased greatly the observed cytotoxicity. However, even with microsomal activation, pretreatment of cells with 1 mM alpha-difluoromethylornithine for 48 hr produced a significant decrease in AZQ cytotoxicity; dose modification factors were 2.4 and 2.2 at 10 and 1% survival levels, respectively. Addition of putrescine to polyamine-depleted cells 24 hr before AZQ treatment prevented the decrease in cytotoxicity. Pretreatment of 9L cells for 48 hr with 40 microM methylglyoxal bis(guanylhydrazone), a polyamine biosynthesis inhibitor that competitively inhibits S-adenosylmethionine decarboxylase, caused a decrease in the cytotoxicity of AZQ administered without microsomal activation. The dose modification factor was 1.6 at both 10 and 1% survival levels.

Time dependence of the potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity caused by alpha-difluoromethylornithine-induced polyamine depletion in 9L rat brain tumor cells.

Treatment of 9L rat brain tumor cells with 1.0 mM alpha-difluoromethylornithine (DFMO) produced a time-dependent depletion of cellular putrescine and spermidine. An increase in the potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) cytotoxicity, measured with a colony-forming efficiency assay, followed the time course of polyamine depletion, reaching its maximum at 48 hr, the time at which maximum polyamine depletion was achieved. Treatment with DFMO at concentrations as low as 0.05 mM for 48 hr effectively depleted putrescine and spermidine and potentiated BCNU cytotoxicity. Treatment for 96 hr with 0.01 mM DFMO produced a partial decrease in putrescine and spermidine levels and a moderate potentiation of BCNU cytotoxicity. The amount of polyamine depletion in 9L cells treated with 0.05, 0.1, and 0.5 mM DFMO was identical at both 48 and 96 hr, but potentiation of BCNU cytotoxicity was greater at 96 hr than at 48 hr. When 9L cells were treated for 48 hr with 1 mM DFMO and DFMO was then removed from the cultures, polyamine levels did not reach control levels by 96 hr after change of medium. The potentiation of BCNU cytotoxicity during this 96-hr period correlated with the extent of polyamine depletion. When 100 microM putrescine was added to the culture medium after DFMO pretreatment (1 mM), polyamine levels approached those of control cells by 24 hr, and the amount of potentiation of DFMO cytotoxicity decreased. These results show that potentiation of BCNU cytotoxicity correlates closely with the amount of DFMO-induced polyamine depletion in 9L cells.

Sensitization of 9L rat brain gliosarcoma cells to 1,3-bis(2-chloroethyl)-1-nitrosourea by alpha-difluoromethylornithine, an ornithine decarboxylase inhibitor.

alpha-Difluoromethylornithine, a known inhibitor of polyamine biosynthesis, significantly enhanced the cytotoxic effect of 1,3-bis(2-chloroethyl)-1-nitrosourea, a cell cycle-nonspecific agent, in 9L rat brain gliosarcoma cells in vitro. Administered as a single agent, alpha-difluoromethylornithine was not cytotoxic to 9L cells and, compared to untreated control cells, caused no perturbation of cell cycle kinetics. alpha-Difluoromethylornithine-induced depletion of intracellular polyamine levels appears to have caused the observed sensitization of 9L cells to 1,3-bis(2-chloroethyl)-1-nitrosourea. Restoration of intracellular polyamine levels by the addition of exogenous putrescine to treated 9L cells reversed this phenomenon.

Prediction of human tumor cell chemosensitivity using the sister chromatid exchange assay.

The sister chromatid exchange (SCE) assay has been used to predict the chemosensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea of various neoplastic cell subpopulations in eight cell lines derived from human brain tumors. Because the SCE assay is based on analysis of individual cells, data obtained can be plotted as frequency histograms of SCEs per chromosome, and the range of chemosensitivities of cell subpopulations can be identified easily. Results suggest that the SCE assay has predictive value as a clinical assay for drugs for which a strong correlation between cell kill and induction of SCEs has been established.

alpha-Difluoromethylornithine alters calcium signaling in platelet-derived growth factor-stimulated A172 brain tumor cells in culture.

alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, inhibits the growth of brain tumor cell lines and is undergoing clinical trials as a treatment for brain tumors. Platelet-derived growth factor (PDGF) is thought to regulate the growth and development of precursors of both normal and neoplastic astrocytic cells; calcium signaling is thought to play a role in the transduction of PDGF signals. Using laser fluorescence image cytometry, flow cytometry, and spectrofluorometry, we studied the effect of DFMO on the calcium signals induced by PDGF in A172 human glioblastoma cells. Four days of treatment with 5 mM DFMO substantially shortened PDGF-induced calcium signals. The effect was reversed more than 10 h but less than 24 h after putrescine treatment, even though polyamines were repleted 4 h after putrescine and spermidine were added. DFMO did not substantially affect intracellular calcium release or the timing of the opening and closing of plasma membrane calcium channels. These findings support the notion that calcium signaling may be a target for inhibitors of polyamine metabolism.

Reduction in cis-diamminedichloroplatinum(II)-induced cytotoxicity, sister chromatid exchange, and DNA interstrand cross-links in 9L cells treated with the polyamine biosynthesis inhibitor (2R,5R)-6-heptyne-2,5-diamine.

The effects of depletion of intracellular levels of the polyamines putrescine and spermidine on cis-diamminedichloroplatinum(II)-induced cytotoxicity, sister chromatid exchanges, DNA interstrand cross-linking, and intracellular glutathione levels were studied in 9L rat brain tumor cells pretreated with the ornithine decarboxylase inhibitor (2R,5R)-6-heptyne-2,5-diamine (R,R-MAP). Pretreatment of 9L cells with R,R-MAP for 48 h decreased cis-diamminedichloroplatinum(II) cytotoxicity with an average dose reduction ratio of 0.55 at both the 5 and 10% survival levels; addition of putrescine partially prevented this effect. The number of sister chromatid exchanges and DNA interstrand cross-links was also reduced (31 and 38%, respectively). Within 24 h of treatment with R,R-MAP, intracellular glutathione levels began to increase relative to untreated control cells and were significantly elevated in R,R-MAP-treated cells 48-72 h after addition of drug. We discuss several mechanisms by which polyamine depletion could reduce cis-diamminedichloroplatinum(II) toxicity.