Modulation of secreted plasminogen activator activity of human renal carcinoma cells by dimethylsulfoxide, butyrate and retinoate.

Dimethylsulfoxide, butyrate and retinoic acid, agents which induce differentiation of certain malignant cells, were examined for their effect on the activity of plasminogen activator (PA) of serumless conditioned medium (CM) of two human renal carcinoma cell lines. All three agents produced a decrease in PA activity. More than 90% of the PA was secreted in latent form, and this was not altered by the agents. Active PA components of Mr 52,000 and 93,000 were identified in cell secretions by zymography. In the presence of DMSO or butyrate the Mr 52,000 component was markedly reduced. Reversibility of the effect on PA was demonstrated for both DMSO and retinoic acid with activity of CM returning to control level after removal of the agent. That the effect was temporary agrees with most observations of the effects of these and similar agents on cells from solid tumors.

Characterization of plasminogen activator from two human renal carcinoma cell lines.

Plasminogen activator (PA) activity was identified in the conditioned medium of two human renal carcinoma cell lines, Cur and Caki-1. PA activity of medium, following chromatography on Con A-Sepharose, was divided into effluent and eluate fractions, the latter obtained after elution with methyl mannoside. The ratio of PA activity in effluent:eluate was 90:10 for Caki-1 and 60:40 for Cur. The PA of both effluent fractions and the Caki-1 eluate fraction was of the urokinase (UK) type. Identification rested on molecular weight determination by zymography (major component with Mr 52,000 and a less prominent component of 93,000), lack of binding to fibrin, inhibition by anti-UK antibodies, and lack of inhibitory effect of anti-tissue type PA (TPA) antibodies or the Erythrina trypsin inhibitor, which inhibits TPA but not UK. PA of the Cur eluate fraction gave a more complex pattern in that it bound significantly to fibrin (like TPA), was completely inhibited by both anti-UK and anti-TPA antibodies, but was unaffected by Erythrina trypsin inhibitor. These results raise the possibility of an unusual PA-like enzyme that immunologically cross reacts with anti-UK and anti-TPA. Most of the PA of both cell lines was secreted in a latent form that could be activated by trypsin treatment. The latency appears to result largely from secretion of urokinase proenzyme, which is consistent with the Mr 52,000 of the major PA species and the insensitivity to diisopropyl fluorophosphate inhibition prior to trypsin activation. However, in addition, a UK binding component was found in the conditioned medium, which produced an Mr 93,000 component by reaction with UK.

Hymenolepis diminuta: effects of amoscanate on energy metabolism and ultrastructure.

Amoscanate possesses chemotherapeutic activity against schistosomes, and in higher doses against many other helminths including filariids and Hymenolepis diminuta. The primary mode of action of this compound is unknown. Effects of the drug on the carbohydrate metabolism as well as on the tegumental and nephridial epithelia of H. diminuta were examined. At various time intervals after administration of the drug to rats infected with H. diminuta, the parasites were recovered and incubated in glucose-salts medium for 90 min. Chemotherapy resulted in decreases in succinate, lactate, and acetate recoveries, while ATP levels dropped. In addition, glycogen levels were depressed in drug-treated worms which were homogenized immediately upon isolation. Glycogen synthase I activity was inhibited 16-61% in cestodes obtained from Amoscanate-treated animals and homogenized immediately, but returned to normal levels after incubation for 90 min in glucose-salts medium prior to homogenization and assay. Phosphorylase a activity was found to be 25-30% higher in preparations of worms from drug-treated rats, which correlates with the rapid depletion of glycogen in parasites exposed to the drug. However, in contrast with glycogen synthase activity, the elevation of phosphorylase a activity in H. diminuta exposed to the drug was not readily reversible. Attempts to demonstrate activity of the drug in vitro by incubating intact cestodes directly with Amoscanate were unsuccessful. Thin sections of parasites obtained from Amoscanate-treated rats and examined by transmission electron microscopy revealed surface alterations of the tegument and nephridial canals. Alterations included bleb formation and erosion of microtriches from the tegument, as well as disappearance of microvilli from nephridial canals. However, these effects became manifest only after 4 or more hr exposure of the rat to the drug. Biochemical effects, on the other hand, were significant after 3 hr exposure.

Polyamine metabolism in isoproterenol-stimulated mouse salivary glands.

Isoproterenol (0.3 micronmole/gm body weight) was injected intraperitoneally every 24 h for three days. The synthesis of deoxyribonucleic acid, the concentration of putrescine, spermidine and spermine and the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were measured in parotid and submandibular glands at 4 to 8 h after each injection. The parotid glands responded with peaks of DNA synthesis at 24 and 72 h and peaks of putrescine content and decarboxylase activities 8 to 12 h after each injection. Spermidine increased steadily in the parotid, whereas there was little change in the spermine concentration throughout the 72 h. Polyamine metabolism showed much less response in the submandibular gland, and little or no increase in spermidine or spermine levels or in DNA synthesis was observed.

Effects of polyamines and methylglyoxal bis(guanylhydrazone) on Escherichia coli ribonucleic acid polymerase and the template activity of hepatic cell nuclei in vitro.

Escherichia coli RNA polymerase was assayed with 4 mM Mg2+ and 1 mM Mn2+ using native DNA, heat-denatured DNA, histone-nucleate and isolated rat liver nuclei as the template source. With purified DNA and either or both divalent metal ions, 0.1--5 mM amine stimulated enzyme activity. Spermidine resulted in the greatest stimulation (1.7-fold at 5 mM); whereas, spermine or methylglyoxal bis(guanylhydrazone) first stimulated, then above 3 mM inhibited, the reaction. The addition of unfractionated histone to purified DNA inhibited the reaction by 90%. The subsequent addition of amines resulted in a slight stimulation in incorporation (1.5-fold) in the range of 1--3 mM amine. Alternatively, when enzyme was combined with DNA before histone, only a 20% inhibition was observed and this could be completely prevented by 3 mM spermidine. The addition of amines to isolated nuclei resulted in marked alterations in ultrastructure and Mg2+ content; however, relatively small effects on RNA polymerase activity were observed. With the E. coli enzyme, 0.1--1.0 mM amine stimulated RNA synthesis (1.5-fold) whereas, none of the amines stimulated endogeneous activity in the absence or presence of 300 mM (NH4)2SO4.

Effects of polyamines and methylglyoxal bis(guanylhydrazone) on hepatic nuclear structure and deoxyribonucleic acid template activity.

1. The interaction of polyamines and methylglyoxal bis(guanythydrazone) (1, 1'-[(methylethanediylidene)-dinitrilo]diguanidine) with isolated rat liver nuclei was investigated by electron microscopy. 2. At 4mM, putrescine was without effect; however, spermidine, spermine or methylglyoxal bis(guanythydrazone) resulted in dispersed chromatin and alterations in nucleolar structure. In addition, spermidine or methylglyoxal bis(guanylhydrazone) caused marked aggregation of interchromatin granules. 3. The DNA template property of calf thymus DNA was examined by using DNA polymerases from Escherichia coli, Micrococcus lysodeikticus and calf thymus in the presence of 0-5 mM-amine. 4. In the presence of DNA polymerase, spermine or methylglyoxal bis(guanylhydrazone) inhibited activity, whereas putrescine or spermidine had much less effect or in some cases stimulated [3H]dTMP incorporation. 5. Template activity which was inhibited by spermine or methylglyoxal bis(guanylhydrazone) could be partially restored by additional DNA or enzyme. 6. When mixed with calf thymus DNA, calf thymus histone inhibited template activity as measured with E. coli DNA polymerase. The template activity of such a 'histone-nucleate' could not be restored by putrescine, spermidine, spermine or methylglyoxal bis(guanylhydrazone). 7. DNA template activity of isolated rat liver nuclei was tested by using E. coli DNA polymerase. None of the amines was able to increase the template activity of the nuclear DNA in vitro.