Differential toxicity of methylglyoxal-bis [guanylhydrazone].

The toxicity to cultured cells of the cancer chemotherapeutic agent methylglyoxal-bis[guanylhydrazone] (MGBG) varies considerably between different cell lines and is always more toxic in the absence of exogenous polyamine. We have looked at the relative MGBG toxicity of two different murine melanoma tumour cell lines (Harding-Passey and Cloudman) and normal murine fibroblasts, and found wide variation with no correlation in sensitivity to MGBG between tumour and normal cells. High sensitivity to MGBG may be associated with high transport across the cell membrane.

Possible association of radioprotective and chemoprotective aminophosphorothioate drug activity with polyamine oxidase susceptibility.

The aminophosphorothioate drug S-2-(3-aminopropylamino)ethylphosphorothioate (WR-2721) is both radioprotective and chemoprotective, with potential clinical use in cancer therapies. These distinct properties may be associated with its catabolism by polyamine oxidase (EC 1.4.3.4), which is found in varying amounts in different body tissues. Unfortunately, the metabolite is probably a cytotoxic aldehyde, but it should be detoxified in vivo following adduction with tissue sulfhydryls. Whether conversion and adduction benefit or hinder pharmacologic activity is not known, inasmuch as aldehydes may reduce oxygen-dependent free radicals generated by irradiation of tissues. S-2-(3-aminopropylamino)propylphosphorothioate was similarly a substrate for polyamine oxidase, and the product was cytotoxic. Other aminophosphorothioates [S-2-(4-aminobutaneamino)ethylphosphorothioate, S-2-(5-aminopentylamino)ethylphosphorothioate, and S-2-(4-aminobutaneamino)propylphosphorothioate] were poor substrates and less radioprotective.

Hog kidney diamine oxidase conversion of biogenic diamines to inhibitors of cell proliferation.

Hog kidney diamine oxidase (DAO) interacted with 1,3-diaminopropane, putrescine and cadaverine to arrest proliferation of cultured mammalian cells. The byproducts of DAO-substrate interaction, H2O2 and NH4+ are themselves cytotoxic but were apparently not responsible for any significant antiproliferative effect in the experimental system. DAO is known to react with putrescine to generate labile 4-aminobutyraldehyde. This primary product was compatible with a compound (radio-labelled) separated from DAO-putrescine reactive mixtures by ion-exchange chromatography. Its rate of generation, level attained and lability (half-life, 2 hr) in culture simulated conditions were measured. Circumstantial evidence suggested that the amine aldehyde products of diamine oxidation had a potent antiproliferative effect on cells.

Inhibition of proliferation of cultured cells by polyamine and cysteine interaction.

Both L-cysteine and spermidine oxidation inhibited the proliferation of cultured cells when these reagents were added in low concentrations. Higher concentrations (above 1 mM) of cysteine were not cytotoxic. Furthermore, high concentrations of L-cysteine partially protected against spermidine oxidation toxicity. D-cysteine on its own behaved exactly the same as an equimolar concentration of L-cysteine, but instead of protecting against the effects of spermidine oxidation, the inhibition was enhanced. A possible mechanism for the interaction is discussed.

Oxygen-dependent free radicals in spermine oxidation cytostatis and chemiluminescence and the role of superoxide dismutase.

Spermine interacted with serum polyamine oxidase (PAO) to arrest proliferation of cultured Bri8 lymphocytes. Arrest was independent of catalase activity and was not directly due to an H(2)O(2) byproduct. Arrest was averted by 3-hydroxybenzyloxyamine, which inactivates the pyridoxal co-factor of PAO. The oxidation of spermine in the presence of different concentrations of PAO was non-linear, which implied complex intermediate events for conversion of spermine to labile di-oxidized spermine (N,N'-bis(3-propionaldehyde)-1,4-butanediamine) with, perhaps, overall generation of free radicals (O(2) (-·) and ·OH) which are damaging to cells. Exogenous free radicals were apparently neither direct participants in cytostasis, nor in the chemiluminescence demonstrable for spermine oxidation. Thiourea, an ·OH scavenger, protected against both proliferation arrest and luminescence. Many other powerful ·OH scavengers, however, were ineffective. Though reaction mixtures reduced ferricytochrome c initially, reduction was not inhibited by superoxide dismutase (SOD) which indicated that the anion O(2) (-·) had not been generated. The powerful reducing capability of di-oxidized spermine itself could have competed against any O(2) (-·) for ferricytochrome c reduction. Nevertheless, O(2) (-·) was generated during further PAO conversion and/or auto-oxidation of di-oxidized spermine. Curiously, addition of SOD to destroy presumptive O(2) (-·) variably potentiated cytotoxicity. Blockage of any anion channels in the cell plasma membrane by stilbene derivatives did not influence cytotoxicity. Thus, findings support our previous evidence that cationic di-oxidized spermine is a potent G(1) inhibitor of cell proliferation. The possibility of intracellular free-radical and thiol involvement is discussed.

Inhibition of lymphocyte proliferation by polyamines requires ruminant-plasma polyamine oxidase.

Spermine and spermidine in vitro are potent inhibitors of proliferation of phytohaemagglutinin-stimulated rat thymic lymphocytes, lymphoma cells and human lymphoblastic leukaemia cells, but only in media supplemented by foetal calf serum. This inhibition is shown to be due to a bovine plasma polyamine oxidase, with a high specificity for these polyamines. Spontaneously dividing lymphocytes are not subject to this inhibition. This, plus direct evidence from synchronous cultures of EB2 cells demonstrates that the inhibition is expressed in the late G1 or G1/S interface of the cell cycle. Putrescine was not an inhibitor in the presence of foetal calf serum but became so in the presence of human pregnancy serum, possibly due to the action of diamine oxidase.

Evidence for serum binding of oxidized spermine and its potent G1-phase inhibition of cell proliferation.

Serum polyamine oxidase (EC 1.4.3.4) is known to react in vitro with radio-labelled spermine4+ to produce di-oxidized spermine which must incorporate the label. Di-oxidized spermine was compatible with a radio-labelled compound2+ separated from the reaction mixture by ion-exchange chromatography. The compound was measured and had a half-life of about 2.3 h in tissue culture medium. It also rapidly and tightly bound to an unidentified serum component (gel-filtration chromatography indicated a complex of mol. wt 70,000) so that dissociation required treatment with strong acid (10N HCl). Findings suggest that the di-oxidized spermine, in either its free cationic or bound form, potently arrested cell proliferation. This arrest was non-cytotoxic and was confined to the G1 phase of the cell cycle. Products of di-oxidized spermine autodegradation, including trace amounts of stable and cytotoxic acrolein (arrested S phase), were unlikely to have contributed significantly to the arrest.

Polyamine interaction with pregnancy serum in suppression of lymphocyte transformation.

Biogenic polyamines interacting with pregnancy serum elicit potent suppression of lymphocyte transformation in vitro. Within the assay limits, activity was first shown after about 15 weeks' gestation and reached its highest level at about 28 weeks. This level was maintained until term. It is thought that specific humoral amine oxidases were the cause. Fetal-cord serum and non-pregnancy serum were inactive. This system may have an immunoregulatory function in pregnancy.

Maternal-fetal mixed lymphocyte reactivity in pre-eclampsia.

Two-way mixed lymphocyte reactivity (2-way-MLR) between maternal and fetal lymphocytes in vitro, and one-way mixed lymphocyte reactivity (1-way-MLR) of maternal lymphocytes (responders) to stimulation by mitomycin-treated fetal lymphocytes (stimulators), were investigated in normal and pre-eclamptic pregnancies. No relationship was observed between pre-eclampsia and the 2-way-MLR. The 1-way-MLR was lowest in normal pregnancy, higher in mild pre-eclampsia, and highest in severe pre-eclampsia, although none of these differences were statistically significant.

Spontaneous lymphocyte transformation in pregnancies complicated by pre-eclampsia.

There was no significant difference between the mean spontaneous transformation rates of maternal lymphocytes from normal pregnant women and patients with pre-eclampsia.

Immunological control of polyoma virus oncogenesis in mice.

Adult CBA mice thymectomized, treated with antilymphocytic globulin (ALG) and inoculated with human leprosy organisms were accidentally infected with polyoma virus and all developed tumours. After cessation of ALG administration, some animals were given spleen cells from syngeneic donors immunized with polyoma virus; none developed tumours. Similar results were obtained in mice deliberately infected with polyoma virus but not with leprosy organisms. Passive transfer of antibody before but not after virus inoculation prevented tumour formation in immunosuppressed recipients. Virus infection in thymectomized, lethally irradiated and bone marrow reconstituted mice resulted in only a very low incidence of tumours. These results emphasize the role of immunological surveillance in preventing polyoma tumour formation under natural conditions.