MGBG Treatment Research Articles

Exogenous melatonin alleviated chilling injury in harvested plum fruit via affecting the levels of polyamines conjugated to plasma membrane

Polyamines (PAs) are related closely with many abiotic stresses. However, conjugated PA functions are not clear in alleviating chilling injury in fruit. The contents of free PAs in plum flesh cell and conjugated PAs in plasma membrane (PM), the activities of S-adenosylmethionine decarboxylase (SAMDC) and transglutaminase (TGase), and the degree of chilling injury were investigated in harvested plum (Prunus salicina L.) fruit pre-treated with exogenous melatonin. Chilling treatment resulted in the injury of plasma membrane (PM), as judged by the increased PM permeability, chilling injury index and malondialdehyde (MDA) content, and the decreased sulfhydryl group contents. Exogenous melatonin alleviated chilling injury, in parallel with the increased levels of free spermidine (Spd) and spermine (Spm) in cell, conjugated non-covalently (CNC) Spd and Spm, and conjugated covalently (CC) Put and Spd in PM, resulting from the increased activities of SAMDC and TGase. The results of the additional experiments with two inhibitors, methylglyoxyl-bis (guanylhydrazone) (MGBG) and phenanthrolin, which inhibit the activities of SAMDC and TGase, respectively, were complementary evidences for our research. MGBG and TGase treatment decreased the level of CNC PAs (Spd and Spm) and CC PAs (Put and Spd), respectively, coupled with aggravating the chilling injury. From these above mentioned-results, it could be suggested that exogenous melatonin alleviated chilling injury by elevating the levels of CNC Spd, CNC Spm, CC Put and CC Spd in PM.

Enhanced salt-induced antioxidative responses involve a contribution of polyamine biosynthesis in grapevine plants

The possible involvement of polyamines in the salt stress adaptation was investigated in grapevine (Vitis vinifera L.) plantlets focusing on photosynthesis and oxidative metabolism. Salt stress resulted in the deterioration of plant growth and photosynthesis, and treatment of plantlets with methylglyoxal-bis(guanylhydrazone) (MGBG), a S-adenosylmethionine decarboxylase (SAMDC) inhibitor, enhanced the salt stress effect. A decrease in PSII quantum yield (Fv/Fm), effective PSII quantum yield (Y(II)) and coefficient of photochemical quenching (qP) as well as increases in non-photochemical quenching (NPQ) and its coefficient (qN) was observed by these treatments. Salt and/or MGBG treatments also triggered an increase in lipid peroxidation and reactive oxygen species (ROS) accumulation as well as an increase of superoxide dismutase (SOD) and peroxidase (POX) activities, but not ascorbate peroxidase (APX) activity. Salt stress also resulted in an accumulation of oxidized ascorbate (DHA) and a decrease in reduced glutathione. MGBG alone or in combination with salt stress increased monodehydroascorbate reductase (MDHAR), SOD and POX activities and surprisingly no accumulation of DHA was noticed following treatment with MGBG. These salt-induced responses correlated with the maintaining of high level of free and conjugated spermidine and spermine, whereas a reduction of agmatine and putrescine levels was observed, which seemed to be amplified by the MGBG treatment. These results suggest that maintaining polyamine biosynthesis through the enhanced SAMDC activity in grapevine leaf tissues under salt stress conditions could contribute to the enhanced ROS scavenging activity and a protection of photosynthetic apparatus from oxidative damages.

Effects of various chemical agents and early ethylene production on floral senescence of Hibiscus syriacus L.

To understand the factors that induce floral senescence in Hibiscus syriacus L., we have investigated the effects of various chemical agents on flower senescence at two different flowering stages, before and after full bloom, as well as the relationship between flower longevity and endogenous ethylene production before full bloom. Treatments with ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), and ethephon enhanced floral senescence, while aminoethoxyvinylglycine (AVG) promoted flower longevity regardless of treatment timing. Although ethanol slightly extended flower longevity, abscisic acid (ABA), nitric oxide, boric acid and sucrose, which have been reported to affect flower longevity or senescence, had no effect on H. syriacus floral senescence. The polyamine spermine (SPM), methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of SPM biosynthesis, and cycloheximide (CHI) accelerated flower senescence when applied before full bloom, but had no effect when applied after full bloom. SPM, MGBG and CHI treatments resulted in enhanced ethylene production during flower opening, and the promotion of flower senescence is mediated by ethylene production prior to full bloom. Furthermore, endogenous ethylene, spontaneously produced before blooming, was closely associated with floral senescence. These results suggest that ethylene production during flower opening plays a key role in determining the timing of Hibiscus flower senescence.

Methylglyoxal-bis(guanylhydrazone), a polyamine analogue, sensitized γ-radiation-induced cell death in HL-60 leukemia cells: Sensitizing effect of MGBG on γ-radiation-induced cell death

Methylglyoxal-bis(guanylhydrazone) (MGBG), a polyamine analogue, has been known to inhibit the biosynthesis of polyamines, which are important in cell proliferation. We showed that MGBG treatment significantly affected γ-radiation-induced cell cycle transition (G 1/G 0 → S → G 2/M) and thus γ-radiation-induced cell death. As determined by micronuclei and comet assay, we showed that it sensitized the cytotoxic effect induced by γ-radiation. One of the reasons is that polyamine depletion by MGBG treatment did not effectively protect against the chemical ( OH) or physical damage to DNA caused by γ-radiation. Through in vitro experiment, we confirmed that DNA strand breaks induced by γ-radiation was prevented more effectively in the presence of polyamines (spermine and spermidine) than in the absence of polyamines. MGBG also blocks the cell cycle transition caused by γ-radiation (G 2 arrest), which helps protect cells by allowing time for DNA repair before entry into mitosis or apoptosis, via the down regulation of cyclin D1, which mediates the transition from G 1 to S phase of cell cycle, and ataxia telangiectasia mutated, which is involved in the DNA sensing, repair and cell cycle check point. Therefore, the abrogation of G 2 arrest sensitizes cells to the effect of γ-radiation. As a result, γ-radiation-induced cell death increased by about 2.5–3.0-fold in cells treated with MGBG. However, exogenous spermidine supplement partially relieved this γ-radiation-induced cytotoxicity and cell death. These findings suggest a potentially therapeutic strategy for increasing the cytotoxic efficacy of γ-radiation.

Increased Polyamines Conjugated to Tonoplast Vesicles Correlate with Maintenance of the H+-ATPase and H+-PPase Activities and Enhanced Osmotic Stress Tolerance in Wheat

The effects of osmotic stress on H+-ATPase and H+-PPase activities and the levels of covalently conjugated polyamines (CC-PAs) and noncovalently conjugated polyamines (NCC-PAs) were investigated using tonoplast vesicles isolated from the roots of wheat (Triticum aestivum L.) seedlings differing in drought-tolerance. The results showed that after polyethylene glycol (PEG) 6,000 (−0.55MPa) treatment for 7 days, seedling leaf relative water content (LRWC), relative dry weight increase rate (RDWIR) and root H+-ATPase and H+-PPase activities from the drought-sensitive cultivar Yangmai No. 9 decreased more markedly than those from the drought-tolerant cultivar Yumai No. 18. At the same time, the increase of the NCC-spermidine (NCC-Spd) and CC-putrescine (CC-Put) levels in root tonoplast vesicles from Yumai No. 18 was more obvious than that from Yangmai No. 9. Exogenous Spd treatment alleviated osmotic stress injury to Yangmai No. 9 seedlings, coupled with marked increases of tonoplast NCC-Spd levels and H+-ATPase and H+-PPase activities. Treatments with methylglyoxyl bis (guanyl hydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), and phenanthrolin, an inhibitor of transglutaminase (TGase), significantly inhibited the osmotically induced increases of NCC-Spd and CC-Put levels, respectively, in root tonoplast vesicles from Yumai No. 18 seedlings. Both MGBG and phenanthrolin treatments markedly promoted osmotically induced decreases of tonoplast H+-ATPase and H+-PPase activities and osmotic stress tolerance of seedlings of this cultivar. These results suggest that the NCC-Spd and CC-Put present in tonoplast vesicles isolated from wheat seedling roots might enhance the adaptation of seedlings to osmotic stress via maintenance of tonoplast H+-ATPase and H+-PPase activities.

The early increase of S-adenosylmethionine decarboxylase activity is essential for the normal germination and tube growth in tomato ( Lycopersicon esculentum Mill.) pollen

The effect of polyamine biosynthesis inhibitors on pollen germination and tube growth and putrescine (Put), spermidine (Spd) and spermine (Spm) levels in germinating tomato ( Lycopersicon esculentum Mill.) pollen was investigated. Pollen germination was initiated 20 min after incubation at 25 °C and completed within 2 h. The activity of arginine decarboxylase (ADC), an enzyme catalyzing Put synthesis, exhibited a marked increase during the initial 2 h and remained high thereafter. The activity of S-adenosylmethionine decarboxylase (SAMDC), an essential enzyme in the synthesis of Spd and Spm, increased transiently during the first hour of incubation. Consequently, the germinating pollen showed elevated ratios of Spd+Spm to Put during the first hour in all of the free, conjugated and bound forms. The addition to the germination medium of either methylglyoxal-bis(guanylhydrazone) (MGBG) or cyclohexylamine, inhibitors of SAMDC and spermidine synthase, respectively, caused a marked decrease of Spd and Spm contents, together with ratios of Spd+Spm to Put, and resulted in inhibition of pollen germination and tube growth. Simultaneous addition of Spd or Spm reversed the adverse effect of MGBG. However, Put failed to reverse the inhibitory effect of d-arginine, a competitive inhibitor of ADC. MGBG treatment 2 h after incubation was still inhibitory to tube growth, which was partially reversed by Spd. The results suggest that the increased activity of SAMDC and the resultant elevation of Spd and Spm contents during the early pollen germination phase are essential for the occurrence of normal germination and tube growth in tomato pollen.

Effects of polyethylene glycol and methylglyoxal bis(guanylhydrazone) on endogenous polyamine levels and somatic embryo maturation in white spruce ( Picea glauca)

In maturation cultures, non-plasmolysing moisture stress induced by polyethylene glycol (PEG) 4000 reduced white spruce embryogenic tissue proliferation and stimulated somatic embryo development. Levels of endogenous putrescine (Put), spermidine (Spd) and spermine (Spm) in the cultures were quantified by high performance liquid chromatography (HPLC). In the cultures, free and conjugated Spd were predominant. Compared with no PEG treatment, levels of free polyamines (PAs) were lower in PEG treatments at days 5 and 10 in the cultures but increased to higher levels thereafter. Although the levels of conjugated polyamine increased early during somatic embryo maturation, the changes of conjugated polyamine levels were similar to those of free PAs. Throughout the maturation process, levels of bound Put and bound Spd in PEG treatments were lower than those in the controls. The level of bound Spm was higher in PEG treatments at day 5, it declined then to a lower level thereafter. Methylglyoxal bis(guanylhydrazone) (MGBG) decreased the levels of endogenous free PAs and reduced tissue growth in the cultures with or without PEG treatment. However MGBG did not inhibit embryo development in these cultures. In 0.2 mM MGBG treatments, although the number of cotyledonary embryos increased on the basis of fresh weight of tissue, the number per dish decreased. In PEG treatments, the maximum concentration which tissue could tolerate was lower than that in the cultures without PEG. Thus, reducing tissue proliferation by PEG may be due to inhibition of endogenous PA levels. However, the stimulation of embryo development by PEG may not solely function through the change of endogenous PA levels as MGBG did not stimulate or inhibit embryo development.

The effects of proline, thioproline and methylglyoxal-bis-(guanylhydrazone) on shoot regeneration frequencies from stem explants of B. napus

Internode segments from aseptic shoot cultures are the most prolific explants for the regeneration of Brassica shoots in vitro. These explants also have the advantage of not being subject to the genotypic variations in regeneration response observed in hypocotyl and cotyledon explants. Despite reports of 80-100% shoot regeneration from stem explants, observed frequencies are typically 50-60%. Three media additives, proline, thioproline and methylglyoxal-bis-(guanylhydrazone) (MGBG), were tested for their efficacy in promoting shoot regeneration from stem explants of two B. napus varieties, Westar and Cobra. The effects of proline and thioproline on both varieties were neutral or deleterious. In Cobra the MGBG treatments caused a uniform reduction in explant regeneration. However, at low concentrations (0.35μM) MGBG resulted in a 50% increase, to 92%, in regeneration from Westar. The potential of MGBG in promoting explant regeneration in B. napus is discussed in the light of its interaction with the explant genotype.

Characterization of a COS cell line deficient in polyamine transport

In the present study, we describe the isolation and characterization of a COS cell line deficient in polyamine uptake that may provide an important tool for the molecular cloning of polymaine transporter(s). The cells were selected by isolation for resistance against the cytotoxic agent, methylglyoxal bis(guanylhydrazone) (MGBG), which is entering the cells using the same transport system as the polyamines. The isolated cell line was capable of growing in the presence of 100 μM MGBG, which totally inhibited the growth of the wild-type cells. The transport of putrescine and spermidine was markedly decreased in the COS-MGBG r cells. The decrease in putrescine transport was mainly a result of a 14-fold decrease in V max , wheras the reduced spermidine uptake was due to a 3–4-fold decrease in V max as well a 12-fold increase in K m , indicating the existence of at least two separate transport systems. No major difference in polyamine content was seen between the parental and the COS-MGBG r cells when grown without MGBG. In the presence of MGBG, both cell lines exhibited an increase in putrescine content. Treatment with MGBG also resulted in a decrease in spermidine and spermine contents in the wild-type cells. In the COS-MGBG r cells, on the other hand, there were no statistically significant effects on the spermidine and spermine contents by MGBG treatment. In the wild-type cells, depletion of polyamines, e.g., by treatment with the ornithine decarboxylase inhibitor 2-difluoromethylornithine (DFMO), stimulated the uptake of polyamines (3–7-fold), whereas in the COS-MGBG r cells the effect of DFMO treatment on polyamine transport was only minor. In contrast to the growth-medium of the wild-type cells, large amounts of polyamines accumulated in the medium of the COS-MGBG r cells, presumably indicating that COS cells normally excrete polyamines and then salvage them using the polyamine transport system.

Effects of Polyamines on the Ice-nucleating Activity ofErwinia uredovoraKUIN-3

The effects of polyamines on the ice-nucleating activity of an ice-nucleating bacterium, Erwinia uredovora KUIN-3, were investigated. The activity in the outer membrane derived from this bacterium was increased by the addition of spermidine (10mM). Also, the activity was stabilized in alkaline pH buffer with the addition of polyamine. Further, the addition of methylglyoxal bis(guanylhydrazone) which inhibited the activity of S-adenosylmethionine decarboxylase, inhibited the ice-nucleating activity of the cells of this bacterium. The amount of polyamine in this bacterium was examined using HPLC. The components of the polyamines were three species, putrescine, cadaverine, and spermidine. The amounts of polyamines in the cells and outer membranes after MGBG treatment decreased. The results suggested that polyamine was the most important component of the ice-nucleating material in this bacterium.

Synergistic antileukemic effect of two polyamine synthesis inhibitors. Host survival and cell-cycle kinetic analysis.

alpha-Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, was used alone and in combination with multiple doses of methylglyoxal-bis(guanylhydrazone) (MGBG) to treat mice with systemic L1210 leukemia. Used as a single agent (administered p.o. as a 3% solution in tap water), DFMO exerted a weak therapeutic effect against this tumor. The therapeutic effect of MGBG (administered i.p. at 50 mg/kg/day) was only slightly better. However, 1-3 days of pretreatment with DFMO strongly potentiated the effect of MGBG treatment. Thus, mice treated with the combination exhibited an increase in life span of up to 138%. The prolonged survival of leukemic mice treated with a combination of DFMO and MGBG was associated with inhibition of polyamine synthesis and a marked decrease in the spermidine and spermine content of the tumor cells as compared to untreated controls. As a consequence, there was a continuous decrease in the S- and G2-phase fractions with a concomitant increase in G1. Used singly, DFMO and MGBG had no significant effect on the cell-cycle distribution. The effects of the combination of DFMO and MGBG on the cell-cycle distribution are consistent with the contention that polyamine deficiency primarily interferes with initiation of DNA synthesis. However, the possibility that selective S-phase kill partly contributes to this change in cell-cycle distribution cannot be excluded.

Regulation of polyamine biosynthesis in tobacco. Effects of inhibitors and exogenous polyamines on arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase.

Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J. (1983) Nature 305, 623-625). MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase. Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical. Precursor incorporation experiments using [3H] methionine and [35S]methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation. The half-life of the enzyme activity was increased when MGBG was present in the growth medium. In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase. The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG. Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine. Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.

Effects of methylglyoxal-bis (guanylhydrazone) and abscisic acid on polyamine metabolism in embryonectomized barley seeds

Incorporation of L-[U-14C] aiginine or L-[U-14C] ornithine into putrescine (Put), spermidine (Spd) and spermine (Spm) in embryonectomized barley seeds(Hordeum vulgare L. cv. Himalaya) was studied following imbibition with methylglyoxal-bis (guanylhydrazone)(MGBG) and abscisic acid (ABA). Both radiolabeled amino acids were incorporated into the amines as a result of: active polyamine biosynthesis in the seed during imbibition. In the aleurone layer, the Spd and Spn existed mainly in the free form (acid soluble). However about 50% of Put was recovered in conjugated form (s) (acid insoluble). Imbibition with 5 and 10μM ABA for 3 days increased the accumulation of the tree form of 14C-Put. probably as a result of inhibition (70%) of 14C-Spd accumulation. The ABA treatment showed no significant effect on levels of the conjugated form of Put and Spd. Imbibition with millimolar concentrations of MGBG resulted in (i) abnormal accumulation of the free form of Put and incorporation of 14C-amino acids into the diamine, (ii) progressive inhibition of the accumulation of the free forms of 14C-Spd and Spm. and (iii) reduction of the 14C incorporation into the conjugated forms of Put and Spd. Uptake of 14C-amino acids was not affected by MGBG treatment. The results indicate that MGBG may inhibit not only the synthesis of Spd and Spm, but the catabolism (e.g. oxidation) of Put in the aleurone layer.

Stabilization of ornithine decarboxylase and N1-spermidine acetyltransferase in rat liver by methylglyoxal bis(guanylhydrazone)

The activities of ornithine decarboxylase and spermidine N 1-acetyltransferase started to rise in normal rat liver 4 h after the intraperitoneal injection of methylglyoxal bis(guanylhydrazone) (MGBG; 80 mg/kg). Ornithine decarboxylase had its greatest activity 24 h after a single injection of MGBG and the acetyltransferase peaked 8 h after the injection. Measurement of the apparent half-life of ornithine decarboxylase after MGBG treatment revealed a clear decrease in the decay rate of the enzyme in both normal and regenerating rat liver. MGBG slowed the decay of the transferase also in normal rat liver, as well as inhibiting its activity in vitro. The stabilization by MGBG of these two short-lived proteins involved in metabolism of polyamines should lead to their accumulation in liver, thus explaining their increased activities. In the case of ornithine decarboxylase, studies with a specific antibody against mouse kidney ornithine decarboxylase showed that the rise in ornithine decarboxylase activity after MGBG application was not due to the appearance of an immunologically different isozyme.

Polyamine Metabolism and Its Relation to Response of the Aleurone Layers of Barley Seeds to Gibberellic Acid

Polyamine metabolism and its relation to the induction of alpha-amylase formation in the aleurone layers of barley seeds (Hordeum vulgare cv Himalaya) in response to gibberellic acid (GA(3)) has been investigated. A high-performance liquid chromatographic system has been employed for qualitative and quantitative analyses of putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm), and agmatine (Agm).Active polyamine metabolism occurs in the aleurone cells of deembryonate barley half seeds during imbibition. The aleurone layers isolated from fully imbibed half seeds contain about 880 nanomoles of Put, 920 nanomoles of Spd, and 610 nanomoles of Spm as free form per gram tissue dry weight while the levels of Cad and Agm are relatively low. The polyamine levels do not change significantly in the aleurone layers in response to added GA(3) (1.5 micromolar) during the 8-hour lag period of the growth substance-induced formation of alpha-amylase. Also, the polyamine levels are not altered by the presence of abscisic acid (3 micromolar) which inhibits the enzyme induction by GA(3). Kinetic studies show that both applied [U-(14)C]ornithine and [U-(14)C]arginine are primarily incorporated into Put during 2 hours of incubation, but the incorporation is not significantly affected by added GA(3). Additionally, added GA(3) does not affect the uptake and turnover of [1,4-(14)C]Put, nor does it affect the conversion of Put --> Spd or Spd --> Spm. Treatment of the aleurone layers with GA(3) for 2 hours results in no significant changes in the total activities or the specific activities of ornithine decarboxylase and arginine decarboxylase.Experiments with polyamine synthesis inhibitors demonstrate that the level of Spd in the aleurone layers could be substantially reduced by the presence of methylglyoxal-bis(guanylhydrazone) (MGBG) during imbibition. MGBG treatment does not affect in vivo incorporation of [8-(14)C] adenosine into ATP. The lower the level of Spd the less alpha-amylase formation is induced by added GA(3). The reduction of GA(3)-induced alpha-amylase formation by MGBG treatment can be either completely or partially overcome by added Spd, depending upon the concentration of MGBG used in the imbibition medium. The results indicate that the early action of GA(3), with respect to induction of alpha-amylase formation in barley aleurone layers, appears to be not on polyamine metabolism. However, polyamines, particularly Spd, may be involved in regulation of the growth substance-dependent enzyme induction.

Potentiation of 1,3-Bis(2-chloroethyl)-1-nitrosourea cytotoxicity in 9L rat brain tumor cells by methylglyoxal-bis(guanylhydrazone), an inhibitor of S-adenosyl- l-methionine decarboxylase

Methylglyoxal-bis(guanylhydrazone) (MGBG), a potent inhibitor of the spermidine and spermine biosynthetic enzyme S-adenosyl- l-methionine decarboxylase, enhanced the cytotoxicity of 1,3-bis-(2-chlorethyl)-1-nitrosourea in 9L rat brain tumor cells in vitro, as measured by a colony-forming efficiency assay, by an amount that was approximately the same as the potentiation caused by the ornithine decarboxylase inhibitor α-difluoromethylornithine. Dose enhancement ratios at 10, 1 and 0.1% survival levels were approximately 1.3 for both inhibitors. 9L cells that were treated for 48 hr with 40 μ M MGBG had putrescine, spermidine and spermine levels that were 112, 41 and 21% respectively, of polyamine levels in control cells. MGBG treatment does not increase intracellular levels of decarboxylated S-adenosyl- l-methionine (AdoMet) as α-difluoromethylornithine treatment does. Elevated levels of decarboxylated AdoMet could modify intracellular methylation reactions and could affect the cytotoxicity of a chloroethylnitrosourea. Despite the fact that MGBG treatment caused a slight increase in intracellular levels of AdoMet, it is unlikely that this elevation will increase the amount of intracellular methylation. Thus it appears that effects caused by the decrease in polyamine levels are responsible for the potentiation of chloroethylnitrosourea cytotoxicity against 9L cells.

Thermosensitization by Methylglyoxal Bis(guanylhydrazone) in Chinese Hamster Cells

The modification of methylglyoxal bis(guanylhydrazone) (MGBG) by 42 degrees C hyperthermia-and/or radiation-induced cell killing was examined in Chinese hamster V-79 cells. At concentrations of more than 10 microM, cell survival decreased exponentially with increased MGBG exposure times. Cell lethality of MGBG (10 microM) was not specific for cell-cycle phases tested from G1/S through G2. When cells were treated with MGBG (10 microM) for 6 hr and then exposed to 42 degrees C hyperthermia with or without a 24-hr interval, cell survival decreased markedly compared with that for 42 degrees C alone. Cells became thermosensitive after MGBG treatment. Cells exposed to MGBG (10 microM) for 6 hr before or after X irradiation were slightly radiosensitive. When X irradiation was combined with MGBG and 42 degrees C hyperthermia, cells became more radiosensitive. From these results, it is suggested that MGBG may change the intracellular state to sensitize cells to the cytotoxic action(s) of hyperthermia.

Inhibition of DNA and Protein Synthesis in UV-Irradiated Mouse Skin by 2-Difluoromethylornithine, Methylglyoxal bis(Guanylhydrazone), and Their Combination

Exposure of mouse skin to UVB irradiation greatly enhanced the biosynthesis and accumulation of putrescine and spermidine before or concomitantly with stimulation of epidermal macromolecular (DNA and protein) synthesis. Topical treatment of UV-exposed skin with 2 inhibitors of polyamine biosynthesis, 2-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG) prevented the enhanced epidermal accumulation of polyamines, especially spermidine, and also inhibited the incorporation of radioactive precursors into DNA and protein. When applied in combination, these 2 antimetabolites of polyamines produced an inhibition of macromolecular synthesis that was at least additive: [3H]thymidine incorporation decreased by 80% and [14C]leucine incorporation by 44% as compared with the UVB-irradiated control mice. A slight decrease in the ratio of [3H]histidine/[14C]leucine incorporation indicated that protein synthesis of the differentiating cell layers was also affected by the inhibitors. The effects of the combined DFMO and MGBG treatment were partially reversed by concomitant topical application of spermidine.

Enhancement of thermal cell killing by methylglyoxal bis(guanylhydrazone) in Chinese hamster V-79 cells.

Using the method of colony formation, the modifying effect of methylglyoxal bis(guanylhydrazone) (MGBG) was examined on 44 degrees hyperthermia- and/or radiation-induced cell killing and on thermotolerance development after 44 degrees exposure in Chinese hamster V-79 cells. After exposure to MGBG (10 microM) for more than 6 hr, cells became more sensitive to 44 degrees hyperthermia. When an interval at 37 degrees (up to 24 hr) was interposed between MGBG and hyperthermic treatments, cell survival was not significantly increased, as compared with that for MGBG immediately followed by 44 degrees. Cells treated with MGBG remained more thermosensitive, even after a 24 hr interval. On the other hand, when cells were exposed to split dose 44 degrees hyperthermia in the presence of MGBG during an interval of 6 hr at 37 degrees, the development of thermotolerance was not inhibited. After the combined treatments of MGBG followed by X-ray irradiation and hyperthermia, cell survival was markedly decreased, as compared with that in the case of X-ray irradiation alone or in combination with hyperthermia.