Diacridines: Bifunctional intercalators II. The biological effects of putrescine,spermidine and spermine diacridines on HeLa cells and on the L-1210 and P-388 leukemia cells

The effect of putrescine, spermidine and spermine diacridines on the growth of HeLa cells and of P-388 and L-1210 leukemia cells has been evaluated and compared to that of the parent compound, 9-aminoacridine. The diacridines are more effective growth inhibitors than 9-aminoacridine. The primary site of action appears to be the inhibition of RNA synthesis.     

Isolation and charaterization of surface glycoproteins from L-1210, P-388 and HeLa cells

A method is described that permits the rapid extration of the cell surface glycoproteins of two murine leukemic cells, the P-388 and the L-1210 cells as well as those of the human adenocarcinoma cells, the HeLa cells.Proof of the surface location of these glycoproteins is provided by labeling the intact cells; (a) with ¹²⁵I by the lactoperoxidase iodination technique; (b) with ³H by the galactose oxidase-reductive tritiation method. Most of these glycoproteins were also shown to incorporate radioactive glucosamine and fucose. By these criteria as well as by the distribution of molecular weights, the surface glycoproteins of the two murine cells are indistinguishable; however, they differ markedly from the surface glycoproteins of HeLa cells. The extracts of the murine cells wee shown to contain lectin receptor activity as determined by their ability to inhibit the lectin-induced agglutination of the intact cells.     

Diacridines: bifunctional intercalators. III. Definition of the general site of action.

Electron microscopy of HeLa cells exposed to spermine diacridine shows nucleolar distortions which disappear after several days despite the persistence of the metabolic changes promoted by spermine diacridine. This compound inhibits ribosomal RNA synthesis and appears to act independently of any particular phase of the cell cycle. The DNA content of the HeLa cells remains unchanged and the cell distribution is not significantly disturbed from its normal distribution in the various phases of the cell cycle. Spermine diacridine and other diacridines inhibit primarily chain initiation but also chain elongation by DNA-directed RNA polymerase of Azotobacter vinelandii.     

Diacridines: bifunctional intercalators. I. Chemistry, physical chemistry and growth inhibitory properties.

The synthesis, as well as the rationale for synthesis of diacridines, double intercalators, as potential inhibitors of nucleic acid synthesis is presented. The syntheses of (9-acridyl)-putrescine and -spermine, and bis(-9-acridyl)-putrescine, -spermidine, -spermine diamines and of bis(6-chloro-2-methoxy-9-acridyl)-putrescine and -spermine diamines, all substituted on the terminal NH2 groups are described. In addition, the homologous series of diacridines connected by the amino groups of the diamines NH2(CH2)nNH2 (where n = 2,3,4,6,8,10,12,14,16,18) to the C-9 of the diacridines has been synthesized. The chemical properties of these compounds as well as their molecular relationship to DNA are presented. The effect of the double intercalators on the Tm of DNA and of (A)n - (U)n, (dA)n - (dT)n, (G)n - (C)n and on (dG)n - (dC)n have been determined. The double acridine intercalators produce a much greater increase of the Tm of these nucleic acids than do the single acridine intercalators. They also profoundly affect the Tm of DNA in physiological salt concentrations; under these latter conditions the single intercalators have no effect. The relationship between the length of the chain connecting the two acridine rings and the inhibition of the growth of P-388 cells in vitro and vivo is presented. Their growth inhibitory properties appear, in general, to parallel their intercalative abilities.     

Action of avermectins on lymphoid leukemia P-388 cells in vitro]

Avermectins are final products in the fermentation process with Streptomyces avermitilis. They have parasitocidic activity and are used as the main substances of insectoacaronematocides. The study of the activity of the natural avermectin complex (aversectin C) and separate avermectins A1, A2, B1 and B2 in the cell culture of lymphoid leukemia P-388 showed that within the concentrations of 0.1 to 1.0 microgram/ml aversectin C inhibited the growth of the tumor cells and induced their death. The inhibition was due to blocking the cell mitosis. The cell death was accompanied by internucleosomal degradation of the DNA nuclei i.e. the death was of the apoptosis type. The sensitivity of the cells to aversectin C was directly proportional to their initial proliferative activity. As for the separate avermectins only avermectin A1 had the cytotoxic activity within the concentrations used, avermectin A2 had the cytostatic activity and avermectins B1 showed no activity under the experimental conditions.     

Decreased sensitivity of an interferon-resistant subline of murine leukemia L-1210 cells to toxic effects of ricin and abrin.

Approximately two to four times higher concentrations of the plant toxins abrin and ricin are required to inhibit protein synthesis in interferon-resistant L-1210R cells to the same degree as in interferon-sensitive L-1210S cells. However, amounts of interferon 10-fold higher than those required for protection from viral infection fail to show any effect on ricin intoxication of L-1210S cells.     

DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators.

The DNA unwinding effects of some 9-aminoacridine derivatives were compared under reaction conditions that could be used to study drug-induced topoisomerase II inhibition. An assay was designed to determine drug-induced DNA unwinding by using L1210 topoisomerase I. 9-aminoacridines could be ranked by decreasing unwinding potency: compound C greater than or equal to 9-aminoacridine greater than o-AMSA greater than or equal to compound A greater than compound B greater than m-AMSA. Ethidium bromide was more potent than any of the 9-aminoacridines. This assay is a fast and simple method to compare DNA unwinding effects of intercalators. It led to the definition of a drug intrinsic unwinding constant (k). An additional finding was that all 9-aminoacridines and ethidium bromide inhibited L1210 topoisomerase I. Enzyme inhibition was detectable at low enzyme concentrations (less than or equal to 1 unit) and when the kinetics of topoisomerase I-mediated DNA relaxation was studied. Topoisomerase I inhibition was not associated with DNA swivelling or cleavage.     

Association of putrescine,spermidine, spermine,and GABA with structural elements of brain cells

The present experiments are the first survey of the association of endogenous and exogenous putrescine, spermidine, and spermine with subcellular structures of rat brain cortex. The differences of distribution in subfractions obtained from salt-free and salt-containing density gradients were studied, with the following results: (1) In contrast with liver preparation, putrescine and the polyamines spermidine and spermine are not distributed in parallel with RNA. (2) In salt-containing media, putrescine and the polyamines were preferentially associated with synaptosomes and with synaptosomal membranes. Significant association with myelin constituents was observed only in salt-free media. (3) Exogenous putrescine and the polyamines were less firmly attached to synaptosomes and to synaptosomal membrane fractions than the endogenous amines. There is good evidence for similar subcellular localizations of putrescine and GABA. Putrescine seems to be entrapped in the nerve endings. (4) Uptake studies with crude mitochondria under conditions of high-affinity uptake showed no temperature-sensitive component of polyamine accumulation in synaptosomes, in contrast with GABA, monoacetylputrescine, and ornithine. (5) Polyamines bound to myelin constituents or mitochondria could be displaced by a 200-fold concentration of nonradioactive amines; this was not the case with polyamines bound to synaptosomes. Mg²⁺ did not effectively compete with spermine for binding sites at synaptic regions. (6) Electrical stimulation and stimulation by mono- and bivalent cations did not change the concentrations of the polyamines and GABA in guinea pig cortex. (7) There is no evidence for a neurotransmitter role of putrescine, spermidine, or spermine, although these compounds might function as modulators of neurotransmission.     

The effect of finoptin on doxorubicin accumulation in leukemia P-388 cells with induced resistance to the combination of finoptin and doxorubicin

Using hybrid mice BDF1 doxorubicin (Dx) accumulation has been determined in leukemia P388 cells (P388/0), P388 cells with induced resistance to Dx (P388/Dx) and P388 cells with induced resistance to the finoptin (Fp) + Dx combination (P388/Fp + Dx). It has been shown that Fp doesn't affect Dx accumulation in or elimination from leukemia cells P388/0 or P388/Fp + Dx. The resistance of P388/Fp + Dx cells to the Fp + Dx combination develops during 6 passages. It can be concluded that Fp application doesn't abolish the problem of tumor cells' resistance to cytostatics.     

The roles of the polyamines, putrescine, spermidine, and spermine in normal and malignant tissues

Despite the initial aversion to polyamine research which can be attributed to their peculiar nomenclature and to the erroneous idea that polyamines are products of bacterial decay, it appears that these ubiquitous amines play important roles in the physiological regulation of growth. Many of the definitive roles are yet to be elucidated, and these areas offer promise to biochemists. The evolution of the multifaceted ramifications of polyamines is not unlike that for cyclic AMP, which has profound effects at the cellular level. Further, we are at a stage at which basic knowledge of the roles of polyamines is becoming clinically relevant. We should begin to measure polyamines routinely to obtain clinical parameters which might allow for more efficacious treatment of cancer. No longer can the discussion of polyamines in biochemical textbooks be limited to a page and a half or no discussion at all, and no longer can well-informed scientists afford to neglect the importance and the far-reaching applications of polyamine research.     

An early enlargement of the putrescine pool is required for growth in L1210 mouse leukemia cells under hypoosmotic stress

Hypoosmotic stress is a potent inducer of ornithine decarboxylase (ODC) activity in a variety of mammalian cells, but the physiological relevance of this response has not been determined. To test whether an increased putrescine content confers a growth advantage at lower osmolarities, we compared the ability of L1210 mouse leukemia cells and of ODC-overproducing variants obtained from this cell line (D-R cells) to proliferate after a hypotonic shock (325----130 mosmol/kg). The growth rate of D-R cells at 130 mosmol/kg was greater than or equal to 5-fold higher than in L1210 cells; and unlike the ODC-overproducing strain, L1210 cells underwent up to a 90% loss of viability over time as seen after restoration of normosmotic growth conditions and by trypan blue exclusion tests. The addition of putrescine or L-ornithine stimulated the proliferation of both cell sublines up to 5-fold in a concentration-dependent manner, with a maximal effect observed at about 10 and 100 microM, respectively. Putrescine restored virtually normal growth rates in both sublines at osmolarities as low as 190 mosmol/kg. No other alpha,omega-diamine was active in that respect whereas spermidine was markedly inhibitory. Furthermore, D-R cells incubated at 130 mosmol/kg showed a marked growth inhibition by 1-aminooxy-3-aminopropane (potent ODC inhibitor to which they are resistant in isotonic media) as a result of putrescine but not spermidine depletion. Whereas ODC was strongly and rapidly induced by hypotonic shock there was a precipitous decline in S-adenosylmethionine decarboxylase activity. Putrescine synthesis and accumulation were nevertheless reduced in D-R cells incubated at 130 mosmol/kg because of a decreased availability of L-ornithine. When either putrescine or L-ornithine was added to hypotonic media, D-R cells accumulated putrescine massively for extended periods together with a reduction in spermidine and spermine contents. Putrescine transport patterns were altered by hypotonic shock, net excretion of the diamine being reduced by about 80%, with a concurrent enlargement of the intracellular pool. Finally, parental L1210 cells incubated with an irreversible inhibitor of S-adenosylmethionine decarboxylase for 24 h until hypotonic shock and supplemented with putrescine in the presence of the drug thereafter exhibited a greatly exaggerated growth stimulation by the diamine. These results demonstrate an essential role for an early increase in putrescine content in the growth adaptation of a mammalian cell line to a lower osmolarity.     

Stimulation of RNA polymerases I and II from mouse L1210 leukemia and Ehrlich ascites carcinoma cells by spermine and spermidine and its modification by ammonium sulfate

Nuclear RNA polymerases from murine L1210 leukemia and Ehrlich carcinoma cells were stimulated more effectively by spermine than by spermidine. Optimal stimulatory concentrations of spermine and spermidine for Ehrlich polymerases Ia and Ib decreased to physiological values and maximal stimulation increased as the concentration of (NH4)2SO4 was reduced from 0.08 to 0 M. In the presence of 0.062-0.074 M (NH4)2SO4 L1210 polymerases Ia, IIa and IIb were stimulated significantly by both polyamines, whereas, at (NH4)2SO4 concentrations of 0.11-0.17 M, stimulation was suppressed and high concentrations of the polyamines were inhibitory. Similarly, stimulation of Ehrlich solubilized polymerase by polyamines was inhibited by 0.064 M (NH4)2SO4.     

High performance liquid chromatography of the dansyl derivatives of putrescine, spermidine, and spermine

A high performance liquid chromatographic (HPLC) method, based on dansylation and fluorescence detection, is described for the estimation of putrescine, spermidine, and spermine in lichen (Evernia prunastri [L.]) samples. Because of the high concentrations of phenols and salts, dansylation was followed by a pre-HPLC purification step. Both flow rate and mobile phase (methanol:water) followed a gradient for optimum resolution on a reverse-phase column. Amounts as small as 0.3 picomole of standard polyamines could be detected. In applying the method to lichens, it was found that 5.45% (w/w) of the exogenous putrescine taken up by the thallus was unbound in the algal partner and that 60% (w/w) was conjugated in the thallus, perhaps to lichen phenolics.     

Purification of topoisomerase II from amsacrine-resistant P388 leukemia cells. Evidence for two forms of the enzyme

Topoisomerase II was purified from an amsacrine-resistant mutant of P388 leukemia. A procedure has been developed which allows the rapid purification of nearly homogeneous enzyme in quantities sufficient for enzyme studies or production of specific antisera. The purified topoisomerase II migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two bands with apparent molecular masses of 180 (p180) and 170 kDa (p170); both proteins unknotted P4 DNA in an ATP-dependent manner and displayed amsacrine-stimulated covalent attachment to DNA. Staphylococcus V8 protease cleavage patterns of p170 and p180 showed distinct differences. Specific polyclonal antibodies to either p170 or p180 recognized very selectively the form of the enzyme used to generate the antibodies. Immunoblotting with these specific antibodies showed that both p180 and p170 were present in cells lysed immediately in boiling sodium dodecyl sulfate. Comparison of the purified topoisomerase II from amsacrine-resistant P388 with that from amsacrine-sensitive P388 demonstrated that each cell type contained both p180 and p170; however, the relative amounts of the two proteins were consistently different in the two cell types. The data strongly suggest that p170 is not a proteolytic fragment of p180. Thus, P388 cells appear to contain two distinct forms of topoisomerase II.     

The effects of nicotine on cell division of HeLa cells.

A time lapse cinemicrographic study shows that, at low concentrations, nicotine can speed up cytokinesis and, at high concentrations, prolong the duration of metaphase in HeLa cells. Based upon the known effects of the alkaloid in other systems, it appears that it may stimulate microfilament activity and inhibit microtubule function by increasing the level of cytosolic calcium ions.