Effects of anticancer drugs on transcription in vitro.

The effects of DNA interacting drugs on: (1) total RNA synthesis catalyzed by E. coli and T7 RNA polymerase; (2) synthesis of the initiating dinucleotide (pppApU) by E. coli RNA polymerase ("abortive initiation"); (3) elongation of RNA chains synthesized by T7 RNA polymerase on pT7-7 plasmid DNA bearing T7 RNA polymerase promoter phi 10 with human Cu/Zn superoxide dismutase coding sequence, (4) interaction of transcription factor Sp1 and its binding site were studied. Intercalating ligands which form quickly dissociating complexes with DNA (anthracyclines, proflavine, ethidium bromide) are compared with the slowly dissociating drug of d(G x C) specificity (actinomycin D), the non-intercalating, d(A x T) specific pyrrole antibiotics (netropsin and distamycin A) and covalently binding to DNA 1-nitroacridine derivative (nitracrine). The obtained results indicate that rapidly dissociating ligands, proflavine and ethidium bromide, inhibit total RNA synthesis in vitro and the abortive initiation to a similar extent while they do not induce discrete elongation stops of RNA polymerase. Actinomycin D and nitracrine exhibit a high inhibitory effect on total RNA synthesis and induce stops of RNA polymerase while not affecting abortive initiation. Pyrrole antibiotics primarily inhibit the initiation, while no elongation stops are induced. Actinomycin D inhibits complex formation between nuclear proteins and the Sp1 binding site. Netropsin, ethidium bromide, proflavine and other intercalating acridines do not affect Sp1 binding. The results indicate that the effects primarily depend on sequence specificity and secondarily on the dissociation rate of ligands from their complexes with DNA.

Inhibition of RNA synthesis in vitro and cell growth by anthracycline antibiotics.

New derivatives of doxorubicin and daunorubicin with amidine group bonded to daunosamine at C-3' atom and bearing the morpholine ring attached to the amidine group have been recently synthesized. Their cytotoxic activities and effects on RNA synthesis in vitro were assayed. The drug concentrations inhibiting mouse leukaemia L1210 cell growth to 50% were about two- and three fold higher for the derivatives compared to doxorubicin and daunorubicin respectively. Inhibition of phage T7 RNA polymerase by the non-covalently interacting derivatives was also slightly lower than that by the parent compounds. As doxorubicin and daunorubicin, their amidine derivatives in the presence of dithiothreitol and Fe(III) ions are activated and covalently bind to DNA. The adducts formed affect RNA polymerase activity. Several bands corresponding to prematurely terminated RNA chains are observed by means of polyacrylamide gel electrophoresis. The patterns of bands are virtually identical for all the anthracyclines studied here and are similar to the terminations induced by actinomycin D. This observation is consistent with a notion that the adducts are formed at guanine in GpC sequences which are also binding sites of actinomycin D. A substantial difference between daunorubicin and its amidine derivative is shown by means of high performance liquid chromatography. The derivative undergoes rapid rearrangements in the presence of dithiothreitol and Fe(III) ions, while daunorubicin is stable for several hours under these conditions. The results presented here indicate that the amidine derivatives despite bulky morpholine substitution exhibit biological activity in the systems used here.

Crosslinking of cellular DNA by nitracrine and furocoumarin derivatives.

The anticancer drug, nitracrine, a 1-nitro-9-aminoalkyl derivative of acridine exhibits potent cytotoxic effects which are due to its metabolic activation, followed by covalent binding to macromolecules--DNA being the target for the drug. The renaturable fraction of DNA from L-1210 cells pretreated with nitracrine is assayed by means of ethidium bromide fluorescence assay and chromatography on hydroxyapatite column. The effect of the drug was compared with furocoumarins of different DNA crosslinking potencies. The existence of crosslinks in DNA upon incubation of cells with nitracrine (1-4 microM) have been confirmed with two different methods under the conditions where 8-methoxypsoralen, a classic crosslinking agent induced the renaturation. The DNA preparation isolated from the drug pretreated cells exhibited decreased transcriptional template activity with E. coli DNA-dependent RNA polymerase.

Effect of DNA-interacting drugs on phage T7 RNA polymerase.

9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands.

Deleterious effects of androstenedione on growth and cell morphology of Schizosaccharomyces pombe.

Androgens (androstenedione and testosterone) belong to the most important compounds in human steroidogenesis. The 17 beta-hydroxysteroid dehydrogenase responsible for interconversion of the oxygenic group on C-17 of androgens ring is involved in steroid hormone synthesis. The fission yeast Schizosaccharomyces pombe 972 h- was found to contain constitutive 17 beta-hydroxysteroid dehydrogenase that was able to reduce androstenedione to testosterone and oxidize testosterone to androstenedione. The reductive pathway was found to be predominant while the oxidative one was carried out with much lower activity. Exogenous androstenedione, contrary to testosterone, inhibited S. pombe growth and stimulated the formation of aberrant swollen cells with slighter cell wall sensitivity to the action of the lytic enzyme Novozym. It is postulated that the 17 beta-hydroxysteroid dehydrogenase prevents the deleterious effects of androstenedione on the morphology and growth of the yeast's cells by androstedione reduction to testosterone.

Inhibition of RNA synthesis in vitro by acridines--relation between structure and activity.

The effects of acridine derivatives (proflavine and 2,7-dialkyl derivatives, diacridines and triacridines, 9-aminoacridine carboxamides, and 9-anilinoacridine, amsacrine and its congeners) on overall RNA synthesis in vitro, on synthesis of initiating oligonucleotides and the binding of the enzyme to DNA were studied. The primary mechanism of action is related to inhibition of the enzyme binding to DNA. The acridines (intercalating or non-intercalating and bis-intercalating ligands) assayed here differ in the properties of their complexes with DNA. Correlation is generally observed between inhibition of RNA synthesis in vitro and cytotoxicity in cell cultures for di- and triacridines and 9-aminoacridine carboxamide derivatives. No relationship was found between the effect on RNA polymerase system and biological effects for amsacrine and its derivatives in contrast to the other series of acridines studied here. The aniline ring seems to decrease the inhibitory potency of a ligand. The discrepancy between the biological effect and RNA synthesis inhibition may be due to a different mechanism of cytotoxicity action of amsacrine which is a potent topoisomerase II poison.

The use of cryopreserved apical protoplasts from Curvularia lunata for electrotransformation.

An electroporation method, utilizing cryopreserved protoplasts, has been developed for the steroid 11-hydroxylating fungus Curvularia lunata strain IM 2901. Protoplasts released from the apical parts of 24- and 48-h-old mycelia were suspended in cryopreservation buffer and stored at -75 degrees C for several weeks. The thawed and freshly prepared (control) protoplasts were electroporated with pAN 7-1 plasmid carrying the Escherichia coli hygromycin B resistance gene (hph) under the control of Aspergillus nidulans sequences. The electroporation efficiency of the control protoplasts with plasmid pAN 7-1 was 7.5 and 12.0 transformants per microgram DNA (protoplasts liberated from 24- and 48-h-old mycelia, respectively). Protoplasts released from the younger mycelium were more stable according to their reversion ability to mycelial form and transformation efficiency. After 16 weeks of cryopreservation the yield of electroporation was 61.3% of the control value. All isolated electrotransformants proved to be stable for a period of > 4 months even without selective pressure.

Transcriptional template activity of covalently modified DNA.

The transcriptional template activity of covalent modified DNA is compared. 8-Methoxypsoralen (MOP), 3,4'dimethyl-8-methoxypsoralen (DMMOP) and benzopsoralen (BP) forming with DNA covalent complexes upon UV irradiation and exhibiting preference to pyrimidines, mostly thymines, differ in their cross-linking potency. MOP and DMMOP form both monoadducts and diadducts while no cross-links are formed by BP. Nitracrine (NC) forms covalent complexes with DNA upon reductive activation with dithiothreitol exhibiting a preference to purines and low cross-linking potency. Semilogarithmic plots of the relative template activity against the number of the drugs molecules covalently bound per 10(3) DNA nucleotides fit to regression lines corresponding to one-hit inactivation characteristics. The number of drug molecules decreasing RNA synthesis to 37% differ from 0.25 to 1.26 depending on the template used and the base preference but no dependence on the cross-linking potency was found.

Photoreaction of furocoumarins with DNA is similarly inhibited by minor and major groove-interacting ligands.

It was found that methyl green, a major groove binding ligand and the minor groove binding ligands, netropsin and 2,7-di-tert-butylproflavine inhibit, to a similar extend a monoadduct forming benzopsoralen and monoadduct and diadduct forming derivatives of psoralen (8-methoxypsoralen and 3,4'-dimethyl-8-methoxypsoralen). Caffeine exhibits an inhibitory effect on furocoumarin photobinding to DNA at 10(3) fold higher concentration. Together with the previously published results it is concluded that both occupancy of the major and minor groove as well as intercalation hinder photobinding of furocoumarins to DNA.

Elimination of by-products in 11 beta-hydroxylation of substance S using Curvularia lunata clones regenerated from NTG-treated protoplasts.

Stable mutants showing improved 11-hydroxylation of Substance S were isolated, following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and regeneration of uninucleate protoplasts of the appropriate fungal strains. This procedure was especially suitable for obtaining more directed 11 beta-hydroxylation of Substance S with Curvularia lunata IM 2901. Apart from producing cortisol (11 beta-hydroxy-S), the parent strain formed several by-products that significantly lowered the yield of the desired 11 beta-hydroxyderivative. Isolated mutants of this microorganism carried out directed 11 beta-hydroxylation with only a small amount of one of the by-products, which resulted in a much higher yield of cortisol.

In vitro binding of nitracrine to DNA in chromatin.

In the presence of sulfhydryl compounds nitracrine, an anticancer drug, binds covalently to DNA. The accessibility of DNA in chromatin both to nitracrine and to 8-methoxypsoralen, which was used as a reference compound in this study, when assayed in NaCl concentrations from 0 to 2 M show similar characteristics. The initial decrease reaches a minimum at 0.15 M NaCl above which dissociation of non-histone proteins and histones at higher ionic strengths is demonstrated by an increase in accessible sites. The relative accessibility of DNA in chromatin to nitracrine is, however, lower than that found for 8-methoxypsoralen. Partial dissociation of chromatin with 0.7 M NaCl increases the accessibility of DNA in chromatin when assayed in the absence of NaCl but has no apparent influence when estimated at ionic strength close to physiological conditions.

DNA binding, cytotoxicity and inhibitory effect on RNA synthesis of two new 1-nitro-9-aminoacridine dimers.

Two 1-nitro-9-aminoacridine dimers were prepared: one bearing a spermine flexible linking chain, compound 4, the other a rigid dipiperidine-type linker, compound 7. Both dimers elicited a higher affinity constant for DNA than the parent monomeric drug nitracrine 2. This affinity was several orders lower than what was found for other dimeric compounds having the same linkers and no nitro group on the acridine ring (3, 5, 6 and 8). Bisintercalation was evidenced for compound 4 by viscosimetric measurements. In the absence of dithiothreitol, an inhibitory effect of RNA synthesis in vitro was observed for all the tested compounds except 2 and 7. In the presence of dithiothreitol, 4 and 7 formed irreversible complexes with DNA of decreased template properties. The level of the dimers binding was lower than that of the parent compound 2. Cross-links were detected by means of hydroxylapatite chromatography in a complex of the dimer bearing a flexible linking chain, compound 4 with DNA, while the compound 7-DNA complex eluted in the single-stranded DNA region. The extent of cytotoxicity of the two 1-nitro-9-aminoacridine dimers against L1210 cultured cells was different.

Inhibition of DNA-dependent RNA synthesis by 8-methoxypsoralen.

The effect of the photobinding of 8-methoxypsoralen to phage T7 DNA on different steps of RNA synthesis in vitro was assayed. Total RNA synthesis is reduced to a few percent and the transcript size is decreased, as shown by means of gel filtration on a Sepharose 4B column when DNA of the adduct content of six drug molecules per 10(3) nucleotides is used. The initiation of RNA chains seems to be less affected, as inferred from an abortive initiation assay. Synthesis of pppApU on DNA of the same adduct content is inhibited to 34% of the corresponding controls, while the overall RNA synthesis is inhibited to 6%. The amount of the enzyme needed for maximal retention of DNA, the kinetics of its binding and the decay of the polymerase-DNA complex at high ionic strength (or on decrease of the temperature) are similar with DNA either irradiated in the absence of the drug or DNA bearing six 8-methoxypsoralen molecules per 10(3) nucleotides. It is concluded from this study that 8-methoxypsoralen partially inhibits initiation and blocks movement of RNA polymerase along the template, inducing premature termination. It does not appear to influence the binding of the enzyme to DNA.

Thyroliberin and other neurohormones have no detectable direct effect on RNA synthesis in isolated nuclei.

Effect of thyroliberin, somatostatin, vasopressin and luliberin on RNA synthesis in isolated calf pituitary, brain cortex, rat liver and rat brain nuclei was assayed. None of the examined neuropeptides affected RNA synthesis.

Potential antitumor agents. 45. Synthesis, DNA-binding interaction, and biological activity of triacridine derivatives.

A series of amide-linked triacridines of varying interchromophore separation were synthesized as potential DNA trisintercalating agents. The corresponding diamide diacridines (lacking the central chromophore) were also prepared, and the DNA-binding and biological activities of both series of compounds were evaluated. Although one of the triacridines shows evidence of a trisintercalative binding mode, most of the triacridines (and all the diacridines) bound by bisintercalation. The diacridines showed great cytotoxicity and higher DNA association constants than the corresponding 9-[[3-(dimethylamino)propyl]amino]acridine monomer, but addition of a third chromophore did not provide corresponding increases in either DNA affinity, inhibition of RNA synthesis, or cytotoxicity. Some members of both series show minimal in vivo antileukemic activity. The results suggest that further development of trimeric molecules as potential antitumor agents should focus on smaller chromophores with lower capacity for nonspecific binding and/or the employment of rigid linker chains to provide true molecular "staples" for DNA.

Effect of intercalating and groove-binding ligands on formation of covalent complexes between nitracrine (Ledakrin, C-283) or 8-methoxypsoralen and DNA.

The effect of ethidium bromide, actinomycin D, distamycin A and netropsin on covalent binding of nitracrine (1-nitro-9-(3,3-N,N-dimethylaminopropylamino)acridine, Ledakrin, C-283) and 8-methoxypsoralen to DNA was examined. The competition was assayed either directly with [3H]- and [14C]nitracrine or indirectly by estimation of transcriptional template activity of nitracrine-DNA and 8-methoxypsoralen-DNA complexes formed in the presence of the ligands. A higher protective effect of ethidium bromide and distamycin on the photo-binding of 8-methoxypsoralen than on the dithiothreitol-dependent attachement of nitracrine to DNA assayed at 0.15 M KCl or NaCl was observed. The non-intercalating antibiotics showed lower competitive effect on 8-methoxypsoralen binding than ethidium bromide. Actinomycin D showed relatively low competition for both drugs with DNA. In contrast to the reaction of 8-methoxypsoralen, the decrease of nitracrine binding in the presence of competing ligands considerably depends on ionic strength. Particularly high inhibition of the adduct formation in the presence of ethidium at 1 M KCl was shown, while the amount of nitracrine bound in the presence of distamycin increases at elevated ionic strength. The results may indicate steric demands of the reaction between nitracrine and DNA.

Factors regulating the steroid 11-hydroxylation by non-germinating spores of Cunninghamella elegans (Lendner).

In the presence of malate or citrate sporangiospores of C. elegans were able to hydroxylate cortexolone with a rate twofold exceeding that of the control, water suspended spores. Analysis of the intracellular nicotinamide coenzyme pools revealed an increased NADPH:(NADP+ + NADPH) ratio, indicating more effective NADPH-generating systems in malate- or citrate-stimulating spores. Swollen spores remaining in the pregermination state, retained higher cortexolone-hydroxylating activity in the absence of malate and citrate. In these spores degradation of endogenous alanine and glutamic acid was observed. Possible NADPH-generating systems in C. elegans sporangiospores were discussed.

Thiol-dependent inhibition of RNA synthesis in vitro by acridines: structure-inhibition relationships.

In the presence of sulfhydryl compounds an anticancer drug, 1-nitro-9-aminoalkylacridine derivative, forms with DNA irreversible, probably covalent, complexes of decreased template properties. Five 9-substituted 1-nitro-9-aminoacridine derivatives of cytostatic activity show irreversible thiol-dependent inhibitory effects on the RNA synthesis in vitro system while equal inhibition is observed both in the presence and in the absence of dithiothreitol with biologically inactive analogues of nitrocrine. In the absence of sulfhydryl compounds the inhibition depends on the planarity of the acridine ring. Hence, both 1-nitro-9-aminoalkylacridine and tetrahydroacridine derivatives show low inhibitory effect.

Substance P isolated from bovine hypothalamus: amino acid composition.

Using the method of Chang and Leeman for isolation the sialogogic undecapeptide from bovine hypothalamus partly purified Substance P was obtained. The final steps of purification were omitted to prevent losing of other peptides possessing biological activity of Substance P. The qualitative and quantitative content of amino acids in the hypothalamic extract was determined and fourteen amino acids were found. Four of them were not present in the sialogogic undecapeptide or neurotensin. It can be concluded that in the hypothalamic tissue apart from sialogogic undecapeptide and neurotensin, there are other peptides related to these two.